Influence of inoculum density on population dynamics and dauer juvenile yields in liquid culture of biocontrol nematodes Steinernema carpocapsae and S. feltiae (Nematoda: Rhabditida).

PubMed

Hirao, Ayako; Ehlers, Ralf-Udo

2010-01-01

For improvement of mass production of the rhabditid biocontrol nematodes Steinernema carpocapsae and Steinernema feltiae in monoxenic liquid culture with their bacterial symbionts Xenorhabdus nematophila and Xenorhabdus bovienii, respectively, the effect of the initial nematode inoculum density on population development and final concentration of dauer juveniles (DJs) was investigated. Symbiotic bacterial cultures are pre-incubated for 1 day prior to inoculation of DJs. DJs are developmentally arrested and recover development as a reaction to food signals provided by their symbionts. After development to adults, the nematodes produce DJ offspring. Inoculum density ranged from 1 to 10 x 10(3) DJ per milliliter for S. carpocapsae and 1 to 8 x 10(3) DJs per milliliter for S. feltiae. No significant influence of the inoculum density on the final DJ yields in both nematode species was recorded, except for S. carpocapsae cultures with a parental female density <2 x 10(3) DJs per milliliter, in which the yields increased with increasing inoculation density. A strong negative response of the parental female fecundity to increasing DJ inoculum densities was recorded for both species with a maximum offspring number per female of >300 for S. carpocapsae and almost 200 for S. feltiae. The compensative adaptation of fecundity to nematode population density is responsible for the lack of an inoculum (or parental female) density effect on DJ yields. At optimal inoculation density of S. carpocapsae, offspring were produced by the parental female population, whereas S. feltiae always developed a F1 female population, which contributed to the DJ yields and was the reason for a more scattered distribution of the yields. The F1 female generation was accompanied by a second peak in X. bovienii density. The optimal DJ inoculum density for S. carpocapsae is 3-6 x 10(3) DJs per milliliter in order to obtain >10(3) parental females per milliliter. Density-dependent effects were neither observed on the DJ recovery nor on the sex ratio in the parental adult generation. As recovery varied between different batches, assessment of the recovery of inoculum DJ batches is recommended. S. feltiae was less variable in DJ recovery usually reaching >90%. The recommended DJ inoculum density is >5 x 10(3) DJs per milliliter to reach >2 x 10(3) parental females per milliliter. The mean yield recorded for S. carpocapsae was 135 x 10(3) and 105 x 10(3) per mililiter for S. feltiae.

Economic and kinetic studies of the biological production of farm energy and chemicals from biomass. Annual progress report, March 1979-August 1980

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gaddy, J.L.

1981-08-01

A system has been designed and built to test the technical and economic feasibility of producing farm energy from cellulosic residues. The system has the capacity to produPropinibactriume 1300 CF of biogas and 180 kwh per day, using four parallel batch anaerobic digesters which are agitated mechanically and heated with waste heat from the engine-generator. This system has been satisfactorily operated for a one year period utilizing native grasses as the raw material. Laboratory reactors have been operated to determine the proper inoculation ratio for starting batch cultures with cellulosic raw materials. Procedures for startup and operation of batch digestionmore » systems have been developed and are presented. Energy and economic analyses of the operation of the test unit have been performed. Studies to determine the fertilizer potential of anaerobic digestor sludge (from agricultural residues) have been conducted. Additional studies reported include determinations of the feasibility of separating the stages of anaerobic digestion, the production of acetic and propionic acids by the micro-organism Propionibacterium acidi-propionici, the production of methane from acid hydrolyzates, and the kinetics of the continuos digestion of corn stover.« less

Comparison of different liquid anaerobic digestion effluents as inocula and nitrogen sources for solid-state batch anaerobic digestion of corn stover

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu Fuqing; Shi Jian; Lv Wen

2013-01-15

Highlights: Black-Right-Pointing-Pointer Compared methane production of solid AD inoculated with different effluents. Black-Right-Pointing-Pointer Food waste effluent (FWE) had the largest population of acetoclastic methanogens. Black-Right-Pointing-Pointer Solid AD inoculated with FWE produced the highest methane yield at F/E ratio of 4. Black-Right-Pointing-Pointer Dairy waste effluent (DWE) was rich of cellulolytic and xylanolytic bacteria. Black-Right-Pointing-Pointer Solid AD inoculated with DWE produced the highest methane yield at F/E ratio of 2. - Abstract: Effluents from three liquid anaerobic digesters, fed with municipal sewage sludge, food waste, or dairy waste, were evaluated as inocula and nitrogen sources for solid-state batch anaerobic digestion of cornmore » stover in mesophilic reactors. Three feedstock-to-effluent (F/E) ratios (i.e., 2, 4, and 6) were tested for each effluent. At an F/E ratio of 2, the reactor inoculated by dairy waste effluent achieved the highest methane yield of 238.5 L/kgVS{sub feed}, while at an F/E ratio of 4, the reactor inoculated by food waste effluent achieved the highest methane yield of 199.6 L/kgVS{sub feed}. The microbial population and chemical composition of the three effluents were substantially different. Food waste effluent had the largest population of acetoclastic methanogens, while dairy waste effluent had the largest populations of cellulolytic and xylanolytic bacteria. Dairy waste also had the highest C/N ratio of 8.5 and the highest alkalinity of 19.3 g CaCO{sub 3}/kg. The performance of solid-state batch anaerobic digestion reactors was closely related to the microbial status in the liquid anaerobic digestion effluents.« less

Bacteriophage lytic to Desulfovibrio aespoeensis isolated from deep groundwater.

PubMed

Eydal, Hallgerd S C; Jägevall, Sara; Hermansson, Malte; Pedersen, Karsten

2009-10-01

Viruses were earlier found to be 10-fold more abundant than prokaryotes in deep granitic groundwater at the Aspö Hard Rock Laboratory (HRL). Using a most probable number (MPN) method, 8-30 000 cells of sulphate-reducing bacteria per ml were found in groundwater from seven boreholes at the Aspö HRL. The content of lytic phages infecting the indigenous bacterium Desulfovibrio aespoeensis in Aspö groundwater was analysed using the MPN technique for phages. In four of 10 boreholes, 0.2-80 phages per ml were found at depths of 342-450 m. Isolates of lytic phages were made from five cultures. Using transmission electron microscopy, these were characterized and found to be in the Podoviridae morphology group. The isolated phages were further analysed regarding host range and were found not to infect five other species of Desulfovibrio or 10 Desulfovibrio isolates with up to 99.9% 16S rRNA gene sequence identity to D. aespoeensis. To further analyse phage-host interactions, using a direct count method, growth of the phages and their host was followed in batch cultures, and the viral burst size was calculated to be approximately 170 phages per lytic event, after a latent period of approximately 70 h. When surviving cells from infected D. aespoeensis batch cultures were inoculated into new cultures and reinfected, immunity to the phages was found. The parasite-prey system found implies that viruses are important for microbial ecosystem diversity and activity, and for microbial numbers in deep subsurface groundwater.

Indigenous microbial capability in solid manure residues to start-up solid-phase anaerobic digesters.

PubMed

Yap, S D; Astals, S; Jensen, P D; Batstone, D J; Tait, S

2017-06-01

Batch solid-phase anaerobic digestion is a technology for sustainable on-farm treatment of solid residues, but is an emerging technology that is yet to be optimised with respect to start-up and inoculation. In the present study, spent bedding from two piggeries (site A and B) were batch digested at total solids (TS) concentration of 5, 10 and 20% at mesophilic (37°C) and thermophilic (55°C) temperatures, without adding an external inoculum. The results showed that the indigenous microbial community present in spent bedding was able to recover the full methane potential of the bedding (140±5 and 227±6L CH 4 kgVS fed -1 for site A and B, respectively), but longer treatment times were required than for digestion with an added external inoculum. Nonetheless, at high solid loadings (i.e. TS level>10%), the digestion performance was affected by chemical inhibition due to ammonia and/or humic acid. Thermophilic temperatures did not influence digestion performance but did increase start-up failure risk. Further, inoculation of residues from the batch digestion to subsequent batch enhanced start-up and achieved full methane potential recovery of the bedding. Inoculation with liquid residue (leachate) was preferred over a solid residue, to preserve treatment capacity for fresh substrate. Overall, the study highlighted that indigenous microbial community in the solid manure residue was capable of recovering full methane potential and that solid-phase digestion was ultimately limited by chemical inhibition rather than lack of suitable microbial community. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comparative analysis of microbial community of novel lactic acid fermentation inoculated with different undefined mixed cultures.

PubMed

Liang, Shaobo; Gliniewicz, Karol; Mendes-Soares, Helena; Settles, Matthew L; Forney, Larry J; Coats, Erik R; McDonald, Armando G

2015-03-01

Three undefined mixed cultures (activated sludge) from different municipal wastewater treatment plants were used as seeds in a novel lactic acid fermentation process fed with potato peel waste (PPW). Anaerobic sequencing batch fermenters were run under identical conditions to produce predominantly lactic acid. Illumina sequencing was used to examine the 16S rRNA genes of bacteria in the three seeds and fermenters. Results showed that the structure of microbial communities of three seeds were different. All three fermentation products had unique community structures that were dominated (>96%) by species of the genus Lactobacillus, while members of this genus constituted <0.1% in seeds. The species of Lactobacillus sp. differed among the three fermentations. Results of this study suggest the structure of microbial communities in lactic acid fermentation of PPW with undefined mixed cultures were robust and resilient, which provided engineering prospects for the microbial utilization of carbohydrate wastes to produce lactic acid. Copyright © 2014 Elsevier Ltd. All rights reserved.

Model-based strategy for cell culture seed train layout verified at lab scale.

PubMed

Kern, Simon; Platas-Barradas, Oscar; Pörtner, Ralf; Frahm, Björn

2016-08-01

Cell culture seed trains-the generation of a sufficient viable cell number for the inoculation of the production scale bioreactor, starting from incubator scale-are time- and cost-intensive. Accordingly, a seed train offers potential for optimization regarding its layout and the corresponding proceedings. A tool has been developed to determine the optimal points in time for cell passaging from one scale into the next and it has been applied to two different cell lines at lab scale, AGE1.HN AAT and CHO-K1. For evaluation, experimental seed train realization has been evaluated in comparison to its layout. In case of the AGE1.HN AAT cell line, the results have also been compared to the formerly manually designed seed train. The tool provides the same seed train layout based on the data of only two batches.

Production of nattokinase by batch and fed-batch culture of Bacillus subtilis.

PubMed

Cho, Young-Han; Song, Jae Yong; Kim, Kyung Mi; Kim, Mi Kyoung; Lee, In Young; Kim, Sang Bum; Kim, Hyeon Shup; Han, Nam Soo; Lee, Bong Hee; Kim, Beom Soo

2010-09-30

Nattokinase was produced by batch and fed-batch culture of Bacillus subtilis in flask and fermentor. Effect of supplementing complex media (peptone, yeast extract, or tryptone) was investigated on the production of nattokinase. In flask culture, the highest cell growth and nattokinase activity were obtained with 50 g/L of peptone supplementation. In this condition, nattokinase activity was 630 unit/ml at 12 h. In batch culture of B. subtilis in fermentor, the highest nattokinase activity of 3400 unit/ml was obtained at 10h with 50 g/L of peptone supplementation. From the batch kinetics data, it was shown that nattokinase production was growth-associated and culture should be harvested before stationary phase for maximum nattokinase production. In fed-batch culture of B. subtilis using pH-stat feeding strategy, cell growth (optical density monitored at 600 nm) increased to ca. 100 at 22 h, which was 2.5 times higher than that in batch culture. The highest nattokinase activity was 7100 unit/ml at 19 h, which was also 2.1 times higher than that in batch culture. Copyright 2010 Elsevier B.V. All rights reserved.

ENVIRONMENTAL BENIGN MITIGATION OF MICROBIOLOGICALLY INFLUENCED CORROSION (MIC)

DOE Office of Scientific and Technical Information (OSTI.GOV)

J.R. Paterek; G. Husmillo; V. Trbovic

The overall program objective is to develop and evaluate environmental benign agents or products that are effective in the prevention, inhibition, and mitigation of microbially influenced corrosion (MIC) in the internal surfaces of metallic natural gas pipelines. The goal is one or more environmental benign, a.k.a. ''green'' products that can be applied to maintain the structure and dependability of the natural gas infrastructure. The technical approach for this quarter were isolation and cultivation of MIC-causing microorganisms from corroded pipeline samples, optimizing parameters in the laboratory-scale corrosion test loop system and testing the effective concentrations of Capsicum sp. extracts to verifymore » the extent of corrosion on metal coupons by batch culture method. A total of 22 strains from the group of heterotrophic, acid producing, denitrifying and sulfate reducing bacteria were isolated from the gas pipeline samples obtained from Northern Indiana Public Service Company in Trenton, Indiana. They were purified and will be sent out for identification. Bacterial strains of interest were used in antimicrobial screenings and test loop experiments. Parameters for the laboratory-scale test loop system such as gas and culture medium flow rate; temperature; inoculation period; and length of incubation were established. Batch culture corrosion study against Desulfovibrio vulgaris showed that one (S{sub 1}M) out of the four Capsicum sp. extracts tested was effective in controlling the corrosion rate in metal coupons by 33.33% when compared to the untreated group.« less

Inhibition of ochratoxigenic moulds by Debaryomyces hansenii strains for biopreservation of dry-cured meat products.

PubMed

Andrade, Maria J; Thorsen, Line; Rodríguez, Alicia; Córdoba, Juan J; Jespersen, Lene

2014-01-17

The ability of the osmotolerant yeast Debaryomyces hansenii to inhibit Penicillium nordicum, the most common ochratoxigenic mould encountered in dry-cured meat products, was evaluated. The antagonistic effect of ten D. hansenii strains isolated from dry-cured ham was screened in vitro using malt extract media and meat extract peptone media with the water activity (a(w)) adjusted to 0.97 and 0.90. A significant inhibition of the two tested P. nordicum strains by D. hansenii cells and cell-free supernatants was observed. At 0.97 a(w), increasing D. hansenii inoculum concentrations significantly improved the inhibition of mould growth on solid medium, whereas at 0.90 a(w) this was not always the case. As observed by bright field microscopy, most D. hansenii strains were able to delay P. nordicum spore germination when co-cultured in malt extract broth. D. hansenii FHSCC 253H showed the highest overall in vitro inhibition of ochratoxigenic mould growth, and was therefore chosen for co-cultivation assays in dry-cured ham slices incubated at 0.94 and 0.84 a(w) simulating ham ripening. Regardless of the experimental conditions tested, lower levels of the inoculated P. nordicum strain were detected in co-cultivation batches compared with batches without D. hansenii. The highest level of mould growth inhibition was observed in batches at 0.94 a(w). Ochratoxin A (OTA) production in ham samples was detected by HPLC-MS. Co-culturing of P. nordicum with D. hansenii FHSCC 253H resulted in lower OTA levels compared with control samples without D. hansenii. The decrease of the mycotoxin presence due to D. hansenii FHSCC 253H was more efficient at 0.94 a(w) (OTA was below the detection limit). In conclusion, D. hansenii is potentially suitable as a biopreservative agent for preventing ochratoxigenic mould growth and OTA accumulation in dry-cured meat products. The inoculation of D. hansenii should be made at the beginning of processing (at the end of post salting) when the a(w) of the product is still high (near 0.94). This action in addition to application of appropriate hygienic actions and control of temperature and relative humidity throughout ripening is required to reduce health risks due to OTA exposure. Copyright © 2013 Elsevier B.V. All rights reserved.

An Automated Sample Preparation Instrument to Accelerate Positive Blood Cultures Microbial Identification by MALDI-TOF Mass Spectrometry (Vitek®MS).

PubMed

Broyer, Patrick; Perrot, Nadine; Rostaing, Hervé; Blaze, Jérome; Pinston, Frederic; Gervasi, Gaspard; Charles, Marie-Hélène; Dachaud, Fabien; Dachaud, Jacques; Moulin, Frederic; Cordier, Sylvain; Dauwalder, Olivier; Meugnier, Hélène; Vandenesch, Francois

2018-01-01

Sepsis is the leading cause of death among patients in intensive care units (ICUs) requiring an early diagnosis to introduce efficient therapeutic intervention. Rapid identification (ID) of a causative pathogen is key to guide directed antimicrobial selection and was recently shown to reduce hospitalization length in ICUs. Direct processing of positive blood cultures by MALDI-TOF MS technology is one of the several currently available tools used to generate rapid microbial ID. However, all recently published protocols are still manual and time consuming, requiring dedicated technician availability and specific strategies for batch processing. We present here a new prototype instrument for automated preparation of Vitek ® MS slides directly from positive blood culture broth based on an "all-in-one" extraction strip. This bench top instrument was evaluated on 111 and 22 organisms processed using artificially inoculated blood culture bottles in the BacT/ALERT ® 3D (SA/SN blood culture bottles) or the BacT/ALERT Virtuo TM system (FA/FN Plus bottles), respectively. Overall, this new preparation station provided reliable and accurate Vitek MS species-level identification of 87% (Gram-negative bacteria = 85%, Gram-positive bacteria = 88%, and yeast = 100%) when used with BacT/ALERT ® 3D and of 84% (Gram-negative bacteria = 86%, Gram-positive bacteria = 86%, and yeast = 75%) with Virtuo ® instruments, respectively. The prototype was then evaluated in a clinical microbiology laboratory on 102 clinical blood culture bottles and compared to routine laboratory ID procedures. Overall, the correlation of ID on monomicrobial bottles was 83% (Gram-negative bacteria = 89%, Gram-positive bacteria = 79%, and yeast = 78%), demonstrating roughly equivalent performance between manual and automatized extraction methods. This prototype instrument exhibited a high level of performance regardless of bottle type or BacT/ALERT system. Furthermore, blood culture workflow could potentially be improved by converting direct ID of positive blood cultures from a batch-based to real-time and "on-demand" process.

Survival and growth of Listeria monocytogenes and enterohemorrhagic Escherichia coli O157:H7 in minimally processed artichokes.

PubMed

Sanz, Susana; Giménez, Mercedes; Olarte, Carmen

2003-12-01

The ability of Listeria monocytogenes and Escherichia coli O157:H7 inoculated by immersion (at 4.6 and 5.5 log CFU/ g, respectively) to survive on artichokes during various stages of preparation was determined. Peeling, cutting, and disinfecting operations (immersion in 50 ppm of a free chlorine solution at 4 degrees C for 5 min) reduced populations of L. monocytogenes and E. coli O157:H7 by only 1.6 and 0.8 log units, respectively. An organic acid rinse (0.02% citric acid and 0.2% ascorbic acid) was more effective than a tap water rinse in removing these pathogens. Given the possibility of both pathogens being present on artichokes at the packaging stage, their behavior during the storage of minimally processed artichokes was investigated. For this purpose, batches of artichokes inoculated with L. monocytogenes or E. coli O157:H7 (at 5.5 and 5.2 log CFU/g, respectively) were packaged in P-Plus film bags and stored at 4 degrees C for 16 days. During this period, the equilibrium atmosphere composition and natural background microflora (mesophiles, psychrotrophs, anaerobes, and fecal coliforms) were also analyzed. For the two studied pathogens, the inoculum did not have any effect on the final atmospheric composition (10% O2, 13% CO2) or on the survival of the natural background microflora of the artichokes. L. monocytogenes was able to survive during the entire storage period in the inoculated batches, while the E. coli O157:H7 level increased by 1.5 log units in the inoculated batch during the storage period. The modified atmosphere was unable to control the behavior of either pathogen.

Sanitizer efficacy in preventing cross-contamination of heads of lettuce during retail crisping.

PubMed

Jung, Yangjin; Jang, Hyein; Guo, Mengqi; Gao, Jingwen; Matthews, Karl R

2017-06-01

This study was conducted to provide information regarding mitigation of cross-contamination through the use of sanitizer during crisping at retail outlets. Seven non-inoculated heads and one inoculated head (≈5 log CFU/g) of lettuce were placed into commercial sink filled with 76 L of tap water (TW), electrolyzed water (EW, free chlorine: 43 ± 6 ppm), lactic acid and phosphoric acid-based sanitizer (LPA, pH 2.89), or citric acid-based sanitizer (CA, pH 2.78) and soaked for 5 min. Two subsequent batches (eight non-inoculated heads per batch) were soaked in the same solution. Soaking with EW significantly reduced the population of S. enterica (2.8 ± 1.5 log CFU/g), E. coli O157:H7 (3.4 ± 1.1 log CFU/g), and L. monocytogenes (2.6 ± 0.7 log CFU/g) inoculated on Romaine lettuce compared to TW, LPA, and CA (p < 0.05). On Red leaf lettuce, EW significantly reduced populations of S. enterica and E. coli O157:H7, but not L. monocytogenes compared to other treatments. No significant difference was noted between TW, LPA, and CA in reducing foodborne pathogens (p > 0.05) or preventing cross-contamination. Soaking with EW prevented cross-contamination among lettuce heads and controlled bacterial populations in crisping water for three consecutive batches. EW may be an effective option as a sanitizer to minimizing the cross-contamination of leafy greens during the retail crisping. Copyright © 2017 Elsevier Ltd. All rights reserved.

The start-up of denitrifying phosphorus removal system by using nitrite as electron acceptor

NASA Astrophysics Data System (ADS)

Li, W.; Liu, J.; Sun, H. Z.; Fu, J. X.; Gao, Y.; Sun, J.

2017-08-01

The inoculation of short-cut denitrifying polyphosphate-accumulating organisms (DPAOs) mainly included two-phase inoculation and three-phase inoculation. The short-cut denitrifying phosphorus removal bacteria were quickly inoculated by sequencing batch reactor (SBR) to treatment domestic wastewater. The results showed that the average effluent concentration of TP was 0.85 mg/L after 132 cycles by 44 days in two-phase inoculation. The removal rates of NO2 --N, TP and COD were 94.73%, 95.47% and 89.96% after 126 cycles by 42 days in three-phase inoculation, and the effluent concentrations were separately 1.31 mg/L, 0.45 mg/L and 17.07 mg/L, which reached the first A class requirement of Urban sewage treatment plant pollutant discharge standard. It was indicated that the efficiency of three-phase inoculation was higher. Anoxic phosphorus uptake was influenced seriously by anaerobic residual carbon, and it was the difference of the two inoculations.

Hydrogen production from microbial strains

DOEpatents

Harwood, Caroline S; Rey, Federico E

2012-09-18

The present invention is directed to a method of screening microbe strains capable of generating hydrogen. This method involves inoculating one or more microbes in a sample containing cell culture medium to form an inoculated culture medium. The inoculated culture medium is then incubated under hydrogen producing conditions. Once incubating causes the inoculated culture medium to produce hydrogen, microbes in the culture medium are identified as candidate microbe strains capable of generating hydrogen. Methods of producing hydrogen using one or more of the microbial strains identified as well as the hydrogen producing strains themselves are also disclosed.

Detection of reticuloendotheliosis virus as a contaminant of fowl pox vaccines.

PubMed

Awad, A M; Abd El-Hamid, H S; Abou Rawash, A A; Ibrahim, H H

2010-11-01

This study was designed to detect reticuloendotheliosis virus (REV) as a contaminant in fowl pox vaccines. A total of 30 fowl pox vaccine samples were examined for the presence of REV using both in vitro and in vivo methods. In in vitro testing, the fowl pox vaccine samples were inoculated into chicken embryo fibroblast cultures prepared from specific-pathogen-free embryonated chicken eggs, and the cultures were examined using PCR to detect REV. In in vivo testing, each fowl pox vaccine sample was inoculated into 5-d-old specific-pathogen-free chicks, which were kept under observation for up to 12 wk postinoculation; serum samples were collected at 15, 30, and 45 d postinoculation for the detection of REV-specific antibodies using ELISA. Tissue samples were collected at 8 and 12 wk postinoculation for histopathological examination. Of the tested vaccines, only one imported vaccine sample tested positive for REV using PCR. Serum samples collected from chicks infected with the PCR-positive vaccine batch also tested positive for REV-specific antibodies using ELISA. Histopathological examination of the liver, spleen, and bursa of Fabricius demonstrated the presence of tumor cells in these organs, confirming the results obtained using PCR and ELISA, and indicating that the sample was contaminated with REV. These data clearly indicate that the screening of all commercial poultry vaccines for viruses is an important factor in assuring the biosafety of animal vaccines.

Studies on antibacterial activity of ZnO nanoparticles by ROS induced lipid peroxidation.

PubMed

Dutta, R K; Nenavathu, Bhavani P; Gangishetty, Mahesh K; Reddy, A V R

2012-06-01

Recent studies indicated the role of ROS toward antibacterial activity. In our study we report ROS mediated membrane lipid oxidation of Escherichia coli treated with ZnO nanoparticles (NPs) as supported by detection and spectrophotometric measurement of malondialdehyde (MDA) by TBARS (thiobarbituric acid-reactive species) assay. The antibacterial effects of ZnO NPs were studied by measuring the growth curve of E. coli, which showed concentration dependent bacteriostatic and bacteriocidal effects of ZnO NPs. The antibacterial effects were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Further, antibacterial effect of ZnO NPs was found to decrease by introducing histidine to the culture medium treated with ZnO NPs. The ROS scavenging action of histidine was confirmed by treating histidine to the batch of Escherichia coli+ZnO NPs at the end of the lag phase of the growth curve (Set-I) and during inoculation (Set-II). A moderate bacteriostatic effect (lag in the E. coli growth) was observed in Set-II batch while Set-I showed no bacteriostatic effect. From these evidences we confirmed that the antibacterial effect of bare as well as TG capped ZnO NPs were due to membrane lipid peroxidation caused by the ROS generated during ZnO NPs interaction in culture medium. Copyright © 2012 Elsevier B.V. All rights reserved.

In vitro azadirachtin production by hairy root cultivation of Azadirachta indica in nutrient mist bioreactor.

PubMed

Srivastava, Smita; Srivastava, A K

2012-01-01

Azadirachtin, a well-known biopesticide is a secondary metabolite conventionally extracted from the seeds of Azadirachta indica. The present study involved in vitro azadirachtin production by developing hairy roots of A. indica via Agrobacterium rhizogenes-mediated transformation of A. indica explants. Liquid culture of hairy roots was established in shake flask to study the kinetics of growth and azadirachtin production. A biomass production of 13.3 g/L dry weight (specific growth rate of 0.7 day(-1)) was obtained after 25 days of cultivation period with an azadirachtin yield of 3.3 mg/g root biomass. To overcome the mass transfer limitation in conventionally used liquid-phase reactors, batch cultivation of hairy roots was carried out in gas-phase reactors (nutrient spray and nutrient mist bioreactor) to investigate the possible scale-up of A. indica hairy root culture. The nano-size nutrient mist particles generated from the nozzle of the nutrient mist bioreactor could penetrate till the inner core of the inoculated root matrix, facilitating uniform growth during high-density cultivation of hairy roots. A biomass production of 9.8 g/L dry weight with azadirachtin accumulation of 2.8 mg/g biomass (27.4 mg/L) could be achieved in 25 days of batch cultivation period, which was equivalent to a volumetric productivity of 1.09 mg/L per day of azadirachtin.

Contribution of selected fungi to the reduction of cyanogen levels during solid substrate fermentation of cassava.

PubMed

Essers, A J; Jurgens, C M; Nout, M J

1995-07-01

The effect of six individual strains of the dominant microflora in solid substrate fermenting cassava on cyanogen levels was examined. Six out of eight batches of disinfected cassava root pieces were incubated for 72 h after inoculation with either of the fungi Geotrichum candidum, Mucor racemosus, Neurospora sitophila, Rhizopus oryzae and Rhizopus stolonifer, or a Bacillus sp., isolated from on-farm fermented cassava flours from Uganda. One non-inoculated batch was incubated as a reference. Levels of initial and final moisture and cyanogens were assayed. The experiment was done in quadruplicate. Incubation of disinfected root pieces reduced cyanogenic glucoside levels significantly to 62.7% (SD 2.8) of the initial value. Microbial growth resulted in significant additional reduction of the cyanogenic glucoside levels to 29.8% (SD 18.9) of the levels which were obtained after non-inoculated incubation. Among the tested strains, N. sitophila reduced cyanogenic glucoside levels most effectively, followed by R. stolonifer and R. oryzae. Of all fermented samples, both Rhizopus spp. showed highest proportion of residual cyanogens in the cyanohydrin form. Flours showed similar patterns of cyanogens as the batches they were prepared from. Cyanogenic glucoside level reduction was significantly correlated (r = 0.86) with the extent of root softening. It is concluded that both incubation and microbial activity are instrumental in reducing the potential toxicity of cassava during the solid substrate fermentation and that effectiveness varies considerably between the species of microorganisms applied.

Co-inoculating ruminal content neither provides active hydrolytic microbes nor improves methanization of ¹³C-cellulose in batch digesters.

PubMed

Chapleur, Olivier; Bize, Ariane; Serain, Thibaut; Mazéas, Laurent; Bouchez, Théodore

2014-03-01

Cellulose hydrolysis often limits the kinetics and efficiency of anaerobic degradation in industrial digesters. In animal digestive systems, specialized microorganisms enable cellulose biodegradation at significantly higher rates. This study aims to assess the potential of ruminal microbial communities to settle and to express their cellulolytic properties in anaerobic digesters. Cellulose-degrading batch incubations were co-inoculated with municipal solid waste digester sludge and ruminal content. ¹³C-labeled cellulose degradation was described over time with Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry. Results were linked to the identification of the microorganisms assimilating ¹³C and to the monitoring of their relative dynamics. Cellulose degradation in co-inoculated incubations was efficient but not significantly improved. Transient disturbances in degradation pathways occurred, as revealed by propionate accumulation. Automated Ribosomal Intergenic Spacer Analysis dynamics and pyrosequencing revealed that expected classes of Bacteria and Archaea were active and degraded cellulose. However, despite the favorable co-inoculation conditions, molecular tools also revealed that no ruminal species settled in the bioreactors. Other specific parameters were probably needed for this to happen. This study shows that exploiting the rumen's cellulolytic properties in anaerobic digesters is not straightforward. Co-inoculation can only be successful if ruminal microorganisms manage to thrive in the anaerobic digester and outcompete native microorganisms, which requires specific nutritional and environmental parameters, and a meticulous reproduction of the selection pressure encountered in the rumen. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Lactic acid production with undefined mixed culture fermentation of potato peel waste.

PubMed

Liang, Shaobo; McDonald, Armando G; Coats, Erik R

2014-11-01

Potato peel waste (PPW) as zero value byproduct generated from food processing plant contains a large quantity of starch, non-starch polysaccharide, lignin, protein, and lipid. PPW as one promising carbon source can be managed and utilized to value added bioproducts through a simple fermentation process using undefined mixed cultures inoculated from wastewater treatment plant sludge. A series of non-pH controlled batch fermentations under different conditions such as pretreatment process, enzymatic hydrolysis, temperature, and solids loading were studied. Lactic acid (LA) was the major product, followed by acetic acid (AA) and ethanol under fermentation conditions without the presence of added hydrolytic enzymes. The maximum yields of LA, AA, and ethanol were respectively, 0.22 g g(-1), 0.06 g g(-1), and 0.05 g g(-1). The highest LA concentration of 14.7 g L(-1) was obtained from a bioreactor with initial solids loading of 60 g L(-1) at 35°C. Copyright © 2014 Elsevier Ltd. All rights reserved.

A mesophilic Clostridium species that produces butanol from monosaccharides and hydrogen from polysaccharides.

PubMed

Bramono, Sandhi Eko; Lam, Yuen Sean; Ong, Say Leong; He, Jianzhong

2011-10-01

A unique mesophilic Clostridium species strain BOH3 is obtained in this study, which is capable of fermenting monosaccharides to produce butanol and hydrolyzing polysaccharides to produce hydrogen (H(2)) and volatile fatty acids (VFAs). From 30 g/L of glucose and xylose each, batch culture BOH3 was able to produce 4.67 and 4.63 g/L of butanol. Enhancement treatments by increasing the inoculated cells improved butanol production to 7.05 and 7.41 g/L, respectively. Hydrogen production (2.47 and 1.93 mmol) was observed when cellulose and xylan (10 g/L each) were used, suggesting that strain BOH3 possesses xylanolytic and cellulolytic capabilities. These unique features reveal the strain's novelty as most wild-type solventogenic strains have not been reported to have such properties. Therefore, culture BOH3 is promising in generating butanol and hydrogen from renewable feedstock. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

Establishing a laboratory model of dental unit waterlines bacterial biofilms using a CDC biofilm reactor.

PubMed

Yoon, Hye Young; Lee, Si Young

2017-11-01

In this study, a laboratory model to reproduce dental unit waterline (DUWL) biofilms was developed using a CDC biofilm reactor (CBR). Bacteria obtained from DUWLs were filtered and cultured in Reasoner's 2A (R2A) for 10 days, and were subsequently stored at -70°C. This stock was cultivated on R2A in batch mode. After culturing for five days, the bacteria were inoculated into the CBR. Biofilms were grown on polyurethane tubing for four days. Biofilm accumulation and thickness was 1.3 × 10 5  CFU cm -2 and 10-14 μm respectively, after four days. Bacteria in the biofilms included cocci and rods of short and medium lengths. In addition, 38 bacterial genera were detected in biofilms. In this study, the suitability and reproducibility of the CBR model for DUWL biofilm formation were demonstrated. The model provides a foundation for the development of bacterial control methods for DUWLs.

Biodegradation of PAHs in Soil: Influence of Initial PAHs Concentration

NASA Astrophysics Data System (ADS)

Kamil, N. A. F. M.; Talib, S. A.

2016-07-01

Most studies on biodegradation of Polycyclic Aromatic Hydrocarbons (PAHs) evaluate the effect of initial PAHs concentration in liquid medium. There are limited studies on evaluation in solid medium such as contaminated soil. This study investigated the potential of the bacteria, Corynebacterium urealyticum isolated from municipal sludge in degrading phenanthrene contaminated soil in different phenanthrene concentration. Batch experiments were conducted over 20 days in reactors containing artificially contaminated phenanthrene soil at different concentration inoculated with a bacterial culture. This study established the optimum condition for phenanthrene degradation by the bacteria under nonindigenous condition at 500 mg/kg of initial phenanthrene concentration. High initial concentration required longer duration for biodegradation process compared to low initial concentration. The bacteria can survive for three days for all initial phenanthrene concentrations.

Growth kinetics and physiological behavior of co-cultures of Saccharomyces cerevisiae and Kluyveromyces lactis, fermenting carob sugars extracted with whey.

PubMed

Rodrigues, B; Lima-Costa, M E; Constantino, A; Raposo, S; Felizardo, C; Gonçalves, D; Fernandes, T; Dionísio, L; Peinado, J M

2016-10-01

Alcoholic fermentation of carob waste sugars (sucrose, glucose and fructose) extracted with cheese whey, by co-cultures of Saccharomyces cerevisiae and Kluyveromyces lactis has been analyzed. Growth and fermentation of S. cerevisiae in the carob-whey medium showed an inhibition of about 30% in comparison with water-extracted carob. The inhibition of K. lactis on carob-whey was greater (70%) when compared with the whey medium alone, due to osmolarity problems. Oxygen availability was a very important factor for K. lactis, influencing its fermentation performance. When K. lactis was grown alone on carob-whey medium, lactose was always consumed first, and glucose and fructose were consumed afterwards, only at high aeration conditions. In co-culture with S. cerevisiae, K. lactis was completely inhibited and, at low aeration, died after 3 days; at high aeration this culture could survive but growth and lactose fermentation were only recovered after S. cerevisiae became stationary. To overcome the osmolarity and K. lactis' oxygen problems, the medium had to be diluted and a sequential fermentative process was designed in a STR-3l reactor. K. lactis was inoculated first and, with low aeration (0.13vvm), consumed all the lactose in 48h. Then S. cerevisiae was inoculated, consuming the total of the carob sugars, and producing ethanol in a fed-batch regime. The established co-culture with K. lactis increased S. cerevisiae ethanol tolerance. This fermentation process produced ethanol with good efficiency (80g/l final concentration and a conversion factor of 0.4g ethanol/g sugar), eliminating all the sugars of the mixed waste. These efficient fermentative results pointed to a new joint treatment of agro-industrial wastes which may be implemented successfully, with economic and environmental sustainability for a bioethanol industrial proposal. Copyright © 2016 Elsevier Inc. All rights reserved.

Investigation of vinegar production using a novel shaken repeated batch culture system.

PubMed

Schlepütz, Tino; Büchs, Jochen

2013-01-01

Nowadays, bioprocesses are developed or optimized on small scale. Also, vinegar industry is motivated to reinvestigate the established repeated batch fermentation process. As yet, there is no small-scale culture system for optimizing fermentation conditions for repeated batch bioprocesses. Thus, the aim of this study is to propose a new shaken culture system for parallel repeated batch vinegar fermentation. A new operation mode - the flushing repeated batch - was developed. Parallel repeated batch vinegar production could be established in shaken overflow vessels in a completely automated operation with only one pump per vessel. This flushing repeated batch was first theoretically investigated and then empirically tested. The ethanol concentration was online monitored during repeated batch fermentation by semiconductor gas sensors. It was shown that the switch from one ethanol substrate quality to different ethanol substrate qualities resulted in prolonged lag phases and durations of the first batches. In the subsequent batches the length of the fermentations decreased considerably. This decrease in the respective lag phases indicates an adaptation of the acetic acid bacteria mixed culture to the specific ethanol substrate quality. Consequently, flushing repeated batch fermentations on small scale are valuable for screening fermentation conditions and, thereby, improving industrial-scale bioprocesses such as vinegar production in terms of process robustness, stability, and productivity. Copyright © 2013 American Institute of Chemical Engineers.

Forensic Application of Microbiological Culture Analysis To Identify Mail Intentionally Contaminated with Bacillus anthracis Spores†

PubMed Central

Beecher, Douglas J.

2006-01-01

The discovery of a letter intentionally filled with dried Bacillus anthracis spores in the office of a United States senator prompted the collection and quarantine of all mail in congressional buildings. This mail was subsequently searched for additional intentionally contaminated letters. A microbiological sampling strategy was used to locate heavy contamination within the 642 separate plastic bags containing the mail. Swab sampling identified 20 bags for manual and visual examination. Air sampling within the 20 bags indicated that one bag was orders of magnitude more contaminated than all the others. This bag contained a letter addressed to Senator Patrick Leahy that had been loaded with dried B. anthracis spores. Microbiological sampling of compartmentalized batches of mail proved to be efficient and relatively safe. Efficiency was increased by inoculating culture media in the hot zone rather than transferring swab samples to a laboratory for inoculation. All mail sampling was complete within 4 days with minimal contamination of the sampling environment or personnel. However, physically handling the intentionally contaminated letter proved to be exceptionally hazardous, as did sorting of cross-contaminated mail, which resulted in generation of hazardous aerosol and extensive contamination of protective clothing. Nearly 8 × 106 CFU was removed from the most highly cross-contaminated piece of mail found. Tracking data indicated that this and other heavily contaminated envelopes had been processed through the same mail sorting equipment as, and within 1 s of, two intentionally contaminated letters. PMID:16885280

Does zero-water discharged technology enhance culture performance of pacific white shrimp (Litopenaeus vannamei Boone.)?

NASA Astrophysics Data System (ADS)

Suantika, Gede; Anggraeni, Jayanty; Hasby, Fahri Azhari; Yanuwiarti, Ni Putu Indah

2014-03-01

Litopenaeus vannamei or white leg shrimp is an introduced shrimp which has successfully cultured in Indonesia. In Indonesia, L. vannamei is commonly cultured on outdoor/earthen pond that requires renewal of water, less control in term of water quality and disease and attributed to unpredictable yield production. Based on the existing culture condition, a system that enable to minimize water consumption, improve the hygiene of the culture and at the same time maintain a more stable yield production is urgent to be developed by using a zero water discharge system. The system consists of: (a) culture tank - to retain and culture the shrimp; (b) CaCO3 grained - buffering agent and substrate of nitrifying bacteria; (c) aeration line - to provide O2 and homogenize the culture; (d) ancho (feeding) - to control an appropriate feed; (e) nitrifying bacteria adding - to consume ammonium and nitrite then convert it to nitrate, and also control pathogen Vibrio sp.; (f) diatom microalgae (Chaetoceros gracilis) - to uptake nitrate, bacteriostatic agent, feed source, provide O2 and shading. In this study, there were 2 treatments: the static culture (batch) system was set as control (K) (in 70 PL/m2), and culture system with zero-water discharge system which was inoculated by 0.02% v/v 106 CFU/ml of mixed culture nitrifying bacteria and diatom microalgae in 70 PL/m2 (P1). The white leg shrimp used in this experiment was at post larvae (PL) 10 and cultured in a batch system (1 × 1 × 0.5 m3 pond) during 2 months. Several parameters including survival rate, mean body weight, and water quality (salinity, temperature, pH, DO, ammonium, nitrite, and nitrate) were measured. Based on the results, biomass of P1 (237.12 ± 31.11) gram is significantly higher than control (K) (180.80 ± 12.26) gram (P< 0,05). Water quality during the culture period in all treatments were still in tolerance range of white leg shrimp post larvae, except ammonium concentration in control (K) (2.612 ± 0.56) mg/L which is significantly different from P1 (1.287 ± 0.49) mg/L. Based on this research, zero-water discharge technology using nitrifying bacteria and diatom microalgae can improve productivity of white shrimp by increasing the biomass and maintaining a stable water quality especially ammonium concentration.

Transformation of Mycelial and Yeast Forms of Paracoccidioides brasiliensis in Cultures and in Experimental Inoculations

PubMed Central

Carbonell, Luis M.; Rodríguez, Joaquín

1965-01-01

Carbonell, Luis M. (Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela), and Joaquín Rodríguez. Transformation of mycelial and yeast forms of Paracoccidioides brasiliensis in cultures and in experimental inoculations. J. Bacteriol. 90:504–510. 1965.—Experimental transformations of mycelial to yeast and yeast to mycelial forms in culture, and mycelial to yeast forms in tissue, were studied. All the transitional forms that appeared in culture were also seen in tissue, but in fewer number. Most of the hyphae in culture were transformed into yeast, but only a few in tissue. Yeast appeared in testicle around the 3rd day after inoculation, but on the 10th day in subcutaneous tissue. Pathogenicity of mycelium was high, since yeast was found in almost all of the organs inoculated with mycelium. Histologically, an acute inflammation occurred first, owing to the inoculation of mycelium, followed by a giant-cell granuloma with abundant hyphae detritus. These giant cells almost disappeared about 10 days after inoculation, giving place to a second giant-cell granuloma with yeast forms. Images PMID:14329466

Biofiltration of waste gases with the fungi Exophiala oligosperma and Paecilomyces variotii.

PubMed

Estévez, Elena; Veiga, María C; Kennes, Christian

2005-06-01

Two biofilters fed toluene-polluted air were inoculated with new fungal isolates of either Exophiala oligosperma or Paecilomyces variotii, while a third bioreactor was inoculated with a defined consortium composed of both fungi and a co-culture of a Pseudomonas strain and a Bacillus strain. Elimination capacities of 77 g m(-3) h(-1) and 55 g m(-3) h(-1) were reached in the fungal biofilters (with removal efficiencies exceeding 99%) in the case of, respectively, E. oligosperma and Paecilomyces variotii when feeding air with a relative humidity (RH) of 85%. The inoculated fungal strains remained the single dominant populations throughout the experiment. Conversely, in the biofilter inoculated with the bacterial-fungal consortium, the bacteria were gradually overgrown by the fungi, reaching a maximum elimination capacity around 77 g m(-3) h(-1). Determination of carbon dioxide concentrations both in batch assays and in biofiltration studies suggested the near complete mineralization of toluene. The non-linear toluene removal along the height of the biofilters resulted in local elimination capacities of up to 170 g m(-3) h(-1) and 94 g m(-3) h(-1) in the reactors inoculated, respectively, with E. oligosperma and P. variotii. Further studies with the most efficient strain, E. oligosperma, showed that the performance was highly dependent on the RH of the air and the pH of the nutrient solution. At a constant 85% RH, the maximum elimination capacity either dropped to 48.7 g m(-3) h(-1) or increased to 95.6 g m(-3) h(-1), respectively, when modifying the pH of the nutrient solution from 5.9 to either 4.5 or 7.5. The optimal conditions were 100% RH and pH 7.5, which allowed a maximum elimination capacity of 164.4 g m(-3) h(-1) under steady-state conditions, with near-complete toluene degradation.

Liquid culture mass production of biocontrol nematodes, Heterorhabditis bacteriophora (Nematoda: Rhabditida): improved timing of dauer juvenile inoculation.

PubMed

Johnigk, S-A; Ecke, F; Poehling, M; Ehlers, R-U

2004-06-01

Heterorhabditis bacteriophora is used in biological control of soil-borne insect pests in horticulture and turf. Mass production is carried out in monoxenic liquid cultures pre-incubated with the symbiont of the nematodes, the bacterium Photorhabdus luminescens, before nematode dauer juveniles (DJ) are inoculated. As a response to bacterial food signals, the DJ recover from the developmentally arrested dauer stage, grow to adults and produce DJ offspring. Variable DJ recovery after inoculation into cultures of P. luminescens often causes process failure due to low numbers of adult nematodes in the medium. In order to enhance DJ recovery, improve nematode population management and increase yields, the optimal timing for DJ inoculation was sought. The process parameter pH and respiration quotient (RQ) were recorded in order to test whether changes can be used to identify the best moment for DJ inoculation. When DJ were inoculated during the lag and early logarithmic growth phases of P. luminescens cultures, DJ recovery was low and almost no nematode reproduction was obtained. High populations of P. luminescens phase variants were recorded. Recovery and yields increased when DJ were inoculated during the latter log phase during which the RQ dropped to values <0.8 and the pH reached a maximum. The highest DJ recovery and yields were observed in cultures that were inoculated during the late stationary growth phase. This period started with the increase of the pH after its distinct minimum at pH <8.0. Thus optimal timing for DJ inoculation can be defined through monitoring of the pH in the P. luminescens culture.

Transformation of carbon tetrachloride and chloroform by trichloroethene respiring anaerobic mixed cultures and supernatant.

PubMed

Vickstrom, Kyle E; Azizian, Mohammad F; Semprini, Lewis

2017-09-01

Carbon tetrachloride (CT) and chloroform (CF) were transformed in batch reactor experiments conducted with anaerobic dechlorinating cultures and supernatant (ADC + S) harvested from continuous flow reactors. The Evanite (EV) and Victoria/Stanford (VS) cultures, capable of respiring trichloroethene (TCE), 1,2-cis-dichloroethene (cDCE), and vinyl chloride (VC) to ethene (ETH), were grown in continuous flow reactors receiving an influent feed of saturated TCE (10 mM; 60 mEq) and formate (45 mM; 90 mEq) but no CT or CF. Cells and supernatant were harvested from the chemostats and inoculated into batch reactors at the onset of each experiment. CT transformation was complete following first order kinetics with CF, DCM and CS 2 as the measurable transformation products, representing 20-40% of the original mass of CT, with CO 2 likely the unknown transformation product. CF was transformed to DCM and likely CO 2 at an order of magnitude rate lower than CT, while DCM was not further transformed. An analytical first order model including multiple key reactions effectively simulated CT transformation, product formation and transformation, and provided reasonable estimates of transformation rate coefficients. Biotic and abiotic treatments indicated that CT was mainly transformed via abiotic processes. However, the presence of live cells was associated with the transformation of CF to DCM. In biotic tests both TCE and CT were simultaneously transformed, with TCE transformed to ETH and approximately 15-53% less CF formed via CT transformation. A 14-day exposure to CF (CF max  = 1.4 μM) reduced all rates of chlorinated ethene respiration by a factor of 10 or greater. Copyright © 2017 Elsevier Ltd. All rights reserved.

Modeling of Fusarium redolens Dzf2 mycelial growth kinetics and optimal fed-batch fermentation for beauvericin production.

PubMed

Xu, Li-Jian; Liu, Yuan-Shuai; Zhou, Li-Gang; Wu, Jian-Yong

2011-09-01

Beauvericin (BEA) is a cyclic hexadepsipeptide mycotoxin with notable phytotoxic and insecticidal activities. Fusarium redolens Dzf2 is a highly BEA-producing fungus isolated from a medicinal plant. The aim of the current study was to develop a simple and valid kinetic model for F. redolens Dzf2 mycelial growth and the optimal fed-batch operation for efficient BEA production. A modified Monod model with substrate (glucose) and product (BEA) inhibition was constructed based on the culture characteristics of F. redolens Dzf2 mycelia in a liquid medium. Model parameters were derived by simulation of the experimental data from batch culture. The model fitted closely with the experimental data over 20-50 g l(-1) glucose concentration range in batch fermentation. The kinetic model together with the stoichiometric relationships for biomass, substrate and product was applied to predict the optimal feeding scheme for fed-batch fermentation, leading to 54% higher BEA yield (299 mg l(-1)) than in the batch culture (194 mg l(-1)). The modified Monod model incorporating substrate and product inhibition was proven adequate for describing the growth kinetics of F. redolens Dzf2 mycelial culture at suitable but not excessive initial glucose levels in batch and fed-batch cultures.

Evolution of infection in mice inoculated by the oral route with different developmental forms of Trypanosoma cruzi I and II.

PubMed

Dias, Greicy Brisa Malaquias; Gruendling, Ana Paula; Araújo, Silvana Marques; Gomes, Mônica Lúcia; Toledo, Max Jean de Ornelas

2013-11-01

Oral infection has become the most important transmission mechanism of Chagas disease in Brazil. For this study, the development of Trypanosoma cruzi infection in mice, induced by the oral and intraperitoneal (IP) routes, was compared. Four groups of Swiss mice were used to evaluate the influence of parasite genetics, number of parasites, inoculation volume and developmental stages on the development of the orally induced infection: 1 - blood trypomastigotes (BT) via oral; 2 - BT via IP; 3 - culture metacyclic trypomastigotes (MT) via oral; and 4 - culture MT via IP. Animals inoculated orally showed levels of parasitemia, as well as infectivity and mortality rates, lower than animals inoculated via IP, regardless of DTU (discrete typing unit) and inoculum. Animals infected with TcII showed higher levels of these parameters than did animals infected with TcI. The larger volume of inoculum showed a greater capacity to cause an infection when administered via the oral route. BT infection was more virulent than culture MT infection for both routes (oral and IP). However, mice inoculated orally with BT showed lower levels than via IP, while mice inoculated orally with culture MT showed similar levels of infection to those inoculated via IP. Mice inoculated with culture MT showed more histopathological changes than those inoculated with BT, regardless of the inoculation route. These results indicate that this alternative experimental model is useful for evaluating infection by T. cruzi isolates with subpatent parasitemia and low virulence, such as those belonging to the TcI and TcIV DTUs, which are prevalent in outbreaks of orally transmitted Chagas disease. Copyright © 2013 Elsevier Inc. All rights reserved.

Combined moist airtight storage and feed fermentation of barley by the yeast Wickerhamomyces anomalus and a lactic acid bacteria consortium

PubMed Central

Borling Welin, Jenny; Lyberg, Karin; Passoth, Volkmar; Olstorpe, Matilda

2015-01-01

This study combined moist airtight storage of moist grain with pig feed fermentation. Starter cultures with the potential to facilitate both technologies were added to airtight stored moist crimped cereal grain, and the impact on storage microflora and the quality of feed fermentations generated from the grain was investigated. Four treatments were compared: three based on moist barley, either un-inoculated (M), inoculated with Wickerhamomyces anomalus (W), or inoculated with W. anomalus and LAB starter culture, containing Pediococcus acidilactici DSM 16243, Pediococcus pentosaceus DSM 12834 and Lactobacillus plantarum DSM 12837 (WLAB); and one treatment based on dried barley (D). After 6 weeks of storage, four feed fermentations FM, FW, FWLAB, and FD, were initiated from M, W, WLAB, and D, respectively, by mixing the grain with water to a dry matter content of 30%. Each treatment was fermented in batch initially for 7 days and then kept in a continuous mode by adding new feed daily with 50% back-slop. During the 6 week storage period, the average water activity decreased in M, W and WLAB from 0.96 to 0.85, and cereal pH decreased from approximately 6.0 at harvest to 4.5. Feed fermentation conferred a further pH decrease to 3.8–4.1. In M, W and WLAB, molds and Enterobacteriaceae were mostly below detection limit, whereas both organism groups were detected in D. In fermented feed, Enterobacteriaceae were below detection limit in almost all conditions. Molds were detected in FD, for most of the fermentation time in FM and at some sampling points in FW and FWLAB. Starter organisms, especially W. anomalus and L. plantarum comprised a considerable proportion of the yeast and LAB populations, respectively, in both stored grain and fermented feed. However, autochthonous Pichia kudriavzevii and Kazachstania exigua partially dominated the yeast populations in stored grain and fermented feed, respectively. PMID:25954295

Use of autochthonous Pediococcus acidilactici and Staphylococcus vitulus starter cultures in the production of "chorizo" in 2 different traditional industries.

PubMed

Casquete, Rocío; Benito, María J; Martín, Alberto; Ruiz-Moyano, Santiago; Aranda, Emilio; Córdoba, María G

2012-01-01

The present study determined how the different ripening conditions affected the growth and development of 3 autochthonous starter cultures, and the physico-chemical and sensory characteristics of chorizo. Each of 3 strains of Pediococcus acidilactici (MC184, MS198, and MS200) and one of Staphylococcus vitulus (RS34) were associated to prepare the starter cultures, P184S34, P198S34, and P200S34. Then, chorizo was prepared following 2 manufacturing procedures. The autochthonous starter cultures were able to compete and colonize the sausages in both ripening procedures. The use of the starter cultures showed evident differences by the texture analysis, with the control batches being generally tougher than the starter culture batches. Also, the highest biogenic amine (BA) levels were found in control batches and the lowest in P200S34 batches. While the use of these starter cultures does not change the sensory characteristics of these traditional fermented sausages, it improves their homogeneity and safety, except for P184S34 batch in which more BAs are detected in industry 2. The 3 autochthonous starter cultures selected could be used in traditional industries because they are able to compete well and colonize the dry fermented sausages "chorizo." The use of these starter cultures improves the texture and homogeneity of traditional fermented sausages. Biogenic amines decreased in the starter cultures batches improving the safety. © 2011 Institute of Food Technologists®

Comparison of different liquid anaerobic digestion effluents as inocula and nitrogen sources for solid-state batch anaerobic digestion of corn stover.

PubMed

Xu, Fuqing; Shi, Jian; Lv, Wen; Yu, Zhongtang; Li, Yebo

2013-01-01

Effluents from three liquid anaerobic digesters, fed with municipal sewage sludge, food waste, or dairy waste, were evaluated as inocula and nitrogen sources for solid-state batch anaerobic digestion of corn stover in mesophilic reactors. Three feedstock-to-effluent (F/E) ratios (i.e., 2, 4, and 6) were tested for each effluent. At an F/E ratio of 2, the reactor inoculated by dairy waste effluent achieved the highest methane yield of 238.5L/kg VS(feed), while at an F/E ratio of 4, the reactor inoculated by food waste effluent achieved the highest methane yield of 199.6L/kg VS(feed). The microbial population and chemical composition of the three effluents were substantially different. Food waste effluent had the largest population of acetoclastic methanogens, while dairy waste effluent had the largest populations of cellulolytic and xylanolytic bacteria. Dairy waste also had the highest C/N ratio of 8.5 and the highest alkalinity of 19.3g CaCO(3)/kg. The performance of solid-state batch anaerobic digestion reactors was closely related to the microbial status in the liquid anaerobic digestion effluents. Copyright © 2012 Elsevier Ltd. All rights reserved.

Production of branched-chain alcohols by recombinant Ralstonia eutropha in fed-batch cultivation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fei, Q; Brigham, CJ; Lu, JN

Branched-chain alcohols are considered promising green energy sources due to their compatibility with existing infrastructure and their high energy density. We utilized a strain of Ralstonia eutropha capable of producing branched-chain alcohols and examined its production in flask cultures. In order to increase isobutanol and 3-methyl-1-butanol (isoamyl alcohol) productivity in the engineered strain, batch, fed-batch, and two-stage fed-batch cultures were carried out in this work. The effects of nitrogen source concentration on branched-chain alcohol production were investigated under four different initial concentrations in fermenters. A maximum 380 g m(-3) of branched-chain alcohol production was observed with 2 kg m(-3) initialmore » NH4Cl concentration in batch cultures. A pH-stat control strategy was utilized to investigate the optimum carbon source amount fed during fed-batch cultures for higher cell density. In cultures of R. eutropha strains that did not produce polyhydroxyalkanoate or branched-chain alcohols, a maximum cell dry weight of 36 kg m(-3) was observed using a fed-batch strategy, when 10 kg m(-3) carbon source was fed into culture medium. Finally, a total branched-chain alcohol titer of 790 g m(-3), the highest branched-chain alcohol yield of 0.03 g g(-1), and the maximum branched-chain alcohol productivity of 8.23 g m(-3) h(-1) were obtained from the engineered strain Re2410/pJL26 in a two-stage fed-batch culture system with pH-stat control. Isobutanol made up over 95% (mass fraction) of the total branched-chain alcohols titer produced in this study. (C) 2013 Published by Elsevier Ltd.« less

Tolerance of Chemoorganotrophic Bioleaching Microorganisms to Heavy Metal and Alkaline Stresses

PubMed Central

Monballiu, Annick; Cardon, Nele; Tri Nguyen, Minh; Cornelly, Christel; Meesschaert, Boudewijn; Chiang, Yi Wai

2015-01-01

The bioleaching potential of the bacterium Bacillus mucilaginosus and the fungus Aspergillus niger towards industrial residues was investigated by assessing their response towards various heavy metals (including arsenic, cadmium, cobalt, chromium, nickel, lead, and zinc) and elevated pH. The plate diffusion method was performed for each metal to determine the toxicity effect. Liquid batch cultures were set up for more quantitative evaluation as well as for studying the influence of basicity. Growth curves were prepared using bacterial/fungal growth counting techniques such as plate counting, optical density measurement, and dry biomass determination. Cadmium, nickel, and arsenite had a negative influence on the growth of B. mucilaginosus, whereas A. niger was sensitive to cadmium and arsenate. However, it was shown that growth recovered when microorganisms cultured in the presence of these metals were inoculated onto metal-free medium. Based on the findings of the bacteriostatic/fungistatic effect of the metals and the adaptability of the microorganisms to fairly elevated pH values, it is concluded that both strains have potential applicability for further research concerning bioleaching of alkaline waste materials. PMID:26236176

High-Throughput Nuclear Magnetic Resonance Metabolomic Footprinting for Tissue Engineering

PubMed Central

Seagle, Christopher; Christie, Megan A.; Winnike, Jason H.; McClelland, Randall E.; Ludlow, John W.; O'Connell, Thomas M.; Gamcsik, Michael P.

2008-01-01

Abstract We report a high-throughput (HTP) nuclear magnetic resonance (NMR) method for analysis of media components and a metabolic schematic to help easily interpret the data. Spin-lattice relaxation values and concentrations were measured for 19 components and 2 internal referencing agents in pure and 2-day conditioned, hormonally defined media from a 3-dimensional (3D) multicoaxial human bioartificial liver (BAL). The 1H NMR spectral signal-to-noise ratio is 21 for 0.16 mM alanine in medium and is obtained in 12 min using a 400 MHz NMR spectrometer. For comparison, 2D gel cultures and 3D multicoaxial BALs were batch cultured, with medium changed every day for 15 days after inoculation with human liver cells in Matrigel–collagen type 1 gels. Glutamine consumption was higher by day 8 in the BAL than in 2D culture; lactate production was lower through the 15-day culture period. Alanine was the primary amino acid produced and tracked with lactate or urea production. Glucose and pyruvate consumption were similar in the BAL and 2D cultures. NMR analysis permits quality assurance of the bioreactor by identifying contaminants. Ethanol was observed because of a bioreactor membrane “wetting” procedure. A biochemical scheme is presented illustrating bioreactor metabolomic footprint results and demonstrating how this can be translated to modify bioreactor operational parameters or quality assurance issues. PMID:18544027

Cell-controlled hybrid perfusion fed-batch CHO cell process provides significant productivity improvement over conventional fed-batch cultures.

PubMed

Hiller, Gregory W; Ovalle, Ana Maria; Gagnon, Matthew P; Curran, Meredith L; Wang, Wenge

2017-07-01

A simple method originally designed to control lactate accumulation in fed-batch cultures of Chinese Hamster Ovary (CHO) cells has been modified and extended to allow cells in culture to control their own rate of perfusion to precisely deliver nutritional requirements. The method allows for very fast expansion of cells to high density while using a minimal volume of concentrated perfusion medium. When the short-duration cell-controlled perfusion is performed in the production bioreactor and is immediately followed by a conventional fed-batch culture using highly concentrated feeds, the overall productivity of the culture is approximately doubled when compared with a highly optimized state-of-the-art fed-batch process. The technology was applied with near uniform success to five CHO cell processes producing five different humanized monoclonal antibodies. The increases in productivity were due to the increases in sustained viable cell densities. Biotechnol. Bioeng. 2017;114: 1438-1447. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Batch, design optimization, and DNA sequencing study for continuous 1,3-propanediol production from waste glycerol by a soil-based inoculum.

PubMed

Kanjilal, Baishali; Noshadi, Iman; Bautista, Eddy J; Srivastava, Ranjan; Parnas, Richard S

2015-03-01

1,3-propanediol (1,3-PD) was produced with a robust fermentation process using waste glycerol feedstock from biodiesel production and a soil-based bacterial inoculum. An iterative inoculation method was developed to achieve independence from soil and selectively breed bacterial populations capable of glycerol metabolism to 1,3-PD. The inoculum showed high resistance to impurities in the feedstock. 1,3-PD selectivity and yield in batch fermentations was optimized by appropriate nutrient compositions and pH control. The batch yield of 1,3-PD was maximized to ~0.7 mol/mol for industrial glycerol which was higher than that for pure glycerin. 16S rDNA sequencing results show a systematic selective enrichment of 1,3-PD producing bacteria with iterative inoculation and subsequent process control. A statistical design of experiments was carried out on industrial glycerol batches to optimize conditions, which were used to run two continuous flow stirred-tank reactor (CSTR) experiments over a period of >500 h each. A detailed analysis of steady states at three dilution rates is presented. Enhanced specific 1,3-PD productivity was observed with faster dilution rates due to lower levels of solvent degeneration. 1,3-PD productivity, specific productivity, and yield of 1.1 g/l hr, 1.5 g/g hr, and 0.6 mol/mol of glycerol were obtained at a dilution rate of 0.1 h(-1)which is bettered only by pure strains in pure glycerin feeds.

Susceptibility of dogs to infection with Ehrlichia risticii, causative agent of equine monocytic ehrlichiosis (Potomac horse fever).

PubMed

Ristic, M; Dawson, J; Holland, C J; Jenny, A

1988-09-01

Adult dogs 1 to 5 were inoculated IV and/or SC with 3, 5, or 6 ml of a suspension containing 1.2 x 10(4) Ehrlichia risticii-infected cells (derived from primary canine monocyte cell cultures)/ml. Dogs 6 to 8 were inoculated IV and/or SC with 3 or 6 ml of 1.2 x 10(5) organism-free cultured canine monocytes/ml. Ehrlichia risticii was isolated in cultures from inoculated dogs 3, 4, and 5 on postinoculation days (PID) 10 to 16, but not from dogs 6 to 8. Dogs inoculated with E risticii seroconverted between PID 6 and 12. Clinical signs of illness were not observed in these 5 E risticii-inoculated dogs. A pony, inoculated with E risticii isolated from inoculated dog 5, developed clinical signs of equine monocytic ehrlichiosis, including fever, anorexia, depression, and diarrhea, and E risticii was isolated from the pony's blood. This E risticii isolate was then inoculated into susceptible dog 9, and E risticii was repeatedly isolated from dog 9 during PID 6 to 17. Dogs were susceptible to infection with E risticii and may serve as a reservoir of the organism in the field.

Tylosin production by Streptomyces fradiae using raw cornmeal in airlift bioreactor.

PubMed

Choi, Dubok; Choi, On You; Shin, Hyun-Jae; Chung, Dong-Ok; Shin, Dae-Yewn

2007-07-01

Using a 50-l airlift bioreactor, for the effective production of tylosin from Streptomyces fradiae TM-224 using raw cornmeal as the energy source, various environmental factors were studied in flask cultures. The maximum tylosin concentration was obtained at 32 degrees C and pH between 7.0 and 7.5. When seed was inoculated after 24 h of culture, the maximum tylosin concentration, 5.7 g/l, was obtained after 4 days of culture. Various concentrations of raw cornmeal were tested to investigate the optimum initial concentration for the tylosin production. An initial raw cornmeal concentration of 80 g/l gave the highest tylosin concentration, 5.8 g/l, after 5 days of culture. Of the various nitrogen sources, soybean meal and fish meal were found to be the most effective for the production of tylosin. In particular, with the optimal mixing ratio, 12 g/l of soybean meal to 14 g/l of fish meal, 7.2 g/l of tylosin was obtained after 5 days of culture. To compare raw cornmeal and glucose for the production oftylosin in the 50-1 airlift bioreactor for 10 days, fed-batch cultures were carried out under the optimum culture conditions. When raw corn meal was used as the energy source, the tylosin production increased with increasing culture time. The maximum tylosin concentration after 10 days of culture was 13.5 g/l, with a product yield from raw cornmeal of 0.123 g/g of consumed carbon source, which was about 7.2 times higher than that obtained when glucose was used as the carbon source.

Methane biofiltration using autoclaved aerated concrete as the carrier material.

PubMed

Ganendra, Giovanni; Mercado-Garcia, Daniel; Hernandez-Sanabria, Emma; Boeckx, Pascal; Ho, Adrian; Boon, Nico

2015-09-01

The methane removal capacity of mixed methane-oxidizing bacteria (MOB) culture in a biofilter setup using autoclaved aerated concrete (AAC) as a highly porous carrier material was tested. Batch experiment was performed to optimize MOB immobilization on AAC specimens where optimum methane removal was obtained when calcium chloride was not added during bacterial inoculation step and 10-mm-thick AAC specimens were used. The immobilized MOB could remove methane at low concentration (~1000 ppmv) in a biofilter setup for 127 days at average removal efficiency (RE) of 28.7 %. Unlike a plug flow reactor, increasing the total volume of the filter by adding a biofilter in series did not result in higher total RE. MOB also exhibited a higher abundance at the bottom of the filter, in proximity with the methane gas inlet where a high methane concentration was found. Overall, an efficient methane biofilter performance could be obtained using AAC as the carrier material.

Culturability and toxicity of sick building syndrome-related fungi over time.

PubMed

Wilson, Stephen C; Carriker, Curtis G; Brasel, Trevor L; Karunasena, Enusha; Douglas, David R; Wu, Chunfa; Andriychuk, Larysa A; Fogle, Matthew R; Martin, Jared M; Straus, David C

2004-08-01

Two experiments were conducted regarding the culturability and toxicity of fungi located on building materials over time and the efficacy of seven laboratory techniques in recovering culturable fungi from sample swabs. In the first experiment, eight sections of drywall were inoculated with Stachybotrys chartarum and stored at 25 +/- 5 degrees Celsius and 20-60% relative humidity (RH) for up to two years. Another eight sections of ceiling tile were stored at 100% RH for 1 year. Six sections of ceiling tile and 15 swabs were also inoculated with Penicillium chrysogenum and S. chartarum respectively and stored under the same conditions for 8 months and 3.3 years. All materials were tested for culturability at the end of the storage period. S. chartarum-inoculated samples were also tested for toxicity. In the second experiment (replicated twice), S. chartarum and Chaetomium globosum were inoculated onto 84 swabs each. Storage was up to 266 days at 25 +/- 5 degrees Celsius and 20-60% RH. Seven techniques were compared regarding the recovery of culturable fungi from the swabs over different time points. Results for Experiment 1 showed that all samples were culturable after the storage period and that the S. chartarum-inoculated drywall samples were toxic. In Experiment 2, all techniques showed high rates of recovery. These data show that despite being without a water source, these organisms can be culturable and toxic after long periods of time under conditions similar to human-occupied dwellings and that a number of preparation techniques are suitable for the recovery of these fungi from inoculated swabs.

ANAMMOX-like performances for nitrogen removal from ammonium-sulfate-rich wastewater in an anaerobic sequencing batch reactor.

PubMed

Prachakittikul, Pensiri; Wantawin, Chalermraj; Noophan, Pongsak Lek; Boonapatcharoen, Nimaradee

2016-01-01

Ammonium removal by the ANaerobic AMonium OXidation (ANAMMOX) process was observed through the Sulfate-Reducing Ammonium Oxidation (SRAO) process. The same concentration of ammonium (100 mg N L(-1)) was applied to two anaerobic sequencing batch reactors (AnSBRs) that were inoculated with the same activated sludge from the Vermicelli wastewater treatment process, while nitrite was fed in ANAMMOX and sulfate in SRAO reactors. In SRAO-AnSBR, in substrates that were fed with a ratio of NH4(+)/SO4(2-) at 1:0.4 ± 0.03, a hydraulic retention time (HRT) of 48 h and without sludge draining, the Ammonium Removal Rate (ARR) was 0.02 ± 0.01 kg N m(-3).d(-1). Adding specific ANAMMOX substrates to SRAO-AnSBR sludge in batch tests results in specific ammonium and nitrite removal rates of 0.198 and 0.139 g N g(-1) VSS.d, respectively, indicating that the ANAMMOX activity contributes to the removal of ammonium in the SRAO process using the nitrite that is produced from SRAO. Nevertheless, the inability of ANAMMOX to utilize sulfate to oxidize ammonium was also investigated in batch tests by augmenting enriched ANAMMOX culture in SRAO-AnSBR sludge and without nitrite supply. The time course of sulfate in a 24-hour cycle of SRAO-AnSBR showed an increase in sulfate after 6 h. For enriched SRAO culture, the uptake molar ratio of NH4(+)/SO4(2-) at 8 hours in a batch test was 1:0.82 lower than the value of 1:0.20 ± 0.09 as obtained in an SRAO-AnSBR effluent, while the stoichiometric ratio of 1:0.5 that includes the ANAMMOX reaction was in this range. After a longer operation of more than 2 years without sludge draining, the accumulation of sulfate and the reduction of ammonium removal were observed, probably due to the gradual increase in the sulfur denitrification rate and the competitive use of nitrite with ANAMMOX. The 16S rRNA gene PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) and PCR cloning analyses resulted in the detection of the ANAMMOX bacterium (Candidatus Brocadia sinica JPN1) Desulfacinum subterraneum belonging to the genus Desulfacinum and bacteria that are involved in sulfur metabolism (Pseudomonas aeruginosa strain SBTPe-001 and Paracoccus denitrificans strain IAM12479) in SRAO-AnSBR.

Continuous production of monoclonal antibody in a packed-bed bioreactor.

PubMed

Golmakany, Naghmeh; Rasaee, Mohammad Javad; Furouzandeh, Mehdi; Shojaosadati, Seyed Abbas; Kashanian, Soheila; Omidfar, Kobra

2005-06-01

In the present study the growth and MAb (monoclonal antibody) production of a mouse x mouse hybridoma cell producing anti-digoxin MAb was evaluated. The hybridoma cells entrapped within the support matrix Fibra-Cel were cultured in batch and continuous mode following special protocols. Cell-culture studies were performed in a 1-litre spinner basket containing 3 g.litre-1 support matrix. Batch culture was operated with the cell density of 42x10(6) cells. During the 7 days of culture, the medium was sampled daily in order to assess glucose and MAb concentrations and the lactate dehydrogenase released into the culture medium. After a culture period of 72 h, the cell density and MAb concentration were found to be 10.4x10(7) cells/3 g of NWPF (non-woven polyester fibre) discs and 250 microg/ml respectively. This yield gradually decreased to 0.55x10(6) cells/3 g of packaging material and 60 microg/ml respectively at the end of the batch culture. In the continuous-culture studies, the batch culture was initially operated for 64.5 h and then continuous flow was started at the dilution rates of 0.15, 0.2, 0.25 and 0.3 day-1 and finally stabilized at 0.25 day-1 within 288 h (12 days). The MAb concentration at steady state was found to be 116-120 microg/day per ml, and the yield of operation was 62.5 mg/day per ml, which was 3.5 times higher than that of batch culture. In conclusion, a packed-bed bioreactor with the support matrix Fibra-Cel, operated in continuous-feeding mode, is more efficient for large-scale MAb production than a batch culture. On the other hand, by using a continuous-culture system, a better supply of nutrients and removal of inhibitory metabolites and proteolytic enzymes was obtained.

POWASSAN VIRUS: MORPHOLOGY AND CYTOPATHOLOGY.

PubMed

ABDELWAHAB, K S; ALMEIDA, J D; DOANE, F W; MCLEAN, D M

1964-05-02

Powassan virus, a North American tickborne group B arbovirus, multiplied after simultaneous inoculation into bottles or tubes of virus and trypsinized suspension of continuous-line cultures of rhesus monkey kidney cells, strain LLC-MK2. Cytopathic effects comprising cell rounding and cytoplasmic vacuolation were first observed five days after inoculation. Mixture of Powassan antiserum with virus before inoculation into tissue cultures inhibited the appearance of cytopathic effects. Hemagglutinins for rooster erythrocytes, optimally at pH 6.4 and 22 degrees C., first appeared in tissue culture supernatant fluids four days after inoculation.Electron microscopic observation of thin sections of infected tissue culture cells showed virus particles 360-380 A.U. along outer cell membranes and edges of cytoplasmic vacuoles. In phosphotungstic acid negatively stained preparations, intact virus particles, 400-450 A.U. total diameter, were observed inside infected cells. In particles in which the peripheral layer became discontinuous, geometrically arranged subunits compatible with cubic symmetry were observed.

Simultaneous Saccharification and Fermentation of Sugar Beet Pulp with Mixed Bacterial Cultures for Lactic Acid and Propylene Glycol Production.

PubMed

Berlowska, Joanna; Cieciura, Weronika; Borowski, Sebastian; Dudkiewicz, Marta; Binczarski, Michal; Witonska, Izabela; Otlewska, Anna; Kregiel, Dorota

2016-10-17

Research into fermentative production of lactic acid from agricultural by-products has recently concentrated on the direct conversion of biomass, whereby pure sugars are replaced with inexpensive feedstock in the process of lactic acid production. In our studies, for the first time, the source of carbon used is sugar beet pulp, generated as a by-product of industrial sugar production. In this paper, we focus on the simultaneous saccharification of lignocellulosic biomass and fermentation of lactic acid, using mixed cultures with complementary assimilation profiles. Lactic acid is one of the primary platform chemicals, and can be used to synthesize a wide variety of useful products, including green propylene glycol. A series of controlled batch fermentations was conducted under various conditions, including pretreatment with enzymatic hydrolysis. Inoculation was performed in two sequential stages, to avoid carbon catabolite repression. Biologically-synthesized lactic acid was catalytically reduced to propylene glycol over 5% Ru/C. The highest lactic acid yield was obtained with mixed cultures. The yield of propylene glycol from the biological lactic acid was similar to that obtained with a water solution of pure lactic acid. Our results show that simultaneous saccharification and fermentation enables generation of lactic acid, suitable for further chemical transformations, from agricultural residues.

Nisin production in realkalized fed-batch cultures in whey with feeding with lactose- or glucose-containing substrates.

PubMed

Costas Malvido, Mónica; Alonso González, Elisa; Pérez Guerra, Nelson

2016-09-01

Nisin production by Lactococcus lactis CECT 539 was followed in batch cultures in whey supplemented with different concentrations of glucose and in two realkalized fed-batch fermentations in unsupplemented whey, which were fed, respectively, with concentrated solutions of lactose and glucose. In the batch fermentations, supplementation of whey with glucose inhibited both the growth and bacteriocin production. However, fed-batch cultures were characterized with high productions of biomass (1.34 and 1.51 g l(-1)) and nisin (50.6 and 60.3 BU ml(-1)) in comparison to the batch fermentations in unsupplemented whey (0.48 g l(-1) and 22.5 BU ml(-1)) and MRS broth (1.59 g l(-1) and 50.0 BU ml(-1)). In the two realkalized fed-batch fermentations, the increase in bacteriocin production parallels both the biomass production and pH drop generated in each realkalization and feeding cycle, suggesting that nisin was synthesized as a pH-dependent primary metabolite. A shift from homolactic to heterolactic fermentation was observed at the 108 h of incubation, and other metabolites (acetic acid and butane-2,3-diol) in addition to lactic acid accumulated in the medium. On the other hand, the feeding with glucose improved the efficiencies in glucose, nitrogen, and phosphorus consumption as compared to the batch cultures. The realkalized fed-batch fermentations showed to be an effective strategy to enhance nisin production in whey by using an appropriate feeding strategy to avoid the substrate inhibition.

Effect of Growth Conditions and Trehalose Content on Cryotolerance of Bakers' Yeast in Frozen Doughs

PubMed Central

Gélinas, Pierre; Fiset, Gisèle; LeDuy, Anh; Goulet, Jacques

1989-01-01

The cryotolerance in frozen doughs and in water suspensions of bakers' yeast (Saccharomyces cerevisiae) previously grown under various industrial conditions was evaluated on a laboratory scale. Fed-batch cultures were very superior to batch cultures, and strong aeration enhanced cryoresistance in both cases for freezing rates of 1 to 56°C min−1. Loss of cell viability in frozen dough or water was related to the duration of the dissolved-oxygen deficit during fed-batch growth. Strongly aerobic fed-batch cultures grown at a reduced average specific rate (μ = 0.088 h−1 compared with 0.117 h−1) also showed greater trehalose synthesis and improved frozen-dough stability. Insufficient aeration (dissolved-oxygen deficit) and lower growth temperature (20°C instead of 30°C) decreased both fed-batch-grown yeast cryoresistance and trehalose content. Although trehalose had a cryoprotective effect in S. cerevisiae, its effect was neutralized by even a momentary lack of excess dissolved oxygen in the fed-batch growth medium. PMID:16348024

Maize Inoculation with Azospirillum brasilense Ab-V5 Cells Enriched with Exopolysaccharides and Polyhydroxybutyrate Results in High Productivity under Low N Fertilizer Input

PubMed Central

Oliveira, André L. M.; Santos, Odair J. A. P.; Marcelino, Paulo R. F.; Milani, Karina M. L.; Zuluaga, Mónica Y. A.; Zucareli, Claudemir; Gonçalves, Leandro S. A.

2017-01-01

Although Azospirillum strains used in commercial inoculant formulations presents diazotrophic activity, it has been reported that their ability to produce phytohormones plays a pivotal role in plant growth-promotion, leading to a general recommendation of its use in association with regular N-fertilizer doses. In addition, a high variability in the effectiveness of Azospirillum inoculants is still reported under field conditions, contributing to the adoption of the inoculation technology as an additional management practice rather than its use as an alternative practice to the use of chemical inputs in agriculture. To investigate whether the content of stress-resistance biopolymers would improve the viability and performance of Azospirillum inoculants when used as substitute of N-fertilizers, biomass of A. brasilense strain Ab-V5 enriched in exopolysaccharides (EPS) and polyhydroxybutirate (PHB) was produced using a new culture medium developed by factorial mixture design, and the effectiveness of resulting inoculants was evaluated under field conditions. The culture medium formulation extended the log phase of A. brasilense cultures, which presented higher cell counts and increased EPS and PHB contents than observed in the cultures grown in the OAB medium used as control. An inoculation trial with maize conducted under greenhouse conditions and using the biopolymers-enriched Ab-V5 cells demonstrated the importance of EPS and PHB to the long term bacterial viability in soil and to the effectiveness of inoculation. The effectiveness of liquid and peat inoculants prepared with Ab-V5 cells enriched with EPS and PHB was also evaluated under field conditions, using maize as target crop along different seasons, with the inoculants applied directly over seeds or at topdressing under limiting levels of N-fertilization. No additive effect on yield resulted from inoculation under high N fertilizer input, while inoculated plants grown under 80% reduction in N fertilizer showed yields at levels compared to fully fertilized plants, regardless the inoculation method. The presented data highlights the feasibility to partially substitute the N-fertilizer demand in non-legume crops using high-quality inoculant formulations, prepared with diazotrophic bacteria enriched with stress-resistance biopolymers that confer increased viability an effectiveness to the bacterial cells. PMID:29018432

Lactate production as representative of the fermentation potential of Corynebacterium glutamicum 2262 in a one-step process.

PubMed

Khuat, Hoang Bao Truc; Kaboré, Abdoul Karim; Olmos, Eric; Fick, Michel; Boudrant, Joseph; Goergen, Jean-Louis; Delaunay, Stéphane; Guedon, Emmanuel

2014-01-01

The fermentative properties of thermo-sensitive strain Corynebacterium glutamicum 2262 were investigated in processes coupling aerobic cell growth and the anaerobic fermentation phase. In particular, the influence of two modes of fermentation on the production of lactate, the fermentation product model, was studied. In both processes, lactate was produced in significant amount, 27 g/L in batch culture, and up to 55.8 g/L in fed-batch culture, but the specific production rate in the fed-batch culture was four times lower than that in the batch culture. Compared to other investigated fermentation processes, our strategy resulted in the highest yield of lactic acid from biomass. Lactate production by C. glutamicum 2262 thus revealed the capability of the strain to produce various fermentation products from pyruvate.

Fault detection and diagnosis in an industrial fed-batch cell culture process.

PubMed

Gunther, Jon C; Conner, Jeremy S; Seborg, Dale E

2007-01-01

A flexible process monitoring method was applied to industrial pilot plant cell culture data for the purpose of fault detection and diagnosis. Data from 23 batches, 20 normal operating conditions (NOC) and three abnormal, were available. A principal component analysis (PCA) model was constructed from 19 NOC batches, and the remaining NOC batch was used for model validation. Subsequently, the model was used to successfully detect (both offline and online) abnormal process conditions and to diagnose the root causes. This research demonstrates that data from a relatively small number of batches (approximately 20) can still be used to monitor for a wide range of process faults.

Development of a chemically defined platform fed-batch culture media for monoclonal antibody-producing CHO cell lines with optimized choline content.

PubMed

Kuwae, Shinobu; Miyakawa, Ichiko; Doi, Tomohiro

2018-01-11

A chemically defined platform basal medium and feed media were developed using a single Chinese hamster ovary (CHO) cell line that produces a monoclonal antibody (mAb). Cell line A, which showed a peak viable cell density of 5.9 × 10 6  cells/mL and a final mAb titer of 0.5 g/L in batch culture, was selected for the platform media development. Stoichiometrically balanced feed media were developed using glucose as an indicator of cell metabolism to determine the feed rates of all other nutrients. A fed-batch culture of cell line A using the platform fed-batch medium yielded a 6.4 g/L mAb titer, which was 12-fold higher than that of the batch culture. To examine the applicability of the platform basal medium and feed media, three other cell lines (A16, B, and C) that produce mAbs were cultured using the platform fed-batch medium, and they yielded mAb titers of 8.4, 3.3, and 6.2 g/L, respectively. The peak viable cell densities of the three cell lines ranged from 1.3 × 10 7 to 1.8 × 10 7  cells/mL. These results show that the nutritionally balanced fed-batch medium and feeds worked well for other cell lines. During the medium development, we found that choline limitation caused a lower cell viability, a lower mAb titer, a higher mAb aggregate content, and a higher mannose-5 content. The optimal choline chloride to glucose ratio for the CHO cell fed-batch culture was determined. Our platform basal medium and feed media will shorten the medium-development time for mAb-producing cell lines.

Inoculation of starter cultures in a semi-dry coffee (Coffea arabica) fermentation process.

PubMed

Evangelista, Suzana Reis; Miguel, Maria Gabriela da Cruz Pedrozo; Cordeiro, Cecília de Souza; Silva, Cristina Ferreira; Pinheiro, Ana Carla Marques; Schwan, Rosane Freitas

2014-12-01

The aim of this study was to evaluate the use of yeasts as starter cultures in coffee semi-dry processing. Arabica coffee was inoculated with one of the following starter cultures: Saccharomyces cerevisiae UFLA YCN727, S. cerevisiae UFLA YCN724, Candida parapsilosis UFLA YCN448 and Pichia guilliermondii UFLA YCN731. The control was not inoculated with a starter culture. Denaturing gradient gel electrophoresis (DGGE) was used to assess the microbial population, and organic acids and volatile compounds were quantified by HPLC and HS-SPME/GC, respectively. Sensory analyses were evaluated using the Temporal Dominance of Sensations (TDS). DGGE analysis showed that the inoculated yeasts were present throughout the fermentation. Other yeast species were also detected, including Debaryomyces hansenii, Cystofilobasidium ferigula and Trichosporon cavernicola. The bacterial population was diverse and was composed of the following genera: Weissella, Leuconostoc, Gluconobacter, Pseudomonas, Pantoea, Erwinia and Klebsiella. Butyric and propionic acids, were not detected in any treatment A total of 47 different volatiles compounds have been identified. The coffee inoculated with yeast had a caramel flavor that was not detected in the control, as assessed by TDS. The use of starter cultures during coffee fermentation is an interesting alternative for obtaining a beverage quality with distinctive flavor. Copyright © 2014 Elsevier Ltd. All rights reserved.

Acceleration of Antimicrobial Susceptibility Testing of Positive Blood Cultures by Inoculation of Vitek 2 Cards with Briefly Incubated Solid Medium Cultures

PubMed Central

Idelevich, Evgeny A.; Schüle, Isabel; Grünastel, Barbara; Wüllenweber, Jörg; Peters, Georg

2014-01-01

Briefly incubated agar cultures from positive blood cultures were used for antimicrobial susceptibility testing (AST) by Vitek 2. The cultivation time until inoculation was 3.8 h for Gram-positive cocci and 2.4 h for Gram-negative rods. The error rates were low, providing early and reliable AST without additional time or cost expenditure. PMID:25165084

Comparison of Exoelectrogenic Bacteria Detected Using Two Different Methods: U-tube Microbial Fuel Cell and Plating Method

PubMed Central

Yu, Jaecheul; Cho, Sunja; Kim, Sunah; Cho, Haein; Lee, Taeho

2012-01-01

In a microbial fuel cell (MFC), exoelectrogens, which transfer electrons to the electrode, have been regarded as a key factor for electricity generation. In this study, U-tube MFC and plating methods were used to isolate exoelectrogens from the anode of an MFC. Disparate microorganisms were identified depending on isolation methods, despite the use of an identical source. Denaturing gel gradient electrophoresis (DGGE) analysis showed that certain microorganisms became dominant in the U-tube MFC. The predominant bacterium was similar to Ochrobactrum sp., belonging to the Alphaproteobacteria, which was shown to be able to function as an exoelectrogen in a previous study. Three isolates, one affiliated with Bacillus sp. and two with Paenibacillus sp., were identified using the plating method, which belonged to the Gram-positive bacteria, the Firmicutes. The U-tube MFCs were inoculated with the three isolates using the plating method, operated in the batch mode and the current was monitored. All of the U-tube MFCs inoculated with each isolate after isolation from plates produced lower current (peak current density: 3.6–16.3 mA/m2) than those in U-tube MFCs with mixed culture (48.3–62.6 mA/m2). Although the isolates produced low currents, various bacterial groups were found to be involved in current production. PMID:22129603

Linking microbial community structure and product spectrum of rice straw fermentation with undefined mixed culture

NASA Astrophysics Data System (ADS)

Ai, Binling; Chi, Xue; Meng, Jia; Sheng, Zhanwu; Zheng, Lili; Zheng, Xiaoyan; Li, Jianzheng

2017-12-01

Undefined mixed culture-based fermentation is an alternative strategy for biofuels and bioproducts production from lignocellulosic biomass without supplementary cellulolytic enzymes. Mixed culture produces mixed carboxylates. To estimate the relationship between microbial community structure and product spectrum, carboxylate production was initiated by mixed cultures with different microbial community structure. All the inoculum cultures were derived from the same enrichment culture from the combination of cattle manure, pig manure compost, corn field soil and rotten wood. Due to the differences in the preparation method and culture time, the inoculum cultures for batch fermentation had high similarity in microbial community structure, while the community structure of each inoculum culture for repeated batch fermentation differed from that of another. The inoculum cultures with similar community structure led to a similar product spectrum. In batch fermentation, the selectivity of main product butyric acid stabilized around 76%. The inoculum cultures with different community structures resulted in different product spectra. In repeated batch fermentation, the butyric acid content gradually decreased to 27%, and the by-product acetic acid content steadily increased to 56%. The other by-products including propionic, valeric and caproic acids were also increased. It is deduced that keeping the microbial community structure stable makes the basic and key precondition for steady production of specific carboxylic acid with undefined mixed culture.

Switching the mode of sucrose utilization by Saccharomyces cerevisiae

PubMed Central

Badotti, Fernanda; Dário, Marcelo G; Alves, Sergio L; Cordioli, Maria Luiza A; Miletti, Luiz C; de Araujo, Pedro S; Stambuk, Boris U

2008-01-01

Background Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L) initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H+ symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures. Results We have deleted from the genome of a S. cerevisiae strain lacking invertase the high-affinity sucrose-H+ symporter encoded by the AGT1 gene. This strain could still grow efficiently on sucrose due to a low-affinity and low-capacity sucrose-H+ symport activity mediated by the MALx1 maltose permeases, and its further intracellular hydrolysis by cytoplasmic maltases. Although sucrose consumption by this engineered yeast strain was slower than with the parental yeast strain, the cells grew efficiently on sucrose due to an increased respiration of the carbon source. Consequently, this engineered yeast strain produced less ethanol and 1.5 to 2 times more biomass when cultivated in simple batch mode using 20 g/L sucrose as the carbon source. Conclusion Higher cell densities during batch cultures on 20 g/L sucrose were achieved by using a S. cerevisiae strain engineered in the sucrose uptake system. Such result was accomplished by effectively reducing sucrose uptake by the yeast cells, avoiding overflow metabolism, with the concomitant reduction in ethanol production. The use of this modified yeast strain in simpler batch culture mode can be a viable option to more complicated traditional sucrose-limited fed-batch cultures for biomass-directed processes of S. cerevisiae. PMID:18304329

Switching the mode of sucrose utilization by Saccharomyces cerevisiae.

PubMed

Badotti, Fernanda; Dário, Marcelo G; Alves, Sergio L; Cordioli, Maria Luiza A; Miletti, Luiz C; de Araujo, Pedro S; Stambuk, Boris U

2008-02-27

Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L) initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H+ symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures. We have deleted from the genome of a S. cerevisiae strain lacking invertase the high-affinity sucrose-H+ symporter encoded by the AGT1 gene. This strain could still grow efficiently on sucrose due to a low-affinity and low-capacity sucrose-H+ symport activity mediated by the MALx1 maltose permeases, and its further intracellular hydrolysis by cytoplasmic maltases. Although sucrose consumption by this engineered yeast strain was slower than with the parental yeast strain, the cells grew efficiently on sucrose due to an increased respiration of the carbon source. Consequently, this engineered yeast strain produced less ethanol and 1.5 to 2 times more biomass when cultivated in simple batch mode using 20 g/L sucrose as the carbon source. Higher cell densities during batch cultures on 20 g/L sucrose were achieved by using a S. cerevisiae strain engineered in the sucrose uptake system. Such result was accomplished by effectively reducing sucrose uptake by the yeast cells, avoiding overflow metabolism, with the concomitant reduction in ethanol production. The use of this modified yeast strain in simpler batch culture mode can be a viable option to more complicated traditional sucrose-limited fed-batch cultures for biomass-directed processes of S. cerevisiae.

Multivariate statistical process control (MSPC) using Raman spectroscopy for in-line culture cell monitoring considering time-varying batches synchronized with correlation optimized warping (COW).

PubMed

Liu, Ya-Juan; André, Silvère; Saint Cristau, Lydia; Lagresle, Sylvain; Hannas, Zahia; Calvosa, Éric; Devos, Olivier; Duponchel, Ludovic

2017-02-01

Multivariate statistical process control (MSPC) is increasingly popular as the challenge provided by large multivariate datasets from analytical instruments such as Raman spectroscopy for the monitoring of complex cell cultures in the biopharmaceutical industry. However, Raman spectroscopy for in-line monitoring often produces unsynchronized data sets, resulting in time-varying batches. Moreover, unsynchronized data sets are common for cell culture monitoring because spectroscopic measurements are generally recorded in an alternate way, with more than one optical probe parallelly connecting to the same spectrometer. Synchronized batches are prerequisite for the application of multivariate analysis such as multi-way principal component analysis (MPCA) for the MSPC monitoring. Correlation optimized warping (COW) is a popular method for data alignment with satisfactory performance; however, it has never been applied to synchronize acquisition time of spectroscopic datasets in MSPC application before. In this paper we propose, for the first time, to use the method of COW to synchronize batches with varying durations analyzed with Raman spectroscopy. In a second step, we developed MPCA models at different time intervals based on the normal operation condition (NOC) batches synchronized by COW. New batches are finally projected considering the corresponding MPCA model. We monitored the evolution of the batches using two multivariate control charts based on Hotelling's T 2 and Q. As illustrated with results, the MSPC model was able to identify abnormal operation condition including contaminated batches which is of prime importance in cell culture monitoring We proved that Raman-based MSPC monitoring can be used to diagnose batches deviating from the normal condition, with higher efficacy than traditional diagnosis, which would save time and money in the biopharmaceutical industry. Copyright © 2016 Elsevier B.V. All rights reserved.

Batch-to-batch uniformity of bacterial community succession and flavor formation in the fermentation of Zhenjiang aromatic vinegar.

PubMed

Wang, Zong-Min; Lu, Zhen-Ming; Yu, Yong-Jian; Li, Guo-Quan; Shi, Jin-Song; Xu, Zheng-Hong

2015-09-01

Solid-state fermentation of traditional Chinese vinegar is a mixed-culture refreshment process that proceeds for many centuries without spoilage. Here, we investigated bacterial community succession and flavor formation in three batches of Zhenjiang aromatic vinegar using pyrosequencing and metabolomics approaches. Temporal patterns of bacterial succession in the Pei (solid-state vinegar culture) showed no significant difference (P > 0.05) among three batches of fermentation. In all the batches investigated, the average number of community operational taxonomic units (OTUs) decreased dramatically from 119 ± 11 on day 1 to 48 ± 16 on day 3, and then maintained in the range of 61 ± 9 from day 5 to the end of fermentation. We confirmed that, within a batch of fermentation process, the patterns of bacterial diversity between the starter (took from the last batch of vinegar culture on day 7) and the Pei on day 7 were similar (90%). The relative abundance dynamics of two dominant members, Lactobacillus and Acetobacter, showed high correlation (coefficient as 0.90 and 0.98 respectively) among different batches. Furthermore, statistical analysis revealed dynamics of 16 main flavor metabolites were stable among different batches. The findings validate the batch-to-batch uniformity of bacterial community succession and flavor formation accounts for the quality of Zhenjiang aromatic vinegar. Based on our understanding, this is the first study helps to explain the rationality of age-old artistry from a scientific perspective. Copyright © 2015 Elsevier Ltd. All rights reserved.

In vitro approaches to assess the effects of açai (Euterpe oleracea) digestion on polyphenol availability and the subsequent impact on the faecal microbiota.

PubMed

Alqurashi, Randah M; Alarifi, Sehad N; Walton, Gemma E; Costabile, Adele F; Rowland, Ian R; Commane, Daniel M

2017-11-01

A considerable proportion of dietary plant-polyphenols reach the colon intact; determining the effects of these compounds on colon-health is of interest. We hypothesise that both fibre and plant polyphenols present in açai (Euterpe oleracea) provide prebiotic and anti-genotoxic benefits in the colon. We investigated this hypothesis using a simulated in vitro gastrointestinal digestion of açai pulp, and a subsequent pH-controlled, anaerobic, batch-culture fermentation model reflective of the distal region of the human large intestine. Following in vitro digestion, 49.8% of the total initial polyphenols were available. In mixed-culture fermentations with faecal inoculate, the digested açai pulp precipitated reductions in the numbers of both the Bacteroides-Prevotella spp. and the Clostridium-histolyticum groups, and increased the short-chain fatty acids produced compared to the negative control. The samples retained significant anti-oxidant and anti-genotoxic potential through digestion and fermentation. Dietary intervention studies are needed to prove that consuming açai is beneficial to gut health. Copyright © 2017 Elsevier Ltd. All rights reserved.

Proteomic Analysis of Host Cell Protein Dynamics in the Culture Supernatants of Antibody-Producing CHO Cells

PubMed Central

Park, Jin Hyoung; Jin, Jong Hwa; Lim, Myung Sin; An, Hyun Joo; Kim, Jong Won; Lee, Gyun Min

2017-01-01

Chinese hamster ovary (CHO) cells are the most common cell line used for the production of therapeutic proteins including monoclonal antibodies (mAbs). Host cell proteins (HCPs), secreted and released from lysed cells, accumulate extracellularly during the cultures of recombinant CHO (rCHO) cells, potentially impairing product quality. In an effort to maintain good mAb quality during the cultures, HCPs accumulated extracellularly in batch and fed-batch cultures of a mAb-producing rCHO cell line were identified and quantified by nanoflow liquid chromatography-tandem mass spectrometry, followed by their gene ontology and functional analysis. Due to higher cell concentration and longer culture duration, more HCPs were identified and quantitated in fed-batch culture (2145 proteins identified and 1673 proteins quantified) than in batch culture (1934 proteins identified and 1486 proteins quantified). Clustering analysis of HCPs showed that the concentration profiles of HCPs affecting mAb quality (Lgmn, Ctsd, Gbl1, and B4galt1) correlated with changes in mAb quality attributes such as aggregation, charge variants, and N-glycosylation during the cultures. Taken together, the dataset of HCPs obtained in this study provides insights into determining the appropriate target proteins to be removed during both the cultures and purification steps for ensuring good mAb quality. PMID:28281648

Reduction of Biogenic Amines during Miso Fermentation by Lactobacillus plantarum as a Starter Culture.

PubMed

Lee, Yi-Chen; Kung, Hsien-Feng; Huang, Ya-Ling; Wu, Chien-Hui; Huang, Yu-Ru; Tsai, Yung-Hsiang

2016-09-01

Lactobacillus plantarum D-103 isolated from a miso product that possesses amine-degrading activity was used as a starter culture in miso fermentation (25°C for 120 days) in this study. The salt content in control samples (without starter culture) and inoculated samples (inoculated with L. plantarum D-103) remained constant at 10.4% of the original salt concentration throughout fermentation, whereas the pH value decreased from 6.2 to 4.6 during fermentation. The inoculated samples had significantly lower (P < 0.05) levels of total volatile basic nitrogen than control samples after 40 days of fermentation. After 120 days of fermentation, the histamine and overall biogenic amine contents in inoculated samples were reduced by 58 and 27%, respectively, compared with control samples. To our knowledge, this is the first report to demonstrate that application of a starter culture with amine-degrading activity in miso products was effective in reducing the accumulation of biogenic amines.

Powassan Virus: Morphology and Cytopathology

PubMed Central

Abdelwahab, K. S. E.; Almeida, J. D.; Doane, F. W.; McLean, D. M.

1964-01-01

Powassan virus, a North American tickborne group B arbovirus, multiplied after simultaneous inoculation into bottles or tubes of virus and trypsinized suspension of continuous-line cultures of rhesus monkey kidney cells, strain LLC-MK2. Cytopathic effects comprising cell rounding and cytoplasmic vacuolation were first observed five days after inoculation. Mixture of Powassan antiserum with virus before inoculation into tissue cultures inhibited the appearance of cytopathic effects. Hemagglutinins for rooster erythrocytes, optimally at pH 6.4 and 22° C., first appeared in tissue culture supernatant fluids four days after inoculation. Electron microscopic observation of thin sections of infected tissue culture cells showed virus particles 360-380 A.U. along outer cell membranes and edges of cytoplasmic vacuoles. In phosphotungstic acid negatively stained preparations, intact virus particles, 400-450 A.U. total diameter, were observed inside infected cells. In particles in which the peripheral layer became discontinuous, geometrically arranged subunits compatible with cubic symmetry were observed. ImagesFig. 1Fig. 2Fig. 3Fig. 4Fig. 5 PMID:14146854

Influence of 2,4,6-trinitrotoluene (TNT) concentration on the degradation of TNT in explosive-contaminated soils by the white rot fungus Phanerochaete chrysosporium.

PubMed Central

Spiker, J K; Crawford, D L; Crawford, R L

1992-01-01

The ability of Phanerochaete chrysosporium to bioremediate TNT (2,4,6-trinitrotoluene) in a soil containing 12,000 ppm of TNT and the explosives RDX (hexahydro-1,3,5-trinitro-1,3,5- triazine; 3,000 ppm) and HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine; 300 ppm) was investigated. The fungus did not grow in malt extract broth containing more than 0.02% (wt/vol; 24 ppm of TNT) soil. Pure TNT or explosives extracted from the soil were degraded by P. chrysosporium spore-inoculated cultures at TNT concentrations of up to 20 ppm. Mycelium-inoculated cultures degraded 100 ppm of TNT, but further growth was inhibited above 20 ppm. In malt extract broth, spore-inoculated cultures mineralized 10% of added [14C]TNT (5 ppm) in 27 days at 37 degrees C. No mineralization occurred during [14C]TNT biotransformation by mycelium-inoculated cultures, although the TNT was transformed. PMID:1444437

Monochloramine Cometabolism by Mixed-Culture Nitrifiers under Drinking Water Conditions

EPA Science Inventory

The current research investigated monochloramine cometabolism by nitrifying mixed cultures grown under drinking water relevant conditions and harvested from sand-packed reactors before conducting suspended growth batch kinetic experiments. Three batch reactors were used in each ...

Validation of Single and Pooled Manure Drag Swabs for the Detection of Salmonella Serovar Enteritidis in Commercial Poultry Houses.

PubMed

Kinde, Hailu; Goodluck, Helen A; Pitesky, Maurice; Friend, Tom D; Campbell, James A; Hill, Ashley E

2015-12-01

Single swabs (cultured individually) are currently used in the Food and Drug Administration (FDA) official method for sampling the environment of commercial laying hens for the detection of Salmonella enterica ssp. serovar Enteritidis (Salmonella Enteritidis). The FDA has also granted provisional acceptance of the National Poultry Improvement Plan's (NPIP) Salmonella isolation and identification methodology for samples taken from table-egg layer flock environments. The NPIP method, as with the FDA method, requires single-swab culturing for the environmental sampling of laying houses for Salmonella Enteritidis. The FDA culture protocol requires a multistep culture enrichment broth, and it is more labor intensive than the NPIP culture protocol, which requires a single enrichment broth. The main objective of this study was to compare the FDA single-swab culturing protocol with that of the NPIP culturing protocol but using a four-swab pool scheme. Single and multi-laboratory testing of replicate manure drag swab sets (n  =  525 and 672, respectively) collected from a Salmonella Enteritidis-free commercial poultry flock was performed by artificially contaminating swabs with either Salmonella Enteritidis phage type 4, 8, or 13a at one of two inoculation levels: low, x¯  = 2.5 CFU (range 2.5-2.7), or medium, x¯  = 10.0 CFU (range 7.5-12). For each replicate, a single swab (inoculated), sets of two swabs (one inoculated and one uninoculated), and sets of four swabs (one inoculated and three uninoculated), testing was conducted using the FDA or NPIP culture method. For swabs inoculated with phage type 8, the NPIP method was more efficient (P < 0.05) for all swab sets at both inoculation levels than the reference method. The single swabs in the NPIP method were significantly (P < 0.05) better than four-pool swabs in detecting Salmonella Enteritidis at the lower inoculation level. In the collaborative study (n  =  13 labs) using Salmonella Enteritidis phage type 13a inoculated swabs, there was no significant difference (P > 0.05) between the FDA method (single swabs) and the pooled NPIP method (four-pool swabs). The study concludes that the pooled NPIP method is not significantly different from the FDA method for the detection of Salmonella Enteritidis in drag swabs in commercial poultry laying houses. Consequently based on the FDA's Salmonella Enteritidis rule for equivalency of different methods, the pooled NPIP method should be considered equivalent. Furthermore, the pooled NPIP method was more efficient and cost effective.

Effects of dietary roughage and sulfur in diets containing corn dried distillers grains with solubles on hydrogen sulfide production and fermentation by rumen microbes in vitro.

PubMed

Binversie, E Y; Ruiz-Moreno, M; Carpenter, A J; Heins, B J; Crawford, G I; DiCostanzo, A; Stern, M D

2016-09-01

Dried distillers grains with solubles (DDGS) have been used in production animal diets; however, overuse of DDGS can cause toxic concentrations of ruminal hydrogen sulfide gas (HS), resulting in polioencephalomalacia, a deleterious brain disease. Because HS gas requires an acidic rumen environment and diet can influence ruminal pH, it has been postulated that dietary manipulation could help mitigate HS production. The objective of this study was to assess the effect of dietary roughage and sulfur concentrations on HS production and rumen fermentation. In Exp. 1, 7 dual-flow continuous culture fermenters were used in 4 consecutive 9-d periods consisting of 6 d of adaptation followed by 3 d of sampling. At the conclusion of each 9-d continuous culture period, adapted rumen fluid was used for inoculation of 24-h batch culture incubations for Exp. 2. For both experiments, 6 dietary treatments were formulated to consist of 0.3%, 0.4%, or 0.5% dietary sulfur (LS, MS, and HS, respectively) and 3% or 9% dietary roughage (LR and MR, respectively), using grass hay as the roughage source. A corn-based diet without DDGS was used as a control diet. Headspace gas was sampled to determine HS production and concentration. In Exp. 1, greater dietary roughage had no effect ( = 0.14) on HS production but did create a less acidic environment because of an increase ( < 0.01) in the in vitro pH. In Exp. 2, an increase in dietary sulfur caused an increase ( = 0.04) in ruminal HS production, but there was no direct effect ( = 0.25) of dietary roughage on HS production. Greater dietary roughage resulted in a less ( = 0.01) acidic final batch culture pH but a lower ( < 0.01) total VFA concentration. Further investigation is needed to determine a more effective way to mitigate ruminal HS production using dietary manipulation, which could include greater inclusion of dietary roughage or the use of different roughage sources.

Modelling of Batch Lactic Acid Fermentation in
the Presence of Anionic Clay

PubMed Central

Jinescu, Cosmin; Aruş, Vasilica Alisa; Nistor, Ileana Denisa

2014-01-01

Summary Batch fermentation of milk inoculated with lactic acid bacteria was conducted in the presence of hydrotalcite-type anionic clay under static and ultrasonic conditions. An experimental study of the effect of fermentation temperature (t=38–43 °C), clay/milk ratio (R=1–7.5 g/L) and ultrasonic field (ν=0 and 35 kHz) on process dynamics was performed. A mathematical model was selected to describe the fermentation process kinetics and its parameters were estimated based on experimental data. A good agreement between the experimental and simulated results was achieved. Consequently, the model can be employed to predict the dynamics of batch lactic acid fermentation with values of process variables in the studied ranges. A statistical analysis of the data based on a 23 factorial experiment was performed in order to express experimental and model-regressed process responses depending on t, R and ν factors. PMID:27904318

Is whole-culture synchronization biology's 'perpetual-motion machine'?

PubMed

Cooper, Stephen

2004-06-01

Whole-culture or batch synchronization cannot, in theory, produce a synchronized culture because it violates a fundamental law that proposes that no batch treatment can alter the cell-age order of a culture. In analogy with the history of perpetual-motion machines, it is suggested that the study of these whole-culture 'synchronization' methods might lead to an understanding of general biological principles even though these methods cannot be used to study the normal cell cycle.

Improved production of an enzyme that hydrolyses raw yam starch by Penicillium sp. S-22 using fed-batch fermentation.

PubMed

Sun, Hai-Yan; Ge, Xiang-Yang; Zhang, Wei-Guo

2006-11-01

A newly isolated strain, Penicillium sp. S-22, was used to produce an enzyme that hydrolyses raw yam starch [raw yam starch digesting enzyme (RYSDE)]. The enzyme activity and overall enzyme productivity were respectively 16 U/ml and 0.19 U/ml h in the batch culture. The enzyme activity increased to 85 U/ml by feeding of partially hydrolyzed raw yam starch. When a mixture containing partially hydrolyzed raw yam starch and peptone was fed by a pH-stat strategy, the enzyme activity reached 366 U/ml, 23-fold of that obtained in the batch culture, and the overall productivity reached 3.4 U/ml h, which was 18-fold of that in the batch culture.

Perfusion seed cultures improve biopharmaceutical fed-batch production capacity and product quality.

PubMed

Yang, William C; Lu, Jiuyi; Kwiatkowski, Chris; Yuan, Hang; Kshirsagar, Rashmi; Ryll, Thomas; Huang, Yao-Ming

2014-01-01

Volumetric productivity and product quality are two key performance indicators for any biopharmaceutical cell culture process. In this work, we showed proof-of-concept for improving both through the use of alternating tangential flow perfusion seed cultures coupled with high-seed fed-batch production cultures. First, we optimized the perfusion N-1 stage, the seed train bioreactor stage immediately prior to the production bioreactor stage, to minimize the consumption of perfusion media for one CHO cell line and then successfully applied the optimized perfusion process to a different CHO cell line. Exponential growth was observed throughout the N-1 duration, reaching >40 × 10(6) vc/mL at the end of the perfusion N-1 stage. The cultures were subsequently split into high-seed (10 × 10(6) vc/mL) fed-batch production cultures. This strategy significantly shortened the culture duration. The high-seed fed-batch production processes for cell lines A and B reached 5 g/L titer in 12 days, while their respective low-seed processes reached the same titer in 17 days. The shortened production culture duration potentially generates a 30% increase in manufacturing capacity while yielding comparable product quality. When perfusion N-1 and high-seed fed-batch production were applied to cell line C, higher levels of the active protein were obtained, compared to the low-seed process. This, combined with correspondingly lower levels of the inactive species, can enhance the overall process yield for the active species. Using three different CHO cell lines, we showed that perfusion seed cultures can optimize capacity utilization and improve process efficiency by increasing volumetric productivity while maintaining or improving product quality. © 2014 American Institute of Chemical Engineers.

Neonatal blood cultures: effect of delayed entry into the blood culture machine and bacterial concentration on the time to positive growth in a simulated model.

PubMed

Jardine, Luke Anthony; Sturgess, Barbara Ruth; Inglis, Garry Donald Trevor; Davies, Mark William

2009-04-01

To determine if: time from blood culture inoculation to positive growth (total time to positive) and time from blood culture machine entry to positive growth (machine time to positive) is altered by delayed entry into the automated blood culture machine, and if the total time to positive differs by the concentration of organisms inoculated into blood culture bottles. Staphylococcus epidermidis, Escherichia coli and group B beta-haemolytic streptococci were chosen as clinically significant representative organisms. Two concentrations (> or =10 colony-forming units per millilitre and <1 colony-forming units per millilitre) were inoculated into PEDS BacT/Alert blood culture bottles and randomly allocated to one of three delayed automated blood culture machine entry times (30 min/8.5 h/15.5 h). For all organisms at all concentrations, except the Staphylococcus epidermidis, the machine time to positive was significantly decreased by delayed entry. For all organisms at all concentrations, the mean total time to positive significantly increased with increasing delayed entry into the blood culture machine. Higher concentrations of group B beta-haemolytic streptococci and Escherichia coli grew significantly faster than lower concentrations. Bacterial growth in inoculated bottles, stored at room temperature, continues although at a slower rate than in those blood culture bottles immediately entered into the machine. If a blood culture specimen has been stored at room temperature for greater than 15.5 h, the currently allowed safety margin of 36 h (before declaring a result negative) may be insufficient.

Screenhouse and field persistence of nonpathogenic endophytic Fusarium oxysporum in Musa tissue culture plants.

PubMed

Paparu, Pamela; Dubois, Thomas; Gold, Clifford S; Niere, Björn; Adipala, Ekwamu; Coyne, Daniel

2008-04-01

Two major biotic constraints to highland cooking banana (Musa spp., genome group AAA-EA) production in Uganda are the banana weevil Cosmopolites sordidus and the burrowing nematode Radopholus similis. Endophytic Fusarium oxysporum strains inoculated into tissue culture banana plantlets have shown control of the banana weevil and the nematode. We conducted screenhouse and field experiments to investigate persistence in the roots and rhizome of two endophytic Fusarium oxysporum strains, V2w2 and III4w1, inoculated into tissue-culture banana plantlets of highland cooking banana cultivars Kibuzi and Nabusa. Re-isolation of F. oxysporum showed that endophyte colonization decreased faster from the rhizomes than from the roots of inoculated plants, both in the screenhouse and in the field. Whereas rhizome colonization by F. oxysporum decreased in the screenhouse (4-16 weeks after inoculation), root colonization did not. However, in the field (17-33 weeks after inoculation), a decrease was observed in both rhizome and root colonization. The results show a better persistence in the roots than rhizomes of endophytic F. oxysporum strains V2w2 and III4w1.

Adaptation of plastic surfaces for tissue culture by glow discharge.

PubMed Central

Amstein, C F; Hartman, P A

1975-01-01

Plastic petri dishes and microtitration plates were electrically charged by a glow discharge unit installed in a vacuum evaporator. Charged and uncharged plates, as well as plates commercially treated for tissue culture, were inoculated with Vero and BHK-21 cell lines; secondary cultures of monkey kidney, chicken lung, canine kidney, and embryonic bovine kidney; and primary chicken embryo fibroblasts and chicken lung cells. All cell cultures grew normally on petri plates charged with the covers open. Growth rate and cell density compared favorably with growth on the commercial tissue culture plates; cell growth was somewhat less dense, however, on plates charged with the covers closed. Charged plates could be sterilized by ultraviolet light and ethylene oxide with no adverse effects on cell growth. Cells inoculated onto plates charged up to 7 months before inoculation grew as well as on freshly charged plates. Images PMID:818106

Analysis of proteins involved in biodegradation of crop biomass

NASA Technical Reports Server (NTRS)

Crawford, Kamau; Trotman, Audrey

1998-01-01

The biodegradation of crop biomass for re-use in crop production is part of the bioregenerative life support concept proposed by the National Aeronautics and Space Administration (NASA) for long duration, manned space exploration. The current research was conducted in the laboratory to evaluate the use of electrophoretic analysis as a means of rapidly assaying for constitutive and induced proteins associated with the bacterial degradation of crop residue. The proteins involved in crop biomass biodegradation are either constitutive or induced. As a result, effluent and cultures were examined to investigate the potential of using electrophoretic techniques as a means of monitoring the biodegradation process. Protein concentration for optimum banding patterns was determined using the Bio-Rad Protein Assay kit. Four bacterial soil isolates were obtained from the G.W. Carver research Farm at Tuskegee University and used in the decomposition of components of plant biomass. The culture, WDSt3A was inoculated into 500 mL of either Tryptic Soy Broth or Nutrient Broth. Incubation, with shaking of each flask was for 96 hours at 30 C. The cultures consistently gave unique banding patterns under denaturing protein electrophoresis conditions, The associated extracellular enzymes also yielded characteristic banding patterns over a 14-day period, when native electrophoresis techniques were used to examine effluent from batch culture bioreactors. The current study evaluated sample preparation and staining protocols to determine the ease of use, reproducibility and reliability, as well as the potential for automation.

In vitro evaluation of the fermentation properties and potential prebiotic activity of Agave fructans.

PubMed

Gomez, E; Tuohy, K M; Gibson, G R; Klinder, A; Costabile, A

2010-06-01

This study was carried out to evaluate in vitro the fermentation properties and the potential prebiotic activity of Agave-fructans extracted from Agave tequilana (Predilife). Five different commercial prebiotics were compared using 24-h pH-controlled anaerobic batch cultures inoculated with human faecal slurries. Measurement of prebiotic efficacy was obtained by comparing bacterial changes, and the production of short-chain fatty acids (SCFA) was also determined. Effects upon major groups of the microbiota were monitored over 24 h incubations by fluorescence in situ hybridization. SCFA were measured by HPLC. Fermentation of the Agave fructans (Predilife) resulted in a large increase in numbers of bifidobacteria and lactobacilli. Under the in vitro conditions used, this study has shown the differential impact of Predilife on the microbial ecology of the human gut. This is the first study reporting of a potential prebiotic mode of activity for Agave fructans investigated which significantly increased populations of bifidobacteria and lactobacilli compared to cellulose used as a control.

Evaluation of cyanobacteria: Spirulina maxima for growth, nutrient removal, and quality on waste-effluent media in batch cultures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tadros, M.G.; Phillips, J.

1992-01-01

Spirulina maxima, a semi-microscopic filamentous blue-green alga, was inoculated in synthetic and waste media of different sources. The alga was evaluated for growth yield, uptake of nutrients and chemical composition. The removal rate of N and P was rapid during the first week of growth. At the end of the second week, more than 90% of the total -P and -N was removed. The mass of alga was high. The quality of the alga obtained in different media did not show much variations, except when the medium was limited in nutrients. Results indicated that Spirulina may be integrated into themore » effluent treatment system. Recycling waste materials not only minimizes the problem of water pollution but also revitalizes the inherently rich nutrients of waste. The biomass obtained from cultivation of Spirulina in these wastewater media may be used as a pigment-protein supplement in animal feed and as raw material for certain chemicals.« less

Prebiotic evaluation of red seaweed (Kappaphycus alvarezii) using in vitro colon model.

PubMed

Bajury, Dayang Marshitah; Rawi, Muhamad Hanif; Sazali, Iqbal Hakim; Abdullah, Aminah; Sarbini, Shahrul Razid

2017-11-01

Red seaweed (Kappaphycus alvarezii) cultivated from Sabah (RSS) and Langkawi (RSL) were digested using in vitro mouth, gastric and duodenal model. The digested seaweed then fermented in a pH-controlled batch culture system inoculated with human faeces to mimic the distal colon. Bacterial enumeration were monitored using fluorescent in situ hybridisation, and the fermentation end products, the short chain fatty acids (SCFA), were analysed using HPLC. Both RSS and RSL showed significant increase of Bifidobacterium sp.; from log 10 7.96 at 0 h to log 10 8.72 at 24 h, and from log 10 7.96 at 0 h to log 10 8.60 at 24 h, respectively, and shows no significant difference when compared to the Bifidobacterium sp. count at 24 h of inulin fermentation. Both seaweeds also showed significant increase in total SCFA production, particularly acetate and propionate. Overall, this data suggested that K. alvarezii might have the potential as a prebiotic ingredient.

Fate of Campylobacter jejuni in butter.

PubMed

Zhao, T; Doyle, M P; Berg, D E

2000-01-01

An outbreak of Campylobacter enteritis was associated with a restaurant in Louisiana during the summer of 1995. Thirty cases were identified, and four required hospitalization. Campylobacter jejuni was isolated from the patients, and epidemiologic studies revealed illness associated with eating garlic butter served at the restaurant. Three batches of garlic butter prepared by the restaurant associated with the outbreak and a C. jejuni isolate obtained from a patient involved in the outbreak were used for studies to determine the fate of C. jejuni in garlic butter. Studies also were done to determine the efficacy of the heat treatment used by the restaurant to prepare garlic bread to kill C. jejuni. Garlic butter was inoculated with approximately 10(4) and 10(6) CFU/g of C. jejuni and held at 5 or 21 degrees C. Results revealed that the survival of C. jejuni differed greatly, depending on the presence or absence of garlic. At 5 degrees C, C. jejuni populations decreased to an undetectable level (<10 CFU/g) within 3 h for two batches and within 24 h for another batch. In contrast, C. jejuni could survive at 5 degrees C for 13 days in butter with no garlic. At 21 degrees C, C. jejuni populations decreased to an undetectable level within 5 h for two batches and to 50 CFU/g in 5 h for another batch. In contrast, C. jejuni was detected at 500 CFU/g at 28 h after inoculation but was undetectable at 3 days in butter with no garlic held at 21 degrees C. The heating procedure (135 degrees C, 4 min) used to make garlic bread by the implicated restaurant was determined not to be sufficient for killing C. jejuni, with the internal temperature of the buttered bread after heating ranging from 19 to 22 degrees C. This study revealed that C. jejuni can survive for many days in refrigerated butter, but large populations (10(3) to 10(5) CFU/g) are killed within a few hours in butter that contains garlic. Furthermore, the heat treatment used by the restaurant to melt garlic butter in making garlic bread was not adequate to kill C. jejuni.

Increase of fruity aroma during mixed T. delbrueckii/S. cerevisiae wine fermentation is linked to specific esters enhancement.

PubMed

Renault, Philippe; Coulon, Joana; de Revel, Gilles; Barbe, Jean-Christophe; Bely, Marina

2015-08-17

The aim of this work was to study ester formation and the aromatic impact of Torulaspora delbrueckii when used in association with Saccharomyces cerevisiae during the alcoholic fermentation of must. In order to evaluate the influence of the inoculation procedure, sequential and simultaneous mixed cultures were carried out and compared to pure cultures of T. delbrueckii and S. cerevisiae. Our results showed that mixed inoculations allowed the increase, in comparison to S. cerevisiae pure culture, of some esters specifically produced by T. delbrueckii and significantly correlated to the maximal T. delbrueckii population reached in mixed cultures. Thus, ethyl propanoate, ethyl isobutanoate and ethyl dihydrocinnamate were considered as activity markers of T. delbrueckii. On the other hand, isobutyl acetate and isoamyl acetate concentrations were systematically increased during mixed inoculations although not correlated with the development of either species but were rather due to positive interactions between these species. Favoring T. delbrueckii development when performing sequential inoculation enhanced the concentration of esters linked to T. delbrueckii activity. On the contrary, simultaneous inoculation restricted the growth of T. delbrueckii, limiting the production of its activity markers, but involved a very important production of numerous esters due to more important positive interactions between species. These results suggest that the ester concentrations enhancement via interactions during mixed modalities was due to S. cerevisiae production in response to the presence of T. delbrueckii. Finally, sensory analyses showed that mixed inoculations between T. delbrueckii and S. cerevisiae allowed to enhance the complexity and fruity notes of wine in comparison to S. cerevisiae pure culture. Furthermore, the higher levels of ethyl propanoate, ethyl isobutanoate, ethyl dihydrocinnamate and isobutyl acetate in mixed wines were found responsible for the increase of fruitiness and complexity. Copyright © 2015 Elsevier B.V. All rights reserved.

Ability of a haloalkaliphilic bacterium isolated from Soap Lake, Washington to generate electricity at pH 11.0 and 7% salinity.

PubMed

Paul, Varun G; Minteer, Shelley D; Treu, Becky L; Mormile, Melanie R

2014-01-01

A variety of anaerobic bacteria have been shown to transfer electrons obtained from organic compound oxidation to the surface of electrodes in microbial fuel cells (MFCs) to produce current. Initial enrichments for iron (III) reducing bacteria were set up with sediments from the haloalkaline environment of Soap Lake, Washington, in batch cultures and subsequent transfers resulted in a culture that grew optimally at 7.0% salinity and pH 11.0. The culture was used to inoculate the anode chamber of a MFC with formate as the electron source. Current densities up to 12.5 mA/m2 were achieved by this bacterium. Cyclic voltammetry experiments demonstrated that an electron mediator, methylene blue, was required to transfer electrons to the anode. Scanning electron microscopic imaging of the electrode surface did not reveal heavy colonization of bacteria, providing evidence that the bacterium may be using an indirect mode of electron transfer to generate current. Molecular characterization of the 16S rRNA gene and restriction fragment length profiles (RFLP) analysis showed that the MFC enriched for a single bacterial species with a 99% similarity to the 16S rRNA gene of Halanaerobium hydrogeniformans. Though modest, electricity production was achieved by a haloalkaliphilic bacterium at pH 11.0 and 7.0% salinity.

Acetylene reduction (nitrogen fixation) associated with corn inoculated with Spirillum.

PubMed

Barber, L E; Tjepkema, J D; Russell, S A; Evans, H J

1976-07-01

Sorghum and corn breeding lines were grown in soil in field and greenhouse experiments with and without an inoculum of N2-fixing in Spirillum strains from Brazil. Estimated rates of N2 fixation associated with field-grown corn and sorghum plants were less than 4 g of N2/ha per day. The mean estimated N2-fixation rates determined on segments of roots from corn inoculated with Spirillum and grown in the greenhouse at 24 to 27 degrees C were 15 g of N2/ha per day (16 inbreds), 25 g of N2/ha per day (six hybrids), and 165 g of N2/ha per day for one hybird which was heavily inoculated. The corresponding mean rates determined from measurements of in situ cultures of the same series of corn plants (i.e., 16 inbreds, six hybrids, and one heavily inoculated hybrid) were 0.4, 2.3, and 1.1 g of N2/ha per day, respectively. Lower rates of C2H2 reduction were associated with control corn cultures which had been treated with autoclaved Spirillum than with cultures inoculated with live Spirillum. No C2H2 reduction was detected in plant cultures treated with ammonium nitrate. Numbers of nitrogen-fixing bacteria on excised roots of corn plants increased an average of about 30-fold during an overnight preincubation period, and as a result acetylene reduction assays of root samples after preincubation failed to serve as a valid basis for estimating N2 fixation by corn in pot cultures. Plants grown without added nitrogen either with or without inoculum exhibited severe symptoms of nitrogen deficiency and in most cases produced significantly less dry weight than those supplied with fixed nitrogen. Although substantial rates of C2H2 reduction by excised corn roots were observed after preincubation under limited oxygen, the yield and nitrogen content of inoculated plants and the C2H2-reduction rates by inoculated pot cultures of corn, in situ, provided no evidence of appreciable N2 fixation.

Experimental inoculation of domestic cats (Felis domesticus) with Sarcocystis neurona or S. neurona-like merozoites.

PubMed

Butcher, M; Lakritz, J; Halaney, A; Branson, K; Gupta, G D; Kreeger, J; Marsh, A E

2002-07-29

Sarcocystis neurona is the parasite most commonly associated with equine protozoal myeloencephalitis (EPM). Recently, cats (Felis domesticus) have been demonstrated to be an experimental intermediate host in the life cycle of S. neurona. This study was performed to determine if cats experimentally inoculated with culture-derived S. neurona merozoites develop tissue sarcocysts infectious to opossums (Didelphis virginiana), the definitive host of S. neurona. Four cats were inoculated with S. neurona or S. neurona-like merozoites and all developed antibodies reacting to S. neurona merozoite antigens, but tissue sarcocysts were detected in only two cats. Muscle tissues from the experimentally inoculated cats with and without detectable sarcocysts were fed to laboratory-reared opossums. Sporocysts were detected in gastrointestinal (GI) scrapings of one opossum fed experimentally infected feline tissues. The study results suggest that cats can develop tissue cysts following inoculation with culture-derived Sarcocystis sp. merozoites in which the particular isolate was originally derived from a naturally infected cat with tissue sarcocysts. This is in contrast to cats which did not develop tissue cysts when inoculated with S. neurona merozoites originally derived from a horse with EPM. These results indicate present biological differences between the culture-derived merozoites of two Sarcocystis isolates, Sn-UCD 1 and Sn-Mucat 2.

Two efficient nitrite-reducing Lactobacillus strains isolated from traditional fermented pork (Nanx Wudl) as competitive starter cultures for Chinese fermented dry sausage.

PubMed

Chen, Xi; Li, Jiapeng; Zhou, Tong; Li, Jinchun; Yang, Junna; Chen, Wenhua; Xiong, Youling L

2016-11-01

Lactic acid bacteria isolated from traditional Dong pork product (Nanx Wudl) were investigated for their potential as starter cultures for Chinese fermented dry sausages. Based on preliminary screening, Lactobacillus plantarum CMRC6 and Lactobacillus sakei CMRC15, both showing excellent nitrite-reducing capacity, were used as single-strain starter cultures. For comparison, a commercial composite starter was also tested. In CMRC6 and CMRC15-inoculated sausages, lactic acid bacteria dominated the microflora and improved the microbiological safety by suppression of Enterobacteriaceae growth. Nitrite content of all inoculated sausages declined rapidly during ripening compared to non-inoculated. Texture profiles analysis showed inoculated sausages had more pronounced textural development during ripening. Sensory evaluation indicated CMRC6 and CMRC15-fermented sausages had comparable or more desirable organoleptic characteristics than sausage made with commercial starters. Therefore, CMRC6 and CMRC15 are promising candidates as multi-functional starter cultures for microbiological safety and residual nitrite control in gourmet Chinese dry sausage production. Copyright © 2016 Elsevier Ltd. All rights reserved.

Efficacy of a commercial probiotic relative to oxytetracycline as Gram-negative bacterial control agents in a rotifer (Brachionus plicatilis) batch culture

USDA-ARS?s Scientific Manuscript database

Two trials were conducted to evaluate two gram-negative bacterial control strategies in batch cultures of the rotifer Brachionus plicatilis. In the first trial, rotifers at an initial density of 47/mL were cultured for 5 d and dosed with a 10-mg/L solution of either oxytetracycline or a commercial p...

Significantly enhanced production of acarbose in fed-batch fermentation with the addition of S-adenosylmethionine.

PubMed

Sun, Li-Hui; Li, Ming-Gang; Wang, Yuan-Shan; Zheng, Yu-Guo

2012-06-01

Acarbose, a pseudo-oligosaccharide, is widely used clinically in therapies for non-insulin-dependent diabetes. In the present study, S-adenosylmethionine (SAM) was added to selected media in order to investigate its effect on acarbose fermentation by Actinoplanes utahensis ZJB- 08196. Acarbose titer was seen to increase markedly when concentrations of SAM were added over a period of time. The effects of glucose and maltose on the production of acarbose were investigated in both batch and fed-batch fermentation. Optimal acarbose production was observed at relatively low glucose levels and high maltose levels. Based on these results, a further fed-batch experiment was designed so as to enhance the production of acarbose. Fed-batch fermentation was carried out at an initial glucose level of 10 g/l and an initial maltose level of 60 g/l. Then, 12 h post inoculation, 100 micromol/l SAM was added. In addition, 8 g/l of glucose was added every 24 h, and 20 g/l of maltose was added at 96 h. By way of this novel feeding strategy, the maximum titer of acarbose achieved was 6,113 mg/l at 192 h. To our knowledge, the production level of acarbose achieved in this study is the highest ever reported.

Perfusion cell culture decreases process and product heterogeneity in a head-to-head comparison with fed-batch.

PubMed

Walther, Jason; Lu, Jiuyi; Hollenbach, Myles; Yu, Marcella; Hwang, Chris; McLarty, Jean; Brower, Kevin

2018-05-30

In this study, we compared the impacts of fed-batch and perfusion platforms on process and product attributes for IgG1- and IgG4-producing cell lines. A "plug-and-play" approach was applied to both platforms at bench scale, using commercially available basal and feed media, a standard feed strategy for fed-batch, and ATF filtration for perfusion. Product concentration in fed-batch was 2.5 times greater than perfusion, while average productivity in perfusion was 7.5 times greater than fed-batch. PCA revealed more variability in the cell environment and metabolism during the fed-batch run. LDH measurements showed that exposure of product to cell lysate was 7-10 times greater in fed-batch. Product analysis shows larger abundances of neutral species in perfusion, likely due to decreased bioreactor residence times and extracellular exposure. The IgG1 perfusion product also had higher purity and lower half-antibody. Glycosylation was similar across both culture modes. The first perfusion harvest slice for both product types showed different glycosylation than subsequent harvests, suggesting that product quality lags behind metabolism. In conclusion, process and product data indicate that intra-lot heterogeneity is decreased in perfusion cultures. Additional data and discussion is required to understand the developmental, clinical and commercial implications, and in what situations increased uniformity would be beneficial. This article is protected by copyright. All rights reserved.

Exploiting the metabolism of PYC expressing HEK293 cells in fed-batch cultures.

PubMed

Vallée, Cédric; Durocher, Yves; Henry, Olivier

2014-01-01

The expression of recombinant yeast pyruvate carboxylase (PYC) in animal cell lines was shown in previous studies to reduce significantly the formation of waste metabolites, although it has translated into mixed results in terms of improved cellular growth and productivity. In this work, we demonstrate that the unique phenotype of PYC expressing cells can be exploited through the application of a dynamic fed-batch strategy and lead to significant process enhancements. Metabolically engineered HEK293 cells stably producing human recombinant IFNα2b and expressing the PYC enzyme were cultured in batch and fed-batch modes. Compared to parental cells, the maximum cell density in batch was increased 1.5-fold and the culture duration was extended by 2.5 days, but the product yield was only marginally increased. Further improvements were achieved by developing and implementing a dynamic fed-batch strategy using a concentrated feed solution. The feeding was based on an automatic control-loop to maintain a constant glucose concentration. This strategy led to a further 2-fold increase in maximum cell density (up to 10.7×10(6)cells/ml) and a final product titer of 160mg/l, representing nearly a 3-fold yield increase compared to the batch process with the parental cell clone. Copyright © 2013 Elsevier B.V. All rights reserved.

Heterotrophic growth and lipid accumulation of Chlorella protothecoides in whey permeate, a dairy by-product stream, for biofuel production.

PubMed

Espinosa-Gonzalez, Isabel; Parashar, Archana; Bressler, David C

2014-03-01

This study proposes a novel alternative for the utilization of whey permeate, a by-product stream from the dairy industry, as the feedstock for the biomass and lipid production of the microalgae Chlorella protothecoides. Glucose and galactose from the pre-hydrolyzed whey permeate were used as main carbon sources in a base mineral media for establishing batch and fed batch cultures. Batch cultures reached a biomass production of 9.1±0.2g/L with a total lipid accumulation of 42.0±6.6% (dry weight basis), while in the fed batch cultures 17.2±1.3g/L of biomass with 20.5±0.3% lipid accumulation (dry weight basis) were obtained. A third strategy for the direct utilization of whey permeate was investigated by simultaneous saccharification and fermentation (SSF), wherein, 7.3±1.3g/L of biomass with 49.9±3.3% lipid accumulation (dry weight basis) was obtained in batch mode using immobilized enzyme. Copyright © 2013 Elsevier Ltd. All rights reserved.

Sequential culture with Torulaspora delbrueckii and Saccharomyces cerevisiae and management of fermentation temperature to improve cherry wine quality.

PubMed

Sun, Shu Yang; Gong, Han Sheng; Zhao, Yu Ping; Liu, Wen Li; Jin, Cheng Wu

2016-04-01

There has been limited research on the use of non-Saccharomyces yeasts for the production of cherry wines. This work used an autochthonous Torulaspora delbrueckii strain 49 (TD49) in association with a commercial S. cerevisiae RC212 yeast, to investigate the effect of multi-starter culture (sequential inoculation and simultaneous inoculation) and fermentation temperature on the quality of cherry wines. Both TD49 and RC212 proliferated during alcoholic fermentation (AF) under sequential inoculation conditions, whereas in the case of simultaneous inoculation, TD49 increased slowly at first and then declined sharply near the fermentation end. The analytical profile showed that both mixed fermentations produced lower levels of volatile acidy and higher levels of aromatic compounds than those from RC212 mono-culture. During sensory analysis, wines from sequential fermentation obtained the highest score, mainly due to the higher intensity in 'fruity' and 'floral' characters. As for the influence of temperature, a low temperature (20 °C) enhanced TD49 persistence during AF, but the sensory quality decreased anyway; 30 °C resulted in decreases in most measured descriptors. Therefore, 25 °C was selected as the best culture temperature. TD49/RC212 sequential inoculation and fermentation at 25 °C significantly enhanced the cherry wine quality. © 2015 Society of Chemical Industry.

A Simple Method to Reduce both Lactic Acid and Ammonium Production in Industrial Animal Cell Culture

PubMed Central

Freund, Nathaniel W.; Croughan, Matthew S.

2018-01-01

Fed-batch animal cell culture is the most common method for commercial production of recombinant proteins. However, higher cell densities in these platforms are still limited due to factors such as excessive ammonium production, lactic acid production, nutrient limitation, and/or hyperosmotic stress related to nutrient feeds and base additions to control pH. To partly overcome these factors, we investigated a simple method to reduce both ammonium and lactic acid production—termed Lactate Supplementation and Adaptation (LSA) technology—through the use of CHO cells adapted to a lactate-supplemented medium. Using this simple method, we achieved a reduction of nearly 100% in lactic acid production with a simultaneous 50% reduction in ammonium production in batch shaker flasks cultures. In subsequent fed-batch bioreactor cultures, lactic acid production and base addition were both reduced eight-fold. Viable cell densities of 35 million cells per mL and integral viable cell days of 273 million cell-days per mL were achieved, both among the highest currently reported for a fed-batch animal cell culture. Investigating the benefits of LSA technology in animal cell culture is worthy of further consideration and may lead to process conditions more favorable for advanced industrial applications. PMID:29382079

A Simple Method to Reduce both Lactic Acid and Ammonium Production in Industrial Animal Cell Culture.

PubMed

Freund, Nathaniel W; Croughan, Matthew S

2018-01-28

Fed-batch animal cell culture is the most common method for commercial production of recombinant proteins. However, higher cell densities in these platforms are still limited due to factors such as excessive ammonium production, lactic acid production, nutrient limitation, and/or hyperosmotic stress related to nutrient feeds and base additions to control pH. To partly overcome these factors, we investigated a simple method to reduce both ammonium and lactic acid production-termed Lactate Supplementation and Adaptation (LSA) technology-through the use of CHO cells adapted to a lactate-supplemented medium. Using this simple method, we achieved a reduction of nearly 100% in lactic acid production with a simultaneous 50% reduction in ammonium production in batch shaker flasks cultures. In subsequent fed-batch bioreactor cultures, lactic acid production and base addition were both reduced eight-fold. Viable cell densities of 35 million cells per mL and integral viable cell days of 273 million cell-days per mL were achieved, both among the highest currently reported for a fed-batch animal cell culture. Investigating the benefits of LSA technology in animal cell culture is worthy of further consideration and may lead to process conditions more favorable for advanced industrial applications.

A Community-Based Culture Collection for Targeting Novel Plant Growth-Promoting Bacteria from the Sugarcane Microbiome

PubMed Central

Armanhi, Jaderson Silveira Leite; de Souza, Rafael Soares Correa; Damasceno, Natália de Brito; de Araújo, Laura M.; Imperial, Juan; Arruda, Paulo

2018-01-01

The soil-plant ecosystem harbors an immense microbial diversity that challenges investigative approaches to study traits underlying plant-microbe association. Studies solely based on culture-dependent techniques have overlooked most microbial diversity. Here we describe the concomitant use of culture-dependent and -independent techniques to target plant-beneficial microbial groups from the sugarcane microbiome. The community-based culture collection (CBC) approach was used to access microbes from roots and stalks. The CBC recovered 399 unique bacteria representing 15.9% of the rhizosphere core microbiome and 61.6–65.3% of the endophytic core microbiomes of stalks. By cross-referencing the CBC (culture-dependent) with the sugarcane microbiome profile (culture-independent), we designed a synthetic community comprised of naturally occurring highly abundant bacterial groups from roots and stalks, most of which has been poorly explored so far. We then used maize as a model to probe the abundance-based synthetic inoculant. We show that when inoculated in maize plants, members of the synthetic community efficiently colonize plant organs, displace the natural microbiota and dominate at 53.9% of the rhizosphere microbial abundance. As a result, inoculated plants increased biomass by 3.4-fold as compared to uninoculated plants. The results demonstrate that abundance-based synthetic inoculants can be successfully applied to recover beneficial plant microbes from plant microbiota. PMID:29354144

Identification and Susceptibility Testing of Enterobacteriaceae and Pseudomonas aeruginosa by Direct Inoculation from Positive BACTEC Blood Culture Bottles into Vitek 2

PubMed Central

Bruins, Marjan J.; Bloembergen, Peter; Ruijs, Gijs J. H. M.; Wolfhagen, Maurice J. H. M.

2004-01-01

Inoculation of an automated system for rapid identification (ID) and antimicrobial susceptibility testing (AST) directly from positive blood culture bottles will reduce the turnaround time of laboratory diagnosis of septicemic patients, which benefits clinical outcome and decreases patient costs. Direct test results, however, must always be confirmed by testing a pure overnight culture, which is the “gold standard.” We studied the accuracy of direct testing versus repeat testing in order to investigate the possibility of refraining from repeat testing. We also assessed the clinical risk of reporting results based on direct testing only. We inoculated Vitek 2 (bioMérieux) directly from 410 positive BACTEC 9240 (BD) blood culture bottles containing gram-negative rods and studied the ID and AST results. In a comparison of direct inoculation with the standard method, a total of 344 isolates of Enterobacteriaceae and Pseudomonas aeruginosa were tested, and 93.0% were correctly identified. Of the 39 (10.2%) samples that contained bacilli not identifiable by Vitek 2, only 1 gave a conclusive, correct result. The overall MIC agreement among 312 isolates was 99.2%, with 0.8% very major and 0.02% major error rates. Of only three (polymicrobial) samples, the direct susceptibility pattern would be reported to the clinician as too sensitive. Vitek 2 results obtained from direct inoculation of blood culture bottles containing gram-negative bacilli are safe enough for immediate reporting, provided that ID and AST are consistent. Repeat testing is not necessary, unless Gram stain or overnight subculture results raise doubt about the purity of the culture. PMID:14715724

Dynamic behavior of Yarrowia lipolytica in response to pH perturbations: dependence of the stress response on the culture mode.

PubMed

Timoumi, Asma; Cléret, Mégane; Bideaux, Carine; Guillouet, Stéphane E; Allouche, Yohan; Molina-Jouve, Carole; Fillaudeau, Luc; Gorret, Nathalie

2017-01-01

Yarrowia lipolytica, a non-conventional yeast with a promising biotechnological potential, is able to undergo metabolic and morphological changes in response to environmental conditions. The effect of pH perturbations of different types (pulses, Heaviside) on the dynamic behavior of Y. lipolytica W29 strain was characterized under two modes of culture: batch and continuous. In batch cultures, different pH (4.5, 5.6 (optimal condition), and 7) were investigated in order to identify the pH inducing a stress response (metabolic and/or morphologic) in Y. lipolytica. Macroscopic behavior (kinetic parameters, yields, viability) of the yeast was slightly affected by pH. However, contrary to the culture at pH 5.6, a filamentous growth was induced in batch experiments at pH 4.5 and 7. Proportions of the filamentous subpopulation reached 84 and 93 % (v/v) under acidic and neutral conditions, respectively. Given the significant impact of neutral pH on morphology, pH perturbations from 5.6 to 7 were subsequently assayed in batch and continuous bioreactors. For both process modes, the growth dynamics remained fundamentally unaltered during exposure to stress. Nevertheless, morphological behavior of the yeast was dependent on the culture mode. Specifically, in batch bioreactors where cells proliferated at their maximum growth rate, mycelia were mainly formed. Whereas, in continuous cultures at controlled growth rates (from 0.03 to 0.20 h -1 ) even closed to the maximum growth rate of the stain (0.24 h -1 ), yeast-like forms predominated. This pointed out differences in the kinetic behavior of filamentous and yeast subpopulations, cell age distribution, and pH adaptive mechanisms between both modes of culture.

Anaerobic Biodegradation Of Methyl tert-Butyl Ether Under Iron-Reducing Conditions In Batch And Continuous-Flow Cultures

EPA Science Inventory

The feasibility of biodegradation of the fuel oxygenate methyl tert-butyl ether (MTBE) under iron-reducing conditions was explored in batch and continuous-flow systems. A porous pot completely-mixed reactor was seeded with diverse cultures and operated under iron-reducing...

Bacteriocin Production with Lactobacillus amylovorus DCE 471 Is Improved and Stabilized by Fed-Batch Fermentation

PubMed Central

Callewaert, Raf; De Vuyst, Luc

2000-01-01

Amylovorin L471 is a small, heat-stable, and hydrophobic bacteriocin produced by Lactobacillus amylovorus DCE 471. The nutritional requirements for amylovorin L471 production were studied with fed-batch fermentations. A twofold increase in bacteriocin titer was obtained when substrate addition was controlled by the acidification rate of the culture, compared with the titers reached with constant substrate addition or pH-controlled batch cultures carried out under the same conditions. An interesting feature of fed-batch cultures observed under certain culture conditions (constant feed rate) is the apparent stabilization of bacteriocin activity after obtaining maximum production. Finally, a mathematical model was set up to simulate cell growth, glucose and complex nitrogen source consumption, and lactic acid and bacteriocin production kinetics. The model showed that bacterial growth was dependent on both the energy and the complex nitrogen source. Bacteriocin production was growth associated, with a simultaneous bacteriocin adsorption on the producer cells dependent on the lactic acid accumulated and hence the viability of the cells. Both bacteriocin production and adsorption were inhibited by high concentrations of the complex nitrogen source. PMID:10653724

A novel process-based model of microbial growth: self-inhibition in Saccharomyces cerevisiae aerobic fed-batch cultures.

PubMed

Mazzoleni, Stefano; Landi, Carmine; Cartenì, Fabrizio; de Alteriis, Elisabetta; Giannino, Francesco; Paciello, Lucia; Parascandola, Palma

2015-07-30

Microbial population dynamics in bioreactors depend on both nutrients availability and changes in the growth environment. Research is still ongoing on the optimization of bioreactor yields focusing on the increase of the maximum achievable cell density. A new process-based model is proposed to describe the aerobic growth of Saccharomyces cerevisiae cultured on glucose as carbon and energy source. The model considers the main metabolic routes of glucose assimilation (fermentation to ethanol and respiration) and the occurrence of inhibition due to the accumulation of both ethanol and other self-produced toxic compounds in the medium. Model simulations reproduced data from classic and new experiments of yeast growth in batch and fed-batch cultures. Model and experimental results showed that the growth decline observed in prolonged fed-batch cultures had to be ascribed to self-produced inhibitory compounds other than ethanol. The presented results clarify the dynamics of microbial growth under different feeding conditions and highlight the relevance of the negative feedback by self-produced inhibitory compounds on the maximum cell densities achieved in a bioreactor.

Method and automated apparatus for detecting coliform organisms

NASA Technical Reports Server (NTRS)

Dill, W. P.; Taylor, R. E.; Jeffers, E. L. (Inventor)

1980-01-01

Method and automated apparatus are disclosed for determining the time of detection of metabolically produced hydrogen by coliform bacteria cultured in an electroanalytical cell from the time the cell is inoculated with the bacteria. The detection time data provides bacteria concentration values. The apparatus is sequenced and controlled by a digital computer to discharge a spent sample, clean and sterilize the culture cell, provide a bacteria nutrient into the cell, control the temperature of the nutrient, inoculate the nutrient with a bacteria sample, measures the electrical potential difference produced by the cell, and measures the time of detection from inoculation.

Implantation ability of the potential probiotic strain, Lactobacillus reuteri PL519, in "Salchichón," a traditional Iberian dry fermented sausage.

PubMed

Ruiz-Moyano, Santiago; Martín, Alberto; Benito, María José; Aranda, Emilio; Casquete, Rocío; Córdoba, María de Guia

2011-01-01

The purpose of this study was to investigate the potential of incorporating the probiotic L. reuteri PL519 into the manufacturing of Iberian dry fermented sausages, and to observe its effect on the sensory properties of these meat products. Specific polymerase chain reaction (PCR) was carried out to detect the presence of probiotic strain at high counts in the inoculated sausages. Changes due to probiotic inoculation on physicochemical parameters were determined and the impact on sensory quality evaluated. Dry fermented sausages inoculated with L. reuteri PL519 may be considered as functional products according to the counts of this strain found at the end of processing. Inoculation with L. reuteri PL519 increased the amount of acetic acid, protein, and lipid degradation products in dry fermented sausages. The differences observed in the descriptive sensorial analysis corresponded, however, to a little impact on overall acceptability since no significant changes were found between the control and L. reuteri PL519 batch in the hedonic test. Processing and marketing of Iberian dry fermented sausages with functional characteristics. © 2011 Institute of Food Technologists®

Fate of Yersinia enterocolitica during manufacture, ripening and storage of Lighvan cheese.

PubMed

Hanifian, Shahram; Khani, Sajjad

2012-05-15

This study aimed to evaluate the behavior of virulent Yersinia enterocolitica (YE) during the manufacture, ripening and storage of Lighvan cheese with particular reference to strains of YE, initial inoculation level, and storage time. Three strains of YE with low (1 log cfu/ml) and high (3 log cfu/ml) inoculation levels were inoculated to raw whole ewe's milk which was then used for manufacturing of Lighvan cheese. Throughout the manufacturing, ripening and storage periods the number of YE was counted on selective media. Enumerated colonies were then confirmed by duplex PCR using ail and virF genes. Moreover, some microbial and physiochemical characteristics of the cheese samples were examined. According to the results, initial inoculation level and storage time had statistically significant (P<0.01) effects on persistency of YE, while strain type exhibited no statistically significant (P>0.01) impact on survival of the pathogen. Results showed a rapid increase in the number of YE during manufacturing, however, in the ripening and storage periods the number of YE was decreased and eventually it was eliminated in all cheese batches after 4 months of storage. Copyright © 2012 Elsevier B.V. All rights reserved.

Bioelectrochemical ethanol production through mediated acetate reduction by mixed cultures.

PubMed

Steinbusch, Kirsten J J; Hamelers, Hubertus V M; Schaap, Joris D; Kampman, Christel; Buisman, Cees J N

2010-01-01

Biological acetate reduction with hydrogen is a potential method to convert wet biomass waste into ethanol. Since the ethanol concentration and reaction rates are low, this research studies the feasibility of using an electrode, in stead of hydrogen, as an electron donor for biological acetate reduction in conjunction of an electron mediator. Initially, the effect of three selected mediators on metabolic flows during acetate reduction with hydrogen was explored; subsequently, the best performing mediator was used in a bioelectrochemical system to stimulate acetate reduction at the cathode with mixed cultures at an applied cathode potential of -550 mV. In the batch test, methyl viologen (MV) was found to accelerate ethanol production 6-fold and increased ethanol concentration 2-fold to 13.5 +/- 0.7 mM compared to the control. Additionally, MV inhibited n-butyrate and methane formation, resulting in high ethanol production efficiency (74.6 +/- 6%). In the bioelectrochemical system, MV addition to an inoculated cathode led directly to ethanol production (1.82 mM). Hydrogen was coproduced at the cathode (0.0035 Nm(3) hydrogen m(-2) d(-1)), so it remained unclear whether acetate was reduced to ethanol by electrons supplied by the mediator or by hydrogen. As MV reacted irreversibly at the cathode, ethanol production stopped after 5 days.

Regulatory design governing progression of population growth phases in bacteria.

PubMed

Martínez-Antonio, Agustino; Lomnitz, Jason G; Sandoval, Santiago; Aldana, Maximino; Savageau, Michael A

2012-01-01

It has long been noted that batch cultures inoculated with resting bacteria exhibit a progression of growth phases traditionally labeled lag, exponential, pre-stationary and stationary. However, a detailed molecular description of the mechanisms controlling the transitions between these phases is lacking. A core circuit, formed by a subset of regulatory interactions involving five global transcription factors (FIS, HNS, IHF, RpoS and GadX), has been identified by correlating information from the well- established transcriptional regulatory network of Escherichia coli and genome-wide expression data from cultures in these different growth phases. We propose a functional role for this circuit in controlling progression through these phases. Two alternative hypotheses for controlling the transition between the growth phases are first, a continuous graded adjustment to changing environmental conditions, and second, a discontinuous hysteretic switch at critical thresholds between growth phases. We formulate a simple mathematical model of the core circuit, consisting of differential equations based on the power-law formalism, and show by mathematical and computer-assisted analysis that there are critical conditions among the parameters of the model that can lead to hysteretic switch behavior, which--if validated experimentally--would suggest that the transitions between different growth phases might be analogous to cellular differentiation. Based on these provocative results, we propose experiments to test the alternative hypotheses.

Kinetics of D-lactic acid production by Sporolactobacillus sp. strain CASD using repeated batch fermentation.

PubMed

Zhao, Bo; Wang, Limin; Li, Fengsong; Hua, Dongliang; Ma, Cuiqing; Ma, Yanhe; Xu, Ping

2010-08-01

D-lactic acid was produced by Sporolactobacillus sp. strain CASD in repeated batch fermentation with one- and two-reactor systems. The strain showed relatively high energy consumption in its growth-related metabolism in comparison with other lactic acid producers. When the fermentation was repeated with 10% (v/v) of previous culture to start a new batch, D-lactic acid production shifted from being cell-maintenance-dependent to cell-growth-dependent. In comparison with the one-reactor system, D-lactic acid production increased approximately 9% in the fourth batch of the two-reactor system. Strain CASD is an efficient D-lactic acid producer with increased growth rate at the early stage of repeated cycles, which explains the strain's physiological adaptation to repeated batch culture and improved performance in the two-reactor fermentation system. From a kinetic point of view, two-reactor fermentation system was shown to be an alternative for conventional one-reactor repeated batch operation. Copyright 2010 Elsevier Ltd. All rights reserved.

Magnet-Facilitated Selection of Electrogenic Bacteria from Marine Sediment

PubMed Central

Kiseleva, Larisa; Briliute, Justina; Khilyas, Irina V.; Simpson, David J. W.; Fedorovich, Viacheslav; Cohen, M.; Goryanin, Igor

2015-01-01

Some bacteria can carry out anaerobic respiration by depositing electrons on external materials, such as electrodes, thereby creating an electrical current. Into the anode chamber of microbial fuel cells (MFCs) having abiotic air-cathodes we inoculated microorganisms cultured from a magnetic particle-enriched portion of a marine tidal sediment, reasoning that since some external electron acceptors are ferromagnetic, electrogenic bacteria should be found in their vicinity. Two MFCs, one inoculated with a mixed bacterial culture and the other with an axenic culture of a helical bacterium isolated from the magnetic particle enrichment, termed strain HJ, were operated for 65 d. Both MFCs produced power, with production from the mixed culture MFC exceeding that of strain HJ. Strain HJ was identified as a Thalassospira sp. by transmission electron microscopic analysis and 16S rRNA gene comparisons. An MFC inoculated with strain HJ and operated in open circuit produced 47% and 57% of the maximal power produced from MFCs inoculated with the known electrogen Geobacter daltonii and the magnetotactic bacterium Desulfamplus magnetomortis, respectively. Further investigation will be needed to determine whether bacterial populations associated with magnetic particles within marine sediments are enriched for electrogens. PMID:26504814

Magnet-Facilitated Selection of Electrogenic Bacteria from Marine Sediment.

PubMed

Kiseleva, Larisa; Briliute, Justina; Khilyas, Irina V; Simpson, David J W; Fedorovich, Viacheslav; Cohen, M; Goryanin, Igor

2015-01-01

Some bacteria can carry out anaerobic respiration by depositing electrons on external materials, such as electrodes, thereby creating an electrical current. Into the anode chamber of microbial fuel cells (MFCs) having abiotic air-cathodes we inoculated microorganisms cultured from a magnetic particle-enriched portion of a marine tidal sediment, reasoning that since some external electron acceptors are ferromagnetic, electrogenic bacteria should be found in their vicinity. Two MFCs, one inoculated with a mixed bacterial culture and the other with an axenic culture of a helical bacterium isolated from the magnetic particle enrichment, termed strain HJ, were operated for 65 d. Both MFCs produced power, with production from the mixed culture MFC exceeding that of strain HJ. Strain HJ was identified as a Thalassospira sp. by transmission electron microscopic analysis and 16S rRNA gene comparisons. An MFC inoculated with strain HJ and operated in open circuit produced 47% and 57% of the maximal power produced from MFCs inoculated with the known electrogen Geobacter daltonii and the magnetotactic bacterium Desulfamplus magnetomortis, respectively. Further investigation will be needed to determine whether bacterial populations associated with magnetic particles within marine sediments are enriched for electrogens.

A novel fed-batch based strategy for enhancing cell-density and recombinant cyprosin B production in bioreactors.

PubMed

Sampaio, P N; Pais, M S; Fonseca, L P

2014-12-01

Nowadays, the dairy industry is continuously looking for new and more efficient clotting enzymes to create innovative products. Cyprosin B is a plant aspartic protease characterized by clotting activity that was previously cloned in Saccharomyces cerevisiae BJ1991 strain. The production of recombinant cyprosin B by a batch and fed-batch culture was compared using glucose and galactose as carbon sources. The strategy for fed-batch cultivation involved two steps: in the first batch phase, the culture medium presented glucose 1 % (w/v) and galactose 0.5 % (w/v), while in the feed step the culture medium was constituted by 5 % (w/v) galactose with the aim to minimize the GAL7 promoter repression. Based on fed-batch, in comparison to batch growth, an increase in biomass (6.6-fold), protein concentration (59 %) and cyprosin B activity (91 %) was achieved. The recombinant cyprosin B was purified by a single hydrophobic chromatography, presenting a specific activity of 6 × 10(4) U·mg(-1), corresponding to a purification degree of 12.5-fold and a recovery yield of 16.4 %. The SDS-PAGE analysis showed that recovery procedure is suitable for achieving the purified recombinant cyprosin B. The results show that the recombinant cyprosin B production can be improved based on two distinct steps during the fed-batch, presenting that this strategy, associated with a simplified purification procedure, could be applied to large-scale production, constituting a new and efficient alternative for animal and fungal enzymes widely used in cheese making.

Phenylalanine ammonia-lyase activity in suspension cultures of Ulmus pumila and U. campestris treated with spores of Ceratocystis ulmi.

PubMed

Corchete, M P; Diez, J J; Valle, T

1993-12-01

Cell suspension cultures of a Ceratocystis ulmi-resistant (Ulmus pumila) and a -susceptible elm (U.campestris) were established from leaf callus tissue. Treatment of cultures with spores of C.ulmi induced a large increase in the activity of phenylalanine ammonialyase, only in the cells of the resistant species U.pumila with a maximum after 24 h. Inoculated U.pumila cells also excreted a red unidentified chemical into the culture medium. Neither responses were induced in inoculated U.campestris cultures. The results are discussed in relation to the development of the elm cell culture system as a model for studying the differential biochemical mechanisms of disease resistance in elms.

Glucose metabolism in batch and continuous cultures of Gluconacetobacter diazotrophicus PAL 3.

PubMed

Luna, María F; Bernardelli, Cecilia E; Galar, María L; Boiardi, José L

2006-03-01

Periplasmic glucose oxidation (by way of a pyrrolo-quinoline-quinone [PQQ]-linked glucose dehydrogenase [GDH]) was observed in continuous cultures of Gluconacetobacter diazotrophicus regardless of the carbon source (glucose or gluconate) and the nitrogen source (N(2) or NH(3)). Its synthesis was stimulated by conditions of high energetic demand (i.e., N(2)-fixation) and/or C-limitation. Under C-excess conditions, PQQ-GDH synthesis increased with the glucose concentration in the culture medium. In batch cultures, PQQ-GDH was actively expressed in very early stages with higher activities under conditions of N(2)-fixation. Hexokinase activity was almost absent under any culture condition. Cytoplasmic nicotinamide adenine dinucleotide (NAD)-linked glucose dehydrogenase (GDH) was expressed in continuous cultures under all tested conditions, and its synthesis increased with the glucose concentration. In contrast, low activities of this enzyme were detected in batch cultures. Periplasmic oxidation, by way of PQQ-GDH, seems to be the principal pathway for metabolism of glucose in G. Diazotrophicus, and NAD-GDH is an alternative route under certain environmental conditions.

Effect of physical characteristics on bioleaching using indigenous acidophilic bacteria for recovering the valuable resources

NASA Astrophysics Data System (ADS)

Wi, D.; Kim, B.; Cho, K.; Choi, N.; Park, C.

2011-12-01

Bioleaching technology which is based on the ability of bacteria to transform solid compounds into soluble or extractable elements that can be recovered, has developed rapidly in recent decades for its advantages, such as mild reaction, low energy consumption, simple process, environmentally friendly and suitable for low-grade mine tailing and residues. This study investigated the bioleaching efficiency of copper matte under batch experimental conditions (various mineral particle size) using the indigenous acidophilic bacteria collected from acidic hot spring in Hatchnobaru, Japan. We conducted the batch experiments at three different mineral particle sizes: 0.06, 0.16 and 1.12mm. The results showed that the pH in the bacteria inoculating sample increased than initial condition, possibly due to buffer effects by phosphate ions in growth medium. After 22 days from incubation the leached accumulation content of Cu was 0.06 mm - 1,197 mg/L, 0.16 mm - 970 mg/L and 1.12 mm - 704 mg/L. Additionally, through SEM analysis we found of gypsum formed crystals which coated the copper matte surface 6 days after inoculation in 1.12mm case. This study informs basic knowledge when bacteria apply to eco-/economic resources utilization studies including the biomining and the recycling of mine waste system.

[Effect of ectomycorrhizae on the growth of Picea koraiensis seedlings].

PubMed

Song, Rui-Qing; Wu, Ke

2005-12-01

Basidioscarps of Agaricales in different Picea koraiensis forest plantations were collected during August-October, 2000. 36 isolaters of species of Agaricales were obtained by isolating and culturing to the basidioscarps. Through indoor inoculation test on seedlings of Picea koraiensis, 6 ectomycorrhizae fungi cultures were obtained from 36 isolaters. The inoculation results show that the period for ectomycorrhizae inoculation to 1-year seedlings of Picea koraiensis should be about 30 days after seedlings emerging, the suitable temperature for ectomycorrhizae forming is about 20 degrees C. 6 ectomycorrhizae strains all have growth-promoting effect to the seedlings of Picea koraiensis. The contents of chlorophyll a of the seedlings inoculated strains of Agaricus silvaticus, 031 and L15 were significantly higher than other strains and control. The contents of chlorophyll b in the seedlings inoculated strains 009, 004, Agaricus silvaticus and L15 were significantly higher than other strains and control. The weights of seedlings which inoculated strains 009, 025, 031, Agaricus silvaticus and L15 were significantly different to control, the weight of seedlings inoculated strains of Agaricus silvaticus and L15 are 19.23% and 23.08% more than control; The heights of the seedlings inoculated 6 strains all have significant difference to control, the weight of seedlings inoculated strains of Agaricus silvaticus and L15 are 17.83% and 16.37% more than control. The results of outdoor inoculation show that the seedlings inoculated Agaricus silvaticus grow best on height, 9.25% more than control after inoculated 70 days; the seedlings inoculated strain L15 grow best on collar diameter, 9.92% more than control after inoculated 70 days; the lateral root numbers of seedlings inoculated strain 009 is largest, 51.91% more than control after inoculated 70 days; the main roots of seedlings inoculated strain 009 are longest, 3.36% more than control after inoculated 70 days; the ectomycorrhizae fungi infestation percentage of seedlings inoculated strain 031 is highest, 13.90% more than control after inoculated 70 days; the photosynthesis of seedlings inoculated strain 031 is more powerful, 20.02% more than control after inoculated 70 days. To sum up, strain 009, strain 031 and strain L15 should be fine strains to seedlings of Picea koraiensis.

Growth and enzymological characteristics of a pink-pigmented facultative methylotroph Methylobacterium sp. MB1.

PubMed

Baev, M V; Kuznetsov, E V; Skladnev, D A; Govorukhina, N I; Sterkin, V E; Tsygankov, Y D

1992-01-01

Growth characteristics of batch and continuous cultures of the pink facultative methylotroph Methylobacterium sp. MB1 were determined. The response of a chemostat culture to a pulse increase of methanol concentration was studied. Malate, succinate and oxaloacetate additions to the methanol-supplemented medium decreased batch culture growth inhibition by methanol. The carotenoid content in cells grown in a chemostat decreased with increasing growth rate. The key enzyme activities of C1-metabolism were measured in a chemostat culture at different dilution rates.

Effects of plants and essential oils on ruminal in vitro batch culture methane production and fermentation

USDA-ARS?s Scientific Manuscript database

In this study, plants (14) and essential oils (EO; 88) from plants that are naturalized to, or can be successfully grown in North America were evaluated in a batch culture in vitro screening experiments with ruminal fluid as potential anti-methanogenic additives for ruminant diets. Essential oils we...

CHANGES IN CHLOROPHYLL A FLOURESCENCE AND PIGMENT RATIOS DURING DIFFERENT GROWTH PHASES OF A UNICELLULAR MARINE CHAETOCEROS (BACILLAROPHYCEAE) IN BATCH CULTURE

EPA Science Inventory

Interpretations of chlorophyll a fluorescence data are based largely on application with green algae and higher plants. This study evaluated the interpretation of fluorescence data for a unicellular marine diatom. Chaetoceros sp. was grown in 4-liter batch cultures on a 16:8, L:D...

Novel Production Protocol for Small-scale Manufacture of Probiotic Fermented Foods

PubMed Central

Westerik, Nieke; Wacoo, Alex Paul; Sybesma, Wilbert; Kort, Remco

2016-01-01

A novel dried bacterial consortium of Lactobacillus rhamnosus yoba 2012 and Streptococcus thermophilus C106 is cultured in 1 L of milk. This fresh starter can be used for the production of fermented milk and other fermented foods either at home or at small-scale in rural settings. For the fresh starter, 1 L of milk is pasteurized in a pan that fits into a larger pan containing water, placed on a source of heat. In this water bath, the milk is heated and incubated at 85 °C for 30 min. Thereafter, the milk is cooled down to 45 °C, transferred to a vacuum flask, inoculated with the dried bacteria and left for at least 16 hr between 30 °C and 45 °C. For the purpose of frequent home production, the fresh starter is frozen into ice cubes, which can be used for the production of small volumes of up to 2 L of fermented milk. For the purpose of small-scale production in resource-poor countries, pasteurization of up to 100 L of milk is conducted in milk cans that are placed in a large sauce pan filled with water and heated on a fire at 85 °C for 30 min, and subsequently cooled to 45 °C. Next, the 100 L batch is inoculated with the 1 L freshly prepared starter mentioned before. To assure an effective fermentation at a temperature between 30 and 45 °C, the milk can is covered with a blanket for 12 hr. For the production of non-dairy fermented foods, the fresh starter is left in a cheese cloth for 12 hr, and the drained-off whey can be subsequently used for the inoculation of a wide range of food raw materials, including vegetables and cereal-based foods. PMID:27684196

Novel Production Protocol for Small-scale Manufacture of Probiotic Fermented Foods.

PubMed

Westerik, Nieke; Wacoo, Alex Paul; Sybesma, Wilbert; Kort, Remco

2016-09-10

A novel dried bacterial consortium of Lactobacillus rhamnosus yoba 2012 and Streptococcus thermophilus C106 is cultured in 1 L of milk. This fresh starter can be used for the production of fermented milk and other fermented foods either at home or at small-scale in rural settings. For the fresh starter, 1 L of milk is pasteurized in a pan that fits into a larger pan containing water, placed on a source of heat. In this water bath, the milk is heated and incubated at 85 °C for 30 min. Thereafter, the milk is cooled down to 45 °C, transferred to a vacuum flask, inoculated with the dried bacteria and left for at least 16 hr between 30 °C and 45 °C. For the purpose of frequent home production, the fresh starter is frozen into ice cubes, which can be used for the production of small volumes of up to 2 L of fermented milk. For the purpose of small-scale production in resource-poor countries, pasteurization of up to 100 L of milk is conducted in milk cans that are placed in a large sauce pan filled with water and heated on a fire at 85 °C for 30 min, and subsequently cooled to 45 °C. Next, the 100 L batch is inoculated with the 1 L freshly prepared starter mentioned before. To assure an effective fermentation at a temperature between 30 and 45 °C, the milk can is covered with a blanket for 12 hr. For the production of non-dairy fermented foods, the fresh starter is left in a cheese cloth for 12 hr, and the drained-off whey can be subsequently used for the inoculation of a wide range of food raw materials, including vegetables and cereal-based foods.

Bioprocess development for the production of mouse-human chimeric anti-epidermal growth factor receptor vIII antibody C12 by suspension culture of recombinant Chinese hamster ovary cells.

PubMed

Hu, Suwen; Deng, Lei; Wang, Huamao; Zhuang, Yingping; Chu, Ju; Zhang, Siliang; Li, Zhonghai; Guo, Meijin

2011-05-01

The mouse-human chimeric anti-epidermal growth factor receptor vIII (EGFRvIII) antibody C12 is a promising candidate for the diagnosis of hepatocellular carcinoma (HCC). In this study, 3 processes were successfully developed to produce C12 by cultivation of recombinant Chinese hamster ovary (CHO-DG44) cells in serum-free medium. The effect of inoculum density was evaluated in batch cultures of shaker flasks to obtain the optimal inoculum density of 5 × 10(5) cells/mL. Then, the basic metabolic characteristics of CHO-C12 cells were studied in stirred bioreactor batch cultures. The results showed that the limiting concentrations of glucose and glutamine were 6 and 1 mM, respectively. The culture process consumed significant amounts of aspartate, glutamate, asparagine, serine, isoleucine, leucine, and lysine. Aspartate, glutamate, asparagine, and serine were particularly exhausted in the early growth stage, thus limiting cell growth and antibody synthesis. Based on these findings, fed-batch and perfusion processes in the bioreactor were successfully developed with a balanced amino acid feed strategy. Fed-batch and especially perfusion culture effectively maintained high cell viability to prolong the culture process. Furthermore, perfusion cultures maximized the efficiency of nutrient utilization; the mean yield coefficient of antibody to consumed glucose was 44.72 mg/g and the mean yield coefficient of glutamine to antibody was 721.40 mg/g. Finally, in small-scale bioreactor culture, the highest total amount of C12 antibody (1,854 mg) was realized in perfusion cultures. Therefore, perfusion culture appears to be the optimal process for small-scale production of C12 antibody by rCHO-C12 cells.

Enhancing enterovirus A71 vaccine production yield by microcarrier profusion bioreactor culture.

PubMed

Liu, Chia-Chyi; Wu, Suh-Chin; Wu, Shang-Rung; Lin, Hsiao-Yu; Guo, Meng-Shin; Yung-Chih Hu, Alan; Chow, Yen-Hung; Chiang, Jen-Ron; Shieh, Dar-Bin; Chong, Pele

2018-05-24

Hand, foot and mouth diseases (HFMD) are mainly caused by Enterovirus A71 (EV-A71) infections. Clinical trials in Asia conducted with formalin-inactivated EV-A71 vaccine candidates produced from serum-free Vero cell culture using either roller bottle or cell factory technology, are found to be safe and highly efficacious. To increase vaccine yields and reduce the production costs, the bioprocess improvement for EV-A71 vaccine manufacturing is currently being investigated. The parameters that could affect and enhance the production yields of EV-A71 virus growth in the microcarrier bioreactor were investigated. The medium replacement culture strategy included a multi-harvested semi-batch process and perfusion technology and was found to increase the production yields more than 7-14 folds. Based on the western blot and cryo-EM analyses of the EV-A71 virus particles produced from either the multi-harvested semi-batch (MHSBC) or perfusion cultures were found to be similar to those virus particles obtained from the single batch culture. Mouse immunogenicity studies indicate that the EV-A71 vaccine candidates produced from the perfusion culture have similar potency to those obtained from single batch bioprocess. The physical structures of the EV-A71 particles revealed by the cryo-EM analysis were found to be spherical capsid particles. These results provide feasible technical bioprocesses for increasing virus yields and the scale up of EV-A71 vaccine manufacturing using the bioreactor cell culture methods. Copyright © 2017 Elsevier Ltd. All rights reserved.

Fed-batch culture of Escherichia coli for L-valine production based on in silico flux response analysis.

PubMed

Park, Jin Hwan; Kim, Tae Yong; Lee, Kwang Ho; Lee, Sang Yup

2011-04-01

We have previously reported the development of a 100% genetically defined engineered Escherichia coli strain capable of producing L-valine from glucose with a high yield of 0.38 g L-valine per gram glucose (0.58 mol L-valine per mol glucose) by batch culture. Here we report a systems biological strategy of employing flux response analysis in bioprocess development using L-valine production by fed-batch culture as an example. Through the systems-level analysis, the source of ATP was found to be important for efficient L-valine production. There existed a trade-off between L-valine production and biomass formation, which was optimized for the most efficient L-valine production. Furthermore, acetic acid feeding strategy was optimized based on flux response analysis. The final fed-batch cultivation strategy allowed production of 32.3 g/L L-valine, the highest concentration reported for E. coli. This approach of employing systems-level analysis of metabolic fluxes in developing fed-batch cultivation strategy would also be applicable in developing strategies for the efficient production of other bioproducts. Copyright © 2010 Wiley Periodicals, Inc.

Effects of fungal elicitors on seed germination and tissue culture of Cymbidium goeringii

NASA Astrophysics Data System (ADS)

Gong, Mingfu; Guan, Qinlan; Lin, Tianxing; Lan, Juming; Liu, Si

2018-04-01

The endophytic fungi were isolated from the roots of Cymbidium goeringii and Cymbidium faberi and were made into liquid fungal elicitors used to co-fungus germination of seeds of C. goeringii and C. faberi. The results showed that the germination rate of the seeds of C. goeringii was increased 15% to 30% by inoculating the mixed fungal elicitors of C. goeringii and was no significant change by inoculating the mixed fungal elicitors of C. faberi. Mixed fungal elicitor promoted the acclimation of tissue culture seedlings. Mixed fungal elicitor prepared mycorrhizal inoculant made new seedlings easier to survive when transplanted.

Production of nattokinase by high cell density fed-batch culture of Bacillus subtilis.

PubMed

Kwon, Eun-Yeong; Kim, Kyung Mi; Kim, Mi Kyoung; Lee, In Young; Kim, Beom Soo

2011-09-01

Bacillus subtilis was cultivated to high cell density for nattokinase production by pH-stat fed-batch culture. A concentrated mixture solution of glucose and peptone was automatically added by acid-supplying pump when culture pH rose above high limit. Effect of the ratio of glucose to peptone in feeding solution was investigated on cell growth and nattokinase production by changing the ratio from 0.2 to 5 g glucose/g peptone. The highest cell concentration was 77 g/L when the ratio was 0.2 g glucose/g peptone. Cell concentration decreased with increasing the ratio of glucose to peptone in feeding solution, while the optimum condition existed for nattokinase production. The highest nattokinase activity was 14,500 unit/mL at a ratio of 0.33 g glucose/g peptone, which was 4.3 times higher than that in batch culture.

Butanol production under microaerobic conditions with a symbiotic system of Clostridium acetobutylicum and Bacillus cereus.

PubMed

Wu, Pengfei; Wang, Genyu; Wang, Gehua; Børresen, Børre Tore; Liu, Hongjuan; Zhang, Jianan

2016-01-14

One major problem of ABE (acetone, butanol and ethanol) fermentation is high oxygen sensitivity of Clostridium acetobutylicum. Currently, no single strain has been isolated or genetically engineered to produce butanol effectively under aerobic conditions. In our previous work, a symbiotic system TSH06 has been developed successfully by our group, and two strains, C. acetobutylicum TSH1 and Bacillus cereus TSH2, were isolated from TSH06. Compared with single culture, TSH06 showed promotion on cell growth and solvent accumulation under microaerobic conditions. To simulate TSH06, a new symbiotic system was successfully re-constructed by adding living cells of B. cereus TSH2 into C. acetobutylicum TSH1 cultures. During the fermentation process, the function of B. cereus TSH2 was found to deplete oxygen and provide anaerobic environment for C. acetobutylicum TSH1. Furthermore, inoculation ratio of C. acetobutylicum TSH1 and B. cereus TSH2 affected butanol production. In a batch fermentation with optimized inoculation ratio of 5 % C. acetobutylicum TSH1 and 0.5 % B. cereus TSH2, 11.0 g/L butanol and 18.1 g/L ABE were produced under microaerobic static condition. In contrast to the single culture of C. acetobutylicum TSH1, the symbiotic system became more aerotolerant and was able to produce 11.2 g/L butanol in a 5 L bioreactor even with continuous 0.15 L/min air sparging. In addition, qPCR assay demonstrated that the abundance of B. cereus TSH2 increased quickly at first and then decreased sharply to lower than 1 %, whereas C. acetobutylicum TSH1 accounted for more than 99 % of the whole population in solventogenic phase. The characterization of a novel symbiotic system on butanol fermentation was studied. The new symbiotic system re-constructed by co-culture of C. acetobutylicum TSH1 and B. cereus TSH2 showed excellent performance on butanol production under microaerobic conditions. B. cereus TSH2 was a good partner for C. acetobutylicum TSH1 by providing an anaerobic environment. During fermentation process, the high ratio of Clostridium and low ratio of Bacillus composition indicated that this symbiotic system was an effective and easily controlled cultivation model for ABE fermentation under microaerobic conditions.

Swine dysentery: inoculation of gnotobiotic pigs with Treponema hyodysenteriae and Vibrio coli and a Peptostreptococcus.

PubMed Central

Brandenburg, A C; Miniats, O P; Geissinger, H D; Ewert, E

1977-01-01

Pure cultures of Treponema hyodysenteriae given orally to conventional pigs resulted in the development of swine dysentery, whereas identical cultures given to gnotobiotic pigs did not produce the disease. Oral inoculation of gnotobiotic pigs with Vibrio coli and/or a peptostreptococcus in addition to T. hyodysenteriae did not result in dysentery. Neutralization of gastric secretions with NaHCO3 immediately prior to inoculation with T. hyodysenteriae increased the period during which treponemes were evident in the feces, as did the inoculation of this organism via the intracecal route. None of the gnotobiotic pigs with a persistent fecal Treponema population developed signs of dysentery. Factors other than those investigated in this work must play a part in the etiology of swine dysentery. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:907906

Features of Saccharomyces cerevisiae as a culture starter for the production of the distilled sugar cane beverage, cachaça in Brazil.

PubMed

Campos, C R; Silva, C F; Dias, D R; Basso, L C; Amorim, H V; Schwan, R F

2010-06-01

To evaluate the dominance and persistence of strains of Saccharomyces cerevisiae during the process of sugar cane fermentation for the production of cachaça and to analyse the microbial compounds produced in each fermentative process. Three S. cerevisiae strains were evaluated during seven consecutive 24-h fermentation batches using recycled inocula. The UFLA CA 116 strain had the largest population of viable organisms, and the maximum population was achieved in the fourth batch after 96 h of fermentation. The UFLA CA 1162 and UFLA CA 1183 strains grew more slowly, and the maximum population was reached in the seventh batch. Molecular characterization of isolated yeast cells using PFGE (pulse field gel electrophoresis) revealed that more than 86% of the isolates corresponded to the initially inoculated yeast strain. The concentration of aldehydes, esters, methanol, alcohol and volatile acids in the final-aged beverages were within the legal limits. Cachaça produced by select yeast strains exhibits analytical differences. UFLA CA 1162 and UFLA CA 116 S. cerevisiae isolates can be considered the ideal strains for the artisanal production of cachaça in Brazil. The use of select yeast strains can improve the quality and productivity of cachaça production. Our findings are important for the appropriate monitoring of yeast during sugar cane fermentation. In addition, we demonstrate that UFLA CA 116 and UFLA CA 1162, the ideal yeast strains for cachaça production, are maintained at a high population density. The persistence of these yeast strains in the fermentation of sugar cane juice promotes environmental conditions that prevent or decrease bacterial contamination. Thus, the use of select yeast strains for the production of cachaça is a viable economic alternative to standardize the production of this beverage.

Multi-stage high cell continuous fermentation for high productivity and titer.

PubMed

Chang, Ho Nam; Kim, Nag-Jong; Kang, Jongwon; Jeong, Chang Moon; Choi, Jin-dal-rae; Fei, Qiang; Kim, Byoung Jin; Kwon, Sunhoon; Lee, Sang Yup; Kim, Jungbae

2011-05-01

We carried out the first simulation on multi-stage continuous high cell density culture (MSC-HCDC) to show that the MSC-HCDC can achieve batch/fed-batch product titer with much higher productivity to the fed-batch productivity using published fermentation kinetics of lactic acid, penicillin and ethanol. The system under consideration consists of n-serially connected continuous stirred-tank reactors (CSTRs) with either hollow fiber cell recycling or cell immobilization for high cell-density culture. In each CSTR substrate supply and product removal are possible. Penicillin production is severely limited by glucose metabolite repression that requires multi-CSTR glucose feeding. An 8-stage C-HCDC lactic acid fermentation resulted in 212.9 g/L of titer and 10.6 g/L/h of productivity, corresponding to 101 and 429% of the comparable lactic acid fed-batch, respectively. The penicillin production model predicted 149% (0.085 g/L/h) of productivity in 8-stage C-HCDC with 40 g/L of cell density and 289% of productivity (0.165 g/L/h) in 7-stage C-HCDC with 60 g/L of cell density compared with referring batch cultivations. A 2-stage C-HCDC ethanol experimental run showed 107% titer and 257% productivity of the batch system having 88.8 g/L of titer and 3.7 g/L/h of productivity. MSC-HCDC can give much higher productivity than batch/fed-batch system, and yield a several percentage higher titer as well. The productivity ratio of MSC-HCDC over batch/fed-batch system is given as a multiplication of system dilution rate of MSC-HCDC and cycle time of batch/fed-batch system. We suggest MSC-HCDC as a new production platform for various fermentation products including monoclonal antibody.

Growth of Streptococcus mutans on various selective media.

PubMed

Emilson, C G; Bratthall, D

1976-07-01

The ability of Streptococcus mutans to grow on mitis-salivarius (MS) agar, MC agar, mitis-sucrose-bacitracin (MSB), BCY agar, and MM10 sucrose agar was studied. Batch cultures of S. mutans serotype a demonstrated no growth on MSB agar. Certain serotype d and g strains did not grow on MC agar. The yield for most strains of other serotypes on these selective media was lower compared with that on MS agar. The number of total colony-forming units on BCY and MM10 sucrose agar was similar to the blood agar results. Similar data were obtained when fermenter-grown strains, harvested in the middle or the end of the logarithmic growth phase, were used for inoculation of the various media. Enumeration of S. mutans from plaque samples plated on MC and MSB agar yielded about 75% of the counts obtained on MS or the nonselective medium. When the proportions of S. mutans were expressed as a percentage of the total cultivable flora, the selective media (MC and MSB agar) showed approximately 10% lower values than the MS, BCY, and MM10 sucrose agar.

Formation of ectomycorrhizae following inoculation of containerized Sitka spruce seedlings.

Treesearch

C.G. Shaw; R. Molina

1980-01-01

Containerized Sitka spruce, [Picea sitchensis (Bong.) Carr.] were inoculated at sowing with pure cultures of either Pisolithus tinctorius (Pers.) Coker & Couch, Laccaria laccata (Scop. ex Fr.) Berk. & Br., Astraeus pteridis (Shear) Feller, Amanita pantherina...

Effects of co-inoculating rice straw with ruminal microbiota and anaerobic sludge: digestion performance and spatial distribution of microbial communities.

PubMed

Deng, Yuying; Huang, Zhenxing; Zhao, Mingxing; Ruan, Wenquan; Miao, Hengfeng; Ren, Hongyan

2017-07-01

Ruminal microbiota (RM) were co-inoculated with anaerobic sludge (AS) at different ratios to study the digestion of rice straw in batch experiments. The CH 4 yield reached 273.64 mL/g volatile solid (VS) at a co-inoculum ratio of 1:1. The xylanase and cellulase activities were 198.88-212.88 and 24.51-29.08 U/mL in co-inoculated samples, respectively, and were significantly different compared to the results for single inoculum (p < 0.05). Higher ratios of AS enhanced acetoclastic methanogenesis, and propionate accumulation could be the main reason for the longer lag phase observed in samples with a higher RM ratio. The microbial compositions were clearly altered after digestion. Fibrobacter, Ruminococcus and Butyrivibrio from the rumen did not settle in the co-inoculated system, whereas Clostridiales members became the main polysaccharide degraders. Microbial interactions involving hydrolytic bacteria and acetoclastic methanogens in the residue were considered to be significant for hydrolysis activities and methane production. Syntrophy involving propionate oxidizers with associated methanogens occurred in the liquid phase. Our findings provide a better understanding of the anaerobic digestion of rice straw that is driven by specific microbial populations.

Butyric acid fermentation of sodium hydroxide pretreated rice straw with undefined mixed culture.

PubMed

Ai, Binling; Li, Jianzheng; Chi, Xue; Meng, Jia; Liu, Chong; Shi, En

2014-05-01

This study describes an alternative mixed culture fermentation technology to anaerobically convert lignocellulosic biomass into butyric acid, a valuable product with wide application, without supplementary cellulolytic enzymes. Rice straw was soaked in 1% NaOH solution to increase digestibility. Among the tested pretreatment conditions, soaking rice straw at 50°C for 72 h removed ~66% of the lignin, but retained ~84% of the cellulose and ~71% of the hemicellulose. By using an undefined cellulose-degrading butyrate-producing microbial community as butyric acid producer in batch fermentation, about 6 g/l of butyric acid was produced from the pretreated rice straw, which accounted for ~76% of the total volatile fatty acids. In the repeated-batch operation, the butyric acid production declined batch by batch, which was most possibly caused by the shift of microbial community structure monitored by denaturing gradient gel electrophoresis. In this study, batch operation was observed to be more suitable for butyric acid production.

Bactec™ blood culture bottles allied to MALDI-TOF mass spectrometry: rapid etiologic diagnosis of bacterial endophthalmitis.

PubMed

Tanaka, Tatiana; Oliveira, Luiza Manhezi de Freitas; Ferreira, Bruno Fortaleza de Aquino; Kato, Juliana Mika; Rossi, Flavia; Correa, Karoline de Lemes Giuntini; Pimentel, Sergio Luis Gianotti; Yamamoto, Joyce Hisae; Almeida Junior, João Nóbrega

2017-07-01

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has been used for direct identification of pathogens from blood-inoculated blood culture bottles (BCBs). We showed that MALDI-TOF MS is an useful technique for rapid identification of the causative agents of endophthalmitis from vitreous humor-inoculated BCBs with a simple protocol. Copyright © 2017 Elsevier Inc. All rights reserved.

[Improved method of studying the blood for sterility].

PubMed

Talapa, A I

1983-05-01

The technique used for the inoculation and subculturing of blood samples in testing them for sterility is described. This technique eliminates the possibility of contaminating the culture medium and the blood sample under test with extraneous bacterial flora. Blood samples were inoculated without opening the containers with the culture medium. Inoculation was made with the syringe and the needle used for taking the blood sample through the punctured rubber stopper closing the container. Subculturing on solid culture media was also carried out without opening the containers: the rubber stopper was punctured and the contents of the container withdrawn with a pipette needle. The use of this new technique made it possible to detect bacteremia in 12.8% of cases, only in persons with purulent and septic diseases, whereas by using the existing technique bacteremia was detected both in sick and healthy persons, in 38.6% and 26.6% of cases, respectively.

High solid fed-batch butanol fermentation with simultaneous product recovery: part II - process integration.

USDA-ARS?s Scientific Manuscript database

In these studies liquid hot water (LHW) pretreated and enzymatically hydrolyzed Sweet Sorghum Bagasse (SSB) hydrolyzates were fermented in a fed-batch reactor. As reported in the preceding paper, the culture was not able to ferment the hydrolyzate I in a batch process due to presence of high level o...

"RCL-Pooling Assay": A Simplified Method for the Detection of Replication-Competent Lentiviruses in Vector Batches Using Sequential Pooling.

PubMed

Corre, Guillaume; Dessainte, Michel; Marteau, Jean-Brice; Dalle, Bruno; Fenard, David; Galy, Anne

2016-02-01

Nonreplicative recombinant HIV-1-derived lentiviral vectors (LV) are increasingly used in gene therapy of various genetic diseases, infectious diseases, and cancer. Before they are used in humans, preparations of LV must undergo extensive quality control testing. In particular, testing of LV must demonstrate the absence of replication-competent lentiviruses (RCL) with suitable methods, on representative fractions of vector batches. Current methods based on cell culture are challenging because high titers of vector batches translate into high volumes of cell culture to be tested in RCL assays. As vector batch size and titers are continuously increasing because of the improvement of production and purification methods, it became necessary for us to modify the current RCL assay based on the detection of p24 in cultures of indicator cells. Here, we propose a practical optimization of this method using a pairwise pooling strategy enabling easier testing of higher vector inoculum volumes. These modifications significantly decrease material handling and operator time, leading to a cost-effective method, while maintaining optimal sensibility of the RCL testing. This optimized "RCL-pooling assay" ameliorates the feasibility of the quality control of large-scale batches of clinical-grade LV while maintaining the same sensitivity.

Efficacy of Detergent and Water Versus Bleach for the Disinfection of Direct Contact Ophthalmic Lenses

PubMed Central

Abbey, Ashkan M.; Gregori, Ninel Z.; Surapaneni, Krishna; Miller, Darlene

2014-01-01

Purpose While manufacturers recommend cleaning ophthalmic lenses with detergent and water and then a specific disinfectant, disinfectants are rarely used in ophthalmic practices. The aim of this pilot study was to evaluate the efficacy of detergent and water versus bleach, a recommended disinfectant, to eliminate common ocular bacteria and viruses from ophthalmic lenses. Methods Three bacterial strains (Staphylococcus epidermidis, Corynebacterium straitum, and methicillin-resistant Staphylococcus aureus (MRSA) and two viral strains (adenovirus and herpes simplex virus (HSV) type-1) were individually inoculated to 20 gonioscopy and laser lenses. Lenses were washed with detergent and water and then disinfected with 10% bleach. All lenses were cultured after inoculation, after detergent and water, and after the bleach. Bacterial cultures in thioglycollate broth were observed for 3 weeks and viral cultures for 2 weeks. The presence of viruses was also detected by multiplex polymerase chain reaction (PCR). Results All 20 lenses inoculated with Staphylococcus epidermidis, Corynebacterium straitum, adenovirus, and HSV-1 showed growth after inoculation, but no growth after detergent/water and after the bleach. All lenses showed positive HSV and adenovirus PCR after inoculation and negative PCR after detergent/water and after bleach. All MRSA contaminated lenses showed growth after inoculation and no growth after detergent and water. However, one lens showed positive growth after bleach. Conclusions Cleaning with detergent and water appeared to effectively eliminate bacteria and viruses from the surface of contaminated ophthalmic lenses. Further studies are warranted to design practical disinfection protocols that minimize lens damage. PMID:24747806

Side effects of immunization with liver attenuated Trypanosoma cruzi in mice and rabbits.

PubMed Central

Basombrío, M A; Besuschio, S; Cossio, P M

1982-01-01

Immunity against lethal, bloodstream forms of Trypanosoma cruzi was achieved in mice by preinoculation of approximately equal to 10(5) culture epimastigotes of an attenuated T. cruzi strain (TCC). The risks of TCC inoculation in terms of pathogenicity or eventual increase in virulence of TCC progeny were evaluated. No pathogenic parasites could be selected from TCC progeny by either mouse, triatome, or culture passages. Immunizing doses of live TCC did not induce in adult mice alterations resembling chronic Chagas' disease, as judged by patterns of mortality, tissue damage, autoantibodies, or parasite recovery. On the basis of the same criteria, However, a remarkable similarity could be established between the disease caused in mice by inoculation of low numbers (10(2)) of pathogenic trypomastigotes and human chronic Chagas' disease. Although patent parasitemias were never revealed in fresh blood mounts obtained from TCC-inoculated mice, a few hemocultures and xenodiagnoses gave positive results, particularly soon after inoculations at birth. The parasites recovered by either method remained in the attenuated, epimastigote stage. In rabbits, no local lesions, fever, weight loss, or histopathological alterations were detected after subcutaneous inoculation of 10(7) TCC organisms, although one fifth of the animals yielded positive hemocultures of epimastigotes. The contrasting host response to cultured epimastigotes as compared with blood trypomastigotes indicates that, in experimental Chagas' disease, immunoprotection is not necessarily associated with immunopathology. Images PMID:6804389

Gravimetric enrichment of high lipid and starch accumulating microalgae.

PubMed

Hassanpour, Morteza; Abbasabadi, Mahsa; Ebrahimi, Sirous; Hosseini, Maryam; Sheikhbaglou, Ahmad

2015-11-01

This study presents gravimetric enrichment of mixed culture to screen starch and lipid producing species separately in a sequencing batch reactor. In the enriched starch-producing mixed culture photobioreactor, the starch content at the end of steady state batch became 3.42 times the beginning of depletion. Whereas in the enriched lipid-producing photobioreactor, the lipid content at the end of steady state batch became 3 times the beginning of famine phase. The obtained results revealed that the gravimetric enrichment is a suitable screening method for specific production of storage compounds in none-sterile large-scaled condition. Copyright © 2015 Elsevier Ltd. All rights reserved.

In vitro growth of Curcuma longa L. in response to five mineral elements and plant density in fed-batch culture systems.

PubMed

El-Hawaz, Rabia F; Bridges, William C; Adelberg, Jeffrey W

2015-01-01

Plant density was varied with P, Ca, Mg, and KNO3 in a multifactor experiment to improve Curcuma longa L. micropropagation, biomass and microrhizome development in fed-batch liquid culture. The experiment had two paired D-optimal designs, testing sucrose fed-batch and nutrient sucrose fed-batch techniques. When sucrose became depleted, volume was restored to 5% m/v sucrose in 200 ml of modified liquid MS medium by adding sucrose solutions. Similarly, nutrient sucrose fed-batch was restored to set points with double concentration of treatments' macronutrient and MS micronutrient solutions, along with sucrose solutions. Changes in the amounts of water and sucrose supplementations were driven by the interaction of P and KNO3 concentrations. Increasing P from 1.25 to 6.25 mM increased both multiplication and biomass. The multiplication ratio was greatest in the nutrient sucrose fed-batch technique with the highest level of P, 6 buds/vessel, and the lowest level of Ca and KNO3. The highest density (18 buds/vessel) produced the highest fresh biomass at the highest concentrations of KNO3 and P with nutrient sucrose fed-batch, and moderate Ca and Mg concentrations. However, maximal rhizome dry biomass required highest P, sucrose fed-batch, and a moderate plant density. Different media formulations and fed-batch techniques were identified to maximize the propagation and storage organ responses. A single experimental design was used to optimize these dual purposes.

In Vitro Growth of Curcuma longa L. in Response to Five Mineral Elements and Plant Density in Fed-Batch Culture Systems

PubMed Central

El-Hawaz, Rabia F.; Bridges, William C.; Adelberg, Jeffrey W.

2015-01-01

Plant density was varied with P, Ca, Mg, and KNO3 in a multifactor experiment to improve Curcuma longa L. micropropagation, biomass and microrhizome development in fed-batch liquid culture. The experiment had two paired D-optimal designs, testing sucrose fed-batch and nutrient sucrose fed-batch techniques. When sucrose became depleted, volume was restored to 5% m/v sucrose in 200 ml of modified liquid MS medium by adding sucrose solutions. Similarly, nutrient sucrose fed-batch was restored to set points with double concentration of treatments’ macronutrient and MS micronutrient solutions, along with sucrose solutions. Changes in the amounts of water and sucrose supplementations were driven by the interaction of P and KNO3 concentrations. Increasing P from 1.25 to 6.25 mM increased both multiplication and biomass. The multiplication ratio was greatest in the nutrient sucrose fed-batch technique with the highest level of P, 6 buds/vessel, and the lowest level of Ca and KNO3. The highest density (18 buds/vessel) produced the highest fresh biomass at the highest concentrations of KNO3 and P with nutrient sucrose fed-batch, and moderate Ca and Mg concentrations. However, maximal rhizome dry biomass required highest P, sucrose fed-batch, and a moderate plant density. Different media formulations and fed-batch techniques were identified to maximize the propagation and storage organ responses. A single experimental design was used to optimize these dual purposes. PMID:25830292

Auto-aggregation properties of a novel aerobic denitrifier Enterobacter sp. strain FL.

PubMed

Wang, Xia; An, Qiang; Zhao, Bin; Guo, Jin Song; Huang, Yuan Sheng; Tian, Meng

2018-02-01

Enterobacter sp. strain FL was newly isolated from activated sludge and exhibited significant capability of auto-aggregation as well as aerobic denitrification. The removal efficiencies of NO 3 - -N, total nitrogen (TN), and TOC by strain FL in batch culture reached 94.6, 63.9, and 72.5% in 24 h, respectively. The production of N 2 O and N 2 in the presence of oxygen demonstrated the occurrence of aerobic denitrification. The auto-aggregation index of strain FL reached 54.3%, suggesting a high tendency that the cells would agglomerate into aggregates. The production of extracellular polymeric substances (EPSs), which were mainly composed of proteins followed by polysaccharides, was considered to be related to the cell aggregation according to Fourier transform infrared (FT-IR) and confocal laser scanning microscopy (CLSM). The proteins in EPS were evenly and tightly combined to cells and altered the protein secondary structures of cell surface from random coils to β-sheets and three-turn helices. The alteration of protein secondary structures of cell surface caused by the proteins in EPS might play a dominant role in the auto-aggregation of strain FL. To further assess the feasibility of strain FL for synthetic wastewater treatment, a sequencing batch reactor (SBR), solely inoculated with strain FL, was conducted. During the 16 running cycles, the removal efficiency of NO 3 - -N was 90.2-99.7% and the auto-aggregation index was stabilized at 35.0-41.5%. The EPS promoted the biomass of strain FL to aggregate in the SBR.

Bacterial communities and metabolic activity of faecal cultures from equol producer and non-producer menopausal women under treatment with soy isoflavones.

PubMed

Guadamuro, Lucía; Dohrmann, Anja B; Tebbe, Christoph C; Mayo, Baltasar; Delgado, Susana

2017-04-17

Isoflavones are polyphenols with estrogenic activity found mainly in soy and soy-derived products that need to be metabolised in the intestine by the gut bacteria to be fully active. There is little knowledge about isoflavone bioconversion and equol production in the human intestine. In this work, we developed an in vitro anaerobic culture model based on faecal slurries to assess the impact of isoflavone supplementation on the overall intestinal bacterial composition changes and associated metabolic transformations. In the faecal anaerobic batch cultures of this study bioconversion of isoflavones into equol was possible, suggesting the presence of viable equol-producing bacterial taxa within the faeces of menopausal women with an equol producer phenotype. The application of high-throughput DNA sequencing of 16S rRNA gene amplicons revealed the composition of the faecal cultures to be modified by the addition of isoflavones, with enrichment of some bacterial gut members associated with the metabolism of phenolics and/or equol production, such as Collinsella, Faecalibacterium and members of the Clostridium clusters IV and XIVa. In addition, the concentration of short-chain fatty acids (SCFAs) detected in the isoflavone-containing faecal cultures was higher in those inoculated with faecal slurries from equol-producing women. This study constitutes the first step in the development of a faecal culturing system with isoflavones that would further allow the selection and isolation of intestinal bacterial types able to metabolize these compounds and produce equol in vitro. Although limited by the low number of faecal cultures analysed and the inter-individual bacterial diversity, the in vitro results obtained in this work tend to indicate that soy isoflavones might provide an alternative energy source for the increase of equol-producing taxa and enhancement of SCFAs production. SCFAs and equol are both considered pivotal bacterial metabolites in the triggering of intestinal health-related beneficial effects.

Enhanced kefiran production by mixed culture of Lactobacillus kefiranofaciens and Saccharomyces cerevisiae.

PubMed

Cheirsilp, Benjamas; Shimizu, Hiroshi; Shioya, Suteaki

2003-01-09

In a batch mixed culture of Lactobacillus kefiranofaciens and Saccharomyces cerevisiae, which could assimilate lactic acid, cell growth and kefiran production rates of L. kefiranofaciens significantly increased, compared with those in pure cultures. The kefiran production rate was 36 mg l(-1) h(-1) in the mixed culture under the anaerobic condition, which was greater than that in the pure culture (24 mg l(-1) h(-1)). Under the aerobic condition, a more intensive interaction between these two strains was observed and higher kefiran production rate (44 mg l(-1) h(-1)) was obtained compared with that under the anaerobic condition. Kefiran production was further enhanced by an addition of fresh medium in the fed-batch mixed culture. In the fed-batch mixed culture, a final kefiran concentration of 5.41 g l(-1) was achieved at 87 h, thereby attaining the highest productivity at 62 mg l(-1) h(-1). Simulation study considered the reduction of lactic acid in pure culture was performed to estimate the additional effect of coculture with S. cerevisiae. Slightly higher cell growth and kefiran production rates in the mixed culture than those expected from pure culture by simulation were observed. These results suggest that coculture of L. kefiranofaciens and S. cerevisiae not only reduces the lactic acid concentration by consumption but also stimulates cell growth and kefiran production of L. kefiranofaciens.

An optimized fed-batch culture strategy integrated with a one-step fermentation improves L-lactic acid production by Rhizopus oryzae.

PubMed

Fu, Yongqian; Sun, Xiaolong; Zhu, Huayue; Jiang, Ru; Luo, Xi; Yin, Longfei

2018-05-21

In previous work, we proposed a novel modified one-step fermentation fed-batch strategy to efficiently generate L-lactic acid (L-LA) using Rhizopus oryzae. In this study, to further enhance efficiency of L-LA production through one-step fermentation in fed-batch cultures, we systematically investigated the initial peptone- and glucose-feeding approaches, including different initial peptone and glucose concentrations and maintained residual glucose levels. Based on the results of this study, culturing R. oryzae with initial peptone and glucose concentrations of 3.0 and 50.0 g/l, respectively, using a fed-batch strategy is an effective approach of producing L-LA through one-step fermentation. Changing the residual glucose had no obvious effect on the generation of L-LA. We determined the maximum LA production and productivity to be 162 g/l and 6.23 g/(l·h), respectively, during the acid production stage. Compared to our previous work, there was almost no change in L-LA production or yield; however, the productivity of L-LA increased by 14.3%.

Kinetics and modeling of hexavalent chromium reduction in Enterobacter cloacae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamamoto, Koji; Kato, Junichi; Yano, Takuo

1993-01-05

Kinetics of bacterial reduction of toxic hexavalent chromium (chromate: CrO[sub 4][sup [minus]2]) was investigated using batch and fed-batch cultures of Enterobacter cloacae strain HO1. In fed-batch cultures, the CrO[sub 4][sup [minus]2] feed was controlled on the basis of the rate of pH change. This control strategy has proven to be useful for avoiding toxic CrO[sub 3][sup [minus]2] overload. A simple mathematical model was developed to describe the bacterial process of CrO[sub 4][sup [minus]2] reduction. In this model, two types of bacterial cells were considered: induced, CrO[sub 4][sup [minus]2]-resistant cells and uninduced, sensitive ones. Only resistant cells were assumed to bemore » able to reduce CrO[sub 4][sup [minus]2]. These fundamental ideas were supported by the model predictions which well approximated all experimental data. In a simulation study, the model was also used to optimize fed-batch cultures, instead of lengthy and expensive laboratory experiments.« less

Microbial conversion of synthetic and food waste-derived volatile fatty acids to lipids.

PubMed

Vajpeyi, Shashwat; Chandran, Kartik

2015-01-01

Lipid accumulation in the oleaginous yeast Cryptococcus albidus was evaluated using mixtures of volatile fatty acids (VFA) as substrates. In general, batch growth under nitrogen limitation led to higher lipid accumulation using synthetic VFA. During batch growth, an initial COD:N ratio of 25:1mg COD:mg N led to maximum intracellular lipid accumulation (28.3 ± 0.7% g/g dry cell weight), which is the maximum reported for C. albidus using VFA as the carbon source, without compromising growth kinetics. At this feed COD:N ratio, chemostat cultures fed with synthetic VFA yielded statistically similar intracellular lipid content as batch cultures (29.9 ± 1.9%, g/g). However, batch cultures fed with VFA produced from the fermentation of food waste, yielded a lower lipid content (14.9 ± 0.1%, g/g). The lipid composition obtained with synthetic and food-waste-derived VFA was similar to commercial biodiesel feedstock. We therefore demonstrate the feasibility of linking biochemical waste treatment and biofuel production using VFA as key intermediates. Copyright © 2015 Elsevier Ltd. All rights reserved.

Continuous production of ethanol with Zymomonas mobilis growing on Jerusalem artichoke juice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Allais, J.J.; Torres, E.F.; Baratti, J.

1987-04-01

Recent work from the authors laboratory has shown that, compared to yeasts, much higher ethanol productivity and yield can be obtained in batch or continuous cultures using the bacterium Zymomonas mobilis grown on fructose media. In batch culture, hydrolyzed Jerusalem artichoke juice with sugar concentrations ranging from 100 to 250 g/L can be converted efficiently to ethanol. The present work describes the conversion of the hydrolyzed juice to ethanol in continuous culture. The extraction and enzymatic hydrolysis of inulin from the tubers of Jerusalem artichoke is also reported.

High stability of yellow fever 17D-204 vaccine: a 12-year restrospective analysis of large-scale production.

PubMed

Barban, V; Girerd, Y; Aguirre, M; Gulia, S; Pétiard, F; Riou, P; Barrere, B; Lang, J

2007-04-12

We have retrospectively analyzed 12 bulk lots of yellow fever vaccine Stamaril, produced between 1990 and 2002 and prepared from the same seed lot that has been in continuous use since 1990. All vaccine batches displayed identical genome sequence. Only four nucleotide substitutions were observed, compared to previously published sequence, with no incidence at amino-acid level. Fine analysis of viral plaque size distribution was used as an additional marker for genetic stability and demonstrated a remarkable homogeneity of the viral population. The total virus load, measured by qRT-PCR, was also homogeneous pointing out reproducibility of the vaccine production process. Mice inoculated intracerebrally with the different bulks exhibited a similar average survival time, and ratio between in vitro potency and mouse LD(50) titers remained constant from batch-to-batch. Taken together, these data demonstrate the genetic stability of the strain at mass production level over a period of 12 years and reinforce the generally admitted idea of the safety of YF17D-based vaccines.

Sol-gel immobilization as a suitable technique for enhancement of α-amylase activity of Aspergillus oryzae PP.

PubMed

Evstatieva, Yana; Yordanova, Mariya; Chernev, Georgi; Ruseva, Yanislava; Nikolova, Dilyana

2014-07-04

Bioencapsulation of microbial cells in silica-based matrices has proved to be a good strategy to enhance the biosynthetic capabilities and viability of bioproducers. In the present study, mycelium and pellet cultures of strain Aspergillus oryzae PP were successfully immobilized in sol-gel hybrid matrices composed of tetraethylorthosilicate as an inorganic precursor, 5% (w/v) starch and 10 or 15% (w/v) polyethylene oxide, or 10% (w/v) calcium alginate as organic compounds. Biosynthetic activity of immobilized cultures was investigated by batch and fed-batch cultivation and the obtained results of 3042.04 IU cm -3 were comparable with the enzyme activity of the free cell culture. Immobilized cultures retained their viability and biosynthetic capabilities up to the 744th h during fed-batch fermentation processes. Consequently, sol-gel encapsulation in hybrid matrices could be considered as a promising technique for immobilization of Aspergillus oryzae PP in order to increase the α-amylase production.

Sensitivity of bhk-21 cells supplemented with diethylaminoethyl-dextran for detection of street rabies virus in saliva samples.

PubMed Central

Larghi, O P; Nebel, A E; Lazaro, L; Savy, V L

1975-01-01

A tissue culture system for detecting rabies virus from saliva samples of suspected animals was developed and compared to suckling mouse inoculation. Swab samples were obtained from the mouth of the animal heads received for rabies diagnosis; these swabs were submerged in maintenance medium. The maintenance medium was inoculated intracerebrally into suckling mice and onto BHK-21 cells with diethylaminoethyl (DEAE)-dextran (BHK/DEAE) and without (BHK). Rabies immunofluorescence was performed on the brain of the mice dying during the observation period and also on both tissue culture systems every day after infection. The BHK-DEAE system detected 28 positive samples obtained from 48 rabid animals and the BHK system detected 18. By suckling mouse inoculation only 11 of the same positive samples were detected. A total of 90 samples was studied by the three methods. Rabies virus was detected by the tissue culture methods earlier than by suckling mouse inoculation. The BHK-DEAE method was an economic and fast method for rabies virus detection in saliva samples, which could be used for ecological and pathogenesis studies, as well for rabies diagnosis before the death of the suspected animal. PMID:1100655

In vitro culture of coconut (Cocos nucifera L.) zygotic embryos.

PubMed

Engelmann, Florent; Malaurie, Bernard; N'Nan, Oulo

2011-01-01

Coconut is a very important crop for millions of people in tropical countries. With coconut, in vitro culture protocols have been developed with two main objectives, viz. the large scale production of particular types of coconuts and the international exchange and conservation of coconut germplasm. The methods described in this chapter have been developed in the framework of collaborative activities between research institutes in Côte d'Ivoire and France. Two coconut embryo in vitro collecting protocols have been established, one consisting of storing the disinfected embryos in a KCl solution until they are brought back to the laboratory, where they are re-disinfected and inoculated in vitro under sterile conditions, and the other including in vitro inoculation of the embryos in the field. For international germplasm exchange, zygotic embryos inoculated in vitro in plastic test tubes or endosperm cylinders containing embryos in plastic bags are used. For in vitro culture, embryos are inoculated on semi-solid medium supplemented with sucrose and activated charcoal and placed in the dark, and then transferred to light conditions with the same (solid or liquid) medium once the first true leaf is visible and the root system has started developing.

Isolation, Identification and Characterization of Yeasts from Fermented Goat Milk of the Yaghnob Valley in Tajikistan

PubMed Central

Qvirist, Linnea A.; De Filippo, Carlotta; Strati, Francesco; Stefanini, Irene; Sordo, Maddalena; Andlid, Thomas; Felis, Giovanna E.; Mattarelli, Paola; Cavalieri, Duccio

2016-01-01

The geographically isolated region of the Yaghnob Valley, Tajikistan, has allowed its inhabitants to maintain a unique culture and lifestyle. Their fermented goat milk constitutes one of the staple foods for the Yaghnob population, and is produced by backslopping, i.e., using the previous fermentation batch to inoculate the new one. This study addresses the yeast composition of the fermented milk, assessing genotypic, and phenotypic properties. The 52 isolates included in this study revealed small species diversity, belonging to Kluyveromyces marxianus, Pichia fermentans, Saccharomyces cerevisiae, and one Kazachstania unispora. The K. marxianus strains showed two different genotypes, one of which never described previously. The two genetically different groups also differed significantly in several phenotypic characteristics, such as tolerance toward high temperatures, low pH, and presence of acid. Microsatellite analysis of the S. cerevisiae strains from this study, compared to 350 previously described strains, attributed the Yaghnobi S. cerevisiae to two different ancestry origins, both distinct from the wine and beer strains, and similar to strains isolated from human and insects feces, suggesting a peculiar origin of these strains, and the existence of a gut reservoir for S. cerevisiae. Our work constitutes a foundation for strain selection for future applications as starter cultures in food fermentations. This work is the first ever on yeast diversity from fermented milk of the previously unexplored area of the Yaghnob Valley. PMID:27857705

Derivation of host and pathogen genotypes in the fusiform rust pathosystem on slash pine using a complimentary genetics model and diallel data

Treesearch

H.E. Stelzel; Robert L. Doudrick; Thomas L. Kubisiak

1997-01-01

Seedlings from 20, full-sib families five-parent slash pine diallel were inoculated using two, single urediniospore-derived cultures of the fusiform rust fungus on two different dates during the 1994 growing season. Presence or absence of fusiform rust galls was recorded for each inoculated seedling at nine months post-inoculation and percent infection levels for each...

Autochthonous starter cultures and indigenous grape variety for regional wine production.

PubMed

Garofalo, C; El Khoury, M; Lucas, P; Bely, M; Russo, P; Spano, G; Capozzi, V

2015-06-01

To characterize Oenococcus oeni strains isolated from North-Apulian wines where malic acid degradation is usually achieved by spontaneous fermentations, and to determine the influence of bacterial inoculation time on the malolactic performances in 'Nero di Troia' wine using a complete autochthonous microbial regime. Oenococcus oeni strains from wines produced with the autochthonous (Apulia Region, southern Italy) grape variety 'Uva di Troia' were isolated, selected and characterized. Multilocus sequence typing and variable number tandem repeat analysis were used to investigate intraspecific diversity. Oenococcus oeni strains were tested in co-inoculation and in sequential inoculation, with two autochthonous yeast strains previously isolated from 'Nero di Troia' wine. After a preliminary screening using co-inoculation regime, the O. oeni strains were grouped in reason of the different behaviour in malic acid performances. Results suggested that the efficient degradation of malic acid in co-inoculation is a strain-dependent characteristic. Autochthonous yeast/bacterium combinations were identified as starter culture, and used in a co-inoculation approach, for vinification of regional wines. The 'microbial terroir' of typical fermented food and beverage production represents a dynamic sector of applied research in food microbiology. In this work, we propose the use of autochthonous bacteria and yeast for wine production from an indigenous grape variety. © 2015 The Society for Applied Microbiology.

Essential oils have different effects on human pathogenic and commensal bacteria in mixed faecal fermentations compared with pure cultures.

PubMed

Thapa, Dinesh; Louis, Petra; Losa, Riccardo; Zweifel, Béatrice; Wallace, R John

2015-02-01

A static batch culture system inoculated with human faeces was used to determine the influence of essential oil compounds (EOCs) on mixed faecal microbiota. Bacteria were quantified using quantitative PCR of 16S rRNA genes. Incubation for 24 h of diluted faeces from six individuals caused enrichment of Bifidobacterium spp., but proportions of other major groups were unaffected. Thymol and geraniol at 500 p.p.m. suppressed total bacteria, resulting in minimal fermentation. Thymol at 100 p.p.m. had no effect, nor did eugenol or nerolidol at 100 or 500 p.p.m. except for a slight suppression of Eubacterium hallii. Methyl isoeugenol at 100 or 500 p.p.m. suppressed the growth of total bacteria, accompanied by a large fall in the molar proportion of propionate formed. The relative abundance of Faecalibacterium prausnitzii was unaffected except with thymol at 500 p.p.m. The ability of EOCs to control numbers of the pathogen Clostridium difficile was investigated in a separate experiment, in which the faecal suspensions were amended by the addition of pure culture of C. difficile. Numbers of C. difficile were suppressed by thymol and methyl isoeugenol at 500 p.p.m. and to a lesser extent at 100 p.p.m. Eugenol and geraniol gave rather similar suppression of C. difficile numbers at both 100 and 500 p.p.m. Nerolidol had no significant effect. It was concluded from these and previous pure-culture experiments that thymol and geraniol at around 100 p.p.m. could be effective in suppressing pathogens in the small intestine, with no concern for beneficial commensal colonic bacteria in the distal gut. © 2015 The Authors.

Improvement of hydrogen fermentation of galactose by combined inoculation strategy.

PubMed

Sivagurunathan, Periyasamy; Anburajan, Parthiban; Kumar, Gopalakrishnan; Arivalagan, Pugazhendhi; Bakonyi, Péter; Kim, Sang-Hyoun

2017-03-01

This study evaluated the feasibility of anaerobic hydrogen fermentation of galactose, a red algal biomass sugar, using individual and combined mixed culture inocula. Heat-treated (90°C, 30 min) samples of granular sludge (GS) and suspended digester sludge (SDS) were used as inoculum sources. The type of mixed culture inoculum played an important role in hydrogen production from galactose. Between two inocula, granular sludge showed higher hydrogen production rate (HPR) and hydrogen yield (HY) of 2.2 L H 2 /L-d and 1.09 mol H 2 /mol galactose added , respectively. Combined inoculation (GS + SDS) led to an elevated HPR and HY of 3.1 L H 2 /L-d and 1.28 mol H 2 /mol galactose added , respectively. Acetic and butyric acids are the major organic acids during fermentation. Quantitative polymerase chain reaction (qPCR) revealed that the mixed culture generated using the combined inoculation contained a higher cluster I Clostridium abundance than the culture produced using the single inoculum. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Susceptibility of calves to porcine circovirus-2 (PCV2).

PubMed

Halami, Mohammad Y; Freick, Markus; Shehata, Awad A; Müller, Hermann; Vahlenkamp, Thomas W

2014-09-17

Circoviruses are known to infect pigs and birds and cause severe diseases with various clinical signs. Porcine circovirus-2 (PCV2), associated with severe economic losses, was detected in rodents, mosquitoes, cattle, and in calves affected with bovine neonatal pancytopenia (BNP). However, molecular and serological investigations on circovirus infections in cattle revealed inconsistent results. The aim of the study was to investigate the susceptibility and immune response of calves to experimental PCV2 inoculation. Animals were either intravenously inoculated with tissue-culture grown PCV2, with bone marrow from PCV2 positive and negative calves or immunized with a commercial inactivated PCV2 vaccine. The results showed that the animals inoculated with tissue-culture grown PCV2 and with PCV2 positive bone marrow displayed clinical signs including lymph node swelling, reddening of oral and ocular mucosa, and diarrhoea 7-18 days post inoculation (p.i.). PCV2-specific antibodies were detected in the tissue-culture grown PCV2-infected animals and in the PCV2-immunized animals from day 11 and 7 p.i. onwards, respectively, but were absent in both bone marrow inoculated groups. PCV2 was detected by real-time quantitative PCR only in blood samples of the tissue-culture grown PCV2-infected animals and in various tissues (e.g. spleen, lymph nodes, thymus), with high copy numbers in blood between day 4 (5.16log10 genomic copy number/ml) and 46 (5.33log10 genomic copy number/ml) p.i. In conclusion, the seroconversion and the detection of PCV2 in lymphoid tissues for more than five weeks p.i. revealed that host susceptibility of PCV2 is not solely restricted to pigs. Copyright © 2014 Elsevier B.V. All rights reserved.

Continuous culture methodology for the screening of microalgae for oil.

PubMed

Del Río, Esperanza; Armendáriz, Ana; García-Gómez, Elena; García-González, Mercedes; Guerrero, Miguel G

2015-02-10

A basic criterion in the selection of microalgae suitable as source of oil for biodiesel should be their actual capacity to produce lipids or, more properly, the fatty acid yield. Performance assessment of 10 preselected microalgae under both batch and continuous culture points to the latter approach as the most adequate for evaluating fatty acid productivity. Differences were patent in continuous culture among strains that otherwise had analogous oil accumulation potential under batch culture. Some promising strains under batch culture (like Muriella aurantiaca and Monoraphidium braunii) exhibited, however, values for actual fatty acid productivity lower than 40 mgL(-1)d(-1) in continuous regime. The analysis performed in photochemostat under continuous culture regime revealed the great potential of Chlorococcum olefaciens, Pseudokirchneriella subcapitata and Scenedesmus almeriensis as oil producing microalgae. Fatty acid productivity levels over 90 mgL(-1)d(-1) were recorded for the latter strains under moderate nitrogen limitation, conditions which led to an enrichment in saturated and monounsaturated fatty acids, a more suitable profile as raw material for biodiesel. The continuous culture methodology employed represents a sound procedure for screening microalgae for biofuel production, providing a reliable evaluation of their fatty acid production capacity, under conditions close to those of outdoor production systems. Copyright © 2015 Elsevier B.V. All rights reserved.

Enhanced recombinant factor VII expression in Chinese hamster ovary cells by optimizing signal peptides and fed-batch medium.

PubMed

Peng, Lin; Yu, Xiao; Li, Chengyuan; Cai, Yanfei; Chen, Yun; He, Yang; Yang, Jianfeng; Jin, Jian; Li, Huazhong

2016-04-01

Signal peptides play an important role in directing and efficiently transporting secretory proteins to their proper locations in the endoplasmic reticulum of mammalian cells. The aim of this study was to enhance the expression of recombinant coagulation factor VII (rFVII) in CHO cells by optimizing the signal peptides and type of fed-batch culture medium used. Five sub-clones (O2, I3, H3, G2 and M3) with different signal peptide were selected by western blot (WB) analysis and used for suspension culture. We compared rFVII expression levels of 5 sub-clones and found that the highest rFVII expression level was obtained with the IgK signal peptide instead of Ori, the native signal peptide of rFVII. The high protein expression of rFVII with signal peptide IgK was mirrored by a high transcription level during suspension culture. After analyzing culture and feed media, the combination of M4 and F4 media yielded the highest rFVII expression of 20 mg/L during a 10-day suspension culture. After analyzing cell density and cell cycle, CHO cells feeding by F4 had a similar percentage of cells in G0/G1 and a higher cell density compared to F2 and F3. This may be the reason for high rFVII expression in M4+F4. In summary, rFVII expression was successfully enhanced by optimizing the signal peptide and fed-batch medium used in CHO suspension culture. Our data may be used to improve the production of other therapeutic proteins in fed-batch culture.

Optimization of culture conditions for gamma-aminobutyric acid production in fermented adzuki bean milk.

PubMed

Song, Hung Yi; Yu, Roch Chui

2018-01-01

γ-Aminobutyric acid (GABA), a nonprotein amino acid, is widely distributed in nature and fulfills several physiological functions. In this study, various lactic acid strains commonly used to produce fermented milk products were inoculated into adzuki bean milk for producing GABA. The high GABA producing strain was selected in further experiment to improve the GABA production utilizing culture medium optimization. The results demonstrated that adzuki bean milk inoculated with Lactobacillus rhamnosus GG increased GABA content from 0.05 mg/mL to 0.44 mg/mL after 36 hours of fermentation, which showed the greatest elevation in this study. Furthermore, the optimal cultural condition to adzuki bean milk inoculated with L. rhamnosus GG to improve the GABA content was performed using response surface methodology. The results showed that GABA content was dependent on the addition of galactose, monosodium glutamate, and pyridoxine with which the increasing ratios of GABA were 23-38%, 24-68%, and 8-36%, respectively. The optimal culture condition for GABA production of adzuki bean milk was found at the content of 1.44% galactose, 2.27% monosodium glutamate, and 0.20% pyridoxine. Under the optimal cultural condition, the amount of GABA produced in the fermented adzuki bean milk was 1.12 mg/mL, which was 22.4-fold higher than that of the unfermented adzuki bean milk (0.05 mg/100 mL). The results suggested that the optimized cultural condition of adzuki bean milk inoculated with L. rhamnosus GG can increase GABA content for consumers as a daily supplement as suggested. Copyright © 2017. Published by Elsevier B.V.

Lactobacillus plantarum (KACC 92189) as a Potential Probiotic Starter Culture for Quality Improvement of Fermented Sausages

PubMed Central

2018-01-01

This study was conducted to evaluate the effects of fermenting temperature on the applicability of Lactobacillus plantarum for production of fermented sausages as starter cultures, and its applicable efficiency was also compared with those inoculated with commercial starter culture or non-inoculated control. The L. plantarum isolated from a naturally-fermented meat, identified by 16S rDNA sequencing and again identified by de novo Assembly Analysis method was used as a starter culture. Six treatments: 3 with L. plantarum at different fermenting temperatures (20, 25 and 30°C), and other 3 treatments (1 with commercial starter culture, 1 with its mixture with L. plantarum and 1 non-inoculated control) fermented under the same conditions (25°C) were prepared. Results revealed that the fermenting temperature considerably affected the pH change in samples added with L. plantarum; the highest pH drop rate (1.57 unit) was obtained on the samples fermented at 30°C, followed by those at 25°C (1.3 unit) and 20°C (0.99 unit) after 4 days fermentation. Increasing the temperature up to 30°C resulted in significantly lower spoilage bacteria count (5.15 log CFU/g) and lipid oxidation level in the products inoculated with L. plantarum. The sensory analysis also showed that the samples added with L. plantarum at 30°C had significantly higher odor, taste and acceptability scores than those fermented at lower temperatures. Under the same processing condition, although the L. plantarum showed slightly lower acidification than the commercial starter culture, however, it significantly improved the eating quality of the product. PMID:29725237

Quantitative modeling of viable cell density, cell size, intracellular conductivity, and membrane capacitance in batch and fed-batch CHO processes using dielectric spectroscopy.

PubMed

Opel, Cary F; Li, Jincai; Amanullah, Ashraf

2010-01-01

Dielectric spectroscopy was used to analyze typical batch and fed-batch CHO cell culture processes. Three methods of analysis (linear modeling, Cole-Cole modeling, and partial least squares regression), were used to correlate the spectroscopic data with routine biomass measurements [viable packed cell volume, viable cell concentration (VCC), cell size, and oxygen uptake rate (OUR)]. All three models predicted offline biomass measurements accurately during the growth phase of the cultures. However, during the stationary and decline phases of the cultures, the models decreased in accuracy to varying degrees. Offline cell radius measurements were unsuccessfully used to correct for the deviations from the linear model, indicating that physiological changes affecting permittivity were occurring. The beta-dispersion was analyzed using the Cole-Cole distribution parameters Deltaepsilon (magnitude of the permittivity drop), f(c) (critical frequency), and alpha (Cole-Cole parameter). Furthermore, the dielectric parameters static internal conductivity (sigma(i)) and membrane capacitance per area (C(m)) were calculated for the cultures. Finally, the relationship between permittivity, OUR, and VCC was examined, demonstrating how the definition of viability is critical when analyzing biomass online. The results indicate that the common assumptions of constant size and dielectric properties used in dielectric analysis are not always valid during later phases of cell culture processes. The findings also demonstrate that dielectric spectroscopy, while not a substitute for VCC, is a complementary measurement of viable biomass, providing useful auxiliary information about the physiological state of a culture. (c) 2010 American Institute of Chemical Engineers

The Oenological Potential of Hanseniaspora uvarum in Simultaneous and Sequential Co-fermentation with Saccharomyces cerevisiae for Industrial Wine Production

PubMed Central

Tristezza, Mariana; Tufariello, Maria; Capozzi, Vittorio; Spano, Giuseppe; Mita, Giovanni; Grieco, Francesco

2016-01-01

In oenology, the utilization of mixed starter cultures composed by Saccharomyces and non-Saccharomyces yeasts is an approach of growing importance for winemakers in order to enhance sensory quality and complexity of the final product without compromising the general quality and safety of the oenological products. In fact, several non-Saccharomyces yeasts are already commercialized as oenological starter cultures to be used in combination with Saccharomyces cerevisiae, while several others are the subject of various studies to evaluate their application. Our aim, in this study was to assess, for the first time, the oenological potential of H. uvarum in mixed cultures (co-inoculation) and sequential inoculation with S. cerevisiae for industrial wine production. Three previously characterized H. uvarum strains were separately used as multi-starter together with an autochthonous S. cerevisiae starter culture in lab-scale micro-vinification trials. On the basis of microbial development, fermentation kinetics and secondary compounds formation, the strain H. uvarum ITEM8795 was further selected and it was co- and sequentially inoculated, jointly with the S. cerevisiae starter, in a pilot scale wine production. The fermentation course and the quality of final product indicated that the co-inoculation was the better performing modality of inoculum. The above results were finally validated by performing an industrial scale vinification The mixed starter was able to successfully dominate the different stages of the fermentation process and the H. uvarum strain ITEM8795 contributed to increasing the wine organoleptic quality and to simultaneously reduce the volatile acidity. At the best of our knowledge, the present report is the first study regarding the utilization of a selected H. uvarum strain in multi-starter inoculation with S. cerevisiae for the industrial production of a wine. In addition, we demonstrated, at an industrial scale, the importance of non-Saccharomyces in the design of tailored starter cultures for typical wines. PMID:27242698

The Oenological Potential of Hanseniaspora uvarum in Simultaneous and Sequential Co-fermentation with Saccharomyces cerevisiae for Industrial Wine Production.

PubMed

Tristezza, Mariana; Tufariello, Maria; Capozzi, Vittorio; Spano, Giuseppe; Mita, Giovanni; Grieco, Francesco

2016-01-01

In oenology, the utilization of mixed starter cultures composed by Saccharomyces and non-Saccharomyces yeasts is an approach of growing importance for winemakers in order to enhance sensory quality and complexity of the final product without compromising the general quality and safety of the oenological products. In fact, several non-Saccharomyces yeasts are already commercialized as oenological starter cultures to be used in combination with Saccharomyces cerevisiae, while several others are the subject of various studies to evaluate their application. Our aim, in this study was to assess, for the first time, the oenological potential of H. uvarum in mixed cultures (co-inoculation) and sequential inoculation with S. cerevisiae for industrial wine production. Three previously characterized H. uvarum strains were separately used as multi-starter together with an autochthonous S. cerevisiae starter culture in lab-scale micro-vinification trials. On the basis of microbial development, fermentation kinetics and secondary compounds formation, the strain H. uvarum ITEM8795 was further selected and it was co- and sequentially inoculated, jointly with the S. cerevisiae starter, in a pilot scale wine production. The fermentation course and the quality of final product indicated that the co-inoculation was the better performing modality of inoculum. The above results were finally validated by performing an industrial scale vinification The mixed starter was able to successfully dominate the different stages of the fermentation process and the H. uvarum strain ITEM8795 contributed to increasing the wine organoleptic quality and to simultaneously reduce the volatile acidity. At the best of our knowledge, the present report is the first study regarding the utilization of a selected H. uvarum strain in multi-starter inoculation with S. cerevisiae for the industrial production of a wine. In addition, we demonstrated, at an industrial scale, the importance of non-Saccharomyces in the design of tailored starter cultures for typical wines.

Efficacy of detergent and water versus bleach for disinfection of direct contact ophthalmic lenses.

PubMed

Abbey, Ashkan M; Gregori, Ninel Z; Surapaneni, Krishna; Miller, Darlene

2014-06-01

Although manufacturers recommend cleaning ophthalmic lenses with detergent and water and then with a specific disinfectant, disinfectants are rarely used in ophthalmic practices. The aim of this pilot study was to evaluate the efficacy of detergent and water versus that of bleach, a recommended disinfectant, to eliminate common ocular bacteria and viruses from ophthalmic lenses. Three bacterial strains (Staphylococcus epidermidis, Corynebacterium straitum, and methicillin-resistant Staphylococcus aureus and 2 viral strains (adenovirus and herpes simplex virus [HSV] type-1) were individually inoculated onto 20 gonioscopy and laser lenses. The lenses were washed with detergent and water and then disinfected with 10% bleach. All the lenses were cultured after inoculation, after washing with detergent and water, and after disinfecting with the bleach. Bacterial cultures in thioglycollate broth were observed for 3 weeks, and viral cultures were observed for 2 weeks. The presence of viruses was also detected by multiplex polymerase chain reaction (PCR). All 20 lenses inoculated with S. epidermidis, C. straitum, adenovirus, and HSV-1 showed growth after inoculation but no growth after washing with detergent/water and after disinfecting with the bleach. All lenses showed positive HSV and adenovirus PCR results after inoculation and negative PCR results after washing with detergent/water and after disinfecting with bleach. All methicillin-resistant S. aureus-contaminated lenses showed growth after inoculation and no growth after washing with detergent and water. However, 1 lens showed positive growth after disinfecting with bleach. Cleaning with detergent and water seemed to effectively eliminate bacteria and viruses from the surface of contaminated ophthalmic lenses. Further studies are warranted to design practical disinfection protocols that minimize lens damage.

Optimization of glutathione production in batch and fed-batch cultures by the wild-type and recombinant strains of the methylotrophic yeast Hansenula polymorpha DL-1

PubMed Central

2011-01-01

Background Tripeptide glutathione (gamma-glutamyl-L-cysteinyl-glycine) is the most abundant non-protein thiol that protects cells from metabolic and oxidative stresses and is widely used as medicine, food additives and in cosmetic industry. The methylotrophic yeast Hansenula polymorpha is regarded as a rich source of glutathione due to the role of this thiol in detoxifications of key intermediates of methanol metabolism. Cellular and extracellular glutathione production of H. polymorpha DL-1 in the wild type and recombinant strains which overexpress genes of glutathione biosynthesis (GSH2) and its precursor cysteine (MET4) was studied. Results Glutathione producing capacity of H. polymorpha DL-1 depending on parameters of cultivation (dissolved oxygen tension, pH, stirrer speed), carbon substrate (glucose, methanol) and type of overexpressed genes of glutathione and its precursor biosynthesis during batch and fed-batch fermentations were studied. Under optimized conditions of glucose fed-batch cultivation, the glutathione productivity of the engineered strains was increased from ~900 up to ~ 2300 mg of Total Intracellular Glutathione (TIG) or GSH+GSSGin, per liter of culture medium. Meantime, methanol fed-batch cultivation of one of the recombinant strains allowed achieving the extracellular glutathione productivity up to 250 mg of Total Extracellular Glutathione (TEG) or GSH+GSSGex, per liter of the culture medium. Conclusions H. polymorpha is an competitive glutathione producer as compared to other known yeast and bacteria strains (Saccharomyces cerevisiae, Candida utilis, Escherichia coli, Lactococcus lactis etc.) with good perspectives for further improvement especially for production of extracellular form of glutathione. PMID:21255454

Impact of diatom growth on trace metal dynamics (Mn, Mo, V, U)

NASA Astrophysics Data System (ADS)

Osterholz, Helena; Simon, Heike; Beck, Melanie; Maerz, Joeran; Rackebrandt, Siri; Brumsack, Hans-Jürgen; Feudel, Ulrike; Simon, Meinhard

2014-03-01

In order to examine the specific role of diatoms in cycling of the trace metals manganese (Mn), molybdenum (Mo), vanadium (V), and uranium (U) Thalassiosira rotula, Skeletonema marinoi, Chaetoceros decipiens, and Rhizosolenia setigera were grown in batch cultures axenically and inoculated with three different bacterial strains isolated from the North Sea. Algal and bacterial growth, concentrations of trace metals and dissolved organic carbon (DOC) were monitored over time and showed that Mn and V were removed from the dissolved phase whereas Mo and U were not. R. setigera and T. rotula exhibited lowest growth and lowest removal whereas S. marinoi grew best and removed highest fractions of Mn and V. The high potential of Mn removal by S. marinoi was also evident from its 7 × higher Mn/P elemental ratio relative to T. rotula. The presence of bacteria modified the timing of the growth of S. marinoi but not directly trace metal removal whereas bacteria enhanced trace metal removal in the cultures of T. rotula and C. decipiens. Modeling of phytoplankton growth, concentrations of Mn and DOC fraction in axenic T. rotula cultures indicated that processes of binding and desorption of Mn to excreted organic components are important to explain the varying proportions of dissolved Mn and thus must be considered as an active component in Mn cycling. The results show distinct differences in the potential of the diatoms in the removal of Mn and V and that bacteria can play an active role in this context. S. marinoi presumably is an important player in Mn and V dynamics in coastal marine systems.

Purification of Encephalitozoon Cultures Contaminated by Mycoplasmas by Murine Intraperitoneal Inoculation

PubMed Central

Ridoux, Olivier; Foucault, Cédric; Drancourt, Michel

1998-01-01

Encephalitozoon species are strict intracellular microsporidia. Cocultures with eukaryotic cell lines can become accidently contaminated by mycoplasmas. We propose a decontamination protocol based on differential cell targeting after intraperitoneal inoculation in mice. Mycoplasma-free microsporidia were isolated from the brains and spleens of inoculated mice 24 h postinoculation by using the centrifugation shell vial system. Identification was confirmed by direct sequencing of PCR-amplified 16S rRNA. PMID:9666031

Oxygen availability strongly affects chronological lifespan and thermotolerance in batch cultures of Saccharomyces cerevisiae

PubMed Central

Bisschops, Markus M.; Vos, Tim; Martínez-Moreno, Rubén; Cortés, Pilar T.; Pronk, Jack T.; Daran-Lapujade, Pascale

2015-01-01

Stationary-phase (SP) batch cultures of Saccharomyces cerevisiae, in which growth has been arrested by carbon-source depletion, are widely applied to study chronological lifespan, quiescence and SP-associated robustness. Based on this type of experiments, typically performed under aerobic conditions, several roles of oxygen in aging have been proposed. However, SP in anaerobic yeast cultures has not been investigated in detail. Here, we use the unique capability of S. cerevisiae to grow in the complete absence of oxygen to directly compare SP in aerobic and anaerobic bioreactor cultures. This comparison revealed strong positive effects of oxygen availability on adenylate energy charge, longevity and thermotolerance during SP. A low thermotolerance of anaerobic batch cultures was already evident during the exponential growth phase and, in contrast to the situation in aerobic cultures, was not substantially increased during transition into SP. A combination of physiological and transcriptome analysis showed that the slow post-diauxic growth phase on ethanol, which precedes SP in aerobic, but not in anaerobic cultures, endowed cells with the time and resources needed for inducing longevity and thermotolerance. When combined with literature data on acquisition of longevity and thermotolerance in retentostat cultures, the present study indicates that the fast transition from glucose excess to SP in anaerobic cultures precludes acquisition of longevity and thermotolerance. Moreover, this study demonstrates the importance of a preceding, calorie-restricted conditioning phase in the acquisition of longevity and stress tolerance in SP yeast cultures, irrespective of oxygen availability. PMID:28357268

42 CFR 493.911 - Bacteriology.

Code of Federal Regulations, 2012 CFR

2012-10-01

... laboratories: (1) Those that interpret Gram stains or perform primary inoculation, or both; and refer cultures...; (2) Those that use direct antigen techniques to detect an organism and may also interpret Gram stains... interpreting Gram stains, performing primary inoculations, and using direct antigen tests, also isolate and...

42 CFR 493.911 - Bacteriology.

Code of Federal Regulations, 2014 CFR

2014-10-01

... laboratories: (1) Those that interpret Gram stains or perform primary inoculation, or both; and refer cultures...; (2) Those that use direct antigen techniques to detect an organism and may also interpret Gram stains... interpreting Gram stains, performing primary inoculations, and using direct antigen tests, also isolate and...

42 CFR 493.911 - Bacteriology.

Code of Federal Regulations, 2013 CFR

2013-10-01

... laboratories: (1) Those that interpret Gram stains or perform primary inoculation, or both; and refer cultures...; (2) Those that use direct antigen techniques to detect an organism and may also interpret Gram stains... interpreting Gram stains, performing primary inoculations, and using direct antigen tests, also isolate and...

Increased acidification in the rhizosphere of cactus seedlings induced by Azospirillum brasilense

NASA Astrophysics Data System (ADS)

Carrillo, Angel; Li, Ching; Bashan, Yoav

2002-08-01

Acidification of the rhizosphere of cactus seedlings (giant cardon, Pachycereus pringlei) after inoculation with the plant growth-promoting bacterium Azospirillum brasilense Cd, in the presence or absence of ammonium and nitrate, was studied to understand how to increase growth of cardon seedlings in poor desert soils. While ammonium enhanced rhizosphere and liquid culture acidification, inoculation with the bacteria enhanced it further. On the other hand, nitrate increased pH of the rhizosphere, but combined with the bacterial inoculation, increase in pH was significantly smaller. Bacterial inoculation with ammonium enhanced plant growth.

Pathogen propagation in cultured three-dimensional tissue mass

NASA Technical Reports Server (NTRS)

Wolf, David A. (Inventor); Spaulding, Glenn F. (Inventor); Goodwin, Thomas J. (Inventor)

2000-01-01

A process for propagating a pathogen in a three-dimensional tissue mass cultured at microgravity conditions in a culture vessel containing culture media and a culture matrix is provided. The three-dimensional tissue mass is inoculated with a pathogen and pathogen replication in the cells of the tissue mass achieved.

Cocultivation of Legionella pneumophila and free-living amoebae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tyndall, R.L.; Domingue, E.L.

1982-10-01

Studies of the interaction of Legionella pneumophila with free-living amoebae showed that Naegleria lovaniensis and Acanthamoeba royreba could use L. pneumophia as a sole food source. However, growth of the amoebae on nonnutrient agar plates seeded with L. pneumophila was slower than growth on nonnutrient agar plates seeded with Escherichia coli. On inoculation of L. pneumophila into axenic cultures of N. lovaniensis and A. roryba, 99.9% of the L. pneumophila was destroyed within 24 h. After several weeks, however, some amoeba cultures became chronically infected and supported the growth of L. pneumophila. Amoebae exposed to L. pneumophila and containing adheredmore » L. pneumophila, L. pneumophila antigens, or both, showed no increased pathogenic potential on intranasal inoculation of weanling mice. Similarly, L. pneumophila propagated in chronically infected amoeba cultures showed no increase in virulence on intraperitoneal inoculation of guinea pigs relative to L. pneumophila grown in yeast extract broth. 20 references, 1 figure, 4 tables.« less

Ethanol production from glycerol using immobilized Pachysolen tannophilus during microaerated repeated-batch fermentor culture.

PubMed

Cha, Hye-Geun; Kim, Yi-Ok; Choi, Woon Yong; Kang, Do-Hyung; Lee, Hyeon-Yong; Jung, Kyung-Hwan

2015-03-01

Herein, we established a repeated-batch process for ethanol production from glycerol by immobilized Pachysolen tannophilus. The aim of this study was to develop a more practical and applicable ethanol production process for biofuel. In particular, using industrial-grade medium ingredients, the microaeration rate was optimized for maximization of the ethanol production, and the relevant metabolic parameters were then analyzed. The microaeration rate of 0.11 vvm, which is far lower than those occurring in a shaking flask culture, was found to be the optimal value for ethanol production from glycerol. In addition, it was found that, among those tested, Celite was a more appropriate carrier for the immobilization of P. tannophilus to induce production of ethanol from glycerol. Finally, through a repeated-batch culture, the ethanol yield (Ye/g) of 0.126 ± 0.017 g-ethanol/g-glycerol (n = 4) was obtained, and this value was remarkably comparable with a previous report. In the future, it is expected that the results of this study will be applied for the development of a more practical and profitable long-term ethanol production process, thanks to the industrial-grade medium preparation, simple immobilization method, and easy repeated-batch operation.

Improved Mannanase Production from Penicillium occitanis by Fed-Batch Fermentation Using Acacia Seeds

PubMed Central

Blibech, Monia; Ellouz Ghorbel, Raoudha; Chaari, Fatma; Dammak, Ilyes; Bhiri, Fatma; Neifar, Mohamed; Ellouz Chaabouni, Semia

2011-01-01

By applying a fed-batch strategy, production of Penicillium occitanis mannanases could be almost doubled as compared to a batch cultivation on acacia seeds (76 versus 41 U/mL). Also, a 10-fold increase of enzyme activities was observed from shake flask fermentation to the fed-batch fermentation. These production levels were 3-fold higher than those obtained on coconut meal. The high mannanase production using acacia seeds powder as inducer substrate showed the suitability of this culture process for industrial-scale development. PMID:23724314

Photoproduction of hydrogen by marine blue-green algae. Progress report 15 August 80-14 February 81

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mitsui, A.

The growth of Miami BG 7 (Oscillatoria sp.) in seawater was studied using 2.8 liters of Fernbach batch cultures, 14 liters of Carboy batch cultures, and 7 liters of a controlled environment system. In the batch culture tests, both 'instant ocean' and natural seawater from the local environment were used. The effects of adding supplemental nitrate and ammonia to the seawater bases were also examined. The 7-liter controlled environment culture system provides for the control and monitoring of physical and chemical parameters, and can be used for continuous culture experiments. Work on the comparative study of Anabaena cylindrica and Miamimore » BG 7 indicates that their hydrogen metabolism is quite different having a direct bearing on their applied potential. In particular, this study has shown that there is a strong uptake hydrogenase activity in Anabaena cylindrica in a closed system. This differs sharply with the high rates of production achieved by Anabaena cylindrica in 'flow through systems,' where hydrogen gas is continuously eliminated from the experimental chamber. In contrast, Miami BG 7 exhibits no uptake hydrogenase activity. Consequently, a high rate of hydrogen evolution is maintained.« less

Enrichment of denitrifying methane-oxidizing microorganisms using up-flow continuous reactors and batch cultures.

PubMed

Hatamoto, Masashi; Kimura, Masafumi; Sato, Takafumi; Koizumi, Masato; Takahashi, Masanobu; Kawakami, Shuji; Araki, Nobuo; Yamaguchi, Takashi

2014-01-01

Denitrifying anaerobic methane oxidizing (DAMO) microorganisms were enriched from paddy field soils using continuous-flow and batch cultures fed with nitrate or nitrite as a sole electron acceptor. After several months of cultivation, the continuous-flow cultures using nitrite showed remarkable simultaneous methane oxidation and nitrite reduction and DAMO bacteria belonging to phylum NC10 were enriched. A maximum volumetric nitrite consumption rate of 70.4±3.4 mg-N·L(-1)·day(-1) was achieved with very short hydraulic retention time of 2.1 hour. In the culture, about 68% of total microbial cells were bacteria and no archaeal cells were detected by fluorescence in situ hybridization. In the nitrate-fed continuous-flow cultures, 58% of total microbial cells were bacteria while archaeal cells accounted for 7% of total cell numbers. Phylogenetic analysis of pmoA gene sequence showed that enriched DAMO bacteria in the continuous-flow cultivation had over 98% sequence similarity to DAMO bacteria in the inoculum. In contrast, for batch culture, the enriched pmoA gene sequences had 89-91% sequence similarity to DAMO bacteria in the inoculum. These results indicate that electron acceptor and cultivation method strongly affect the microbial community structures of DAMO consortia.

Benzoate-induced stress enhances xylitol yield in aerobic fed-batch culture of Candida mogii TISTR 5892.

PubMed

Wannawilai, Siwaporn; Sirisansaneeyakul, Sarote; Chisti, Yusuf

2015-01-20

Production of the natural sweetener xylitol from xylose via the yeast Candida mogii TISTR 5892 was compared with and without the growth inhibitor sodium benzoate in the culture medium. Sodium benzoate proved to be an uncompetitive inhibitor in relatively poorly oxygenated shake flask aerobic cultures. In a better controlled aerobic environment of a bioreactor, the role of sodium benzoate could equally well be described as competitive, uncompetitive or noncompetitive inhibitor of growth. In intermittent fed-batch fermentations under highly aerobic conditions, the presence of sodium benzoate at 0.15gL(-1) clearly enhanced the xylitol titer relative to the control culture without the sodium benzoate. The final xylitol concentration and the average xylitol yield on xylose were nearly 50gL(-1) and 0.57gg(-1), respectively, in the presence of sodium benzoate. Both these values were substantially higher than reported for the same fermentation under microaerobic conditions. Therefore, a fed-batch aerobic fermentation in the presence of sodium benzoate is promising for xylitol production using C. mogii. Copyright © 2014 Elsevier B.V. All rights reserved.

Evaluation of the use of malic acid decarboxylase-deficient starter culture in NaCl-free cucumber fermentations to reduce bloater incidence.

PubMed

Zhai, Y; Pérez-Díaz, I M; Diaz, J T; Lombardi, R L; Connelly, L E

2018-01-01

Accumulation of carbon dioxide (CO 2 ) in cucumber fermentations is known to cause hollow cavities inside whole fruits or bloaters, conducive to economic losses for the pickling industry. This study focused on evaluating the use of a malic acid decarboxylase (MDC)-deficient starter culture to minimize CO 2 production and the resulting bloater index in sodium chloride-free cucumber fermentations brined with CaCl 2 . Attempts to isolate autochthonous MDC-deficient starter cultures from commercial fermentations, using the MD medium for screening, were unsuccessful. The utilization of allochthonous MDC-deficient starter cultures resulted in incomplete utilization of sugars and delayed fermentations. Acidified fermentations were considered, to suppress the indigenous microbiota and favour proliferation of the allochthonous MDC-deficient Lactobacillus plantarum starter cultures. Inoculation of acidified fermentations with L. plantarum alone or in combination with Lactobacillus brevis minimally improved the conversion of sugars. However, inoculation of the pure allochthonous MDC-deficient starter culture to 10 7 CFU per ml in acidified fermentations resulted in a reduced bloater index as compared to wild fermentations and those inoculated with the mixed starter culture. Although use of an allochthonous MDC-deficient starter culture reduces bloater index in acidified cucumber fermentations brined with CaCl 2 , an incomplete conversion of sugars is observed. Economical losses due to the incidence of bloaters in commercial cucumber fermentations brined with CaCl 2 may be reduced utilizing a starter culture to high cell density. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

Bio-protective potential of lactic acid bacteria: Effect of Lactobacillus sakei and Lactobacillus curvatus on changes of the microbial community in vacuum-packaged chilled beef.

PubMed

Zhang, Yimin; Zhu, Lixian; Dong, Pengcheng; Liang, Rongrong; Mao, Yanwei; Qiu, Shubing; Luo, Xin

2018-04-01

This study was to determine the bacterial diversity and monitor the community dynamic changes during storage of vacuum-packaged sliced raw beef as affected by Lactobacillus sakei and Lactobacillus curvatus . L. sakei and L. curvatus were separately incubated in vacuumed-packaged raw beef as bio-protective cultures to inhibit the naturally contaminating microbial load. Dynamic changes of the microbial diversity of inoculated or non-inoculated (control) samples were monitored at 4°C for 0 to 38 days, using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The DGGE profiles of DNA directly extracted from non-inoculated control samples highlighted the order of appearance of spoilage bacteria during storage, showing that Enterbacteriaceae and Pseudomonas fragi emerged early, then Brochothrix thermosphacta shared the dominant position, and finally, Pseudomonas putida showed up became predominant. Compared with control, the inoculation of either L. sakei or L. curvatus significantly lowered the complexity of microbial diversity and inhibited the growth of spoilage bacteria (p<0.05). Interestingly, we also found that the dominant position of L. curvatus was replaced by indigenous L. sakei after 13 d for L. curvatus -inoculated samples. Plate counts on selective agars further showed that inoculation with L. sakei or L. curvatus obviously reduced the viable counts of Enterbacteraceae , Pseudomonas spp. and B. thermosphacta during later storage (p< 0.05), with L. sakei exerting greater inhibitory effect. Inoculation with both bio-protective cultures also significantly decreased the total volatile basic nitrogen values of stored samples (p<0.05). Taken together, the results proved the benefits of inoculation with lactic acid bacteria especially L. sakei as a potential way to inhibit growth of spoilage-related bacteria and improve the shelf life of vacuum-packaged raw beef.

Understanding the interplay of carbon and nitrogen supply for ectoines production and metabolic overflow in high density cultures of Chromohalobacter salexigens.

PubMed

Salar-García, María J; Bernal, Vicente; Pastor, José M; Salvador, Manuel; Argandoña, Montserrat; Nieto, Joaquín J; Vargas, Carmen; Cánovas, Manuel

2017-02-08

The halophilic bacterium Chromohalobacter salexigens has been proposed as promising cell factory for the production of the compatible solutes ectoine and hydroxyectoine. This bacterium has evolved metabolic adaptations to efficiently grow under high salt concentrations by accumulating ectoines as compatible solutes. However, metabolic overflow, which is a major drawback for the efficient conversion of biological feedstocks, occurs as a result of metabolic unbalances during growth and ectoines production. Optimal production of ectoines is conditioned by the interplay of carbon and nitrogen metabolisms. In this work, we set out to determine how nitrogen supply affects the production of ectoines. Chromohalobacter salexigens was challenged to grow in media with unbalanced carbon/nitrogen ratio. In C. salexigens, overflow metabolism and ectoines production are a function of medium composition. At low ammonium conditions, the growth rate decreased importantly, up to 80%. Shifts in overflow metabolism were observed when changing the C/N ratio in the culture medium. 13 C-NMR analysis of ectoines labelling revealed a high metabolic rigidity, with almost constant flux ratios in all conditions assayed. Unbalanced C/N ratio led to pyruvate accumulation, especially upon N-limitation. Analysis of an ect - mutant demonstrated the link between metabolic overflow and ectoine biosynthesis. Under non ectoine synthesizing conditions, glucose uptake and metabolic overflow decreased importantly. Finally, in fed-batch cultures, biomass yield was affected by the feeding scheme chosen. High growth (up to 42.4 g L -1 ) and volumetric ectoine yields (up to 4.21 g L -1 ) were obtained by minimizing metabolite overflow and nutrient accumulation in high density cultures in a low nitrogen fed-batch culture. Moreover, the yield coefficient calculated for the transformation of glucose into biomass was 30% higher in fed-batch than in the batch culture, demonstrating that the metabolic efficiency of C. salexigens can be improved by careful design of culture feeding schemes. Metabolic shifts observed at low ammonium concentrations were explained by a shift in the energy required for nitrogen assimilation. Carbon-limited fed-batch cultures with reduced ammonium supply were the best conditions for cultivation of C. salexigens, supporting high density growth and maintaining high ectoines production.

Role of Thiobacillus thioparus in the biodegradation of carbon disulfide in a biofilter packed with a recycled organic pelletized material.

PubMed

Prenafeta-Boldú, Francesc X; Rojo, Naiara; Gallastegui, Gorka; Guivernau, Miriam; Viñas, Marc; Elías, Ana

2014-07-01

This study reports the biodegradation of carbon disulfide (CS2) in air biofilters packed with a pelletized mixture of composted manure and sawdust. Experiments were carried out in two lab-scale (1.2 L) biofiltration units. Biofilter B was seeded with activated sludge enriched previously on CS2-degrading biomass under batch conditions, while biofilter A was left as a negative inoculation control. This inoculum was characterized by an acidic pH and sulfate accumulation, and contained Achromobacter xylosoxidans as the main putative CS2 biodegrading bacterium. Biofilter operation start-up was unsuccessfully attempted under xerophilic conditions and significant CS2 elimination was only achieved in biofilter A upon the implementation of an intermittent irrigation regime. Sustained removal efficiencies of 90-100 % at an inlet load of up to 12 g CS2 m(-3) h(-1) were reached. The CS2 removal in this biofilter was linked to the presence of the chemolithoautotrophic bacterium Thiobacillus thioparus, known among the relatively small number of species with a reported capacity of growing on CS2 as the sole energy source. DGGE molecular profiles confirmed that this microbe had become dominant in biofilter A while it was not detected in samples from biofilter B. Conventional biofilters packed with inexpensive organic materials are suited for the treatment of low-strength CS2 polluted gases (IL <12 g CS2 m(-3) h(-1)), provided that the development of the adequate microorganisms is favored, either upon enrichment or by inoculation. The importance of applying culture-independent techniques for microbial community analysis as a diagnostic tool in the biofiltration of recalcitrant compounds has been highlighted.

Evaluation of Mixed Probiotic Starter Cultures Isolated from Kimchi on Physicochemical and Functional Properties, and Volatile Compounds of Fermented Hams.

PubMed

Kim, Young Joo; Park, Sung Yong; Lee, Hong Chul; Yoo, Seung Seok; Oh, Sejong; Kim, Kwang Hyun; Chin, Koo Bok

2016-01-01

The objective of this study was to investigate the effects of mixed starter cultures isolated from kimchi on physicochemical properties, functionality and flavors of fermented ham. Physicochemical properties, microbial counts, shear force, cholesterol contents and volatile compounds of fermented ham were investigated during processing (curing and ripening time). Curing process for 7 d increased saltiness, however, decreased hunter color values (L, a, and b values). Ripening process for 21 d increased most parameters, such as saltiness, color values, weight loss, shear force and cholesterol content due to the drying process. The mixed starter culture had higher lactic acid bacteria than the commercial one. While eight volatile compounds were identified from fermented hams during curing process, total fiftyeight volatile compounds were identified from fermented hams during ripening process. The main volatile compounds were alcohols, esters and furans. However, no differences in volatile compounds were observed between two batches. Fermented hams (batch B) manufactured with probiotic starter culture (LPP) had higher sensory score in texture, color and overall acceptability than counterparts (batch A), while the opposite trend was observed in flavor. Therefore, mixed probiotic starter culture isolated from kimchi might be used as a starter culture to be able to replace with commercial starter culture (LK-30 plus) for the manufacture of fermented ham.

Evaluation of Mixed Probiotic Starter Cultures Isolated from Kimchi on Physicochemical and Functional Properties, and Volatile Compounds of Fermented Hams

PubMed Central

Yoo, Seung Seok

2016-01-01

The objective of this study was to investigate the effects of mixed starter cultures isolated from kimchi on physicochemical properties, functionality and flavors of fermented ham. Physicochemical properties, microbial counts, shear force, cholesterol contents and volatile compounds of fermented ham were investigated during processing (curing and ripening time). Curing process for 7 d increased saltiness, however, decreased hunter color values (L, a, and b values). Ripening process for 21 d increased most parameters, such as saltiness, color values, weight loss, shear force and cholesterol content due to the drying process. The mixed starter culture had higher lactic acid bacteria than the commercial one. While eight volatile compounds were identified from fermented hams during curing process, total fiftyeight volatile compounds were identified from fermented hams during ripening process. The main volatile compounds were alcohols, esters and furans. However, no differences in volatile compounds were observed between two batches. Fermented hams (batch B) manufactured with probiotic starter culture (LPP) had higher sensory score in texture, color and overall acceptability than counterparts (batch A), while the opposite trend was observed in flavor. Therefore, mixed probiotic starter culture isolated from kimchi might be used as a starter culture to be able to replace with commercial starter culture (LK-30 plus) for the manufacture of fermented ham. PMID:27499673

Improved productivity of poly (3-hydroxybutyrate) (PHB) in thermophilic Chelatococcus daeguensis TAD1 using glycerol as the growth substrate in a fed-batch culture.

PubMed

Cui, Bin; Huang, Shaobin; Xu, Fuqian; Zhang, Ruijian; Zhang, Yongqing

2015-07-01

A particularly successful polyhydroxyalkanoate (PHA) in industrial applications is poly (3-hydroxybutyrate) (PHB). However, one of the major obstacles for wider application of PHB is the cost of its production and purification. Therefore, it is desirable to discover a method for producing PHB in large quantities at a competitive price. Glycerol is a cheap and widely used carbon source that can be applied in PHB production process. There are numerous advantages to operating fermentation at elevated temperatures; only several thermophilic bacteria are able to accumulate PHB when glycerol is the growth substrate. Here, we report on the possibility of increasing PHB production at low cost using thermophilic Chelatococcus daeguensis TAD1 when glycerol is the growth substrate in a fed-batch culture. We found that (1) excess glycerol inhibited PHB accumulation and (2) organic nitrogen sources, such as tryptone and yeast extract, promoted the growth of C. daeguensis TAD1. In the batch fermentation experiments, we found that using glycerol at low concentrations as the sole carbon source, along with the addition of mixed nitrate (NH4Cl, tryptone, and yeast extract), stimulated PHB accumulation in C. daeguensis TAD1. The results showed that the PHB productivity decreased in the following order: two-stage fed-batch fermentation > fed-batch fermentation > batch fermentation. In optimized culture conditions, a PHB amount of 17.4 g l(-1) was obtained using a two-stage feeding regimen, leading to a productivity rate of 0.434 g l(-1) h(-1), which is the highest productivity rate reported for PHB to date. This high PHB biosynthetic productivity could decrease the total production cost, allowing for further development of industrial applications of PHB.

Strategies for the startup of methanogenic inverse fluidized-bed reactors using colonized particles.

PubMed

Alvarado-Lassman, A; Sandoval-Ramos, A; Flores-Altamirano, M G; Vallejo-Cantú, N A; Méndez-Contreras, J M

2010-05-01

One of the inconveniences in the startup of methanogenic inverse fluidized-bed reactors (IFBRs) is the long period required for biofilm formation and stabilization of the system. Previous researchers have preferred to start up in batch mode to shorten stabilization times. Much less work has been done with continuous-mode startup for the IFBR configuration of reactors. In this study, we prepared two IFBRs with similar characteristics to compare startup times for batch- and continuous-operation modes. The reactors were inoculated with a small quantity of colonized particles and run for a period of 3 months, to establish the optimal startup strategy using synthetic media as a substrate (glucose as a source of carbon). After the startup stage, the continuous- and batch-mode reactors removed more than 80% of the chemical oxygen demand (COD) in 51 and 60 days of operation, respectively; however, at the end of the experiments, the continuous-mode reactor had more biomass attached to the support media than the batch-mode reactor. Both reactors developed fully covered support media, but only the continuous-mode reactor had methane yields close to the theoretical value that is typical of stable reactors. Then, a combined startup strategy was proposed, with industrial wastewater as the substrate, using a sequence of batch cycles followed by continuous operation, which allows stable operation at an organic loading rate of 20 g COD/L x d in 15 days. Using a fraction of colonized support as an inoculum presents advantages, with respect to previously reported strategies.

The role of heterotrophic microorganism Galactomyces sp. Z3 in improving pig slurry bioleaching.

PubMed

Zhou, Jun; Zheng, Guanyu; Zhou, Lixiang; Liu, Fenwu; Zheng, Chaocheng; Cui, Chunhong

2013-01-01

The feasibility of removing heavy metals and eliminating pathogens from pig slurry through bioleaching involving the fungus Galactomyces sp. Z3 and two acidophilic thiobacillus (A. ferrooxidans LX5 and A. thiooxidans TS6) was investigated. It was found that the isolated pig slurry dissolved organic matter (DOM) degrader Z3 was identified as Galactomyces sp. Z3, which could grow well at pH 2.5-7 and degrade pig slurry DOM from 1973 to 942 mg/l within 48 h. During the successive multi-batch bioleaching systems, the co-inoculation of pig slurry degrader Galactomyces sp. Z3 and the two Acidithiobacillus species could improve pig slurry bioleaching efficiency compared to the single system without Galactomyces sp. Z3. The removal efficiency of Zn and Cu exceeded 94% and 85%, respectively. In addition, the elimination efficiencies of pathogens, including both total coliform and faecal coliform counts, exceeded 99% after bioleaching treatment. However, the counts of Galactomyces sp. Z3 decreased with the fall of pH and did not restore to the initial level during successive multi-batch bioleaching systems, and it is necessary to re-inoculate Galactomyces sp. Z3 cells into the bioleaching system to maintain its role in degrading pig slurry DOM. Therefore, a bioleaching technique involving both Galactomyces sp. Z3 and Acidithiobacillus species is an efficient method for removing heavy metals and eliminating pathogens from pig slurry.

Acidification of In-Storage-Psychrophilic-Anaerobic-Digestion (ISPAD) process to reduce ammonia volatilization: Model development and validation.

PubMed

Madani-Hosseini, Mahsa; Mulligan, Catherine N; Barrington, Suzelle

2016-06-01

In-Storage-Psychrophilic-Anaerobic-Digestion (ISPAD) is an ambient temperature treatment system for wastewaters stored for over 100days under temperate climates, which produces a nitrogen rich digestate susceptible to ammonia (NH3) volatilization. Present acidification techniques reducing NH3 volatilization are not only expensive and with secondary environmental effects, but do not apply to ISPAD relying on batch-to-batch inoculation. The objectives of this study were to identify and validate sequential organic loading (OL) strategies producing imbalances in acidogen and methanogen growth, acidifying ISPAD content one week before emptying to a pH of 6, while also preserving the inoculation potential. This acidification process is challenging as wastewaters often offer a high buffering capacity and ISPAD operational practices foster low microbial populations. A model simulating the ISPAD pH regime was used to optimize 3 different sequential OLs to decrease the ISPAD pH to 6.0. All 3 strategies were compared in terms of biogas production, volatile fatty acid (VFA) concentration, microbial activity, glucose consumption, and pH decrease. Laboratory validation of the model outputs confirmed that a sequential OL of 13kg glucose/m(3) of ISPAD content over 4days could indeed reduce the pH to 6.0. Such OL competes feasibly with present acidification techniques. Nevertheless, more research is required to explain the 3-day lag between the model results and the experimental data. Copyright © 2016 Elsevier Ltd. All rights reserved.

The visual assessment of broth cultures for tissue bank samples.

PubMed

Varettas, Kerry

2017-09-01

The bioburden screening process of allograft musculoskeletal tissue samples received at the South Eastern Area Laboratory Services includes the routine use of solid agar and cooked meat (CM) broth media. CM has been routinely sub-cultured onto solid agar plates after aerobic incubation at 35 °C. This study will evaluate whether a visual assessment of CM can replace sub-culture by an in vitro inoculation and a prospective study. Eight challenge organisms were serially diluted and inoculated into CM. The average inoculum of 0.5-5.5 CFU produced visible turbidity of CM after 24-h incubation for 7 of the challenge organisms with one organism producing turbidity after 48-h incubation. The prospective study evaluated 222 CM of which 213 were visually clear and no-growth on sub-culture and 9 turbid CM which were culture positive. Broth cultures are an integral part of the bioburden screening process of allograft musculoskeletal tissue and swab samples and visual assessment of CM can replace sub-culture.

Strategies for improving production performance of probiotic Pediococcus acidilactici viable cell by overcoming lactic acid inhibition.

PubMed

Othman, Majdiah; Ariff, Arbakariya B; Wasoh, Helmi; Kapri, Mohd Rizal; Halim, Murni

2017-11-27

Lactic acid bacteria are industrially important microorganisms recognized for fermentative ability mostly in their probiotic benefits as well as lactic acid production for various applications. Fermentation conditions such as concentration of initial glucose in the culture, concentration of lactic acid accumulated in the culture, types of pH control strategy, types of aeration mode and different agitation speed had influenced the cultivation performance of batch fermentation of Pediococcus acidilactici. The maximum viable cell concentration obtained in constant fed-batch fermentation at a feeding rate of 0.015 L/h was 6.1 times higher with 1.6 times reduction in lactic acid accumulation compared to batch fermentation. Anion exchange resin, IRA 67 was found to have the highest selectivity towards lactic acid compared to other components studied. Fed-batch fermentation of P. acidilactici coupled with lactic acid removal system using IRA 67 resin showed 55.5 and 9.1 times of improvement in maximum viable cell concentration compared to fermentation without resin for batch and fed-batch mode respectively. The improvement of the P. acidilactici growth in the constant fed-batch fermentation indicated the use of minimal and simple process control equipment is an effective approach for reducing by-product inhibition. Further improvement in the cultivation performance of P. acidilactici in fed-bath fermentation with in situ addition of anion-exchange resin significantly helped to enhance the growth of P. acidilactici by reducing the inhibitory effect of lactic acid and thus increasing probiotic production.

BIOLAB experiment development status 2005

NASA Astrophysics Data System (ADS)

Brinckmann, Enno; Manieri, Pierfilippo

2005-08-01

BIOLAB, ESA's major facility for biological Space research on the International Space Station (ISS), will accommodate the first two batches of experiments after its launch with the "Columbus" Laboratory (spring 2007). Seven experiments have been selected for development: three of the first batch have concluded Phase A/B with the testing of the breadboards, in which the main functions of the scientific studies can be simulated and defined for further inputs to the final design of the experiment hardware. The biological specimens of the first batch are scorpions, plant seedlings, bacteria suspensions and cell cultures of mammalian and invertebrate origin. The experiment protocols request demanding resources ranging from life support for the entire mission (90 days) to skilled crew operations and transport/storage in deep freezers. Even more sophisticated experiments are in preparation for the second batch, dealing with various cell culture systems. This presentation gives an overview about the experiment development status, whilst the science background and breadboard test results will be presented by the respective experiment teams.

Kinetic modelling of a diesel-polluted clayey soil bioremediation process.

PubMed

Fernández, Engracia Lacasa; Merlo, Elena Moliterni; Mayor, Lourdes Rodríguez; Camacho, José Villaseñor

2016-07-01

A mathematical model is proposed to describe a diesel-polluted clayey soil bioremediation process. The reaction system under study was considered a completely mixed closed batch reactor, which initially contacted a soil matrix polluted with diesel hydrocarbons, an aqueous liquid-specific culture medium and a microbial inoculation. The model coupled the mass transfer phenomena and the distribution of hydrocarbons among four phases (solid, S; water, A; non-aqueous liquid, NAPL; and air, V) with Monod kinetics. In the first step, the model simulating abiotic conditions was used to estimate only the mass transfer coefficients. In the second step, the model including both mass transfer and biodegradation phenomena was used to estimate the biological kinetic and stoichiometric parameters. In both situations, the model predictions were validated with experimental data that corresponded to previous research by the same authors. A correct fit between the model predictions and the experimental data was observed because the modelling curves captured the major trends for the diesel distribution in each phase. The model parameters were compared to different previously reported values found in the literature. Pearson correlation coefficients were used to show the reproducibility level of the model. Copyright © 2016. Published by Elsevier B.V.

Monochloramine Cometabolism by Nitrifying Biofilm Relevant to Drinking Water

EPA Science Inventory

Recently, biological monochloramine removal (i.e., cometabolism) by a pure culture ammonia–oxidizing bacteria, Nitrosomonas europaea, and a nitrifying mixed–culture have been shown to increase monochloramine demand. Although important, these previous suspended culture batch kine...

Improving lactate metabolism in an intensified CHO culture process: productivity and product quality considerations.

PubMed

Xu, Sen; Hoshan, Linda; Chen, Hao

2016-11-01

In this study, we discussed the development and optimization of an intensified CHO culture process, highlighting medium and control strategies to improve lactate metabolism. A few strategies, including supplementing glucose with other sugars (fructose, maltose, and galactose), controlling glucose level at <0.2 mM, and supplementing medium with copper sulfate, were found to be effective in reducing lactate accumulation. Among them, copper sulfate supplementation was found to be critical for process optimization when glucose was in excess. When copper sulfate was supplemented in the new process, two-fold increase in cell density (66.5 ± 8.4 × 10(6) cells/mL) and titer (11.9 ± 0.6 g/L) was achieved. Productivity and product quality attributes differences between batch, fed-batch, and concentrated fed-batch cultures were discussed. The importance of process and cell metabolism understanding when adapting the existing process to a new operational mode was demonstrated in the study.

A simple method to determine evaporation and compensate for liquid losses in small-scale cell culture systems.

PubMed

Wiegmann, Vincent; Martinez, Cristina Bernal; Baganz, Frank

2018-04-24

Establish a method to indirectly measure evaporation in microwell-based cell culture systems and show that the proposed method allows compensating for liquid losses in fed-batch processes. A correlation between evaporation and the concentration of Na + was found (R 2  = 0.95) when using the 24-well-based miniature bioreactor system (micro-Matrix) for a batch culture with GS-CHO. Based on these results, a method was developed to counteract evaporation with periodic water additions based on measurements of the Na + concentration. Implementation of this method resulted in a reduction of the relative liquid loss after 15 days of a fed-batch cultivation from 36.7 ± 6.7% without volume corrections to 6.9 ± 6.5% with volume corrections. A procedure was established to indirectly measure evaporation through a correlation with the level of Na + ions in solution and deriving a simple formula to account for liquid losses.

Improving Embryonic Stem Cell Expansion through the Combination of Perfusion and Bioprocess Model Design

PubMed Central

Yeo, David; Kiparissides, Alexandros; Cha, Jae Min; Aguilar-Gallardo, Cristobal; Polak, Julia M.; Tsiridis, Elefterios; Pistikopoulos, Efstratios N.; Mantalaris, Athanasios

2013-01-01

Background High proliferative and differentiation capacity renders embryonic stem cells (ESCs) a promising cell source for tissue engineering and cell-based therapies. Harnessing their potential, however, requires well-designed, efficient and reproducible expansion and differentiation protocols as well as avoiding hazardous by-products, such as teratoma formation. Traditional, standard culture methodologies are fragmented and limited in their fed-batch feeding strategies that afford a sub-optimal environment for cellular metabolism. Herein, we investigate the impact of metabolic stress as a result of inefficient feeding utilizing a novel perfusion bioreactor and a mathematical model to achieve bioprocess improvement. Methodology/Principal Findings To characterize nutritional requirements, the expansion of undifferentiated murine ESCs (mESCs) encapsulated in hydrogels was performed in batch and perfusion cultures using bioreactors. Despite sufficient nutrient and growth factor provision, the accumulation of inhibitory metabolites resulted in the unscheduled differentiation of mESCs and a decline in their cell numbers in the batch cultures. In contrast, perfusion cultures maintained metabolite concentration below toxic levels, resulting in the robust expansion (>16-fold) of high quality ‘naïve’ mESCs within 4 days. A multi-scale mathematical model describing population segregated growth kinetics, metabolism and the expression of selected pluripotency (‘stemness’) genes was implemented to maximize information from available experimental data. A global sensitivity analysis (GSA) was employed that identified significant (6/29) model parameters and enabled model validation. Predicting the preferential propagation of undifferentiated ESCs in perfusion culture conditions demonstrates synchrony between theory and experiment. Conclusions/Significance The limitations of batch culture highlight the importance of cellular metabolism in maintaining pluripotency, which necessitates the design of suitable ESC bioprocesses. We propose a novel investigational framework that integrates a novel perfusion culture platform (controlled metabolic conditions) with mathematical modeling (information maximization) to enhance ESC bioprocess productivity and facilitate bioprocess optimization. PMID:24339957

Development and application of a quantitative PCR assay to study equine herpesvirus 5 invasion and replication in equine tissues in vitro and in vivo.

PubMed

Zarski, Lila M; High, Emily A; Nelli, Rahul K; Bolin, Steven R; Williams, Kurt J; Hussey, Gisela

2017-10-01

Equine herpesvirus 5 (EHV-5) infection is associated with pulmonary fibrosis in horses, but further studies on EHV-5 persistence in equine cells are needed to fully understand viral and host contributions to disease pathogenesis. Our aim was to develop a quantitative PCR (qPCR) assay to measure EHV-5 viral copy number in equine cell cultures, blood lymphocytes, and nasal swabs of horses. Furthermore, we used a recently developed equine primary respiratory cell culture system to study EHV-5 pathogenesis at the respiratory tract. PCR primers and a probe were designed to target gene E11 of the EHV-5 genome. Sensitivity and repeatability were established, and specificity was verified by testing multiple isolates of EHV-5, as well as DNA from other equine herpesviruses. Four-week old fully differentiated (mature), newly seeded (immature) primary equine respiratory epithelial cell (ERECs), and equine dermal cell cultures were inoculated with EHV-5 and the cells and supernatants collected daily for 14days. Blood lymphocytes and nasal swabs were collected from horses experimentally infected with equine herpesvirus 1 (EHV-1). The qPCR assay detected EHV-5 at stable concentrations throughout 14days in inoculated mature EREC and equine dermal cell cultures (peaking at 202 and 5861 viral genomes per 10 6 cellular β actin, respectively). EHV-5 copies detected in the immature EREC cultures increased over 14days and reached levels greater than 10,000 viral genomes per 10 6 cellular β actin. Moreover, EHV-5 was detected in the lymphocytes of 76% of horses and in the nasal swabs of 84% of horses experimentally infected with EHV-1 pre-inoculation with EHV-1. Post-inoculation with EHV-1, EHV-5 was detected in lymphocytes of 52% of horses while EHV-5 levels in nasal swabs were not significantly different from pre-inoculation levels. In conclusion, qPCR was a reliable technique to investigate viral load in in vivo and in vitro samples, and EHV-5 replication in equine epithelial cells may be influenced by cellular stages of differentiation. Copyright © 2017 Elsevier B.V. All rights reserved.

Aspergillus oryzae NRRL 35191 from coffee, a non-toxigenic endophyte with the ability to synthesize kojic acid

USDA-ARS?s Scientific Manuscript database

Aspergillus oryzae was isolated as an endophyte from coffee leaves and found to produce kojic acid in culture. When inoculated in cacao seedlings (Theobroma cacao L.), A. oryzae grew endophytically and synthesize kojic acid in planta. Cacao seedlings inoculated with A. oryzae produced higher levels...

Use of Tetragenococcus halophilus as a starter culture for flavor improvement in fish sauce fermentation.

PubMed

Udomsil, Natteewan; Rodtong, Sureelak; Choi, Yeung Joon; Hua, Yanglin; Yongsawatdigul, Jirawat

2011-08-10

The potential of Tetragenococcus halophilus as a starter culture for flavor improvement in fish sauce fermentation was elucidated. Four strains of T. halophilus isolated from fish sauce mashes were inoculated to anchovy mixed with 25% NaCl with an approximate cell count of 10(6) CFU/mL. The α-amino content of 6-month-old fish sauce samples inoculated with T. halophilus was 780-784 mM. The addition of T. halophilus MRC10-1-3 and T. halophilus MCD10-5-10 resulted in a reduction of histamine (P < 0.05). Fish sauce inoculated with T. halophilus showed high contents of total amino acids with predominantly high glutamic acid. Major volatile compounds in fish sauce were 2-methylpropanal, 2-methylbutanal, 3-methylbutanal, and benzaldehyde. T. halophilus-inoculated fish sauce samples demonstrated the ability to reduce dimethyl disulfide, a compound contributing to a fecal note. The use of T. halophilus for fish sauce fermentation improves amino acid profiles and volatile compounds as well as reduces biogenic amine content of a fish sauce product.

Kinetics of styrene biodegradation by Pseudomonas sp. E-93486.

PubMed

Gąszczak, Agnieszka; Bartelmus, Grażyna; Greń, Izabela

2012-01-01

The research into kinetics of styrene biodegradation by bacterial strain Pseudomonas sp. E-93486 coming from VTT Culture Collection (Finland) was presented in this work. Microbial growth tests in the presence of styrene as the sole carbon and energy source were performed both in batch and continuous cultures. Batch experiments were conducted for initial concentration of styrene in the liquid phase changed in the range of 5-90 g m(-3). The Haldane model was found to be the best to fit the kinetic data, and the estimated constants of the equation were: μ (m) = 0.1188 h(-1), K(S) = 5.984 mg l(-1), and K (i) = 156.6 mg l(-1). The yield coefficient mean value [Formula in text] for the batch culture was 0.72 g(dry cells weight) (g(substrate))(-1). The experiments conducted in a chemostat at various dilution rates (D = 0.035-0.1 h(-1)) made it possible to determine the value of the coefficient for maintenance metabolism m (d) = 0.0165 h(-1) and the maximum yield coefficient value [Formula in text]. Chemostat experiments confirmed the high value of yield coefficient [Formula in text] observed in the batch culture. The conducted experiments showed high activity of the examined strain in the styrene biodegradation process and a relatively low sensitivity to inhibition of its growth at higher concentrations of styrene in the solution. Such exceptional features of Pseudomonas sp. E-93486 make this bacterial strain the perfect candidate for technical applications.

An experimental infection model to induce digital dermatitis infection in cattle.

PubMed

Gomez, A; Cook, N B; Bernardoni, N D; Rieman, J; Dusick, A F; Hartshorn, R; Socha, M T; Read, D H; Döpfer, D

2012-04-01

Bovine digital dermatitis (DD), also known as papillomatous digital dermatitis (foot warts), has been recognized as a major cause of lameness in cattle, with important economic and welfare consequences. The evaluation of therapeutic and preventive interventions aiming to control DD infections in dairy cattle is often challenged by the complex multifactorial etiology of the disease. An experimental infection model to induce acute DD lesions in a controlled environment is proposed. The goal was to provide a standard way of reproducing DD infections independent of external factors that could confound the natural course of the disease, such as management practices or infection pressure, resulting in transmission of DD between animals. A group of 4 yearling Holstein heifers free of any clinical evidence of hoof disease was recruited from a commercial dairy farm and housed in an experimental facility in 1 pen with slatted flooring. The hind feet were wrapped to mimic conditions of prolonged moisture (maceration) and reduced access to air (closure) and inoculated at the heel and dewclaw areas with a homogenate of a naturally occurring DD lesion skin biopsy or a culture broth of Treponema spp. After a period of 12 to 25 d, 4 of 6 and 1 of 4 dewclaw areas inoculated with biopsied DD lesion or a Treponema spp. culture, respectively, had gross lesions compatible with DD. Histopathology confirmed the gross diagnosis in the sites inoculated with tissue homogenate. In the site inoculated with Treponema spp. culture broth, histopathology revealed an incipient DD lesion. Treponema spp. were detected by PCR in both naturally occurring DD homogenate and Treponema spp. culture broth inoculation sites. An experimental infection model to induce acute DD in cattle was developed, which may be used to evaluate interventions to control DD and study the pathogenesis of this infectious hoof disease in a controlled manner. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Valorization of crude glycerol and eggshell biowaste as media components for hydrogen production: A scale-up study using co-culture system.

PubMed

Pachapur, Vinayak Laxman; Das, Ratul Kumar; Brar, Satinder Kaur; Le Bihan, Yann; Buelna, Gerardo

2017-02-01

The properties of eggshells (EGS) as neutralizing and immobilizing agent were investigated for hydrogen (H 2 ) production using crude glycerol (CG) by co-culture system. Eggshells of different sizes and concentrations were used during batch and repeated-batch fermentation. For batch and repeated-batch fermentation, the maximum H 2 production (36.53±0.53 and 41.16±0.95mmol/L, respectively) was obtained with the EGS size of 33μm

Metabolic switches and adaptations deduced from the proteomes of Streptomyces coelicolor wild type and phoP mutant grown in batch culture.

PubMed

Thomas, Louise; Hodgson, David A; Wentzel, Alexander; Nieselt, Kay; Ellingsen, Trond E; Moore, Jonathan; Morrissey, Edward R; Legaie, Roxane; Wohlleben, Wolfgang; Rodríguez-García, Antonio; Martín, Juan F; Burroughs, Nigel J; Wellington, Elizabeth M H; Smith, Margaret C M

2012-02-01

Bacteria in the genus Streptomyces are soil-dwelling oligotrophs and important producers of secondary metabolites. Previously, we showed that global messenger RNA expression was subject to a series of metabolic and regulatory switches during the lifetime of a fermentor batch culture of Streptomyces coelicolor M145. Here we analyze the proteome from eight time points from the same fermentor culture and, because phosphate availability is an important regulator of secondary metabolite production, compare this to the proteome of a similar time course from an S. coelicolor mutant, INB201 (ΔphoP), defective in the control of phosphate utilization. The proteomes provide a detailed view of enzymes involved in central carbon and nitrogen metabolism. Trends in protein expression over the time courses were deduced from a protein abundance index, which also revealed the importance of stress pathway proteins in both cultures. As expected, the ΔphoP mutant was deficient in expression of PhoP-dependent genes, and several putatively compensatory metabolic and regulatory pathways for phosphate scavenging were detected. Notably there is a succession of switches that coordinately induce the production of enzymes for five different secondary metabolite biosynthesis pathways over the course of the batch cultures.

Effect of Selenate on Viability and Selenomethionine Accumulation of Chlorella sorokiniana Grown in Batch Culture

PubMed Central

Vílchez, Carlos; Torronteras, Rafael; Vigara, Javier; Gómez-Jacinto, Veronica; Janzer, Nora; Gómez-Ariza, José-Luis; Márová, Ivana

2014-01-01

The aim of this work was to study the effect of Se(+VI) on viability, cell morphology, and selenomethionine accumulation of the green alga Chlorella sorokiniana grown in batch cultures. Culture exposed to sublethal Se concentrations of 40 mg·L−1 (212 μM) decreased growth rates for about 25% compared to control. A selenate EC50 value of 45 mg·L−1 (238.2 μM) was determined. Results showed that chlorophyll and carotenoids contents were not affected by Se exposure, while oxygen evolution decreased by half. Ultrastructural studies revealed granular stroma, fingerprint-like appearance of thylakoids which did not compromise cell activity. Unlike control cultures, SDS PAGE electrophoresis of crude extracts from selenate-exposed cell cultures revealed appearance of a protein band identified as 53 kDa Rubisco large subunit of Chlorella sorokiniana, suggesting that selenate affects expression of the corresponding chloroplast gene as this subunit is encoded in the chloroplast DNA. Results revealed that the microalga was able to accumulate up to 140 mg·kg−1 of SeMet in 120 h of cultivation. This paper shows that Chlorella sorokiniana biomass can be enriched in the high value aminoacid SeMet in batch cultures, while keeping photochemical viability and carbon dioxide fixation activity intact, if exposed to suitable sublethal concentrations of Se. PMID:24688385

High cell density fed-batch fermentations for lipase production: feeding strategies and oxygen transfer.

PubMed

Salehmin, M N I; Annuar, M S M; Chisti, Y

2013-11-01

This review is focused on the production of microbial lipases by high cell density fermentation. Lipases are among the most widely used of the enzyme catalysts. Although lipases are produced by animals and plants, industrial lipases are sourced almost exclusively from microorganisms. Many of the commercial lipases are produced using recombinant species. Microbial lipases are mostly produced by batch and fed-batch fermentation. Lipases are generally secreted by the cell into the extracellular environment. Thus, a crude preparation of lipases can be obtained by removing the microbial cells from the fermentation broth. This crude cell-free broth may be further concentrated and used as is, or lipases may be purified from it to various levels. For many large volume applications, lipases must be produced at extremely low cost. High cell density fermentation is a promising method for low-cost production: it allows a high concentration of the biomass and the enzyme to be attained rapidly and this eases the downstream recovery of the enzyme. High density fermentation enhances enzyme productivity compared with the traditional submerged culture batch fermentation. In production of enzymes, a high cell density is generally achieved through fed-batch operation, not through perfusion culture which is cumbersome. The feeding strategies used in fed-batch fermentations for producing lipases and the implications of these strategies are discussed. Most lipase-producing microbial fermentations require oxygen. Oxygen transfer in such fermentations is discussed.

Comparison of a modified shell vial culture procedure with conventional mouse inoculation for rabies virus isolation.

PubMed

Ribas Antúnez, María de los Angeles; Girón, Blanca; Monsalvez, Iraima; Morier, Luis; Acosta, Gretel; Tejero, Yahisel; Cordero, Yanislet; Piedra, Dainelyd

2013-04-01

Rabies is a neurotropic disease that is often lethal. The early diagnosis of rabies infection is important and requires methods that allow for the isolation of the virus from animals and humans. The present study compared a modified shell vial (MSV) procedure using 24-well tissue culture plates with the mouse inoculation test (MIT), which is considered the gold standard for rabies virus isolation. Thirty brain samples (25 positive and 5 negative by the fluorescent antibody test) obtained from different animal species at the National Institute of Hygiene Rafael Rangel in Caracas, Venezuela, were studied by the MIT and MSV assays. Nine samples (36%) were positive at 24 h, 10 (40%) were positive at 48 h and six (24%) were positive at 72 h by the MSV assay. With the MIT assay, 76% were positive at six days post inoculation and 12% were positive at 12 and 18 days post inoculation. One sample that was negative according to the MSV assay was positive with MIT on the 12th day. The MSV procedure exhibited a sensitivity of 96.2%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value 80%. This procedure allowed for rapid rabies virus detection. MIT can be employed as an alternative method in laboratories without tissue culture facilities.

Induction of tumor necrosis factor by Legionella pneumophila.

PubMed Central

Blanchard, D K; Djeu, J Y; Klein, T W; Friedman, H; Stewart, W E

1987-01-01

Mice were inoculated with Legionella pneumophila via an intratracheal route to establish an experimental model of infection. Lung lavage fluid obtained from infected mice contained a cytolytic factor identified as tumor necrosis factor (TNF). Peak levels of TNF were produced at about 24 h postinfection and rapidly declined thereafter. Treatment of the mice with dextran sulfate before inoculation with the bacteria resulted in lowered amounts of TNF in the lung lavage fluid, suggesting that macrophages were responsible for production of the cytokine. Furthermore, cultures of adherent lung leukocytes and a macrophage cell line, PU 5-1.8, were stimulated to produce TNF by exposure to Legionella antigens. In addition, adherent lung leukocytes from Legionella-infected mice spontaneously released TNF into the culture supernatant. Inoculation of mice with saline or latex particles failed to induce TNF in vivo, indicating that bacterial antigens or products were the stimulating signals. Since there was no detectable TNF activity in sera at any time after intratracheal inoculation, TNF production appeared to be confined to the site of infection. Pretreatment of PU 5-1.8 cultures with gamma interferon, which was detected in the lung lavage fluid before TNF, resulted in augmented TNF production, suggesting cooperativity may exist between the two cytokines, either in the pathogenicity of the bacterium or in a possible immunomodulatory function of TNF and interferon during infection. PMID:2433220

Methylobacterium sp. resides in unculturable state in potato tissues in vitro and becomes culturable after induction by Pseudomonas fluorescens IMGB163.

PubMed

Podolich, O; Laschevskyy, V; Ovcharenko, L; Kozyrovska, N; Pirttilä, A M

2009-03-01

To induce growth of endophytic bacteria residing in an unculturable state in tissues of in vitro-grown potato plantlets. To isolate and identify the induced bacteria and to localize the strains in tissues of in vitro-grown potato plantlets. The inoculation of in vitro-grown potato plants with Pseudomonas fluorescens IMBG163 led to induction of another bacterium, a pink-pigmented facultative methylotroph that was identified as Methylobacterium sp. using phylogenetic 16S rDNA approach. Two molecular methods were used for localizing methylobacteria in potato plantlets: PCR and in situ hybridization (ISH/FISH). A PCR product specific for the Methylobacterium genus was found in DNA isolated from the surface-sterilized plantlet leaves. Presence of Methylobacterium rRNA was detected by ISH/FISH in leaves and stems of inoculated as well as axenic potato plantlets although the bacterium cannot be isolated from the axenic plants. Methylobacterium sp. resides in unculturable state within tissues of in vitro-grown potato plants and becomes culturable after inoculation with P. fluorescens IMBG163. In order to develop endophytic biofertilizers and biocontrol agents, a detailed knowledge of the life-style of endophytes is essential. To our knowledge, this is the first report on increase of the culturability of endophytes in response to inoculation by nonpathogenic bacteria.

Exploitation of Endophytic Bacteria to Enhance the Phytoremediation Potential of the Wetland Helophyte Juncus acutus

PubMed Central

Syranidou, Evdokia; Christofilopoulos, Stavros; Gkavrou, Georgia; Thijs, Sofie; Weyens, Nele; Vangronsveld, Jaco; Kalogerakis, Nicolas

2016-01-01

This study investigated the potential of indigenous endophytic bacteria to improve the efficiency of the wetland helophyte Juncus acutus to deal with a mixed pollution consisting of emerging organic contaminants (EOCs) and metals. The beneficial effect of bioaugmentation with selected endophytic bacteria was more prominent in case of high contamination: most of the inoculated plants (especially those inoculated with the mixed culture) removed higher percentages of organics and metals from the liquid phase in shorter times compared to the non-inoculated plants without exhibiting significant oxidative stress. When exposed to the lower concentrations, the tailored mixed culture enhanced the performance of the plants to decrease the organics and metals from the water. The composition of the root endophytic community changed in response to increased levels of contaminants while the inoculated bacteria did not modify the community structure. Our results indicate that the synergistic relationships between endophytes and the macrophyte enhance plants’ performance and may be exploited in constructed wetlands treating water with mixed contaminations. Taking into account that the concentrations of EOCs used in this study are much higher than the average contents of typical wastewaters, we can conclude that the macrophyte J. acutus with the aid of a mixed culture of tailored endophytic bacteria represents a suitable environmentally friendly alternative for treating pharmaceuticals and metals. PMID:27458433

Counting Yeast.

ERIC Educational Resources Information Center

Bealer, Jonathan; Welton, Briana

1998-01-01

Describes changes to a traditional study of population in yeast colonies. Changes to the procedures include: (1) only one culture per student team; (2) cultures are inoculated only once; and (3) the same tube is sampled daily. (DDR)

Identification and susceptibility testing of microorganism by direct inoculation from positive blood culture bottles by combining MALDI-TOF and Vitek-2 Compact is rapid and effective.

PubMed

Romero-Gómez, María-Pilar; Gómez-Gil, Rosa; Paño-Pardo, Jose Ramón; Mingorance, Jesús

2012-12-01

The objective of this study was to evaluate the reliability and accuracy of the combined use of MALDI-TOF MS bacterial identification and the Vitek-2 Compact antimicrobial susceptibility testing (AST) directly from positive blood cultures. Direct identification by MALDI-TOF MS and AST were performed in parallel to the standard methods in all positively flagged blood cultures bottles during the study period. Three hundred and twenty four monomicrobial positive blood cultures were included in the present study, with 257 Gram-negative and 67 Gram-positive isolates. MALDI-TOF MS identification directly from blood bottles reported the correct identification for Enterobacteriaceae in 97.7%, non-fermentative Gram-negative bacilli 75.0%, Staphylococcus aureus 75.8%, coagulase negative staphylococci 63.3% and enterococci 63.3%. A total 6156 isolate/antimicrobial agent combinations were tested. Enterobacteriaceae group and non-fermentative Gram-negative Bacilli showed an agreement of 96.67% and 92.30%, respectively, for the Gram-positive cocci the overall agreement found was 97.84%. We conclude that direct identification by MALDI-TOF and inoculation of Vitek-2 Compact AST with positive blood culture bottles yielded very good results and decreased time between initial inoculation of blood culture media and determination of the antibiotic susceptibility for Gram-negative rods and Gram-positive cocci causing bacteremia. Copyright © 2012 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Influence of oxygen availability on the metabolism and morphology of Yarrowia lipolytica: insights into the impact of glucose levels on dimorphism.

PubMed

Timoumi, Asma; Bideaux, Carine; Guillouet, Stéphane E; Allouche, Yohan; Molina-Jouve, Carole; Fillaudeau, Luc; Gorret, Nathalie

2017-10-01

Dynamic behavior of Yarrowia lipolytica W29 strain under conditions of fluctuating, low, and limited oxygen supply was characterized in batch and glucose-limited chemostat cultures. In batch cultures, transient oscillations between oxygen-rich and -deprived environments induced a slight citric acid accumulation (lower than 29 mg L -1 ). By contrast, no citric acid was detected in continuous fermentations for all stress conditions: full anoxia (zero pO 2 value, 100% N 2 ), limited (zero pO 2 value, 75% of cell needs), and low (pO 2 close to 2%) dissolved oxygen (DO) levels. The macroscopic behavior (kinetic parameters, yields, viability) of Y. lipolytica was not significantly affected by the exposure to DO fluctuations under both modes of culture. Nevertheless, conditions of oxygen limitation resulted in the destabilization of the glucose-limited growth during the continuous cultivations. Morphological responses of Y. lipolytica to DO oscillations were different between batch and chemostat runs. Indeed, a yeast-to-mycelium transition was induced and progressively intensified during the batch fermentations (filamentous subpopulation reaching 74% (v/v)). While, in chemostat bioreactors, the culture consisted mainly of yeast-like cells (mean diameter not exceeding 5.7 μm) with a normal size distribution. During the continuous cultures, growth at low DO concentration did not induce any changes in Y. lipolytica morphology. Dimorphism (up to 80.5% (v/v) of filaments) was only detected under conditions of oxygen limitation in the presence of a residual glucose excess (more than 0.75 g L -1 ). These data suggest an impact of glucose levels on the signaling pathways regulating dimorphic responses in Y. lipolytica.

Effect of bioaugmentation by cellulolytic bacteria enriched from sheep rumen on methane production from wheat straw.

PubMed

Ozbayram, E Gozde; Kleinsteuber, Sabine; Nikolausz, Marcell; Ince, Bahar; Ince, Orhan

2017-08-01

The aim of this study was to determine the potential of bioaugmentation with cellulolytic rumen microbiota to enhance the anaerobic digestion of lignocellulosic feedstock. An anaerobic cellulolytic culture was enriched from sheep rumen fluid using wheat straw as substrate under mesophilic conditions. To investigate the effects of bioaugmentation on methane production from straw, the enrichment culture was added to batch reactors in proportions of 2% (Set-1) and 4% (Set-2) of the microbial cell number of the standard inoculum slurry. The methane production in the bioaugmented reactors was higher than in the control reactors. After 30 days of batch incubation, the average methane yield was 154 mL N CH 4 g VS -1 in the control reactors. Addition of 2% enrichment culture did not enhance methane production, whereas in Set-2 the methane yield was increased by 27%. The bacterial communities were examined by 454 amplicon sequencing of 16S rRNA genes, while terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of mcrA genes was applied to analyze the methanogenic communities. The results highlighted that relative abundances of Ruminococcaceae and Lachnospiraceae increased during the enrichment. However, Cloacamonaceae, which were abundant in the standard inoculum, dominated the bacterial communities of all batch reactors. T-RFLP profiles revealed that Methanobacteriales were predominant in the rumen fluid, whereas the enrichment culture was dominated by Methanosarcinales. In the batch rectors, the most abundant methanogens were affiliated to Methanobacteriales and Methanomicrobiales. Our results suggest that bioaugmentation with sheep rumen enrichment cultures can enhance the performance of digesters treating lignocellulosic feedstock. Copyright © 2017 Elsevier Ltd. All rights reserved.

Fermentative and growth performances of Dekkera bruxellensis in different batch systems and the effect of initial low cell counts in co-cultures with Saccharomyces cerevisiae.

PubMed

Meneghin, Maria Cristina; Bassi, Ana Paula Guarnieri; Codato, Carolina Brito; Reis, Vanda Renata; Ceccato-Antonini, Sandra Regina

2013-08-01

Dekkera bruxellensis is a multifaceted yeast present in the fermentative processes used for alcoholic beverage and fuel alcohol production - in the latter, normally regarded as a contaminant. We evaluated the fermentation and growth performance of a strain isolated from water in an alcohol-producing unit, in batch systems with/without cell recycling in pure and co-cultures with Saccharomyces cerevisiae. The ethanol resistance and aeration dependence for ethanol/acid production were verified. Ethanol had an effect on the growth of D. bruxellensis in that it lowered or inhibited growth depending on the concentration. Acid production was verified in agitated cultures either with glucose or sucrose, but more ethanol was produced with glucose in agitated cultures. Regardless of the batch system, low sugar consumption and alcohol production and expressive growth were found with D. bruxellensis. Despite a similar ethanol yield compared to S. cerevisiae in the batch system without cell recycling, ethanol productivity was approximately four times lower. However, with cell recycling, ethanol yield was almost half that of S. cerevisiae. At initial low cell counts of D. bruxellensis (10 and 1000 cells/ml) in co-cultures with S. cerevisiae, a decrease in fermentative efficiency and a substantial growth throughout the fermentative cycles were displayed by D. bruxellensis. Due to the peculiarity of cell repitching in Brazilian fermentation processes, D. bruxellensis is able to establish itself in the process, even when present in low numbers initially, substantially impairing bioethanol production due to the low ethanol productivity, in spite of comparable ethanol yields. Copyright © 2013 John Wiley & Sons, Ltd.

On the way to commercializing plant cell culture platform for biopharmaceuticals: present status and prospect.

PubMed

Xu, Jianfeng; Zhang, Ningning

2014-12-01

Plant cell culture is emerging as an alternative bioproduction system for recombinant pharmaceuticals. Growing plant cells in vitro under controlled environmental conditions allows for precise control over cell growth and protein production, batch-to-batch product consistency and a production process aligned with current good manufacturing practices. With the recent US FDA approval and commercialization of the world's first plant cell-based recombinant pharmaceutical for human use, β-glucocerebrosidase for treatment of Gaucher's disease, a new era has come in which plant cell culture shows high potential to displace some established platform technologies in niche markets. This review updates the progress in plant cell culture processing technology, highlights recent commercial successes and discusses the challenges that must be overcome to make this platform commercially viable.

Detection of bacteriuria and bacteremia in newborn calves by a catalase-based urine test.

PubMed

Raboisson, D; Clément, J; Queney, N; Lebreton, P; Schelcher, F

2010-01-01

Bacteremia occurs frequently in newborn calves. The predictive value of clinical signs is low, suggesting the use of calf-side diagnostic tests. To investigate testing of urine catalase activity (Uriscreen test) for bacteriuria and bacteremia detection. Five colostrum-free calves and 3 colostrum-fed control calves. Controlled experimental trial. Colostrum-free calves were inoculated PO with Escherichia coli O78+. A clinical score was established to define the onset of the illness. Blood and urine (cystocentesis) samplings and cultures, and Uriscreen tests, were performed 4-6 times from inoculation to death. Three control calves received the same management as 3 inoculated calves, but with colostrum and without inoculation. Bacteremia was demonstrated in all of the inoculated colostrum-free calves and in none of the control calves. The E. coli O78+ strain, E. coli, and Klebsiella spp. were recovered from 4/5, 5/5, and 2/5 inoculated colostrum-free calves, respectively. Urine cultures were negative for the 2 groups at the start of the experiment; 5/5 colostrum-deprived inoculated calves were positive for E. coli thereafter whereas 3/3 controls remained negative. Concordance of Uriscreen tests with bacteremia and bacteriuria was 0.86 and 0.88, respectively. Kappa value of agreement between Uriscreen and bacteremia and bacteriuria was 0.73 and 0.76, respectively. Sensitivity of Uriscreen for bacteremia and bacteriuria was 80.0 and 86.6%, respectively, and specificity was 92.8 and 88.8%, respectively. The results suggest that Uriscreen can be used for detection of bacteremia in neonatal calves in connection with a constant bacteriuria. Copyright © 2010 by the American College of Veterinary Internal Medicine.

Comparison of nitrogen removal rates and nitrous oxide production from enriched anaerobic ammonium oxidizing bacteria in suspended and attached growth reactors.

PubMed

Panwivia, Supaporn; Sirvithayapakorn, Sanya; Wantawin, Chalermraj; Noophan, Pongsak Lek; Munakata-Marr, Junko

2014-01-01

Attached growth-systems for the anaerobic ammonium oxidation (anammox) process have been postulated for implementation in the field. However, information about the anammox process in attached growth-systems is limited. This study compared nitrogen removal rates and nitrous oxide (N2O) production of enriched anammox cultures in both suspended and attached growth sequencing batch reactors (SBRs). Suspended growth reactors (SBR-S) and attached growth reactors using polystyrene sponge as a medium (SBR-A) were used in these experiments. After inoculation with an enriched anammox culture, significant nitrogen removals of ammonium (NH4 (+)) and nitrite (NO2 (-)) were observed under NH4 (+):NO2 (-) ratios ranging from 1:1 to 1:2 in both types of SBRs. The specific rates of total nitrogen removal in SBR-S and SBR-A were 0.52 mg N/mg VSS-d and 0.44 mg N/mg VSS-d, respectively, at an NH4 (+):NO2 (-) ratio of 1:2. N2O production by the enriched anammox culture in both SBR-S and SBR-A was significantly higher at NH4 (+):NO2 (-) ratio of 1:2 than at NH4 (+):NO2 (-) ratios of 1:1 and 1:1.32. In addition, N2O production was higher at a pH of 6.8 than at pH 7.3, 7.8, and 8.3 in both SBR-S and SBR-A. The results of this investigation demonstrate that the anammox process may avoid N2O emission by maintaining an NH4 (+):NO2 (-) ratio of less than 1:2 and pH higher than 6.8.

Patulin Production by Penicillium urticae Bainier in Batch Culture1

PubMed Central

Norstadt, Fred A.; McCalla, T. M.

1969-01-01

A still, batch-culture method, with potato dextrose medium and Penicillium urticae Bainier, produced patulin yields of 1.2 to 1.7 g/liter of medium. Incubation was at 25 C for 14 days. Ethyl acetate extraction of condensed culture filtrate and drying with anhydrous MgSO4, followed by solvent change to dry ethyl ether and purification on alumina (pH 4.5), produced pure crystalline patulin. The use of 2-liter, round-bottom flasks and a rotating vacuum evaporator provided versatile equipment and easy manipulation in the operations. Soil from wheat fields provided a convenient natural P. urticae source. Potato dextrose medium was superior to potato sucrose or Raulin-Thom media. Images PMID:5775903

Assessing the effect of sodium dichloroisocyanurate concentration on transfer of Salmonella enterica serotype Typhimurium in wash water for production of minimally processed iceberg lettuce (Lactuca sativa L.).

PubMed

Maffei, D F; Sant'Ana, A S; Monteiro, G; Schaffner, D W; Franco, B D G M

2016-06-01

This study evaluated the impact of sodium dichloroisocyanurate (5, 10, 20, 30, 40, 50 and 250 mg l(-1) ) in wash water on transfer of Salmonella Typhimurium from contaminated lettuce to wash water and then to other noncontaminated lettuces washed sequentially in the same water. Experiments were designed mimicking the conditions commonly seen in minimally processed vegetable (MPV) processing plants in Brazil. The scenarios were as follows: (1) Washing one inoculated lettuce portion in nonchlorinated water, followed by washing 10 noninoculated portions sequentially. (2) Washing one inoculated lettuce portion in chlorinated water followed by washing five noninoculated portions sequentially. (3) Washing five inoculated lettuce portions in chlorinated water sequentially, followed by washing five noninoculated portions sequentially. (4) Washing five noninoculated lettuce portions in chlorinated water sequentially, followed by washing five inoculated portions sequentially and then by washing five noninoculated portions sequentially in the same water. Salm. Typhimurium transfer from inoculated lettuce to wash water and further dissemination to noninoculated lettuces occurred when nonchlorinated water was used (scenario 1). When chlorinated water was used (scenarios 2, 3 and 4), no measurable Salm. Typhimurium transfer occurred if the sanitizer was ≥10 mg l(-1) . Use of sanitizers in correct concentrations is important to minimize the risk of microbial transfer during MPV washing. In this study, the impact of sodium dichloroisocyanurate in the wash water on transfer of Salmonella Typhimurium from inoculated lettuce to wash water and then to other noninoculated lettuces washed sequentially in the same water was evaluated. The use of chlorinated water, at concentration above 10 mg l(-1) , effectively prevented Salm. Typhimurium transfer under several different washing scenarios. Conversely, when nonchlorinated water was used, Salm. Typhimurium transfer occurred in up to at least 10 noninoculated batches of lettuce washed sequentially in the same water. © 2016 The Society for Applied Microbiology.

Vaccination for the prevention of maternal and fetal infection with guinea pig cytomegalovirus.

PubMed

Bia, F J; Griffith, B P; Tarsio, M; Hsiung, G D

1980-11-01

Live guinea pig cytomegalovirus (CMV) vaccine was prepared after 11 serial passages in tissue culture; noninfectious envelope antigen vaccine was prepared by n-octyl glucoside treatment of CMV-derived dense bodies and virions. Hartley strain guinea pigs immunized with either vaccine were compared with guinea pigs inoculated with virulent, salivary gland-passaged CMV (approximating natural infection), with passively immunized animals, and with nonimmune controls. All vaccinated animals had neutralizing antibodies to CMV. After challenge with virulent CMV, animals previously inoculated with either tissue culture-passaged or virulent CMV were protected against acute viremia and death; pregnant animals previously inoculated with live CMV vaccine had lower incidences of viremia and generalized maternal and fetal infection. Envelope antigen-vaccinated and passively immunized pregnant animals showed acute viremia after similar challenge with virulent virus; however, infection was less generalized than that in control animals, and CMV was not isolated from the fetuses of these vaccinated mothers.

Attempts to Produce Experimental Edema Disease in Swine by Parenterally Injecting Escherichia Coli Serotype 0139:K82:H1*

PubMed Central

Pickrell, J. A.; Link, R. P.; Simon, J.; Rhoades, H. E.; Gossling, J.

1969-01-01

Twenty-two pigs were inoculated parenterally with various E. coli 0139:K82:H1 preparations. Clinical signs of disease in pigs injected with freeze-thaw extract consisted of early listlessness, diarrhea and, later, hyperirritability of varying intensity in some animals. Hemorrhagic gastroenteritis involving the duodenum, spiral colon and the fundic portion of the stomach, and ulceration of the fundic stomach were observed at post-mortem examination of pigs inoculated parenterallly with living culture or freeze-thaw extract. No significant lesions were observed in pigs inoculated with ultrasonic or hypotonic acid-saline extract. In pigs injected with living culture or freeze-thaw extract, the histological alterations consisted of moderate perivascular edema of the brain, marked hepatic parenchymal cell degeneration, hepatic subserosal edema and “toxic” lymph nodes, when compared to the control group. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 4. PMID:4237302

EXPERIMENTAL STUDIES ON YELLOW FEVER IN NORTHERN PERU.

PubMed

Noguchi, H; Kligler, I J

1921-01-31

Fourteen typical cases of yellow fever were studied in northern Peru during an epidemic occurring in 1920, nine in Payta in March and April, and five in Morropon and Piura in April and May. The method of investigation was similar to that previously employed, but as the laboratory facilities were very meager certain changes were required. Although in Payta the work was handicapped by the lack of electric light, the scarcity of water and animal food, the unsuitability of the guinea pigs for inoculation, and the changes in culture media due to age, the results obtained under these adverse conditions were by no means negative. While in no instance was there a typical infection produced in animals, either by direct inoculation of blood or with culture materials, yet certain guinea pigs in each series showed temporary febrile reactions or definite hemorrhagic lesions of the lungs indicative of a mild leptospira infection. Direct search for Leptospira icteroides in the blood of patients or in culture materials was not made because the dark-field microscope could not be used. Subsequently, at Piura, the laboratory facilities were vastly, improved, the use of the dark-field microscope was made possible by means of a storage battery, and a fresh stock of young healthy guinea pigs was received from New York, and fresh rabbit serum obtained in Piura. In the study of the materials obtained from five cases of yellow fever in Morropon all these added facilities were taken advantage of, with the result that the outcome was positive and convincing. Cultures from the five cases were examined after 11, 12, and 13 days, and in those from three cases living leptospiras were found. By inoculation into suitable guinea pigs of culture material from these five cases, irrespective of whether or not leptospiras were detected under the dark-field microscope, a typical Leptospira icteroides infection was produced from four of the five cases. In one of these no leptospira had been detected in the culture tubes. Thus one case only yielded negative results, in that no leptospiras were found under the dark-field microscope and the animal inoculation was negative. The leptospira was demonstrated in the blood or organ emulsions of the infected guinea pigs, and further transmission of each strain to other guinea pigs was obtained and pure cultures were secured. A few points of practical significance appeared in the course of the present investigation. One is the importance of using fresh rabbit serum for culture media. Old rabbit serum, whether in pure form or incorporated with agar, etc., which had been kept for several months in a tropical climate, proved to be unsatisfactory for obtaining a growth of Leptospira icteroides. A second point of interest is the variation in susceptibility of guinea pigs to infection with Leptospira icteroides. In two of four series of positive animal inoculations with the Morropon culture materials only one-half of the guinea pigs inoculated with given materials developed typical symptoms. The other half either suffered from a transient mild infection, as evidenced by a few hemorrhagic foci in the lungs, or escaped infection altogether. From these facts it is highly probable that the lung lesions and febrile reactions observed in certain guinea pigs inoculated with the Payta materials were due to a mild leptospira infection. In a comparative experiment the native guinea pigs procured in Payta were found to be more resistant to the leptospira infection than those recently brought from New York. In fact, only a small portion of the former succumbed to typical infection even when inoculated with a virulent strain of Leptospira icteroides obtained from the Morropon epidemic. In conclusion it may be stated that of fourteen cases of yellow fever studied in Peru, a typical leptospira infection, together with the demonstration of the organism in experimentally infected guinea pigs, was obtained in four, while in the majority of instances indications of a mild, non-fatal leptospira infection were observed. In a few cases only were the results entirely negative. The leptospira isolated from Morropon cases of yellow fever, which is morphologically and culturally identical with the Guayaquil and Merida strains of Leptospira icteroides, was also shown by immunity test to be indistinguishable from the Guayaquil organism.

Growth in liquid media.

PubMed

Elbing, Karen; Brent, Roger

2002-08-01

The procedure for inoculating overnight (starter) cultures of E. coli from a single colony is described along with considerations for growing larger cultures. Also included are two methods for monitoring cell growth using a spectrophotometer or a hemacytometer.

Effect of growth conditions on production of rhamnose-containing cell wall and capsular polysaccharides by strains of Lactobacillus casei subsp. rhamnosus.

PubMed

Wicken, A J; Ayres, A; Campbell, L K; Knox, K W

1983-01-01

Strains of Lactobacillus casei subsp. rhamnosus possessing two cell wall polysaccharides, a hexosamine-containing H-polysaccharide and a rhamnose-containing R-polysaccharide, were examined for the effect of growth conditions on the production of these two components. In strain NCTC 6375, R- and H-polysaccharides accounted for an estimated 44 and 20%, respectively, of the cell wall for organisms grown in batch culture with glucose as the carbohydrate source. Growth on fructose-containing media reduced the amount of R-polysaccharide by approximately 50% without affecting the amount of H-polysaccharide. Subculture of fructose-grown organisms in glucose restored the original proportions of the two polysaccharides. Galactose- and sucrose-grown cells behaved similarly to glucose-grown cells with respect to polysaccharide production, whereas growth in rhamnose or ribose showed values close to those for fructose-grown cells. Continuous culture of strain NCTC 6375 for more than 100 generations showed a gradual and irreversible reduction of the R-polysaccharide to less than 5% of the cell wall and an increase of the H-polysaccharide to 40% of the cell wall. Other type culture strains of L. casei subsp. rhamnosus, NCIB 7473 and ATCC 7469, behaved similarly in batch and continuous culture. In contrast, strains of L. casei subsp. rhamnosus isolated at the Institute of Dental Research showed phenotypic stability with respect to the relative proportions of R- and H-polysaccharides in both batch and continuous culture. Changes in polysaccharide composition of type culture strains were also mirrored in changes in the immunogenicity of the two components and resistance to the rate of enzymic lysis of whole organisms. For L. casei subsp. rhamnosus strain NCTC 10302 the R-polysaccharide is present entirely as capsular material. The amount of R-polysaccharide produced was also markedly dependent on the carbohydrate component of the medium in batch culture and both dilution rate and nature of the limiting carbohydrate in continuous culture, varying over a 10-fold range, whereas the cell wall H-polysaccharide remained constant.

Effect of growth conditions on production of rhamnose-containing cell wall and capsular polysaccharides by strains of Lactobacillus casei subsp. rhamnosus.

PubMed Central

Wicken, A J; Ayres, A; Campbell, L K; Knox, K W

1983-01-01

Strains of Lactobacillus casei subsp. rhamnosus possessing two cell wall polysaccharides, a hexosamine-containing H-polysaccharide and a rhamnose-containing R-polysaccharide, were examined for the effect of growth conditions on the production of these two components. In strain NCTC 6375, R- and H-polysaccharides accounted for an estimated 44 and 20%, respectively, of the cell wall for organisms grown in batch culture with glucose as the carbohydrate source. Growth on fructose-containing media reduced the amount of R-polysaccharide by approximately 50% without affecting the amount of H-polysaccharide. Subculture of fructose-grown organisms in glucose restored the original proportions of the two polysaccharides. Galactose- and sucrose-grown cells behaved similarly to glucose-grown cells with respect to polysaccharide production, whereas growth in rhamnose or ribose showed values close to those for fructose-grown cells. Continuous culture of strain NCTC 6375 for more than 100 generations showed a gradual and irreversible reduction of the R-polysaccharide to less than 5% of the cell wall and an increase of the H-polysaccharide to 40% of the cell wall. Other type culture strains of L. casei subsp. rhamnosus, NCIB 7473 and ATCC 7469, behaved similarly in batch and continuous culture. In contrast, strains of L. casei subsp. rhamnosus isolated at the Institute of Dental Research showed phenotypic stability with respect to the relative proportions of R- and H-polysaccharides in both batch and continuous culture. Changes in polysaccharide composition of type culture strains were also mirrored in changes in the immunogenicity of the two components and resistance to the rate of enzymic lysis of whole organisms. For L. casei subsp. rhamnosus strain NCTC 10302 the R-polysaccharide is present entirely as capsular material. The amount of R-polysaccharide produced was also markedly dependent on the carbohydrate component of the medium in batch culture and both dilution rate and nature of the limiting carbohydrate in continuous culture, varying over a 10-fold range, whereas the cell wall H-polysaccharide remained constant. PMID:6401290

Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: A review

PubMed Central

Cos, Oriol; Ramón, Ramón; Montesinos, José Luis; Valero, Francisco

2006-01-01

The methylotrophic yeast Pichia pastoris has been widely reported as a suitable expression system for heterologous protein production. The use of different phenotypes under PAOX promoter, other alternative promoters, culture medium, and operational strategies with the objective to maximize either yield or productivity of the heterologous protein, but also to obtain a repetitive product batch to batch to get a robust process for the final industrial application have been reported. Medium composition, kinetics growth, fermentation operational strategies from fed-batch to continuous cultures using different phenotypes with the most common PAOX promoter and other novel promoters (GAP, FLD, ICL), the use of mixed substrates, on-line monitoring of the key fermentation parameters (methanol) and control algorithms applied to the bioprocess are reviewed and discussed in detail. PMID:16600031

Bio-protective potential of lactic acid bacteria: Effect of Lactobacillus sakei and Lactobacillus curvatus on changes of the microbial community in vacuum-packaged chilled beef

PubMed Central

Zhang, Yimin; Zhu, Lixian; Dong, Pengcheng; Liang, Rongrong; Mao, Yanwei; Qiu, Shubing

2018-01-01

Objective This study was to determine the bacterial diversity and monitor the community dynamic changes during storage of vacuum-packaged sliced raw beef as affected by Lactobacillus sakei and Lactobacillus curvatus. Methods L. sakei and L. curvatus were separately incubated in vacuumed-packaged raw beef as bio-protective cultures to inhibit the naturally contaminating microbial load. Dynamic changes of the microbial diversity of inoculated or non-inoculated (control) samples were monitored at 4°C for 0 to 38 days, using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Results The DGGE profiles of DNA directly extracted from non-inoculated control samples highlighted the order of appearance of spoilage bacteria during storage, showing that Enterbacteriaceae and Pseudomonas fragi emerged early, then Brochothrix thermosphacta shared the dominant position, and finally, Pseudomonas putida showed up became predominant. Compared with control, the inoculation of either L. sakei or L. curvatus significantly lowered the complexity of microbial diversity and inhibited the growth of spoilage bacteria (p<0.05). Interestingly, we also found that the dominant position of L. curvatus was replaced by indigenous L. sakei after 13 d for L. curvatus-inoculated samples. Plate counts on selective agars further showed that inoculation with L. sakei or L. curvatus obviously reduced the viable counts of Enterbacteraceae, Pseudomonas spp. and B. thermosphacta during later storage (p< 0.05), with L. sakei exerting greater inhibitory effect. Inoculation with both bio-protective cultures also significantly decreased the total volatile basic nitrogen values of stored samples (p<0.05). Conclusion Taken together, the results proved the benefits of inoculation with lactic acid bacteria especially L. sakei as a potential way to inhibit growth of spoilage-related bacteria and improve the shelf life of vacuum-packaged raw beef. PMID:29059725

Benchmarking of commercially available CHO cell culture media for antibody production.

PubMed

Reinhart, David; Damjanovic, Lukas; Kaisermayer, Christian; Kunert, Renate

2015-06-01

In this study, eight commercially available, chemically defined Chinese hamster ovary (CHO) cell culture media from different vendors were evaluated in batch culture using an IgG-producing CHO DG44 cell line as a model. Medium adaptation revealed that the occurrence of even small aggregates might be a good indicator of cell growth performance in subsequent high cell density cultures. Batch experiments confirmed that the culture medium has a significant impact on bioprocess performance, but high amino acid concentrations alone were not sufficient to ensure superior cell growth and high antibody production. However, some key amino acids that were limiting in most media could be identified. Unbalanced glucose and amino acids led to high cell-specific lactate and ammonium production rates. In some media, persistently high glucose concentrations probably induced the suppression of respiration and oxidative phosphorylation, known as Crabtree effect, which resulted in high cell-specific glycolysis rates along with a continuous and high lactate production. In additional experiments, two of the eight basal media were supplemented with feeds from two different manufacturers in six combinations, in order to understand the combined impact of media and feeds on cell metabolism in a CHO fed-batch process. Cell growth, nutrient consumption and metabolite production rates, antibody production, and IgG quality were evaluated in detail. Concentrated feed supplements boosted cell concentrations almost threefold and antibody titers up to sevenfold. Depending on the fed-batch strategy, fourfold higher peak cell concentrations and eightfold increased IgG titers (up to 5.8 g/L) were achieved. The glycolytic flux was remarkably similar among the fed-batches; however, substantially different specific lactate production rates were observed in the different media and feed combinations. Further analysis revealed that in addition to the feed additives, the basal medium can make a considerable contribution to the ammonium metabolism of the cells. The glycosylation of the recombinant antibody was influenced by the selection of basal medium and feeds. Differences of up to 50 % in the monogalacto-fucosylated (G1F) and high mannose fraction of the IgG were observed.

Reductive Dechlorination of Carbon Tetrachloride by Tetrachloroethene and Trichloroethene Respiring Anaerobic Mixed Cultures

NASA Astrophysics Data System (ADS)

Vickstrom, K. E.; Azizian, M.; Semprini, L.

2015-12-01

Carbon tetrachloride (CT) is a toxic and recalcitrant groundwater contaminant with the potential to form a broad range of transformation products. Of the possible biochemical pathways through which CT can be degraded, reductive dehalogenation to less chlorinated compounds and mineralization to carbon dioxide (CO2) appear to be the most frequently utilized pathways by anaerobic organisms. Results will be presented from batch experiments of CT degradation by the Evanite (EV), Victoria Strain (VS) and Point Mugu (PM) anaerobic dechlorinating cultures. The cultures are grown in chemostats and are capable of transforming tetrachloroethene (PCE) or trichloroethene (TCE) to ethene by halorespiration via reductive dehalogenase enzymes. For the batch CT transformation tests, the cells along with supernatant were harvested from chemostats fed PCE or TCE, but never CT. The batch reactors were initially fed 0.0085 mM CT and an excess of formate (EV and VS) or lactate (PM) as electron donor. Transformation of CT was 100% with about 20% converted to chloroform (CF) and undetected products. Multiple additions of CT showed a slowing of pseudo first-order CT transformation rates across all cultures. Batch reactors were then established and fed 0.085 mM CT with an excess of electron donor in order to better quantify the reductive pathway. CT was transformed to CF and dichloromethane (DCM), with trace amounts of chloromethane (CM) detected. Between 60-90% of the mass added to the system was accounted for, showing that the majority of the carbon tetrachloride present is being reductively dehalogenated. Results from batch reactors that were poisoned using sodium azide, and from reactors not provided electron donor will be presented to distinguish between biotic and abiotic reactions. Furthermore, results from reactors prepared with acetylene (a potent, reversible inhibitor of reductive dehalogenases (1)) will be presented as a means of identifying the enzymes involved in the transformation of CT. The results clearly demonstrate that reductive dechlorination of CT can be promoted by anaerobic cultures not previously acclimated to CT. 1. G. Pon, M. R. Hyman, L. Semprini, Environ. Sci. Technol. 37, 3181-3188 (2003).

Continuous and batch cultures of Escherichia coli KJ134 for succinic acid fermentation: metabolic flux distributions and production characteristics.

PubMed

van Heerden, Carel D; Nicol, Willie

2013-09-17

Succinic acid (SA) has become a prominent biobased platform chemical with global production quantities increasing annually. Numerous genetically modified E. coli strains have been developed with the main aim of increasing the SA yield of the organic carbon source. In this study, a promising SA-producing strain, E. coli KJ134 [Biotechnol. Bioeng. 101:881-893, 2008], from the Department of Microbiology and Cell Science of the University of Florida was evaluated under continuous and batch conditions using D-glucose and CO2 in a mineral salt medium. Production characteristics entailing growth and maintenance rates, growth termination points and metabolic flux distributions under growth and non-growth conditions were determined. The culture remained stable for weeks under continuous conditions. Under growth conditions the redox requirements of the reductive tricarboxylic acid (TCA) cycle was solely balanced by acetic acid (AcA) production via the pyruvate dehydrogenase route resulting in a molar ratio of SA:AcA of two. A maximum growth rate of 0.22 h(-1) was obtained, while complete growth inhibition occurred at a SA concentration of 18 g L(-1). Batch culture revealed that high-yield succinate production (via oxidative TCA or glyoxylate redox balancing) occurred under non-growth conditions where a SA:AcA molar ratio of up to five was attained, with a final SA yield of 0.94 g g(-1). Growth termination of the batch culture was in agreement with that of the continuous culture. The maximum maintenance production rate of SA under batch conditions was found to be 0.6 g g(-1) h(-1). This is twice the maintenance rate observed in the continuous runs. The study revealed that the metabolic flux of E. coli KJ134 differs significantly for growth and non-growth conditions, with non-growth conditions resulting in higher SA:AcA ratios and SA yields. Bioreaction characteristics entailing growth and maintenance rates, as well as growth termination markers will guide future fermentor designs and improvements.

A parametric study ot protease production in batch and fed-batch cultures of Bacillus firmus.

PubMed

Moon, S H; Parulekar, S J

1991-03-05

Proteolytic enzymes produced by Bacillus species find a wide variety of applications in brewing, detergent, food, and leather industries. Owing to significant differences normally observed in culture conditions promoting cell growth and those promoting production of metabolites such as enzymes, for increased efficacy of bioreactor operations it is essential to identify these sets of conditions (including medium formulation). This study is focused on formulation of a semidefined medium that substantially enhances synthesis and secretion of an alkaline protease in batch cultures of Bacillus firmus NRS 783, a known superior producer of this enzyme. The series of experiments conducted to identify culture conditions that lead to improved protease production also enables investigation of the regulatory effects of important culture parameters including pH, dissolved oxygen, and concentrations of nitrogen and phosphorous sources and yeast extract in the medium on cell growth, synthesis and secretion of protease, and production of two major nonbiomass products, viz., acetic acid and ethanol. Cell growth and formation of the three nonbiomass products are hampered significantly under nitrogen, phosphorous, or oxygen limitation, with the cells being unable to grow in an oxygen-free environment. Improvement in protease production is achieved with respect to each culture parameter, leading in the process to 80% enhancement in protease activity over that attained using media reported in the literature. Results of a few fed-batch experiments with constant feed rate, conducted to examine possible enhancement in protease production and to further investigate repression of protease synthesis by excess of the principal carbon and nitrogen sources, are also discussed. The detailed investigation of stimulatory and repressory effects of simple and complex nutrients on protease production and metabolism of Bacillus firmus conducted in this study will provide useful guidelines for design of bioreactors for production of protease and bulk chemicals by this bacterium.

Inhibition of Salmonella Typhimurium by Cultures of Cecal Bacteria during Aerobic Incubation

USDA-ARS?s Scientific Manuscript database

Two trials were conducted to examine the ability of cecal bacterial cultures from broilers to inhibit growth of Salmonella Typhimurium during aerobic incubation. Cecal broth media was inoculated with 10 µl of cecal contents from 6 week old broilers taken from 2 separate flocks. Cultures were incubat...

Production of carotenoids and lipids by Rhodococcus opacus PD630 in batch and fed-batch culture.

PubMed

Thanapimmetha, Anusith; Suwaleerat, Tharatron; Saisriyoot, Maythee; Chisti, Yusuf; Srinophakun, Penjit

2017-01-01

Production of carotenoids by Rhodococcus opacus PD630 is reported. A modified mineral salt medium formulated with glycerol as an inexpensive carbon source was used for the fermentation. Ammonium acetate was the nitrogen source. A dry cell mass concentration of nearly 5.4 g/L could be produced in shake flasks with a carotenoid concentration of 0.54 mg/L. In batch culture in a 5 L bioreactor, without pH control, the maximum dry biomass concentration was ~30 % lower than in shake flasks and the carotenoids concentration was 0.09 mg/L. Both the biomass concentration and the carotenoids concentration could be raised using a fed-batch operation with a feed mixture of ammonium acetate and acetic acid. With this strategy, the final biomass concentration was 8.2 g/L and the carotenoids concentration was 0.20 mg/L in a 10-day fermentation. A control of pH proved to be unnecessary for maximizing the production of carotenoids in this fermentation.

Study on Molasses Concentration from Sugarcanne Bagasse for Biohydrogen Production using Enriched Granular Activated Carbon (GAC) Immobilised Cells by Repeated Batch Cultivation

NASA Astrophysics Data System (ADS)

Idris, Norfatiha; Aminah Lutpi, Nabilah; Ruhaizul Che Ridzuan, Che Mohd; Shian, Wong Yee; Nuraiti Tengku Izhar, Tengku

2018-03-01

Repeated batch cultivation is known as most attractive method in improving hydrogen productivity, due to the facts that this approach could minimize the reuse of the cell and the inoculum preparation. In addition, with the combination of attach growth system during the fermentation processes to produce biohydrogen, the density of cells will be increased and the cell washout could be avoided. Therefore, this study aimed to examine the effectiveness of repeated batch cultivation for enrichment of anaerobic mixed culture onto granular activated carbon (GAC) and investigate the effect of molasses concentration during immobilization of mixed culture onto the GAC. The molasses concentration using 50 %, 40 %, 30 %, 20 % and 10 % of diluted molasses were used as feedstock in the fermentation process. The maximum hydrogen production of 60 ml was obtained at 30 % of molasses concentration with 831 ppm of hydrogen concentration. Thus, the kinetic parameter obtained from the batch profiling based on modified Gompertz equation are, Hm= 58 ml for the maximum hydrogen production and Rm= 2.02 ml/h representing the hydrogen production rate.

Heat shock protein 27 overexpression in CHO cells modulates apoptosis pathways and delays activation of caspases to improve recombinant monoclonal antibody titre in fed-batch bioreactors.

PubMed

Tan, Janice G L; Lee, Yih Yean; Wang, Tianhua; Yap, Miranda G S; Tan, Tin Wee; Ng, Say Kong

2015-05-01

CHO cells are major production hosts for recombinant biologics including the rapidly expanding recombinant monoclonal antibodies (mAbs). Heat shock protein 27 (HSP27) expression was observed to be down-regulated towards the late-exponential and stationary phase of CHO fed-batch bioreactor cultures, whereas HSP27 was found to be highly expressed in human pathological cells and reported to have anti-apoptotic functions. These phenotypes suggest that overexpression of HSP27 is a potential cell line engineering strategy for improving robustness of CHO cells. In this work, HSP27 was stably overexpressed in CHO cells producing recombinant mAb and the effects of HSP27 on cell growth, volumetric production titer and product quality were assessed. Concomitantly, HSP27 anti-apoptosis functions in CHO cells were investigated. Stably transfected clones cultured in fed-batch bioreactors displayed 2.2-fold higher peak viable cell density, delayed loss of culture viability by two days and 2.3-fold increase in mAb titer without affecting the N-glycosylation profile, as compared to clones stably transfected with the vector backbone. Co-immunoprecipitation studies revealed HSP27 interactions with Akt, pro-caspase 3 and Daxx and caspase activity profiling showed delayed increase in caspase 2, 3, 8 and 9 activities. These results suggest that HSP27 modulates apoptosis signaling pathways and delays caspase activities to improve performance of CHO fed-batch bioreactor cultures. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Interaction of Vesicular-Arbuscular Mycorrhizae and Cultivars of Alfalfa Susceptible and Resistant to Meloidogyne hapla.

PubMed

Grandison, G S; Cooper, K M

1986-04-01

The interaction between vesicular-arbuscular mycorrhizal (VAM) fungi and the root-knot nematode (Meloidogyne hapla) was investigated using both nematode-susceptible (Grasslands Wairau) and nematode-resistant (Nevada Synthetic XX) cultivars of alfalfa (Medicago sativa) at four levels of applied phosphate. Mycorrhizal inoculation improved plant growth and reduced nematode numbers and adult development in roots in dually infected cultures of the susceptible cultivar. The tolerance of plants to nematode infection and development when preinfected with mycorrhizal fungi was no greater than when they were inoculated with nematodes and mycorrhizal fungi simultaneously. Growth of plants of the resistant cultivar was unaffected by nematode inoculation but was improved by mycorrhizal inoculation. Numbers of nematode juveniles were lower in the roots of the resistant than of the susceptible cultivar and were further reduced by mycorrhizal inoculation, although no adult nematodes developed in any resistant cultivar treatment. Inoculation of alfalfa with VAM fungi increased the tolerance and resistance of a cultivar susceptible to M. hapla and improved the resistance of a resistant cultivar.

Inoculation of Scytalidium thermophilum in Button Mushroom Compost and Its Effect on Yield.

PubMed

Straatsma, G; Olijnsma, T W; Gerrits, J P; Amsing, J G; Op Den Camp, H J; Van Griensven, L J

1994-09-01

Scytalidium thermophilum isolates in culture, as well as the endogenous strain(s) in mushroom compost, were inactivated at 70 degrees C. This temperature was used to pasteurize composts for experiments. Of nine thermophilic fungal species, only S. thermophilum and Myriococcum thermophilum grew well on pasteurized compost in test tubes. The effect of both species on the crop yield of Agaricus bisporus mushrooms was studied. In solid-state fermentation rooms called tunnels, compost was pasteurized and inoculated. After incubation, the inoculated organisms were reisolated and counted, showing their successful colonization. The yield of mushrooms on inoculated composts was almost twice that on the pasteurized control. This result demonstrates the effectiveness of S. thermophilum in compost preparation. Inoculation is not necessary for traditional compost preparation. Naturally occurring strains of S. thermophilum, present in ingredients, readily colonize compost during preparation. Inoculation may be vital if compost is pretreated at a high temperature in tunnels. This finding is of relevance for the environmentally controlled production of high-yielding compost.

Inoculation of Scytalidium thermophilum in Button Mushroom Compost and Its Effect on Yield

PubMed Central

Straatsma, Gerben; Olijnsma, Tineke W.; Gerrits, Jan P. G.; Amsing, Jos G. M.; Op Den Camp, Huub J. M.; Van Griensven, Leo J. L. D.

1994-01-01

Scytalidium thermophilum isolates in culture, as well as the endogenous strain(s) in mushroom compost, were inactivated at 70°C. This temperature was used to pasteurize composts for experiments. Of nine thermophilic fungal species, only S. thermophilum and Myriococcum thermophilum grew well on pasteurized compost in test tubes. The effect of both species on the crop yield of Agaricus bisporus mushrooms was studied. In solid-state fermentation rooms called tunnels, compost was pasteurized and inoculated. After incubation, the inoculated organisms were reisolated and counted, showing their successful colonization. The yield of mushrooms on inoculated composts was almost twice that on the pasteurized control. This result demonstrates the effectiveness of S. thermophilum in compost preparation. Inoculation is not necessary for traditional compost preparation. Naturally occurring strains of S. thermophilum, present in ingredients, readily colonize compost during preparation. Inoculation may be vital if compost is pretreated at a high temperature in tunnels. This finding is of relevance for the environmentally controlled production of high-yielding compost. PMID:16349366

Experimental Clostridium perfringens type D enterotoxemia in goats.

PubMed

Uzal, F A; Kelly, W R

1998-03-01

The effects of intraduodenal administration of Clostridium perfringens cultures and culture products in goats were evaluated to develop a reliable experimental model of enterotoxemia in this species. Five conventionally reared, 11-16-week-old Angora goat kids were dosed intraduodenally with whole cultures of C. perfringens type D; five similar animals were dosed with C. perfringens type D filtered culture supernatant; and a third group of five kids was dosed with C. perfringens type D washed cells. Two kids were used as controls and received sterile, nontoxic culture medium intraduodenally. All animals received starch solution into the abomasum. All five kids inoculated with whole culture and three of five dosed with culture supernatant and with washed cells developed central nervous system signs. Diarrhea was observed in two of five kids inoculated with whole culture, in all five of those dosed with culture supernatant, and in three of five of those that received washed cells. The most striking postmortem findings consisted of lung edema, necrotizing pseudomembranous colitis, and cerebral vasogenic edema. The protocol thus provided a reasonable model of naturally occurring enterotoxemia in goats, producing a range of clinical signs and postmortem changes similar to those observed in the natural disease.

Use of Positive Blood Cultures for Direct Identification and Susceptibility Testing with the Vitek 2 System

PubMed Central

de Cueto, Marina; Ceballos, Esther; Martinez-Martinez, Luis; Perea, Evelio J.; Pascual, Alvaro

2004-01-01

In order to further decrease the time lapse between initial inoculation of blood culture media and the reporting of results of identification and antimicrobial susceptibility tests for microorganisms causing bacteremia, we performed a prospective study in which specially processed fluid from positive blood culture bottles from Bactec 9240 (Becton Dickinson, Cockeysville, Md.) containing aerobic media were directly inoculated into Vitek 2 system cards (bio-Mérieux, France). Organism identification and susceptibility results were compared with those obtained from cards inoculated with a standardized bacterial suspension obtained following subculture to agar; 100 consecutive positive monomicrobic blood cultures, consisting of 50 gram-negative rods and 50 gram-positive cocci, were included in the study. For gram-negative organisms, 31 of the 50 (62%) showed complete agreement with the standard method for species identification, while none of the 50 gram-positive cocci were correctly identified by the direct method. For gram-negative rods, there were 50% categorical agreements between the direct and standard methods for all drugs tested. The very major error rate was 2.4%, and the major error rate was 0.6%. The overall error rate for gram-negatives was 6.6%. Complete agreement in clinical categories of all antimicrobial agents evaluated was obtained for 19 of 50 (38%) gram-positive cocci evaluated; the overall error rate was 8.4%, with 2.8% minor errors, 2.4% major errors, and 3.2% very major errors. These findings suggest that the Vitek 2 cards inoculated directly from positive Bactec 9240 bottles do not provide acceptable bacterial identification or susceptibility testing in comparison with corresponding cards tested by a standard method. PMID:15297523

Inoculated fermentation of green olives with potential probiotic Lactobacillus pentosus and Lactobacillus plantarum starter cultures isolated from industrially fermented olives.

PubMed

Blana, Vasiliki A; Grounta, Athena; Tassou, Chrysoula C; Nychas, George-John E; Panagou, Efstathios Z

2014-04-01

The performance of two strains of lactic acid bacteria (LAB), namely Lactobacillus pentosus B281 and Lactobacillus plantarum B282, previously isolated from industrially fermented table olives and screened in vitro for probiotic potential, was investigated as starter cultures in Spanish style fermentation of cv. Halkidiki green olives. Fermentation was undertaken at room temperature in two different initial salt concentrations (8% and 10%, w/v, NaCl) in the brines. The strains were inoculated as single and combined cultures and the dynamics of their population on the surface of olives was monitored for a period of 114 days. The survival of inoculated strains on olives was determined using Pulsed Field Gel Electrophoresis (PFGE). Both probiotic strains successfully colonized the olive surface at populations ranged from 6.0 to 7.0 log CFU/g throughout fermentation. PFGE analysis revealed that L. pentosus B281 presented higher colonization in both salt levels at the end of fermentation (81.2% and 93.3% in 8% and 10% NaCl brines, respectively). For L. plantarum B282 a high survival rate (83.3%) was observed in 8% NaCl brines, but in 10% NaCl the strain could not colonize the surface of olives. L. pentosus B281 also dominated over L. plantarum B282 in inoculated fermentations when the two strains were used as combined culture. The biochemical profile (pH, organic acids, volatile compounds) attained during fermentation and the sensory analysis of the final product indicated a typical lactic acid fermentation process of green olives. Copyright © 2013 Elsevier Ltd. All rights reserved.

Detection of lymphocystis disease virus (LCDV) in asymptomatic cultured gilt-head seabream (Sparus aurata, L.) using an immunoblot technique.

PubMed

Cano, I; Alonso, M C; Garcia-Rosado, E; Saint-Jean, S Rodriguez; Castro, D; Borrego, Juan J

2006-03-10

An immunoblot technique for the detection of lymphocystis disease virus (LCDV) in naturally infected gilt-head seabream (Sparus aurata, L.) has been developed. A specific antiserum against a 60 kDa viral protein has been proven to be an appropriate tool for LCDV diagnosis either from inoculated cell cultures or from fish tissues using the immunoblot assay. The sensitivity of this technique varied between 10(-1) and 10(2) TCID50. LCDV has also been detected in fish tissues from both, diseased and asymptomatic gilt-head seabream. For the asymptomatic fish detection, a viral amplification step in cell culture and a subsequent viral concentration using polyethylene glycol (PEG) (600 wt) are required. On the contrary, immunoblot allowed the detection of LCDV antigens directly from tissue homogenates of diseased fish. The method described in this study shows higher sensitivity than classical detection techniques based on cell culture inoculation.

Effect of ryegrass (Lolium perenne L.) roots inoculation using different arbuscular mycorrhizal fungi (AMF) species on sorption of iron-cyanide (Fe-CN) complexes

NASA Astrophysics Data System (ADS)

Sut, Magdalena; Boldt-Burisch, Katja; Raab, Thomas

2016-04-01

Soils and groundwater on sites of the former Manufactured Gas Plants (MGPs) are contaminated with various complex iron-cyanides (Fe-CN). Phytoremediation is a promising tool in stabilization and remediation of Fe-CN affected soils, however, it can be a challenging task due to extreme adverse and toxic conditions. Phytoremediation may be enhanced via rhizosphere microbial activity, which can cooperate on the degradation, transformation and uptake of the contaminants. Recently, increasing number of scientist reports improved plants performance in the removal of toxic compounds with the support of arbuscular mycorrhizae fungi (AMF). Series of batch experiments using potassium hexacyanoferrate (II) solutions, in varying concentrations, were used to study the effect of ryegrass roots (Lolium perenne L.) inoculation with Rhizophagus irregularis and a mixture of Rhizophagus irregularis, Funneliformis mosseae, Rhizophagus aggregatus, and Claroideoglomus etunicatum on Fe-CN sorption. Results indicated significantly higher colonization of R. irregularis than for the mixture of AMF species on ryegrass roots. Sorption experiments revealed significantly higher reduction of total CN and free CN content in the mycorrhizal roots, indicating greater cyanide decrease in the treatment inoculated with R. irregularis. Our study indicates contribution of AM fungi in phytoremediation of Fe-CN contaminated soil.

On the way to commercializing plant cell culture platform for biopharmaceuticals: present status and prospect

PubMed Central

Xu, Jianfeng; Zhang, Ningning

2014-01-01

Plant cell culture is emerging as an alternative bioproduction system for recombinant pharmaceuticals. Growing plant cells in vitro under controlled environmental conditions allows for precise control over cell growth and protein production, batch-to-batch product consistency and a production process aligned with current good manufacturing practices. With the recent US FDA approval and commercialization of the world’s first plant cell-based recombinant pharmaceutical for human use, β-glucocerebrosidase for treatment of Gaucher’s disease, a new era has come in which plant cell culture shows high potential to displace some established platform technologies in niche markets. This review updates the progress in plant cell culture processing technology, highlights recent commercial successes and discusses the challenges that must be overcome to make this platform commercially viable. PMID:25621170

The Long Non-Coding RNA Transcriptome Landscape in CHO Cells Under Batch and Fed-Batch Conditions.

PubMed

Vito, Davide; Smales, C Mark

2018-05-21

The role of non-coding RNAs in determining growth, productivity and recombinant product quality attributes in Chinese hamster ovary (CHO) cells has received much attention in recent years, exemplified by studies into microRNAs in particular. However, other classes of non-coding RNAs have received less attention. One such class are the non-coding RNAs known collectively as long non-coding RNAs (lncRNAs). We have undertaken the first landscape analysis of the lncRNA transcriptome in CHO using a mouse based microarray that also provided for the surveillance of the coding transcriptome. We report on those lncRNAs present in a model host CHO cell line under batch and fed-batch conditions on two different days and relate the expression of different lncRNAs to each other. We demonstrate that the mouse microarray was suitable for the detection and analysis of thousands of CHO lncRNAs and validated a number of these by qRT-PCR. We then further analysed the data to identify those lncRNAs whose expression changed the most between growth and stationary phases of culture or between batch and fed-batch culture to identify potential lncRNA targets for further functional studies with regard to their role in controlling growth of CHO cells. We discuss the implications for the publication of this rich dataset and how this may be used by the community. This article is protected by copyright. All rights reserved.

Analysis of key microbial community during the start-up of anaerobic ammonium oxidation process with paddy soil as inoculated sludge.

PubMed

Xu, Xianglong; Liu, Guohua; Wang, Yuanyuan; Zhang, Yuankai; Wang, Hao; Qi, Lu; Wang, Hongchen

2018-02-01

A sequencing batch reactor (SBR)-anaerobic ammonium oxidation (anammox) system was started up with the paddy soil as inoculated sludge. The key microbial community structure in the system along with the enrichment time was investigated by using molecular biology methods (e.g., high-throughput 16S rRNA gene sequencing and quantitative PCR). Meanwhile, the influent and effluent water quality was continuously monitored during the whole start-up stage. The results showed that the microbial diversity decreased as the operation time initially and increased afterwards, and the microbial niches in the system were redistributed. The anammox bacterial community structure in the SBR-anammox system shifted during the enrichment, the most dominant anammox bacteria were CandidatusJettenia. The maximum biomass of anammox bacteria achieved 1.68×10 9 copies/g dry sludge during the enrichment period, and the highest removal rate of TN achieved around 75%. Copyright © 2017. Published by Elsevier B.V.

Occurrence of Anthracnose on Fruits of Asian Pear Tree Caused by Colletotrichum acutatum.

PubMed

Kim, Wan Gyu; Hong, Sung Kee; Park, Yeong Seob

2007-12-01

Anthracnose symptoms often occurred on fruits of Asian pear trees grown in Anseong, Naju, Seonghwan and Pyeongtaek in Korea during the harvesting period from 2000 to 2005. A total of 28 isolates of Colletotrichum sp. were obtained from the anthracnose symptoms. All the isolates were identified as Colletotrichum acutatum based on their morphological and cultural characteristics. Four isolates of the fungus were tested for pathogenicity to fruits of Asian pear tree by artificial inoculation. All the isolates induced anthracnose symptoms on the fruits by wound inoculation but not by unwound inoculation. The anthracnose symptoms induced by artificial inoculation were similar to those observed in the orchard. This is the first report of anthracnose of Asian pear tree caused by Colletotrichum acutatum.

Production and degradation of oxalic acid by brown rot fungi

DOE Office of Scientific and Technical Information (OSTI.GOV)

Espejo, E.; Agosin, E.

1991-07-01

Our results show that all of the brown rot fungi tested produce oxalic acid in liquid as well as in semisolid cultures. Gloeophyllum trabeum, which accumulates the lowest amount of oxalic acid during decay of pine holocellulose, showed the highest polysaccharide-depolymerizing activity. Semisolid cultures inoculated with this fungus rapidly converted {sup 14}C-labeled oxalic acid to CO{sub 2} during cellulose depolymerization. The other brown rot fungi also oxidized {sup 14}C-labeled oxalic acid, although less rapidly. In contrast, semisolid cultures inoculated with the white rot fungus Coriolus versicolor did not significantly catabolize the acid and did not depolymerize the holocellulose during decay.more » Semisolid cultures of G. trabeum amended with desferrioxamine, a specific iron-chelating agent, were unable to lower the degree of polymerization of cellulose or to oxidize {sup 14}C-labeled oxalic acid to the extent or at the rate that control cultures did. These results suggest that both iron and oxalic acid are involved in cellulose depolymerization by brown rot fungi.« less

Responses of Wound-Inoculated Seedlings of Pinus elliottii var. elliottii and Pinus taeda to Mycelial Cultures Derived from Multople and Singel Basidiospores of Cronartium quercuum f. sp. fusiforme

Treesearch

T. Miller; K.P. Gramacho; R.A. Schmidt; H.V. Amerson; E.G. Kuhlman

1998-01-01

In 1991, a series of experiments was initiated to examine the effectiveness and research value of inoculating 6-week-old seedlings of slash (Pinus elliottii var. elliottii) and loblolly pine (P. raeda) with suspensions of basidiospores of Cronartium quercuum f. sp. fusiforme...

MULTIPLICATION OF DIFFERENT VIRUSES AND EVOLUTION OF VIRAL INFECTION IN CELLULAR CULTURES EXPOSED TO THE ACTION OF HEAVY WATER (in French)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lavillaureix, J.; Reeb, E.; Surjus, A.

1961-03-20

The multiplication of West Nile, Coxsackie B3, and polio type II viruses is considerably increased if they are inoculated on cells cultivated in the presence of heavy water 5 hr before inoculation. The viral infection can not evolve in the presence of heavy water. (tr-auth)

Effects of cell culture conditions on antibody N-linked glycosylation--what affects high mannose 5 glycoform.

PubMed

Pacis, Efren; Yu, Marcella; Autsen, Jennifer; Bayer, Robert; Li, Feng

2011-10-01

The glycosylation profile of therapeutic antibodies is routinely analyzed throughout development to monitor the impact of process parameters and to ensure consistency, efficacy, and safety for clinical and commercial batches of therapeutic products. In this study, unusually high levels of the mannose-5 (Man5) glycoform were observed during the early development of a therapeutic antibody produced from a Chinese hamster ovary (CHO) cell line, model cell line A. Follow up studies indicated that the antibody Man5 level was increased throughout the course of cell culture production as a result of increasing cell culture medium osmolality levels and extending culture duration. With model cell line A, Man5 glycosylation increased more than twofold from 12% to 28% in the fed-batch process through a combination of high basal and feed media osmolality and increased run duration. The osmolality and culture duration effects were also observed for four other CHO antibody producing cell lines by adding NaCl in both basal and feed media and extending the culture duration of the cell culture process. Moreover, reduction of Man5 level from model cell line A was achieved by supplementing MnCl2 at appropriate concentrations. To further understand the role of glycosyltransferases in Man5 level, N-acetylglucosaminyltransferase I GnT-I mRNA levels at different osmolality conditions were measured. It has been hypothesized that specific enzyme activity in the glycosylation pathway could have been altered in this fed-batch process. Copyright © 2011 Wiley Periodicals, Inc.

Growth and enzymatic responses of phytopathogenic fungi to glucose in culture media and soil

PubMed Central

Costa, Beatriz de Oliveira; Nahas, Ely

2012-01-01

The effect of inoculation of Aspergillus flavus , Fusarium verticillioides , and Penicillium sp. in Dystrophic Red Latosol (DRL) and Eutroferric Red Latosol (ERL) soils with or without glucose on the total carbohydrate content and the dehydrogenase and amylase activities was studied. The fungal growth and spore production in culture medium with and without glucose were also evaluated. A completely randomized design with factorial arrangement was used. The addition of glucose in the culture medium increased the growth rate of A. flavus and Penicillium sp. but not of F. verticillioides . The number of spores increased 1.2 for F. verticillioides and 8.2 times for A. flavus in the medium with glucose, but was reduced 3.5 times for Penicillium sp. The total carbohydrates contents reduced significantly according to first and second degree equations. The consumption of total carbohydrates by A. flavus and Penicillium sp. was higher than the control or soil inoculated with F. verticillioides . The addition of glucose to soils benefited the use of carbohydrates, probably due to the stimulation of fungal growth. Dehydrogenase activity increased between 1.5 to 1.8 times ( p <0.05) in soils with glucose and inoculated with the fungi (except F. verticillioides ), in relation to soil without glucose. Amylase activity increased 1.3 to 1.5 times due to the addition of glucose in the soil. Increased amylase activity was observed in the DRL soil with glucose and inoculated with A. flavus and Penicillium sp. when compared to control. PMID:24031836

Use of an insect cell culture growth medium to isolate bacteria from horses with effusive, fibrinous pericarditis: a preliminary study.

PubMed

Jones, Samuel L; Valenzisi, Amy; Sontakke, Sushama; Sprayberry, Kimberly A; Maggi, Ricardo; Hegarty, Barbara; Breitschwerdt, Edward

2007-03-31

Effusive, fibrinous pericarditis is an uncommon disease entity in horses. In 2001, pericarditis occurred in conjunction with an epizootic in central Kentucky that was associated with exposure to eastern tent caterpillars (ETCs). Bacterial isolation from equine pericardial fluid samples was attempted using an insect cell culture growth medium (ICCGM). Using previously cultured, stored frozen samples from four horses with fibrinous pericarditis, inoculation of 10% blood agar plates yielded no growth, whereas simultaneous inoculation of ICCGM resulted in the isolation of Proprionibacterium acnes, Staphylococcus equorum, a Streptococcus sp. and Pseudomonas rhodesiae from pericardial fluid samples. A similar or novel caterpillar-associated bacteria was not identified; however, use of an ICCGM might enhance isolation of bacteria from equine pericardial fluid.

Nitrate reductase activity of Staphylococcus carnosus affecting the color formation in cured raw ham.

PubMed

Bosse Née Danz, Ramona; Gibis, Monika; Schmidt, Herbert; Weiss, Jochen

2016-07-01

The influence of the nitrate reductase activity of two Staphylococcus carnosus strains used as starter cultures on the formation of nitrate, nitrite and color pigments in cured raw ham was investigated. In this context, microbiological, chemical and multivariate image analyses were carried out on cured raw hams, which were injected with different brines containing either nitrite or nitrate, with or without the S. carnosus starter cultures. During processing and storage, the viable counts of staphylococci remained constant at 6.5logcfu/g in the hams inoculated with starter cultures, while the background microbiota of the hams processed without the starter cultures developed after 14days. Those cured hams inoculated with S. carnosus LTH 7036 (high nitrate reductase activity) showed the highest decrease in nitrate and high nitrite concentrations in the end product, but were still in the range of the legal European level. The hams cured with nitrate and without starter culture or with the other strain, S. carnosus LTH 3838 (low nitrate reductase activity) showed higher residual nitrate levels and a lower nitrite content in the end product. The multivariate image analysis identified spatial and temporal differences in the meat pigment profiles of the differently cured hams. The cured hams inoculated with S. carnosus LTH 3838 showed an uncured core due to a delay in pigment formation. Therefore, the selection of starter cultures based on their nitrate reductase activity is a key point in the formation of curing compounds and color pigments in cured raw ham manufacture. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cyclic-2,3-diphosphoglycerate levels in Methanobacterium thermoautotrophicum reflect inorganic phosphate availability.

PubMed

Seely, R J; Krueger, R D; Fahrney, D E

1983-11-15

Methanobacterium thermoautotrophicum was grown in phosphate-limited chemostat cultures at a dilution rate corresponding to a doubling time of 13.2 h. The cyclic-2,3-diphospho-D-glycerate content of these cells was 8 to 10-fold lower than that of cells grown in batch cultures having a doubling time of 11.5 h. This metabolite accounted for 5% of cell dry weight during batch growth on 2 mM phosphate. In the chemostat the steady-state concentration of phosphate was 4 microM, showing that this methanogen is adapted to highly efficient growth at low phosphate concentrations. Since growth rates were similar in both cultures, the growth rate clearly does not depend on intracellular levels of cyclic-2,3-diphosphoglycerate.

Cholera Rapid Test with Enrichment Step Has Diagnostic Performance Equivalent to Culture

PubMed Central

Ontweka, Lameck N.; Deng, Lul O.; Rauzier, Jean; Debes, Amanda K.; Tadesse, Fisseha; Parker, Lucy A.; Wamala, Joseph F.; Bior, Bior K.; Lasuba, Michael; But, Abiem Bona; Grandesso, Francesco; Jamet, Christine; Cohuet, Sandra; Ciglenecki, Iza; Serafini, Micaela; Sack, David A.; Quilici, Marie-Laure; Azman, Andrew S.; Luquero, Francisco J.

2016-01-01

Cholera rapid diagnostic tests (RDT) could play a central role in outbreak detection and surveillance in low-resource settings, but their modest performance has hindered their broad adoption. The addition of an enrichment step may improve test specificity. We describe the results of a prospective diagnostic evaluation of the Crystal VC RDT (Span Diagnostics, India) with enrichment step and of culture, each compared to polymerase chain reaction (PCR), during a cholera outbreak in South Sudan. RDTs were performed on alkaline peptone water inoculated with stool and incubated for 4–6 hours at ambient temperature. Cholera culture was performed from wet filter paper inoculated with stool. Molecular detection of Vibrio cholerae O1 by PCR was done from dry Whatman 903 filter papers inoculated with stool, and from wet filter paper supernatant. In August and September 2015, 101 consecutive suspected cholera cases were enrolled, of which 36 were confirmed by PCR. The enriched RDT had 86.1% (95% CI: 70.5–95.3) sensitivity and 100% (95% CI: 94.4–100) specificity compared to PCR as the reference standard. The sensitivity of culture versus PCR was 83.3% (95% CI: 67.2–93.6) for culture performed on site and 72.2% (95% CI: 54.8–85.8) at the international reference laboratory, where samples were tested after an average delay of two months after sample collection, and specificity was 98.5% (95% CI: 91.7–100) and 100% (95% CI: 94.5–100), respectively. The RDT with enrichment showed performance comparable to that of culture and could be a sustainable alternative to culture confirmation where laboratory capacity is limited. PMID:27992488

Development of Safe and Flavor-Rich Doenjang (Korean Fermented Soybean Paste) Using Autochthonous Mixed Starters at the Pilot Plant Scale.

PubMed

Lee, Eun Jin; Hyun, Jiye; Choi, Yong-Ho; Hurh, Byung-Serk; Choi, Sang-Ho; Lee, Inhyung

2018-06-01

Doenjang (Korean fermented soybean paste) with an improved flavor and safety was prepared by the simultaneous fermentation of autochthonous mixed starters at the pilot plan scale. First, whole soybean meju was fermented by coculturing safety-verified starters Aspergillus oryzae MJS14 and Bacillus amyloliquefaciens zip6 or Bacillus subtilis D119C. These fermented whole soybean meju were aged in a brine solution after the additional inoculation of Tetragenococcus halophilus 7BDE22 and Zygosaccharomyces rouxii SMY045 to yield doenjang. Four doenjang batches prepared using a combination of mold, bacilli, lactic acid bacteria, and yeast starters were free of safety issues and had the general properties of traditional doenjang, a rich flavor and taste. All doenjang batches received a high consumer acceptability score, especially the ABsT and ABsTZ batches. This study suggests that flavor-rich doenjang similar to traditional doenjang can be manufactured safely and reproducibly in industry by mimicking the simultaneous fermentation of autochthonous mixed starters as in traditional doenjang fermentation. The development of a pilot plant process for doenjang fermentation using safety-verified autochthonous mixed starter will facilitate the manufacture of flavor-rich doenjang similar to traditional doenjang safely and reproducibly in industry. © 2018 Institute of Food Technologists®.

Denitrifying capability and community dynamics of glycogen accumulating organisms during sludge granulation in an anaerobic-aerobic sequencing batch reactor

PubMed Central

Bin, Zhang; Bin, Xue; Zhigang, Qiu; Zhiqiang, Chen; Junwen, Li; Taishi, Gong; Wenci, Zou; Jingfeng, Wang

2015-01-01

Denitrifying capability of glycogen accumulating organisms (GAOs) has received great attention in environmental science and microbial ecology. Combining this ability with granule processes would be an interesting attempt. Here, a laboratory-scale sequencing batch reactor (SBR) was operated to enrich GAOs and enable sludge granulation. The results showed that the GAO granules were cultivated successfully and the granules had denitrifying capability. The batch experiments demonstrated that all NO3−-N could be removed or reduced, some amount of NO2−-N were accumulated in the reactor, and N2 was the main gaseous product. SEM analysis suggested that the granules were tightly packed with a large amount of tetrad-forming organisms (TFOs); filamentous bacteria served as the supporting structures for the granules. The microbial community structure of GAO granules was differed substantially from the inoculant conventional activated sludge. Most of the bacteria in the seed sludge grouped with members of Proteobacterium. FISH analysis confirmed that GAOs were the predominant members in the granules and were distributed evenly throughout the granular space. In contrast, PAOs were severely inhibited. Overall, cultivation of the GAO granules and utilizing their denitrifying capability can provide us with a new approach of nitrogen removal and saving more energy. PMID:26257096

Denitrifying capability and community dynamics of glycogen accumulating organisms during sludge granulation in an anaerobic-aerobic sequencing batch reactor.

PubMed

Bin, Zhang; Bin, Xue; Zhigang, Qiu; Zhiqiang, Chen; Junwen, Li; Taishi, Gong; Wenci, Zou; Jingfeng, Wang

2015-08-10

Denitrifying capability of glycogen accumulating organisms (GAOs) has received great attention in environmental science and microbial ecology. Combining this ability with granule processes would be an interesting attempt. Here, a laboratory-scale sequencing batch reactor (SBR) was operated to enrich GAOs and enable sludge granulation. The results showed that the GAO granules were cultivated successfully and the granules had denitrifying capability. The batch experiments demonstrated that all NO3(-)-N could be removed or reduced, some amount of NO2(-)-N were accumulated in the reactor, and N2 was the main gaseous product. SEM analysis suggested that the granules were tightly packed with a large amount of tetrad-forming organisms (TFOs); filamentous bacteria served as the supporting structures for the granules. The microbial community structure of GAO granules was differed substantially from the inoculant conventional activated sludge. Most of the bacteria in the seed sludge grouped with members of Proteobacterium. FISH analysis confirmed that GAOs were the predominant members in the granules and were distributed evenly throughout the granular space. In contrast, PAOs were severely inhibited. Overall, cultivation of the GAO granules and utilizing their denitrifying capability can provide us with a new approach of nitrogen removal and saving more energy.

Sexual and transovarian transmission of Crimean-Congo haemorrhagic fever virus in Hyalomma truncatum ticks.

PubMed

Gonzalez, J P; Camicas, J L; Cornet, J P; Faye, O; Wilson, M L

1992-01-01

Male Hyalomma truncatum ticks were inoculated with Crimean-Congo haemorrhagic fever (CCHF) virus, hypostomectomized and then allowed to mate with uninfected females feeding on a naive rabbit. After mating, CCHF virus was reisolated from 2 out of 3 males tested and from 4 of 6 mated, engorged females (titre greater than or equal to 2.2 log LD50/ml). Vertical transmission was then demonstrated by virus reisolation from a portion of 2 of the 6 batches of eggs laid by the positive females. From these 2 positive egg batches, 6 larvae pools were tested with successful virus reisolation from one. Attempts to reisolate CCHF virus from 15 nymph pools of this positive batch of larvae were unsuccessful. Virus reisolation from gonopore-closed female H. truncatum which cofed with preinfected males demonstrated transmission in the absence of copulation. Rabbits that served as bloodmeal sources seroconverted after infestation by infected male ticks. However, CCHF virus was not reisolated from 3 gonopore-closed, engorged females, nor from their eggs, after feeding with hypostomectomized preinfected males. Transmission of CCHF virus during mating or cofeeding of adult H. truncatum, and subsequent transovarial transmission, appear to represent additional mechanisms of infection in the tick population, and may contribute to the maintenance of transmission in nature.

Denitrifying capability and community dynamics of glycogen accumulating organisms during sludge granulation in an anaerobic-aerobic sequencing batch reactor

NASA Astrophysics Data System (ADS)

Bin, Zhang; Bin, Xue; Zhigang, Qiu; Zhiqiang, Chen; Junwen, Li; Taishi, Gong; Wenci, Zou; Jingfeng, Wang

2015-08-01

Denitrifying capability of glycogen accumulating organisms (GAOs) has received great attention in environmental science and microbial ecology. Combining this ability with granule processes would be an interesting attempt. Here, a laboratory-scale sequencing batch reactor (SBR) was operated to enrich GAOs and enable sludge granulation. The results showed that the GAO granules were cultivated successfully and the granules had denitrifying capability. The batch experiments demonstrated that all NO3--N could be removed or reduced, some amount of NO2--N were accumulated in the reactor, and N2 was the main gaseous product. SEM analysis suggested that the granules were tightly packed with a large amount of tetrad-forming organisms (TFOs); filamentous bacteria served as the supporting structures for the granules. The microbial community structure of GAO granules was differed substantially from the inoculant conventional activated sludge. Most of the bacteria in the seed sludge grouped with members of Proteobacterium. FISH analysis confirmed that GAOs were the predominant members in the granules and were distributed evenly throughout the granular space. In contrast, PAOs were severely inhibited. Overall, cultivation of the GAO granules and utilizing their denitrifying capability can provide us with a new approach of nitrogen removal and saving more energy.

Direct Antimicrobial Susceptibility Testing of Gram-Negative Bacilli in Blood Cultures by an Electrochemical Method

PubMed Central

Huang, Ay Huey; Wu, Jiunn Jong; Weng, Yu Mei; Ding, Hwia Cheng; Chang, Tsung Chain

1998-01-01

Nonfastidious aerobic gram-negative bacilli (GNB) are commonly isolated from blood cultures. The feasibility of using an electrochemical method for direct antimicrobial susceptibility testing of GNB in positive blood cultures was evaluated. An aliquot (10 μl) of 1:10-diluted positive blood cultures containing GNB was inoculated into the Bactometer module well (bioMérieux Vitek, Hazelwood, Mo.) containing 1 ml of Mueller-Hinton broth supplemented with an antibiotic. Susceptibility tests were performed in a breakpoint broth dilution format, with the results being categorized as resistant, intermediate, or susceptible. Seven antibiotics (ampicillin, cephalothin, gentamicin, amikacin, cefamandole, cefotaxime, and ciprofloxacin) were used in this study, with each agent being tested at the two interpretive breakpoint concentrations. The inoculated modules were incubated at 35°C, and the change in impedance in each well was continuously monitored for 24 h by the Bactometer. The MICs of the seven antibiotics for each blood isolate were also determined by the standardized broth microdilution method. Of 146 positive blood cultures (1,022 microorganism-antibiotic combinations) containing GNB tested by the direct method, the rates of very major, major, and minor errors were 0, 1.1, and 2.5%, respectively. The impedance method was simple; no centrifugation, preincubation, or standardization of the inocula was required, and the susceptibility results were normally available within 3 to 6 h after inoculation. The rapid method may allow proper antimicrobial treatment almost 30 to 40 h before the results of the standard methods are available. PMID:9738038

Optimization of the microbial synthesis of dihydroxyacetone from glycerol with Gluconobacter oxydans.

PubMed

Hekmat, D; Bauer, R; Fricke, J

2003-12-01

An optimized repeated-fed-batch fermentation process for the synthesis of dihydroxyacetone (DHA) from glycerol utilizing Gluconobacter oxydans is presented. Cleaning, sterilization, and inoculation procedures could be reduced significantly compared to the conventional fed-batch process. A stringent requirement was that the product concentration was kept below a critical threshold level at all times in order to avoid irreversible product inhibition of the cells. On the basis of experimentally validated model calculations, a threshold value of about 60 kg x m(-3) DHA was obtained. The innovative bioreactor system consisted of a stirred tank reactor combined with a packed trickle-bed column. In the packed column, active cells could be retained by in situ immobilization on a hydrophilized Ralu-ring carrier material. Within 17 days, the productivity of the process could be increased by 75% to about 2.8 kg x m(-3) h(-1). However, it was observed that the maximum achievable productivity had not been reached yet.

Characterization of bacterial isolates from rubber dump site and their use in biodegradation of isoprene in batch and continuous bioreactors.

PubMed

Srivastva, Navnita; Shukla, Awadhesh Kumar; Singh, Ram Sharan; Upadhyay, Siddh Nath; Dubey, Suresh Kumar

2015-01-01

Bacterial isolates from contaminated soil of a waste rubber dumping site were isolated and characterized using biochemical and molecular approaches. Isoprene degradation kinetics in batch mode (isoprene concentration: 100-1000 ppm) revealed the degradation efficiency of isolates as: Pseudomonas sp. (83%)>Alcaligenes sp. (70%)>Klebsiella sp. (68.5%). The most efficient isolate Pseudomonas sp. was finally inoculated in a specifically designed bioreactor system comprising a bioscrubber and a biofilter packed with polyurethane foam connected in series. The bioscrubber and biofilter units when operated in a series showed more than 90% removal efficiency up to the inlet loading rate (IL) of 371.1g/m(3)/h. Maximum elimination capacity (EC) of biofilter was found to be an order of magnitude greater than that for bioscrubber. Oxidative cleavage of the double bond of isoprene has been revealed through IR spectra of the leachate. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mass culture of photobacteria to obtain luciferase

NASA Technical Reports Server (NTRS)

Chappelle, E. W.; Picciolo, G. L.; Rich, E., Jr.

1969-01-01

Inoculating preheated trays containing nutrient agar with photobacteria provides a means for mass culture of aerobic microorganisms in order to obtain large quantities of luciferase. To determine optimum harvest time, growth can be monitored by automated light-detection instrumentation.

KINETICS OF CHROMATE REDUCTION DURING NAPHTHALENE DEGRADATION IN A MIXED CULTURE

EPA Science Inventory

A mixed culture of Bacillus sp. K1 and Sphingomonas paucimobilis EPA 505 was exposed to chromate and naphthalene. Batch experiments showed that chromate was reduced and naphthalene was degraded by the mixed culture. Chromate reduction occurred initially at a high rate followed by...

High cell density cultivation and recombinant protein production with Escherichia coli in a rocking-motion-type bioreactor

PubMed Central

2010-01-01

Background Single-use rocking-motion-type bag bioreactors provide advantages compared to standard stirred tank bioreactors by decreased contamination risks, reduction of cleaning and sterilization time, lower investment costs, and simple and cheaper validation. Currently, they are widely used for cell cultures although their use for small and medium scale production of recombinant proteins with microbial hosts might be very attractive. However, the utilization of rocking- or wave-induced motion-type bioreactors for fast growing aerobic microbes is limited because of their lower oxygen mass transfer rate. A conventional approach to reduce the oxygen demand of a culture is the fed-batch technology. New developments, such as the BIOSTAT® CultiBag RM system pave the way for applying advanced fed-batch control strategies also in rocking-motion-type bioreactors. Alternatively, internal substrate delivery systems such as EnBase® Flo provide an opportunity for adopting simple to use fed-batch-type strategies to shaken cultures. Here, we investigate the possibilities which both strategies offer in view of high cell density cultivation of E. coli and recombinant protein production. Results Cultivation of E. coli in the BIOSTAT® CultiBag RM system in a conventional batch mode without control yielded an optical density (OD600) of 3 to 4 which is comparable to shake flasks. The culture runs into oxygen limitation. In a glucose limited fed-batch culture with an exponential feed and oxygen pulsing, the culture grew fully aerobically to an OD600 of 60 (20 g L-1 cell dry weight). By the use of an internal controlled glucose delivery system, EnBase® Flo, OD600 of 30 (10 g L-1 cell dry weight) is obtained without the demand of computer controlled external nutrient supply. EnBase® Flo also worked well in the CultiBag RM system with a recombinant E. coli RB791 strain expressing a heterologous alcohol dehydrogenase (ADH) to very high levels, indicating that the enzyme based feed supply strategy functions well for recombinant protein production also in a rocking-motion-type bioreactor. Conclusions Rocking-motion-type bioreactors may provide an interesting alternative to standard cultivation in bioreactors for cultivation of bacteria and recombinant protein production. The BIOSTAT® Cultibag RM system with the single-use sensors and advanced control system paves the way for the fed-batch technology also to rocking-motion-type bioreactors. It is possible to reach cell densities which are far above shake flasks and typical for stirred tank reactors with the improved oxygen transfer rate. For more simple applications the EnBase® Flo method offers an easy and robust solution for rocking-motion-systems which do not have such advanced control possibilities. PMID:20509968

Production of 2,3-butanediol by a low-acid producing Klebsiella oxytoca NBRF4.

PubMed

Han, Sung-Hyuk; Lee, Jung-Eun; Park, Kyungmoon; Park, Yong-Cheol

2013-01-25

2,3-Butanediol (2,3-BDO) is a value-added chemical with great potential for the industrial production of synthetic rubber, plastic and solvent. For microbial production of 2,3-BDO, in this study, Klebsiella oxytoca NBRF4 was constructed by chemical mutation and screening against NaBr, NaBrO(3) and fluoroacetate. Among metabolic enzymes involved in the production of lactate, acetate and 2,3-BDO, K. oxytoca NBRF4 possessed 1.2 times lower specific activities of lactate dehydrogenase and phosphotransacetylase, and 22% higher specific acetoin reductase activity than the K. oxytoca ATCC43863 control strain. A series of batch fermentations in a defined medium and application of a statistical tool of response surface method led to the determination of optimal culture conditions: 10% dissolved oxygen level, pH 4.3 and 38°C. The actual results of batch fermentation at the optimal conditions using 44 g/L glucose were coincident with the predetermined values: 14.4 g/L 2,3-BDO concentration, 0.32 g/g yield. To increase 2,3-BDO titer, fed-batch fermentation of K. oxytoca NBRF4 was performed by an intermittent feeding of 800 g/L glucose to control its concentration around 5-20 g/L in the culture broth. Finally, 34.2g/L 2,3-BDO concentration and 0.35 g/g yield were obtained without organic acid production in 70 hours of the fed-batch culture, which were 2.4 and 1.2 times higher than those of the batch fermentation using 44 g/L glucose. Copyright © 2012 Elsevier B.V. All rights reserved.

Evaluation of BioFM liquid medium for culture of cerebrospinal fluid in tuberculous meningitis to identify Mycobacterium tuberculosis.

PubMed

Kashyap, R S; Ramteke, S S; Gaherwar, H M; Deshpande, P S; Purohit, H J; Taori, G M; Daginawala, H

2010-01-01

The present study was designed to evaluate the sensitivity and specificity of liquid culture medium (BioFM broth) for the diagnosis of tuberculous meningitis (TBM) in cerebrospinal fluid (CSF). CSF samples from 200 patients (TBM group = 150 and non-TBM group = 50) were tested for culture of Mycobacterium tuberculosis in BioFM liquid culture medium. Out of 150 TBM cases, 120 were found to be culture positive, indicating a sensitivity of 80% in BioFM broth within 2-3 weeks of inoculation. Positive cultures were also observed for CSF from 32 (64%) out of 50 non-TBM patients in BioFM liquid culture medium within 4 days of sample inoculation. Therefore, according to our study, BioFM broth system yielded 80% sensitivity [95% confidence interval (CI): 67-93%] and 36% specificity (95% CI: 57-98%) for TBM diagnosis. Our results indicate that although BioFM broth allows the detection of positive cultures within a shorter time, it has a high potential for contamination or for the coexistence of M. tuberculosis and non-tuberculous meningitis (NTM). This coexistence may go undetected or potentially lead to erroneous reporting of results.

Binding and detoxification of chlorpyrifos by lactic acid bacteria on rice straw silage fermentation.

PubMed

Wang, Yan-Su; Wu, Tian-Hao; Yang, Yao; Zhu, Cen-Ling; Ding, Cheng-Long; Dai, Chuan-Chao

2016-01-01

This investigation examined the reduction of pesticide residues on straw inoculated with lactic acid bacteria (LAB) during ensiling. Lactobacillus casei WYS3 was isolated from rice straw that contained pesticide residues. Non-sterilized rice straw, which was inoculated with L. casei WYS3, showed increased removal of chlorpyrifos after ensiling, compared with rice straw that was not inoculated with L. casei WYS3 or sterilized rice straw. In pure culture, these strains can bind chlorpyrifos as indicated by high-performance liquid chromatography analysis. Viable L. casei WYS3 was shown to bind 33.3-42% of exogenously added chlorpyrifos. These results are similar to those of acid-treated cells but less than those of heat-treated cells, which were found to bind 32.0% and 77.2% of the added chlorpyrifos respectively. Furthermore, gas chromatography-mass spectrometry analysis determined that L. casei WYS3 detoxified chlorpyrifos via P-O-C cleavage. Real-time polymerized chain reaction analysis determined that organophosphorus hydrolase gene expression tripled after the addition of chlorpyrifos to LAB cultures, compared with the control group (without chlorpyrifos). This paper highlights the potential use of LAB starter cultures for the detoxification and removal of chlorpyrifos residues in the environment.

Growth characteristics of a new methylomonad.

PubMed Central

Chen, B J; Hirt, W; Lim, H C; Tsao, G T

1977-01-01

A methylomonad culture was isolated from pond water and examined as a potential source of single-cell protein. A medium containing magnesium sulfate, ammonium hydroxide, sodium phosphate, tap water, and methanol supported the growth of the isolate. Optimal growth conditions in batch cultures for the organism were: temperature, 30 to 33 degrees C; pH 7.1; and phosphate concentration, 0.015 M. The minimum doubling time obtained was 1.6 h. The specific growth rate in batch culture was dependent on the methanol concentration, reaching a maximum around 0.2% (wt/vol). Growth inhibition was apparent above 0.3% (wt/vol), and growth was completely inhibited above 4.6% (wt/vol) methanol. Although the inhibitory effect of formaldehyde on the specific growth rate was much greater than that of formate, the organism utilized formaldehyde, but not formate, as a sole carbon and energy source in batch cultures. The isolate was identified primarily by its inability to utilize any carbon source other than methanol and formaldehyde for growth. Although it is capable of rapid growth on methanol, the organism showed a very weak catalase activity. The amino acid content of the cells compared favorably with the reference levels for the essential amino acids specific by the Food and Agricultural Organization of the United Nations. PMID:15510

Persistence of Escherichia coli O157:H7 during pilot-scale processing of iceberg lettuce using flume water containing peroxyacetic acid-based sanitizers and various organic loads.

PubMed

Davidson, Gordon R; Kaminski-Davidson, Chelsea N; Ryser, Elliot T

2017-05-02

In order to minimize cross-contamination during leafy green processing, chemical sanitizers are routinely added to the wash water. This study assessed the efficacy of peroxyacetic acid and mixed peracid against E. coli O157:H7 on iceberg lettuce, in wash water, and on equipment during simulated commercial production in a pilot-scale processing line using flume water containing various organic loads. Iceberg lettuce (5.4kg) inoculated to contain 10 6 CFU/g of a 4-strain cocktail of non-toxigenic, GFP-labeled, ampicillin-resistant E. coli O157:H7, was shredded using a commercial shredder, step-conveyed to a flume tank, washed for 90s using water alone or two different sanitizing treatments (50ppm peroxyacetic acid or mixed peracid) in water containing organic loads of 0, 2.5, 5 or 10% (w/v) blended iceberg lettuce, and then dried using a shaker table and centrifugal dryer. Thereafter, three 5.4-kg batches of uninoculated iceberg lettuce were identically processed. Various product (25g) and water (50ml) samples collected during processing along with equipment surface samples (100cm 2 ) from the flume tank, shaker table and centrifugal dryer were then assessed for numbers of E. coli O157:H7. Organic load rarely impacted (P>0.05) the efficacy of either peroxyacetic acid or mixed peracid, with typical reductions of >5logCFU/ml in wash water throughout processing for all organic loads. Increases in organic load in the wash water corresponded to changes in total solids, chemical oxygen demand, turbidity, maximum filterable volume, and oxidation/reduction potential. After 90s of exposure to flume water, E. coli O157:H7 reductions on inoculated lettuce ranged from 0.97 to 1.74logCFU/g using peroxyacetic acid, with an average reduction of 1.35logCFU/g for mixed peracid. E. coli O157:H7 persisted on all previously uninoculated lettuce following the inoculated batch, emphasizing the need for improved intervention strategies that can better ensure end-product safety. Copyright © 2017 Elsevier B.V. All rights reserved.

Amino acid and glucose metabolism in fed-batch CHO cell culture affects antibody production and glycosylation.

PubMed

Fan, Yuzhou; Jimenez Del Val, Ioscani; Müller, Christian; Wagtberg Sen, Jette; Rasmussen, Søren Kofoed; Kontoravdi, Cleo; Weilguny, Dietmar; Andersen, Mikael Rørdam

2015-03-01

Fed-batch Chinese hamster ovary (CHO) cell culture is the most commonly used process for IgG production in the biopharmaceutical industry. Amino acid and glucose consumption, cell growth, metabolism, antibody titer, and N-glycosylation patterns are always the major concerns during upstream process optimization, especially media optimization. Gaining knowledge on their interrelations could provide insight for obtaining higher immunoglobulin G (IgG) titer and better controlling glycosylation-related product quality. In this work, different fed-batch processes with two chemically defined proprietary media and feeds were studied using two IgG-producing cell lines. Our results indicate that the balance of glucose and amino acid concentration in the culture is important for cell growth, IgG titer and N-glycosylation. Accordingly, the ideal fate of glucose and amino acids in the culture could be mainly towards energy and recombinant product, respectively. Accumulation of by-products such as NH4(+) and lactate as a consequence of unbalanced nutrient supply to cell activities inhibits cell growth. The levels of Leu and Arg in the culture, which relate to cell growth and IgG productivity, need to be well controlled. Amino acids with the highest consumption rates correlate with the most abundant amino acids present in the produced IgG, and thus require sufficient availability during culture. Case-by-case analysis is necessary for understanding the effect of media and process optimization on glycosylation. We found that in certain cases the presence of Man5 glycan can be linked to limitation of UDP-GlcNAc biosynthesis as a result of insufficient extracellular Gln. However, under different culture conditions, high Man5 levels can also result from low α-1,3-mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase (GnTI) and UDP-GlcNAc transporter activities, which may be attributed to high level of NH4+ in the cell culture. Furthermore, galactosylation of the mAb Fc glycans was found to be limited by UDP-Gal biosynthesis, which was observed to be both cell line and cultivation condition-dependent. Extracellular glucose and glutamine concentrations and uptake rates were positively correlated with intracellular UDP-Gal availability. All these findings are important for optimization of fed-batch culture for improving IgG production and directing glycosylation quality. © 2014 Wiley Periodicals, Inc.

Effects of freezing, drying, ultraviolet irradiation, chlorine, and quaternary ammonium treatments on the infectivity of myxospores of Myxobolus cerebralis for Tubifex tubifex.

PubMed

Hedrick, Ronald P; McDowell, Terry S; Mukkatira, Kaveramma; MacConnell, Elizabeth; Petri, Brian

2008-06-01

The effects of freezing, drying, ultraviolet irradiation (UV), chlorine, and a quaternary ammonium compound on the infectivity of the myxospore stage of Myxobolus cerebralis (the causative agent of whirling disease) for Tubifex tubifex were examined in a series of laboratory trials. Freezing at either -20 degrees C or -80 degrees C for a period of 7 d or 2 months eliminated infectivity as assessed by the absence of production of the actinospore stage (triactinomyxons [TAMs]) from T. tubifex cultures inoculated with treated myxospores over a 4-5-month period. Myxospores retained infectivity when held in well water at 5 degrees C or 22 degrees C for 7 d and when held at 4 degrees C or 10 degrees C d for 2 months. In contrast, no TAMs were produced from T. tubifex cultures inoculated with myxospores held at 20 degrees C for 2 months. Drying of myxospores eliminated any evidence of infectivity for T. tubifex. Doses of UV from 40 to 480 mJ/cm2 were all effective for inactivating myxospores of M. cerebralis, although a few TAMs were detected in one replicate T. tubifex culture at 240 mJ/cm2 and in one replicate culture at 480 mJ/cm2. Treatments of myxospores with chlorine bleach at active concentrations of at least 500 mg/L for 15 min largely inactivated myxospore infectivity for T. tubifex. Likewise, there was no evidence of TAMs produced by T. tubifex inoculated with myxospores treated with alkyl dimethyl benzyl ammonium chloride (ADBAC) at 1,500 mg/L for 10 min. Treatments of myxospores with 1,000-mg/L ADBAC for 10 min reduced TAM production in T. tubifex cultures sevenfold relative to that in cultures inoculated with an equal number of untreated myxospores. These results indicate that myxospores of M. cerebralis demonstrate a selective rather than broad resistance to selected physical and chemical treatments, and this selective resistance is consistent with conditions that myxospores are likely to experience in nature.

Use of muscovy duck embryo fibroblasts for the isolation of viruses from wild birds

USGS Publications Warehouse

Docherty, D.E.; Slota, Paul G.

1988-01-01

Techniques are described for the preparation, cryopreservation, and inoculation of Muscovy duck embryo cell cultures. The procedure yields a susceptible reproducible cell culture system for the isolation and cultivation of viruses from wild birds.

Comparative performance of three sampling techniques to detect airborne Salmonella species in poultry farms.

PubMed

Adell, Elisa; Moset, Verónica; Zhao, Yang; Jiménez-Belenguer, Ana; Cerisuelo, Alba; Cambra-López, María

2014-01-01

Sampling techniques to detect airborne Salmonella species (spp.) in two pilot scale broiler houses were compared. Broilers were inoculated at seven days of age with a marked strain of Salmonella enteritidis. The rearing cycle lasted 42 days during the summer. Airborne Salmonella spp. were sampled weekly using impaction, gravitational settling, and impingement techniques. Additionally, Salmonella spp. were sampled on feeders, drinkers, walls, and in the litter. Environmental conditions (temperature, relative humidity, and airborne particulate matter (PM) concentration) were monitored during the rearing cycle. The presence of Salmonella spp. was determined by culture-dependent and molecular methods. No cultivable Salmonella spp. were recovered from the poultry houses' surfaces, the litter, or the air before inoculation. After inoculation, cultivable Salmonella spp. were recovered from the surfaces and in the litter. Airborne cultivable Salmonella spp. Were detected using impaction and gravitational settling one or two weeks after the detection of Salmonella spp. in the litter. No cultivable Salmonella spp. were recovered using impingement based on culture-dependent techniques. At low airborne concentrations, the use of impingement for the quantification or detection of cultivable airborne Salmonella spp. is not recommended. In these cases, a combination of culture-dependent and culture-independent methods is recommended. These data are valuable to improve current measures to control the transmission of pathogens in livestock environments and for optimising the sampling and detection of airborne Salmonella spp. in practical conditions.

Prescreening slash pine and Cronartium pedigrees for evaluation of complementary gene action in fusiform rust disease.

Treesearch

H.E. Stelzer; Robert L. Doudrick; Thomas L. Kubisiak; C. Dana Nelson

1999-01-01

Single-urediniospore cultures of the fusiform rust fungus were used to inoculate seedlings from 10 full-sib families of a five-parent slash pine diallel at two different times in 1994. The presence or absence of fusiform rust galls was recorded for each inoculated seedling at 9 months postinoculation, and percent infection levels for each family-inoculum-time...

Bioaugmentation for Groundwater Remediation

DTIC Science & Technology

2010-02-01

continually grown and harvested , in fed batch mode where a culture is grown in a vessel and harvested and then another culture begun, or in a hybrid of...the two whereby the culture is maintained in a fermentor until a volume of culture is harvested and then replaced with fresh medium. The primary...producing cultures for bioaugmentation is a semi- continuous process whereby the culture is maintained in the fermentor until needed and then harvested

Improving cellulase productivity of Penicillium oxalicum RE-10 by repeated fed-batch fermentation strategy.

PubMed

Han, Xiaolong; Song, Wenxia; Liu, Guodong; Li, Zhonghai; Yang, Piao; Qu, Yinbo

2017-03-01

Medium optimization and repeated fed-batch fermentation were performed to improve the cellulase productivity by P. oxalicum RE-10 in submerged fermentation. First, Plackett-Burman design (PBD) and central composite design (CCD) were used to optimize the medium for cellulase production. PBD demonstrated wheat bran and NaNO 3 had significant influences on cellulase production. The CCD results showed the maximum filter paper activity (FPA) production of 8.61U/mL could be achieved in Erlenmeyer flasks. The maximal FPA reached 12.69U/mL by submerged batch fermentation in a 7.5-L stirred tank, 1.76-fold higher than that on the original medium. Then, the repeated fed-batch fermentation strategy was performed successfully for increasing the cellulase productivity from 105.75U/L/h in batch fermentation to 158.38U/L/h. The cellulase activity and the glucan conversion of delignined corn cob residue hydrolysis had no significant difference between the enzymes sampled from different cycles of the repeated fed-batch fermentation and that from batch culture. Copyright © 2016 Elsevier Ltd. All rights reserved.

Microorganisms with a Taste for Vanilla: Microbial Ecology of Traditional Indonesian Vanilla Curing

PubMed Central

Röling, Wilfred F. M.; Kerler, Josef; Braster, Martin; Apriyantono, Anton; Stam, Hein; van Verseveld, Henk W.

2001-01-01

The microbial ecology of traditional postharvesting processing of vanilla beans (curing) was examined using a polyphasic approach consisting of conventional cultivation, substrate utilization-based and molecular identification of isolates, and cultivation-independent community profiling by 16S ribosomal DNA based PCR-denaturing gradient gel electrophoresis. At two different locations, a batch of curing beans was monitored. In both batches a major shift in microbial communities occurred after short-term scalding of the beans in hot water. Fungi and yeast disappeared, although regrowth of fungi occurred in one batch during a period in which process conditions were temporarily not optimal. Conventional plating showed that microbial communities consisting of thermophilic and thermotolerant bacilli (mainly closely related to Bacillus subtilis, B. licheniformis,, and B. smithii) developed under the high temperatures (up to 65°C) that were maintained for over a week after scalding. Only small changes in the communities of culturable bacteria occurred after this period. Molecular analysis revealed that a proportion of the microbial communities could not be cultured on conventional agar medium, especially during the high-temperature period. Large differences between both batches were observed in the numbers of microorganisms, in species composition, and in the enzymatic abilities of isolated bacteria. These large differences indicate that the effects of microbial activities on the development of vanilla flavor could be different for each batch of cured vanilla beans. PMID:11319073

Variations in virulence of avian pathogenic Escherichia coli demonstrated by the use of a new in vivo infection model.

PubMed

Pors, Susanne Elisabeth; Olsen, Rikke Heidemann; Christensen, Jens Peter

2014-06-04

Salpingitis and peritonitis are common pathological manifestations observed in egg-laying hens. To improve methods to study these conditions, a surgical model was developed. Initially, eighteen white layers underwent laparotomy with subsequent inoculation of ink, bacteria or sterile broth directly into the oviduct. Eight birds inoculated with 0.1 ml blue ink were euthanized immediately after inoculation and the specific site of inoculation was assessed. In all birds, ink was injected into the oviduct between five and seven cm cranial to the isthmus. To demonstrate the use of this approach to cause infection of the oviduct, five birds were inoculated with 8.6 × 10(6)CFU of a clinical Escherichia coli isolate. Five control birds received broth with no bacteria. Both infected and control birds were euthanized after 48 h followed by a post mortem examination. Infected birds showed diffuse fibrino-purulent peritonitis, E. coli was found in pure culture from one or more positions in the oviduct and the liver. Birds receiving sterile broth did not culture positive and demonstrated no gross lesions. Subsequently, 19 birds were inoculated with an isolate of E. coli ST95 and 20 birds with an isolate of E. coli ST141. Major variation in virulence was observed between the two isolates used in relation to clinical signs, gross lesions and histopathology. In contrast to E. coli ST141, E. coli ST95 caused severe clinical signs, epithelial necrosis of the oviduct and purulent salpingitis. The results of the study show the potential of the model in studies of the pathogenesis of infections and virulence of bacteria of the oviduct. Copyright © 2014 Elsevier B.V. All rights reserved.

Efficient arachidonic acid-rich oil production by Mortierella alpina through a repeated fed-batch fermentation strategy.

PubMed

Ji, Xiao-Jun; Zhang, Ai-Hui; Nie, Zhi-Kui; Wu, Wen-Jia; Ren, Lu-Jing; Huang, He

2014-10-01

Arachidonic acid (ARA)-rich oil production by Mortierella alpina is a long fermentation period needed process due to the low growth rate of the filamentous fungus used. This causes the low productivity of ARA-rich oil and hinders its industrial mass scale production. In the present study, different fed-batch strategies were conducted to shorten the fermentation period. The result showed that compared with the batch culture, the fermentation period was shortened from 7days to 5days with the productivity of ARA-rich oil increased from 0.9g/(L·d) to 1.3g/(L·d) by using the fed-batch fermentation strategy. Furthermore, repeated fed-batch fermentation strategy was adopted to achieve the purpose of continuous production. By using this strategy, the fermentation period was shortened from 40days to 26days in a four cycle repeated fed-batch fermentation. This strategy proved to be convenient and economical for ARA-rich oil commercial production process. Copyright © 2014 Elsevier Ltd. All rights reserved.

Lipid Content and Cryotolerance of Bakers' Yeast in Frozen Doughs †

PubMed Central

Gélinas, Pierre; Fiset, Gisèle; Willemot, Claude; Goulet, Jacques

1991-01-01

The relationship between lipid content and tolerance to freezing at −50°C was studied in Saccharomyces cerevisiae grown under batch or fed-batch mode and various aeration and temperature conditions. A higher free-sterol-to-phospholipid ratio as well as higher free sterol and phospholipid contents correlated with the superior cryoresistance in dough or in water of the fed-batch-grown compared with the batch-grown cells. For both growth modes, the presence of excess dissolved oxygen in the culture medium greatly improved yeast cryoresistance and trehalose content (P. Gélinas, G. Fiset, A. LeDuy, and J. Goulet, Appl. Environ. Microbiol. 26:2453-2459, 1989) without significantly changing the lipid profile. Under the batch or fed-batch modes, no correlation was found between the cryotolerance of bakers' yeast and the total cellular lipid content, the total sterol content, the phospholipid unsaturation index, the phosphate or crude protein content, or the yeast cell morphology (volume and roundness). PMID:16348412

A novel model of invasive fungal rhinosinusitis in rats.

PubMed

Zhang, Fang; An, Yunfang; Li, Zeqing; Zhao, Changqing

2013-01-01

Invasive fungal rhinosinusitis (IFRS) is a life-threatening inflammatory disease that affects immunocompromised patients, but animal models of the disease are scarce. This study aimed to develop an IFRS model in neutropenic rats. The model was established in three consecutive steps: unilateral nasal obstruction with Merocel sponges, followed by administration of cyclophosphamide (CPA), and, finally, nasal inoculation with Aspergillus fumigatus. Fifty healthy Wistar rats were randomly divided into five groups, with group I as the controls, group II undergoing unilateral nasal obstruction alone, group III undergoing nasal obstruction with fungal inoculation, group IV undergoing nasal obstruction with administration of CPA, and group V undergoing nasal obstruction with administration of CPA and fungal inoculation. Hematology, histology, and mycology investigations were performed. The changes in the rat absolute neutrophil counts (ANCs) were statistically different across the groups. The administration of CPA decreased the ANCs, whereas nasal obstruction with fungal inoculation increased the ANCs, and nasal obstruction did not change them. Histological examination of the rats in group V revealed the hyphal invasion of sinus mucosa and bone, thrombosis, and tissue infarction. No pathology indicative of IFRS was observed in the remaining groups. Positive rates of fungal culture in tissue homogenates from the maxillary sinus (62.5%) and lung (25%) were found in group V, whereas groups I, II, III, and IV showed no fungal culture in the homogenates. A rat IFRS model was successfully developed through nasal obstruction, CPA-induced neutropenia, and fungal inoculation. The disease model closely mimics the pathophysiology of anthropic IFRS.

Optimizing N-Fixing cyanobacteria culture to restore arid degraded soils

NASA Astrophysics Data System (ADS)

Roncero-Ramos, Beatriz; Román, Raúl; Gómez, Cintia; Chamizo, Sonia; Rodriguez-Caballero, Emilio; Cantón, Yolanda

2017-04-01

Cyanobacteria present several metabolic activities and mechanisms of adaptation which enable them to colonize different habitats, in almost all biome and continents, especially under extreme environmental conditions, as on the surface of the most arid soils and under the highest temperatures. In drylands, they are usually found among plants, cohabiting with organisms such as algae, lichens, mosses, bacteria and fungi, and in association with soil surface particles, forming communities known as biocrusts. Because they can survive under water stress and are considered ecosystem engineers, facilitating the establishment of other organisms, they can play a key role in the development of a successful restoration approach to recover the functionality of soils in arid and semiarid regions. In addition cyanobacteria can be cultured "ex-situ" obtaining high quantities of biomass to be used as soil inoculum at large scale. For these reasons, the inoculation of degrades soils with cyanobacteria can be considered an alternative to traditional restoration. This approach is expected to promote: the stabilization of the soil surface and the decrease of water and wind erosion; the increase of soil fertility by fixing N and C; and the succession of more developed organisms as mosses or vascular and annual plants. The objectives were: to evaluate the potential of a soil native cyanobacteria strain to be artificially cultured and the optimization of the process, and to analyze the effects of the inoculation of the biomass on soil under laboratory conditions. Cyanobacteria were isolated from biocrusts sampled on a limestone quarry located at the southeastern edge of the Sierra de Gádor massif (Spain). It was genetically and morphological identified as belonging to the nitrogen-fixing genera Nostoc. Essays were accomplished in bubble columns reactors (0.25 L), using different culture media: BG11+N, BG110, and two media made with fertilizers. Illumination simulated a circadian cycle with a maximum irradiance of 1035 μE m-2s-1. Absorbance, chlorophyll fluorescence and dry weight were measured daily. The produced biomass was inoculated (6 g m-2) on Petri dishes with 80g of sterilized soil coming from the limestone quarry. Soils were watered below field capacity twice a week during three months, under constant illumination of 70 μE m-2s-1 and 25oC. The growth rate and biomass productivity obtained for each culture media verified that this strain can be successfully cultured under laboratory conditions. The best results were obtained with BG11+N, nevertheless results from media made with fertilizers were very similar, key to develop a low-cost culture strategy. The culture process has been optimized simulating a continuous mode, in order to produce biomass on a large scale, obtaining an optimal productivity of 0.41 g L-1 d-1, with a dilution rate of 20% and a concentration of 2.06 g L-1. Soils inoculated with cyanobacteria biomass obtained from laboratory culture showed an increase in biocrust cover and soil organic carbon content with time. Thus our results demonstrate that inoculation with native cyanobacteria cultured "ex- situ" represent a very promising and "low-cost" tool for the restoration of arid degraded soils.

Improved Diagnosis of Prosthetic Joint Infection by Culturing Periprosthetic Tissue Specimens in Blood Culture Bottles.

PubMed

Peel, Trisha N; Dylla, Brenda L; Hughes, John G; Lynch, David T; Greenwood-Quaintance, Kerryl E; Cheng, Allen C; Mandrekar, Jayawant N; Patel, Robin

2016-01-05

Despite known low sensitivity, culture of periprosthetic tissue specimens on agars and in broths is routine. Culture of periprosthetic tissue samples in blood culture bottles (BCBs) is potentially more convenient, but it has been evaluated in a limited way and has not been widely adopted. The aim of this study was to compare the sensitivity and specificity of inoculation of periprosthetic tissue specimens into blood culture bottles with standard agar and thioglycolate broth culture, applying Bayesian latent class modeling (LCM) in addition to applying the Infectious Diseases Society of America (IDSA) criteria for prosthetic joint infection. This prospective cohort study was conducted over a 9-month period (August 2013 to April 2014) at the Mayo Clinic, Rochester, MN, and included all consecutive patients undergoing revision arthroplasty. Overall, 369 subjects were studied; 117 (32%) met IDSA criteria for prosthetic joint infection, and 82% had late chronic infection. Applying LCM, inoculation of tissues into BCBs was associated with a 47% improvement in sensitivity compared to the sensitivity of conventional agar and broth cultures (92.1 versus 62.6%, respectively); this magnitude of change was similar when IDSA criteria were applied (60.7 versus 44.4%, respectively; P = 0.003). The time to microorganism detection was shorter with BCBs than with standard media (P < 0.0001), with aerobic and anaerobic BCBs yielding positive results within a median of 21 and 23 h, respectively. Results of our study demonstrate that the semiautomated method of periprosthetic tissue culture in blood culture bottles is more sensitive than and as specific as agar and thioglycolate broth cultures and yields results faster. Prosthetic joint infections are a devastating complication of arthroplasty surgery. Despite this, current microbiological techniques to detect and diagnose infections are imperfect. This study examined a new approach to diagnosing infections, through the inoculation of tissue samples from around the prosthetic joint into blood culture bottles. This study demonstrated that, compared to current laboratory practices, this new technique increased the detection of infection. These findings are important for patient care to allow timely and accurate diagnosis of infection. Copyright © 2016 Peel et al.

Perchlorate remediation using packed-bed bioreactors and electricity generation in microbial fuel cells (MFCs)

NASA Astrophysics Data System (ADS)

Min, Booki

Two pilot-scale fixed bed bioreactors were operated in continuous mode in order to treat groundwater contaminated by perchlorate. The bioreactors were constructed and operated side-by-side at the Texas Street Well Facility in Redlands, California. Each reactor was packed with either sand or plastic media. A perchlorate-reducing bacterium, Dechlorosoma sp. KJ, was used to inoculate the bioreactors. Perchlorate was successfully removed down to a non-detectable level (<4mug/L) in both bioreactors with acetate as a carbon source and nutrients at loading rates less than 0.063 L/s (1 gpm; 0.34 L/m2s). The sand medium bioreactor could achieve complete-perchlorate removal up to flow rate of 0.126 L/s. A regular backwashing cycle (once a week) was an important factor for completely removing perchlorate in groundwater. Power generation directly from pure or mixed organic matter was examined using microbial fuel cells (MFCs), which were run either in batch or continuous mode. In batch experiments, both a pure culture (Geobactor metallireducens) and a mixed culture (wastewater inoculum) were used as the biocatalyst, and acetate was added as substrate in the anode chamber of the MFC. Power output in a membrane MFC with either inoculum was essentially the same, with 40 +/- 1 mW/m2 for G. metallireducens and 38 +/- 1 mW/m2 for mixed culture. A different type of the MFC containing a salt bridge instead of a membrane system was examined to generate power using the same substrate and pure culture as used in the membrane MFC. Power output in the salt bridge MFC was 2.2 mW/m 2. It was found that the lower power output was directly attributed to the higher internal resistance of the salt bridge system (19920 +/- 50 O) in comparison with that of the membrane system (1286 +/- 1 O). Continuous electricity generation was examined in a flat plate microbial fuel cell (FPMFC) using domestic wastewater and specific organic substrates. The FPMFC, containing a combined electrode/proton exchange membrane (PEM), was initially acclimated for one month to domestic wastewater, and then was operated as a plug flow reactor system. Power density using domestic wastewater as a substrate was 72 +/- 1 mW/m2 at a liquid flow rate of 0.39 mL/min (1.1 hr hydraulic retention time, HRT), and COD removal was 42%. At a longer HRT of 4.0 hr, the COD removal increased to 79%, and power density was 43 mW/m2. Several organic compounds (about 1000 mg-COD/L) also generated high power densities including: glucose (212 +/- 2 mW/m2), acetate (286 +/- 3 mW/m2), butyrate (220 +/- 1 mW/m2), dextran (150 +/- 1 mW/m 2), and starch (242 +/- 3 mW/m2). Therefore, it was shown that power could be successfully generated in a continuous-mode MFC with a variety of organic substrates. Animal wastewater was also tested as substrate to generate power in an air-cathode single chamber MFC operated in batch mode. This preliminary experiment demonstrated that power generation could be sustained with animal wastewater and that wastewater strength and odors were substantially reduced in the reactor after only one day of operation.

Analysis of the Anti-Cancer Effects of Cincau Extract (Premna oblongifolia Merr) and Other Types of Non-Digestible Fibre Using Faecal Fermentation Supernatants and Caco-2 Cells as a Model of the Human Colon.

PubMed

Nurdin, Samsu U; Le Leu, Richard K; Young, Graeme P; Stangoulis, James C R; Christophersen, Claus T; Abbott, Catherine A

2017-04-03

Green cincau ( Premna oblongifolia Merr) is an Indonesian food plant with a high dietary fibre content. Research has shown that dietary fibre mixtures may be more beneficial for colorectal cancer prevention than a single dietary fibre type. The aim of this study was to investigate the effects of green cincau extract on short chain fatty acid (SCFA) production in anaerobic batch cultures inoculated with human faecal slurries and to compare these to results obtained using different dietary fibre types (pectin, inulin, and cellulose), singly and in combination. Furthermore, fermentation supernatants (FSs) were evaluated in Caco-2 cells for their effect on cell viability, differentiation, and apoptosis. Cincau increased total SCFA concentration by increasing acetate and propionate, but not butyrate concentration. FSs from all dietary fibre sources, including cincau, reduced Caco-2 cell viability. However, the effects of all FSs on cell viability, cell differentiation, and apoptosis were not simply explainable by their butyrate content. In conclusion, products of fermentation of cincau extracts induced cell death, but further work is required to understand the mechanism of action. This study demonstrates for the first time that this Indonesian traditional source of dietary fibre may be protective against colorectal cancer.

Multisubstrate biodegradation kinetics of naphthalene, phenanthrene, and pyrene mixtures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guha, S.; Peters, C.A.; Jaffe, P.R.

Biodegradation kinetics of naphthalene, phenanthrene and pyrene were studied in sole-substrate systems, and in binary and ternary mixtures to examine substrate interactions. The experiments were conducted in aerobic batch aqueous systems inoculated with a mixed culture that had been isolated from soils contaminated with polycyclic aromatic hydrocarbons (PAHs). Monod kinetic parameters and yield coefficients for the individual parameters and yield coefficients for the individual compounds were estimated from substrate depletion and CO{sub 2} evolution rate data in sole-substrate experiments. In all three binary mixture experiments, biodegradation kinetics were comparable to the sole-substrate kinetics. In the ternary mixture, biodegradation of naphthalenemore » was inhibited and the biodegradation rates of phenanthrene and pyrene were enhanced. A multisubstrate form of the Monod kinetic model was found to adequately predict substrate interactions in the binary and ternary mixtures using only the parameters derived from sole-substrate experiments. Numerical simulations of biomass growth kinetics explain the observed range of behaviors in PAH mixtures. In general, the biodegradation rates of the more degradable and abundant compounds are reduced due to competitive inhibition, but enhanced biodegradation of the more recalcitrant PAHs occurs due to simultaneous biomass growth on multiple substrates. In PAH-contaminated environments, substrate interactions may be very large due to additive effects from the large number of compounds present.« less

Biocontrol of Ralstonia solanacearum by Treatment with Lytic Bacteriophages ▿ †

PubMed Central

Fujiwara, Akiko; Fujisawa, Mariko; Hamasaki, Ryosuke; Kawasaki, Takeru; Fujie, Makoto; Yamada, Takashi

2011-01-01

Ralstonia solanacearum is a Gram-negative bacterium and the causative agent of bacterial wilt in many important crops. We treated R. solanacearum with three lytic phages: φRSA1, φRSB1, and φRSL1. Infection with φRSA1 and φRSB1, either alone or in combination with the other phages, resulted in a rapid decrease in the host bacterial cell density. Cells that were resistant to infection by these phages became evident approximately 30 h after phage addition to the culture. On the other hand, cells infected solely with φRSL1 in a batch culture were maintained at a lower cell density (1/3 of control) over a long period. Pretreatment of tomato seedlings with φRSL1 drastically limited penetration, growth, and movement of root-inoculated bacterial cells. All φRSL1-treated tomato plants showed no symptoms of wilting during the experimental period, whereas all untreated plants had wilted by 18 days postinfection. φRSL1 was shown to be relatively stable in soil, especially at higher temperatures (37 to 50°C). Active φRSL1 particles were recovered from the roots of treated plants and from soil 4 months postinfection. Based on these observations, we propose an alternative biocontrol method using a unique phage, such as φRSL1, instead of a phage cocktail with highly virulent phages. Using this method, φRSL1 killed some but not all bacterial cells. The coexistence of bacterial cells and the phage resulted in effective prevention of wilting. PMID:21498752

Cultivation characteristics of immobilized Aspergillus oryzae for kojic acid production.

PubMed

Kwak, M Y; Rhee, J S

1992-04-15

Aspergillus oryzae in situ grown from spores entrapped in calcium alginate gel beads was used for the production of kojic acid. The immobilized cells in flask cultures produced kojic acid in a linear proportion while maintaining the stable metabolic activity for a prolonged production period. Kojic acid was accumulated up to a high concentration of 83 g/L, at which the kojic acid began to crystallize, and, thus, the culture had to be replaced with fresh media for the next batch culture. The overall productivities of two consecutive cultivations were higher than that of free mycelial fermentation. However, the production rate of kojic acid by the immobilized cells was suddenly decreased with the appearance of central cavernae inside the immobilized gel beads after 12 days of the third batch cultivation.

Combined Effects of Inoculation Level and Sequence on Biochemical and Microbiological Characteristics of Probiotic Doogh

PubMed Central

Rahmati Roudsari, Mohammad; Sohrabvandi, Sara; Homayouni Rad, Aziz; Mortazavian, Amir Mohammad

2013-01-01

The combined effects of inoculation level (4 or 8-fold compared to standard inoculation) and sequence (standard inoculation before fermentation and 3-fold inoculation at the end of fermentation=1+3, Two-fold inoculation before fermentation and the same at the end of fermentation=2+2, 2+6, 4-fold before fermentation=4, 4+4, and 8) of culture inoculum containing probiotics on biochemical and microbiological characteristics of probiotic Doogh during fermentation and over 21 days of refrigerated storage (4˚C) were investigated. The probiotic microorganisms were L. acidophilus LA-5 and Bifidobacterium lactis BB-12. Overall, the treatments 8, 4 and 4+4 resulted in the highest viability at the end of fermentation as well as at early days of refrigerated storage. During the second half of cold storage period, the greatest viability of probiotics was related to the treatment 2+6. The treatment ‘8’ showed the shortest incubation time as well as the highest pH drop rate and acidity increase rate during fermentation and over the storage period. PMID:24250636

Expression of a mutated SPT15 gene in Saccharomyces cerevisiae enhances both cell growth and ethanol production in microaerobic batch, fed-batch, and simultaneous saccharification and fermentations.

PubMed

Seong, Yeong-Je; Park, Haeseong; Yang, Jungwoo; Kim, Soo-Jung; Choi, Wonja; Kim, Kyoung Heon; Park, Yong-Cheol

2017-05-01

The SPT15 gene encodes a Saccharomyces cerevisiae TATA-binding protein, which is able to globally control the transcription levels of various metabolic and regulatory genes. In this study, a SPT15 gene mutant (S42N, S78R, S163P, and I212N) was expressed in S. cerevisiae BY4741 (BSPT15-M3), of which effects on fermentative yeast properties were evaluated in a series of culture types. By applying different nitrogen sources and air supply conditions in batch culture, organic nitrogen sources and microaerobic condition were decided to be more favorable for both cell growth and ethanol production of the BSPT15-M3 strain than the control S. cerevisiae BY4741 strain expressing the SPT15 gene (BSPT15wt). Microaerobic fed-batch cultures of BSPT15-M3 with glucose shock in the presence of high ethanol content resulted in a 9.5-13.4% higher glucose consumption rate and ethanol productivity than those for the BSPT15wt strain. In addition, BSPT15-M3 showed 4.5 and 3.9% increases in ethanol productivity from cassava hydrolysates and corn starch in simultaneous saccharification and fermentation processes, respectively. It was concluded that overexpression of the mutated SPT15 gene would be a potent strategy to develop robust S. cerevisiae strains with enhanced cell growth and ethanol production abilities.

Effects of Cultivating Conditions on the Water Soluble Polysaccharides Content of Ganoderma lucidum Mycelium in Submerged Flask Culture

NASA Astrophysics Data System (ADS)

Rosyida, V. T.; Hayati, S. N.; Apriyana, W.; Darsih, C.; Hernawan; Poeloengasih, C. D.

2017-12-01

The carcinostatic substance in Ganoderma lucidum (Fr.) Karst (Polyporaceae) is a water soluble polysaccharides (WSP) which might be useful in immunotherapy. Attempt to produce effective substances from cultured mycelia is important to carry out since solid cultivation is a time consuming and quality fluctuating. The effects of cultivating conditions on the water soluble polysaccharides content of G. Lucidum mycelium were investigated in submerged flask cultures. Culture from fruiting bodies was maintained on potato dextrose-agar slope. Slopes were inoculated and incubated at 30°C for 7 days, and stored at 4°C. The flask experiments were performed in 100 ml erlenmeyer flasks containing 20 ml of the sterilized media. Actively growing mycelia (1 piece, 5 mm X 5 mm) from a newly prepared slant culture (about 7 days incubation at 30°C) were inoculated into the flask. The pH was measured and adjusted to the desired value by addition of either 4 M HCl or 2.5 M NaOH. Incubation temperature were 20, 25, and 30°C. At the end of inoculation period (14 days) mycelium consisting of individual pellets was harvested and wash for the analysis. WSP content was analysed using phenol-sulfuric acid method. The optimal initial pH for metabolite production would depend on the culture medium. Generally, high values of pH, such as 9, negatively affect both cell growth and WSP production. The optimum temperature range for the high G. lucidum mycelium and WSP production were found to be 25 - 30 °C at pH values 5 - 7 in both of media.

Optimal quality control of bakers' yeast fed-batch culture using population dynamics.

PubMed

Dairaku, K; Izumoto, E; Morikawa, H; Shioya, S; Takamatsu, T

1982-12-01

An optimal quality control policy for the overall specific growth rate of bakers' yeast, which maximizes the fermentative activity in the making of bread, was obtained by direct searching based on the mathematical model proposed previously. The mathematical model had described the age distribution of bakers' yeast which had an essential relationship to the ability of fermentation in the making of bread. The mathematical model is a simple aging model with two periods: Nonbudding and budding. Based on the result obtained by direct searching, the quality control of bakers' yeast fed-batch culture was performed and confirmed to be experimentally valid.

Improved kinetic model of Escherichia coli central carbon metabolism in batch and continuous cultures.

PubMed

Kurata, Hiroyuki; Sugimoto, Yurie

2018-02-01

Many kinetic models of Escherichia coli central metabolism have been built, but few models accurately reproduced the dynamic behaviors of wild type and multiple genetic mutants. In 2016, our latest kinetic model improved problems of existing models to reproduce the cell growth and glucose uptake of wild type, ΔpykA:pykF and Δpgi in a batch culture, while it overestimated the glucose uptake and cell growth rates of Δppc and hardly captured the typical characteristics of the glyoxylate and TCA cycle fluxes for Δpgi and Δppc. Such discrepancies between the simulated and experimental data suggested biological complexity. In this study, we overcame these problems by assuming critical mechanisms regarding the OAA-regulated isocitrate dehydrogenase activity, aceBAK gene regulation and growth suppression. The present model accurately predicts the extracellular and intracellular dynamics of wild type and many gene knockout mutants in batch and continuous cultures. It is now the most accurate, detailed kinetic model of E. coli central carbon metabolism and will contribute to advances in mathematical modeling of cell factories. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Simplifying Collection of Corneal Specimens in Cases of Suspected Bacterial Keratitis

PubMed Central

Kaye, Stephen B.; Rao, Prasad G.; Smith, Godfrey; Scott, John A.; Hoyles, Sharon; Morton, Clare E.; Willoughby, Colin; Batterbury, Mark; Harvey, Graham

2003-01-01

Identification of the causative organisms in suspected bacterial keratitis traditionally involves collecting multiple corneal scrapes, which are plated directly onto different solid agar culture media. Difficulties have been reported with this practice, so the development of a simpler diagnostic method in suspected bacterial keratitis would be useful. It is unclear whether a single corneal scrape sent to the microbiology laboratory in a liquid transport culture medium (indirect method) is as reliable for the diagnosis of bacterial keratitis as inoculation of multiple scrapes directly onto agar plates (direct method). To investigate this, bacterial recovery was assessed following transfer and transport of different concentrations and types of bacteria from an artificially contaminated surgical blade into brain heart infusion (BHI). Bacterial recovery rates between the proposed (indirect) and standard (direct) method were then compared after the in vitro inoculation of pig corneas and following specimen collection in patients with presumed bacterial ulcerative keratitis. Recovery of bacteria from contaminated surgical blades was found to be the same from both solid and liquid culture media. There was no significant difference in the numbers of positive cultures from solid (direct) and liquid (indirect) culture media, both in the experimental pig cornea inoculation study (P = 0.34) and in experiments with patients with clinical infections (P = 0.4), with an 85.2% agreement between methods (kappa = 0.61, P < 0.0001). In conclusion, therefore, the collection of two corneal scrapes, one used for Gram staining and the other transported in BHI followed by plating and subculturing in an enrichment medium, provides a simple method for the investigation of presumed bacterial keratitis. PMID:12843063

The Incidence of Propionibacterium acnes in Shoulder Arthroscopy.

PubMed

Chuang, Michael J; Jancosko, Jason J; Mendoza, Vivian; Nottage, Wesley M

2015-09-01

To document the skin colonization and deep tissue inoculation rates associated with arthroscopic shoulder surgery and how these rates differ with procedural and demographic factors. We prospectively recruited outpatient shoulder arthroscopy patients who agreed to participate and met the inclusion criteria from February 2013 to May 2014. All patients received routine antibiotic prophylaxis intravenously. Initial cultures were obtained before the skin preparation by swabbing the skin at the 3 standard portal sites: posterior, anterosuperior, and anterolateral. The skin preparation used 4% chlorhexidine scrub and 2% chlorhexidine gluconate/70% isopropyl alcohol paint applied to the entire shoulder. After completion of the arthroscopic procedure, a second culture was obtained through a cannula at the surgical site. All cultures were plated for 21 days using Brucella medium. We enrolled 51 patients over a 15-month period. Cultures showed a 72.5% Propionibacterium acnes superficial colonization rate: 46.1% of female and 81.6% of male patients (P = .027). We identified a deep culture-positive inoculation rate of 19.6%, all with positive P acnes skin colonization. No correlation could be made concerning diagnosis, procedure, suture anchor use, age, or sex. The rate of skin colonization with P acnes is high at arthroscopic portals, especially in men. Despite standard skin preparation and prophylactic antibiotics, the rate of deep tissue inoculation with P acnes in shoulder arthroscopy is much higher than the rate of infection reported in the literature. Shoulder arthroscopy introduces a significant amount of P acnes into the deep tissues. Copyright © 2015 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

Biolog(TM) ID as compared to 16S ribosomal RNA ID for environmental isolates from the deep subsurface

DOE Office of Scientific and Technical Information (OSTI.GOV)

McKinsey, P.C.

2000-05-05

The U.S. Dept of Energy (DOE) Subsurface Microbial Culture Collection (SMCC) contains nearly 10,000 strains of microorganisms isolated from terrestrial subsurface environments. Many of the aerobic, gram-negative, chemoheterotrophs isolated from the DOE Savannah River Site (SRS) have previously been identified by phylogenetic analysis of 16S ribosomal RNA (rRNA) gene nucleotide sequences. These SMCC isolates are currently being examined using Biolog GN Microplates and the Biolog Microstation System in order to gain knowledge of their metabolic capabilities and to compare Biolog IDs with 16S IDs. To accommodate the particular needs of these subsurface isolates, which are often incapable of growing undermore » high-nutrient conditions, Biolog's recommendations for inoculating isolates into Biolog GN Microplates have been altered. The isolates are grown on low nutrient media, sodium thioglycolate (3mM) is added to the culture media to inhibit capsule formation, and a low density of bacteria is inoculated into the microplate. Using these altered inoculation criteria, 60 percent of these SMCC isolates have a Biolog genus ID that matches the 16S rRNA ID. These results indicate that the Biolog System can be a good means of identifying unusual environmental isolates, even when recommended inoculation procedures are altered to accommodate particular isolate needs.« less

Antiviral Activity of Polyacrylic and Polymethacrylic Acids

PubMed Central

De Somer, P.; De Clercq, E.; Billiau, A.; Schonne, E.; Claesen, M.

1968-01-01

Polyacrylic acid (PAA) and polymethacrylic acid (PMAA) were investigated for their antiviral properties in tissue culture. Compared to other related polyanions, as dextran sulfate, polystyrene sulfonate, polyvinyl sulfate, and polyphloroglucinol phosphate, PAA and PMAA were found to be significantly more antivirally active and less cytotoxic. PMAA added 24 hr prior to virus inoculation inhibited viral growth most efficiently but it was still effective when added 3 hr after infection. Neither a direct irreversible action on the virus nor inhibition of virus penetration into the cell could explain the antiviral activity of PMAA. PMAA inhibited the adsorption of the virus to the host cell and suppressed the one-cycle viral synthesis in tissue cultures inoculated with infectious RNA. PMID:4302187

Preliminary studies of primary ostrich fibroblasts for the isolation of ratite viruses.

PubMed

Rodgers, S J; Vanhooser, S L; Welsh, R D; Silkwood, T G

1994-01-01

An ostrich egg at 21 days of development was used to propagate primary embryo cell cultures. Primary cultures of skeletal muscle cells (for fibroblasts) were prepared by routine typsinization techniques. The ostrich embryo fibroblasts were tested for their ability to propagate stock avian viruses of infectious bronchitis virus, paramyxiovirus-1 (PMV-1), PMV-2, PMV-3, infectious bursal disease virus, quail bronchitis virus, avian reovirus, turkey coronavirus, and two ostrich-originating specimens (one of which was a possible coronavirus identified by electron microscopy). Cytopathic effects were seen by light microscopy in cell cultures inoculated with PMV-1, turkey coronavirus, and the two ostrich specimens. Hemaglutinating titers of 4 or more were determined for PMV-1, turkey coronavrius, and the two ostrich specimens after inoculation onto monolayers of ostrich embryo fibroblasts. Hemagglutination-inhibition tests confirmed the identification of PMV-1 when homologous antisera were used as the specific inhibitor. Bovine coronavirus antisera inhibited the hemagglutination of one of the cultured ostrich specimens.

Estimation of the Maximum Theoretical Productivity of Fed-Batch Bioreactors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bomble, Yannick J; St. John, Peter C; Crowley, Michael F

2017-10-18

A key step towards the development of an integrated biorefinery is the screening of economically viable processes, which depends sharply on the yields and productivities that can be achieved by an engineered microorganism. In this study, we extend an earlier method which used dynamic optimization to find the maximum theoretical productivity of batch cultures to explicitly include fed-batch bioreactors. In addition to optimizing the intracellular distribution of metabolites between cell growth and product formation, we calculate the optimal control trajectory of feed rate versus time. We further analyze how sensitive the productivity is to substrate uptake and growth parameters.

Bioreactors for high cell density and continuous multi-stage cultivations: options for process intensification in cell culture-based viral vaccine production.

PubMed

Tapia, Felipe; Vázquez-Ramírez, Daniel; Genzel, Yvonne; Reichl, Udo

2016-03-01

With an increasing demand for efficacious, safe, and affordable vaccines for human and animal use, process intensification in cell culture-based viral vaccine production demands advanced process strategies to overcome the limitations of conventional batch cultivations. However, the use of fed-batch, perfusion, or continuous modes to drive processes at high cell density (HCD) and overextended operating times has so far been little explored in large-scale viral vaccine manufacturing. Also, possible reductions in cell-specific virus yields for HCD cultivations have been reported frequently. Taking into account that vaccine production is one of the most heavily regulated industries in the pharmaceutical sector with tough margins to meet, it is understandable that process intensification is being considered by both academia and industry as a next step toward more efficient viral vaccine production processes only recently. Compared to conventional batch processes, fed-batch and perfusion strategies could result in ten to a hundred times higher product yields. Both cultivation strategies can be implemented to achieve cell concentrations exceeding 10(7) cells/mL or even 10(8) cells/mL, while keeping low levels of metabolites that potentially inhibit cell growth and virus replication. The trend towards HCD processes is supported by development of GMP-compliant cultivation platforms, i.e., acoustic settlers, hollow fiber bioreactors, and hollow fiber-based perfusion systems including tangential flow filtration (TFF) or alternating tangential flow (ATF) technologies. In this review, these process modes are discussed in detail and compared with conventional batch processes based on productivity indicators such as space-time yield, cell concentration, and product titers. In addition, options for the production of viral vaccines in continuous multi-stage bioreactors such as two- and three-stage systems are addressed. While such systems have shown similar virus titers compared to batch cultivations, keeping high yields for extended production times is still a challenge. Overall, we demonstrate that process intensification of cell culture-based viral vaccine production can be realized by the consequent application of fed-batch, perfusion, and continuous systems with a significant increase in productivity. The potential for even further improvements is high, considering recent developments in establishment of new (designer) cell lines, better characterization of host cell metabolism, advances in media design, and the use of mathematical models as a tool for process optimization and control.

Experimental infections in Venezuelan lizards by Trypanosoma cruzi.

PubMed

Urdaneta-Morales, S; McLure, I

1981-06-01

Virulent trypomastigotes of the Y strain of Trypanosoma cruzi were administered to Tropidurus hispidus, Ameiva ameiva, Cnemidophorus lemniscatus, Polychrus marmoratus, and Phyllodactylus ventralis (Sauria). Intraperitoneal and subcutaneous inoculations of lizards with mouse blood or with feces of infected Rhodnius prolixus (Reduviidae, Triatominae), as well as forced ingestion of triturated Rhodnius, produced no parasitaemias detectable either directly or by xenodiagnosis, while control mice became parasitized. Pretreatment with the immunosuppressive drug Fluocinolone acetonide led to establishing patent infections in inoculated lizards. Cryptic infections were established by inoculation of 1 X 10(6) parasites from Davis' medium, or by 95 X 10(3) parasites from lizard tissue culture. Parasites were not seen in tissues. Mice inoculated with blood or tissue homogenates from these lizards became parasitized. Parasites from Davis' medium inoculated into the peritoneal cavity of lizards were capable, to a very low degree, of penetrating the free peritoneal macrophages and changing into amastigotes. The factors possibly responsible for the natural resistance of poikilothermic vertebrates to T. cruzi are discussed.

Miso. III. Pure culture fermentation with Saccharomyces rouxii.

PubMed

HESSELTINE, C W; SHIBASAKI, K

1961-11-01

Excellent miso has been prepared with soybean grits inoculated with a pure culture of Saccharomyces rouxii strain NRRL Y-2547. Pure culture inoculum of this osmophilic yeast was prepared by growing the culture in aerated flasks on a yeast extract medium with a salt concentration equal to that used in the manufacture of miso. It has also been found possible to make miso from whole beans with the above culture. The advantages of pure culture fermentation in producing miso are discussed.

Analysis of the role of GADD153 in the control of apoptosis in NS0 myeloma cells.

PubMed

Lengwehasatit, Idsada; Dickson, Alan J

2002-12-30

Apoptosis can limit the maximum production of recombinant protein expression from cultured mammalian cells. This article focuses on the links between nutrient deprivation, ER perturbation, the regulation of (growth arrest and DNA damage inducible gene 153) GADD153 expression and apoptosis. During batch culture, decreases in glucose and glutamine correlated with an increase in apoptotic cells. This event was paralleled by a simultaneous increase in GADD153 expression. The expression of GADD153 in batch culture was suppressed by the addition of nutrients and with fed-batch culture the onset of apoptosis was delayed but not completely prevented. In defined stress conditions, glucose deprivation had the greatest effect on cell death when compared to glutamine deprivation or the addition of tunicamycin (an inhibitor of glycosylation), added to generate endoplasmic reticulum stress. However, the contribution of apoptosis to overall cell death (as judged by morphology) was smaller in conditions of glucose deprivation than in glutamine deprivation or tunicamycin treatment. Transient activation of GADD153 expression was found to occur in response to all stresses and occurred prior to detection of the onset of cell death. These results imply that GADD153 expression is either a trigger for apoptosis or offers a valid indicator of the likelihood of cell death arising from stresses of relevance to the bioreactor environment. Copyright 2002 Wiley Periodicals, Inc.

Stable acetate production in extreme-thermophilic (70°C) mixed culture fermentation by selective enrichment of hydrogenotrophic methanogens

NASA Astrophysics Data System (ADS)

Zhang, Fang; Zhang, Yan; Ding, Jing; Dai, Kun; van Loosdrecht, Mark C. M.; Zeng, Raymond J.

2014-06-01

The control of metabolite production is difficult in mixed culture fermentation. This is particularly related to hydrogen inhibition. In this work, hydrogenotrophic methanogens were selectively enriched to reduce the hydrogen partial pressure and to realize efficient acetate production in extreme-thermophilic (70°C) mixed culture fermentation. The continuous stirred tank reactor (CSTR) was stable operated during 100 days, in which acetate accounted for more than 90% of metabolites in liquid solutions. The yields of acetate, methane and biomass in CSTR were 1.5 +/- 0.06, 1.0 +/- 0.13 and 0.4 +/- 0.05 mol/mol glucose, respectively, close to the theoretical expected values. The CSTR effluent was stable and no further conversion occurred when incubated for 14 days in a batch reactor. In fed-batch experiments, acetate could be produced up to 34.4 g/L, significantly higher than observed in common hydrogen producing fermentations. Acetate also accounted for more than 90% of soluble products formed in these fed-batch fermentations. The microbial community analysis revealed hydrogenotrophic methanogens (mainly Methanothermobacter thermautotrophicus and Methanobacterium thermoaggregans) as 98% of Archaea, confirming that high temperature will select hydrogenotrophic methanogens over aceticlastic methanogens effectively. This work demonstrated a potential application to effectively produce acetate as a value chemical and methane as an energy gas together via mixed culture fermentation.

Stable acetate production in extreme-thermophilic (70°C) mixed culture fermentation by selective enrichment of hydrogenotrophic methanogens

PubMed Central

Zhang, Fang; Zhang, Yan; Ding, Jing; Dai, Kun; van Loosdrecht, Mark C. M.; Zeng, Raymond J.

2014-01-01

The control of metabolite production is difficult in mixed culture fermentation. This is particularly related to hydrogen inhibition. In this work, hydrogenotrophic methanogens were selectively enriched to reduce the hydrogen partial pressure and to realize efficient acetate production in extreme-thermophilic (70°C) mixed culture fermentation. The continuous stirred tank reactor (CSTR) was stable operated during 100 days, in which acetate accounted for more than 90% of metabolites in liquid solutions. The yields of acetate, methane and biomass in CSTR were 1.5 ± 0.06, 1.0 ± 0.13 and 0.4 ± 0.05 mol/mol glucose, respectively, close to the theoretical expected values. The CSTR effluent was stable and no further conversion occurred when incubated for 14 days in a batch reactor. In fed-batch experiments, acetate could be produced up to 34.4 g/L, significantly higher than observed in common hydrogen producing fermentations. Acetate also accounted for more than 90% of soluble products formed in these fed-batch fermentations. The microbial community analysis revealed hydrogenotrophic methanogens (mainly Methanothermobacter thermautotrophicus and Methanobacterium thermoaggregans) as 98% of Archaea, confirming that high temperature will select hydrogenotrophic methanogens over aceticlastic methanogens effectively. This work demonstrated a potential application to effectively produce acetate as a value chemical and methane as an energy gas together via mixed culture fermentation. PMID:24920064

Comparison of the Petrifilm dry rehydratable film and conventional culture methods for enumeration of yeasts and molds in foods: collaborative study.

PubMed

Knight, M T; Newman, M C; Benzinger, M J; Neufang, K L; Agin, J R; McAllister, J S; Ramos, M

1997-01-01

A collaborative study was performed involving 18 laboratories and 6 food types to compare 3M Petrifilm yeast and mold count plates with the method described in the U.S. Food and Drug Administration's Bacteriological Analytical Manual. Four species of mold and 2 species of yeast were used to inoculate the following foods: hot dogs, corn meal, ketchup, orange juice, yogurt, and cake mix. Each collaborator received 15 samples of each food type: 5 low-level inoculations, 5 high-level inoculations, and 5 uninoculated samples. There was no significant difference between the means of the 2 methods for any product or inoculation level. The Petrifilm yeast and mold count plate method for enumeration of yeasts and molds in foods has been adopted first action by AOAC INTERNATIONAL.

Physiological and biochemical characterization of Azospirillum brasilense strains commonly used as plant growth-promoting rhizobacteria.

PubMed

Di Salvo, Luciana P; Silva, Esdras; Teixeira, Kátia R S; Cote, Rosalba Esquivel; Pereyra, M Alejandra; García de Salamone, Inés E

2014-12-01

Azospirillum is a plant growth-promoting rhizobacteria (PGPR) genus vastly studied and utilized as agriculture inoculants. Isolation of new strains under different environmental conditions allows the access to the genetic diversity and improves the success of inoculation procedures. Historically, the isolation of this genus has been performed by the use of some traditional culture media. In this work we characterized the physiology and biochemistry of five different A. brasilense strains, commonly used as cereal inoculants. The aim of this work is to contribute to pose into revision some concepts concerning the most used protocols to isolate and characterize this bacterium. We characterized their growth in different traditional and non-traditional culture media, evaluated some PGPR mechanisms and characterized their profiles of fatty acid methyl esters and carbon-source utilization. This work shows, for the first time, differences in both profiles, and ACC deaminase activity of A. brasilense strains. Also, we show unexpected results obtained in some of the evaluated culture media. Results obtained here and an exhaustive knowledge revision revealed that it is not appropriate to conclude about bacterial species without analyzing several strains. Also, it is necessary to continue developing studies and laboratory techniques to improve the isolation and characterization protocols. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

S. sanguinis adhesion on rough titanium surfaces: effect of culture media.

PubMed

Rodríguez-Hernández, Ana G; Muñoz-Tabares, José A; Godoy-Gallardo, Maria; Juárez, Antonio; Gil, Francisco-Javier

2013-03-01

Bacterial colonization plays a key role in dental implant failure, because they attach directly on implant surface upon implantation. Between different types of bacteria associated with the oral environment, Streptococcus sanguinis is essential in this process since it is an early colonizer. In this work the relationship between titanium surfaces modified by shot blasting treatment and S. sanguinis adhesion; have been studied in approached human mouth environment. Bacteria pre-inoculated with routinary solution were put in contact with titanium samples, shot-blasted with alumina and silicon carbide, and adhesion results were compared with those obtained when bacteria were pre-inoculated with modified artificial saliva medium and on saliva pre-coated titanium samples. Our results showed that bacterial adhesion on titanium samples was influenced by culture conditions. When S. sanguinis was inoculated in routinary culture media, colonies forming unities per square millimeter presented an increment correlated with roughness and surface energy, but separated by the type of particle used during shot-blasting treatment; whereas in modified artificial saliva only a relationship between bacteria adhered and the increment in both roughness and surface energy were observed, regardless of the particle type. Finally, on human saliva pre-coated samples no significant differences were observed among roughness, surface energy or particle. Copyright © 2012 Elsevier B.V. All rights reserved.

Isolation of Coxiella burnetii from serum of patients with acute Q fever.

PubMed

Vincent, Gemma A; Graves, Stephen R; Robson, Jennifer M; Nguyen, Chelsea; Hussain-Yusuf, Hazizul; Islam, Aminul; Fenwick, Stanley G; Stenos, John

2015-12-01

Worldwide there are few isolate collections of the intracellular bacterium Coxiella burnetii, due to the difficulties associated with working with the organism and the scarcity of suitable samples from which to attempt isolation. Particularly lacking are isolates from acute Q fever patients. The aim of this study was to evaluate whether the serum samples taken from patients with confirmed acute Q fever during the early stage of their disease represented a potential source of viable C. burnetii. Isolation was attempted from 65 of these samples by inoculation of the serum into Vero cell culture and was successful in 36 cases (55%). This high success rate was likely due to extended incubation of up to twelve weeks of the inoculated cultures, allowing the growth of the organism to levels detectable by PCR. Retrospective analysis of the time the sera was stored prior to inoculation into culture demonstrated that C. burnetii remained viable for 224 days in samples stored refrigerated and 371 days in samples stored frozen at -20 °C. These results demonstrate that standard serum samples taken from acute Q fever patients are a valuable source of new isolates of C. burnetii, with no special handling of the specimens required to maintain the organism's viability. Copyright © 2015 Elsevier B.V. All rights reserved.

A method to isolate bacterial communities and characterize ecosystems from food products: Validation and utilization in as a reproducible chicken meat model.

PubMed

Rouger, Amélie; Remenant, Benoit; Prévost, Hervé; Zagorec, Monique

2017-04-17

Influenced by production and storage processes and by seasonal changes the diversity of meat products microbiota can be very variable. Because microbiotas influence meat quality and safety, characterizing and understanding their dynamics during processing and storage is important for proposing innovative and efficient storage conditions. Challenge tests are usually performed using meat from the same batch, inoculated at high levels with one or few strains. Such experiments do not reflect the true microbial situation, and the global ecosystem is not taken into account. Our purpose was to constitute live stocks of chicken meat microbiotas to create standard and reproducible ecosystems. We searched for the best method to collect contaminating bacterial communities from chicken cuts to store as frozen aliquots. We tested several methods to extract DNA of these stored communities for subsequent PCR amplification. We determined the best moment to collect bacteria in sufficient amounts during the product shelf life. Results showed that the rinsing method associated to the use of Mobio DNA extraction kit was the most reliable method to collect bacteria and obtain DNA for subsequent PCR amplification. Then, 23 different chicken meat microbiotas were collected using this procedure. Microbiota aliquots were stored at -80°C without important loss of viability. Their characterization by cultural methods confirmed the large variability (richness and abundance) of bacterial communities present on chicken cuts. Four of these bacterial communities were used to estimate their ability to regrow on meat matrices. Challenge tests performed on sterile matrices showed that these microbiotas were successfully inoculated and could overgrow the natural microbiota of chicken meat. They can therefore be used for performing reproducible challenge tests mimicking a true meat ecosystem and enabling the possibility to test the influence of various processing or storage conditions on complex meat matrices. Copyright © 2016 Elsevier B.V. All rights reserved.

Analysis and IbM simulation of the stages in bacterial lag phase: basis for an updated definition.

PubMed

Prats, Clara; Giró, Antoni; Ferrer, Jordi; López, Daniel; Vives-Rego, Josep

2008-05-07

The lag phase is the initial phase of a culture that precedes exponential growth and occurs when the conditions of the culture medium differ from the pre-inoculation conditions. It is usually defined by means of cell density because the number of individuals remains approximately constant or slowly increases, and it is quantified with the lag parameter lambda. The lag phase has been studied through mathematical modelling and by means of specific experiments. In recent years, Individual-based Modelling (IbM) has provided helpful insights into lag phase studies. In this paper, the definition of lag phase is thoroughly examined. Evolution of the total biomass and the total number of bacteria during lag phase is tackled separately. The lag phase lasts until the culture reaches a maximum growth rate both in biomass and cell density. Once in the exponential phase, both rates are constant over time and equal to each other. Both evolutions are split into an initial phase and a transition phase, according to their growth rates. A population-level mathematical model is presented to describe the transitional phase in cell density. INDividual DIScrete SIMulation (INDISIM) is used to check the outcomes of this analysis. Simulations allow the separate study of the evolution of cell density and total biomass in a batch culture, they provide a depiction of different observed cases in lag evolution at the individual-cell level, and are used to test the population-level model. The results show that the geometrical lag parameter lambda is not appropriate as a universal definition for the lag phase. Moreover, the lag phase cannot be characterized by a single parameter. For the studied cases, the lag phases of both the total biomass and the population are required to fully characterize the evolution of bacterial cultures. The results presented prove once more that the lag phase is a complex process that requires a more complete definition. This will be possible only after the phenomena governing the population dynamics at an individual level of description, and occurring during the lag and exponential growth phases, are well understood.

Dynamic genome-scale metabolic modeling of the yeast Pichia pastoris.

PubMed

Saitua, Francisco; Torres, Paulina; Pérez-Correa, José Ricardo; Agosin, Eduardo

2017-02-21

Pichia pastoris shows physiological advantages in producing recombinant proteins, compared to other commonly used cell factories. This yeast is mostly grown in dynamic cultivation systems, where the cell's environment is continuously changing and many variables influence process productivity. In this context, a model capable of explaining and predicting cell behavior for the rational design of bioprocesses is highly desirable. Currently, there are five genome-scale metabolic reconstructions of P. pastoris which have been used to predict extracellular cell behavior in stationary conditions. In this work, we assembled a dynamic genome-scale metabolic model for glucose-limited, aerobic cultivations of Pichia pastoris. Starting from an initial model structure for batch and fed-batch cultures, we performed pre/post regression diagnostics to ensure that model parameters were identifiable, significant and sensitive. Once identified, the non-relevant ones were iteratively fixed until a priori robust modeling structures were found for each type of cultivation. Next, the robustness of these reduced structures was confirmed by calibrating the model with new datasets, where no sensitivity, identifiability or significance problems appeared in their parameters. Afterwards, the model was validated for the prediction of batch and fed-batch dynamics in the studied conditions. Lastly, the model was employed as a case study to analyze the metabolic flux distribution of a fed-batch culture and to unravel genetic and process engineering strategies to improve the production of recombinant Human Serum Albumin (HSA). Simulation of single knock-outs indicated that deviation of carbon towards cysteine and tryptophan formation improves HSA production. The deletion of methylene tetrahydrofolate dehydrogenase could increase the HSA volumetric productivity by 630%. Moreover, given specific bioprocess limitations and strain characteristics, the model suggests that implementation of a decreasing specific growth rate during the feed phase of a fed-batch culture results in a 25% increase of the volumetric productivity of the protein. In this work, we formulated a dynamic genome scale metabolic model of Pichia pastoris that yields realistic metabolic flux distributions throughout dynamic cultivations. The model can be calibrated with experimental data to rationally propose genetic and process engineering strategies to improve the performance of a P. pastoris strain of interest.

Response of dairy calves to vaccinia viruses that express foreign genes.

PubMed Central

Gillespie, J H; Geissinger, C; Scott, F W; Higgins, W P; Holmes, D F; Perkus, M; Mercer, S; Paoletti, E

1986-01-01

Repeated intradermal inoculations of calves with wild-type vaccinia virus and recombinant vaccinia viruses expressing human hepatitis B virus surface antigen and herpes simplex virus, type 1, glycoprotein D produced characteristic pox lesions at each site of injection. In some instances, calves were inoculated as many as five times at intervals from 4 to 7 weeks. The lesions invariably were more severe after the second inoculation. Subsequent inoculations produced a less severe area of redness, swelling, necrosis, and scab formation. No other signs of illness, such as an elevation in temperature, were noted in the calves. Vaccinia virus was isolated in low titers from scabs taken at various times after inoculation. No lesions were formed at the sites injected with tissue culture fluid and cellular debris at the same time that virus inoculations were made. Calf contact controls remained normal through the 8-week exposure in isolation units with calves inoculated twice with vaccinia virus. No neutralizing antibody to vaccinia virus was detected in the contact controls. In contrast, the virus-inoculated calves developed neutralizing antibody to vaccinia virus and to herpes simplex virus glycoprotein D in serum. In all cattle, a second inoculation significantly enhanced the neutralizing antibody response within 1 week, suggesting that an anamnestic response had occurred. No antibody to hepatitis B virus surface antigen was elicited in calves after repeated inoculations with vaccinia recombinants that express hepatitis B virus surface antigen and are known to elicit in rabbits antibodies reactive with hepatitis B virus surface antigen. Images PMID:3700615

Arbuscular mycorrhizal wheat inoculation promotes alkane and polycyclic aromatic hydrocarbon biodegradation: Microcosm experiment on aged-contaminated soil.

PubMed

Ingrid, Lenoir; Lounès-Hadj Sahraoui, Anissa; Frédéric, Laruelle; Yolande, Dalpé; Joël, Fontaine

2016-06-01

Very few studies reported the potential of arbuscular mycorrhizal symbiosis to dissipate hydrocarbons in aged polluted soils. The present work aims to study the efficiency of arbuscular mycorrhizal colonized wheat plants in the dissipation of alkanes and polycyclic aromatic hydrocarbons (PAHs). Our results demonstrated that the inoculation of wheat with Rhizophagus irregularis allowed a better dissipation of PAHs and alkanes after 16 weeks of culture by comparison to non-inoculated condition. These dissipations observed in the inoculated soil resulted from several processes: (i) a light adsorption on roots (0.5% for PAHs), (ii) a bioaccumulation in roots (5.7% for PAHs and 6.6% for alkanes), (iii) a transfer in shoots (0.4 for PAHs and 0.5% for alkanes) and mainly a biodegradation. Whereas PAHs and alkanes degradation rates were respectively estimated to 12 and 47% with non-inoculated wheat, their degradation rates reached 18 and 48% with inoculated wheat. The mycorrhizal inoculation induced an increase of Gram-positive and Gram-negative bacteria by 56 and 37% compared to the non-inoculated wheat. Moreover, an increase of peroxidase activity was assessed in mycorrhizal roots. Taken together, our findings suggested that mycorrhization led to a better hydrocarbon biodegradation in the aged-contaminated soil thanks to a stimulation of telluric bacteria and hydrocarbon metabolization in mycorrhizal roots. Copyright © 2016 Elsevier Ltd. All rights reserved.

21 CFR 680.2 - Manufacture of Allergenic Products.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Manufacture of Allergenic Products. 680.2 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR MISCELLANEOUS PRODUCTS § 680.2 Manufacture of Allergenic Products. (a...) Cultures derived from microorganisms. Culture media into which organisms are inoculated for the manufacture...

Inoculation of soil native cyanobacteria to restore arid degraded soils

NASA Astrophysics Data System (ADS)

Raúl Román Fernández, José; Roncero Ramos, Beatriz; Chamizo de la Piedra, Sonia; Rodríguez Caballero, Emilio; Ángeles Muñoz Martín, M.; Mateo, Pilar; Cantón Castilla, Yolanda

2017-04-01

Restoration projects in semiarid lands often yield poor results. Water scarcity, low soil fertility, and poor soil structure strongly limit the survival and growth of planted seedlings in these areas. Under these conditions, a previous stage that improves edaphic conditions would turn out to a successful plant restoration. By successfully colonizing arid soils, cyanobacteria naturally provide suitable edaphic conditions, enhancing water availability, soil fertility and soil stability. Furthermore, cyanobacteria can be easily isolated and cultured ex-situ to produce high quantities of biomass, representing a potential tool to restore large areas efficiently. The objective of this study was to test the effect of inoculated cyanobacteria on degraded soils at three different semiarid areas from southeast Spain: Tabernas badlands, a limestone quarry located in Gádor, and grazed grassland in Las Amoladeras (Cabo de Gata). Soil native cyanobacteria belonging to three representative N-fixing genera (Nostoc, Scytonema and Tolypothrix) were isolated from such soils and cultured in BG110 medium. Each strain was inoculated (6 g m-2), separately and mixed (all in the same proportion), on Petri dishes with 80 g of each soil. Biocrust development was monitored during 3 months in these soils under laboratory conditions, at a constant temperature of 25oC. During the experiment, two irrigation treatments were applied simulating a dry (180 mm) and a wet (360 mm) rainfall year (average recorded in the study sites). After 3 months, net CO2 flux, spectral response and soil surface microtopography (1 mm spatial resolution) of inoculated and control soils was measured under wet conditions, all of them as a surrogate of biocrust development. Samples of the surface crust were collected in order to determine total soil organic carbon (SOC) content. The inoculated soils showed positive values of net CO2 flux, thus indicating a net CO2 uptake, whereas control soils showed CO2 fluxes closed to zero. This higher CO2 fixation in the inoculated soils was reflected in the higher SOC content found in these soils with respect to the non-inoculated soils. Soil surface roughness increased with biocrust development in the inoculated soils as compared to control soils. From the different treatments, soil inoculation with the mixture of the three strains promoted the highest SOC contents and absorbance at 680 nm (indicative of higher chlorophyll a content) on the three soil types. Therefore, using a consortium of cyanobacteria to inoculate degraded soils seems to be a more promising strategy to restore soils than inoculating individual species. Finally, differences between irrigation treatments were no significant, suggesting that water availability was not a key driver for cyanobacteria development under control laboratory conditions. Our results underline the viability of cyanobacteria inoculation to form an artificial developed biocrust that contribute to CO2 uptake and increase soil fertility which could facilitate further plant cover establishment. However, more studies are necessaries to test the effectiveness of inoculated crust development under field conditions.

Co-Inoculation with Rhizobia and AMF Inhibited Soybean Red Crown Rot: From Field Study to Plant Defense-Related Gene Expression Analysis

PubMed Central

Gao, Xiang; Lu, Xing; Wu, Man; Zhang, Haiyan; Pan, Ruqian; Tian, Jiang; Li, Shuxian; Liao, Hong

2012-01-01

Background Soybean red crown rot is a major soil-borne disease all over the world, which severely affects soybean production. Efficient and sustainable methods are strongly desired to control the soil-borne diseases. Principal Findings We firstly investigated the disease incidence and index of soybean red crown rot under different phosphorus (P) additions in field and found that the natural inoculation of rhizobia and arbuscular mycorrhizal fungi (AMF) could affect soybean red crown rot, particularly without P addition. Further studies in sand culture experiments showed that inoculation with rhizobia or AMF significantly decreased severity and incidence of soybean red crown rot, especially for co-inoculation with rhizobia and AMF at low P. The root colony forming unit (CFU) decreased over 50% when inoculated by rhizobia and/or AMF at low P. However, P addition only enhanced CFU when inoculated with AMF. Furthermore, root exudates of soybean inoculated with rhizobia and/or AMF significantly inhibited pathogen growth and reproduction. Quantitative RT-PCR results indicated that the transcripts of the most tested pathogen defense-related (PR) genes in roots were significantly increased by rhizobium and/or AMF inoculation. Among them, PR2, PR3, PR4 and PR10 reached the highest level with co-inoculation of rhizobium and AMF. Conclusions Our results indicated that inoculation with rhizobia and AMF could directly inhibit pathogen growth and reproduction, and activate the plant overall defense system through increasing PR gene expressions. Combined with optimal P fertilization, inoculation with rhizobia and AMF could be considered as an efficient method to control soybean red crown rot in acid soils. PMID:22442737

Hazards involved in the use of furazolidone for the prevention of salmonellosis in broiler chickens

PubMed Central

Rantala, Marjatta; Nurmi, E.

1974-01-01

The purpose of this work was to study the effects of interrupted, continuous and post-salmonella inoculation treatment with furazolidone in the feed on the colonization of Salmonella infantis in the intestines of chickens, as well as the influence of furazolidone in vitro on the effect of a mixed culture used for the prevention of salmonellosis in chickens. It was shown that chickens given interrupted treatment with 0·01% furazolidone had significantly more salmonellas in the caeca than either chickens fed continuously with this drug or chickens without any treatment. The use of 0·01% furazolidone after inoculation with Salmonella infantis had no effect on Salmonella infantis in the caeca of chickens. The mixed bacterial culture from the normal intestinal flora lost its preventive effect on salmonellosis when cultured with 0·01% furazolidone. PMID:4602035

Agrobacterium tumefaciens-mediated transformation of Lasiodiplodia theobromae, the causal agent of gummosis in cashew nut plants.

PubMed

Muniz, C R; da Silva, G F; Souza, M T; Freire, F C O; Kema, G H J; Guedes, M I F

2014-02-21

Lasiodiplodia theobromae is a major pathogen of many different crop cultures, including cashew nut plants. This paper describes an efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for the successful delivery of T-DNA, transferring the genes of green fluorescent protein (gfp) and hygromycin B phosphotransferase (hph) to L. theobromae. When the fungal pycnidiospores were co-cultured with A. tumefaciens harboring the binary vector with hph-gfp gene, hygromycin-resistant fungus only developed with acetosyringone supplementation. The cashew plants inoculated with the fungus expressing GFP revealed characteristic pathogen colonization by epifluorescence microscopy. Intense and bright green hyphae were observed for transformants in all extensions of mycelium cultures. The penetration of parenchyma cells near to the inoculation site, beneath the epicuticle surface, was observed prior to 25 dpi. Penetration was followed by the development of hyphae within invaded host cells. These findings provide a rapid and reproducible ATMT method for L. theobromae transformation.

Aminogenesis control in fermented sausages manufactured with pressurized meat batter and starter culture.

PubMed

Latorre-Moratalla, M L; Bover-Cid, S; Aymerich, T; Marcos, B; Vidal-Carou, M C; Garriga, M

2007-03-01

The application of high hydrostatic pressure (200MPa) to meat batter just before sausage fermentation and the inoculation of starter culture were studied to improve the safety and quality of traditional Spanish fermented sausages (fuet and chorizo). Higher amounts of biogenic amines were formed in chorizo than in fuet. Without interfering with the ripening performance in terms of acidification, drying and proteolysis, hydrostatic pressure prevented enterobacteria growth but did not affect Gram-positive bacteria significantly. Subsequently, a strong inhibition of diamine (putrescine and cadaverine) accumulation was observed, but that of tyramine was not affected. The inoculated decarboxylase-negative strains, selected from indigenous bacteria of traditional sausages, were resistant to the HHP treatment, being able to lead the fermentation process, prevent enterococci development and significantly reduce enterobacteria counts. In sausages manufactured with either non-pressurized or pressurized meat batter, starter culture was the most protective measure against the accumulation of tyramine and both diamines.

Animal-cell culture media: History, characteristics, and current issues.

PubMed

Yao, Tatsuma; Asayama, Yuta

2017-04-01

Cell culture technology has spread prolifically within a century, a variety of culture media has been designed. This review goes through the history, characteristics and current issues of animal-cell culture media. A literature search was performed on PubMed and Google Scholar between 1880 and May 2016 using appropriate keywords. At the dawn of cell culture technology, the major components of media were naturally derived products such as serum. The field then gradually shifted to the use of chemical-based synthetic media because naturally derived ingredients have their disadvantages such as large batch-to-batch variation. Today, industrially important cells can be cultured in synthetic media. Nevertheless, the combinations and concentrations of the components in these media remain to be optimized. In addition, serum-containing media are still in general use in the field of basic research. In the fields of assisted reproductive technologies and regenerative medicine, some of the medium components are naturally derived in nearly all instances. Further improvements of culture media are desirable, which will certainly contribute to a reduction in the experimental variation, enhance productivity among biopharmaceuticals, improve treatment outcomes of assisted reproductive technologies, and facilitate implementation and popularization of regenerative medicine.

Enhanced single cell oil production by mixed culture of Chlorella pyrenoidosa and Rhodotorula glutinis using cassava bagasse hydrolysate as carbon source.

PubMed

Liu, Lu; Chen, Junhui; Lim, Phaik-Eem; Wei, Dong

2018-05-01

The single cell oil (SCO) production by the mono and mixed culture of microalgae Chlorella pyrenoidosa and red yeast Rhodotorula glutinis was investigated using non-detoxified cassava bagasse hydrolysate (CBH) as carbon source. The results suggested that the two strains were able to tolerate and even degrade some byproducts presented in the CBH, and the mixed culture approach enhanced the degradation of certain byproducts. Biomass (20.37 ± 0.38 g/L) and lipid yield (10.42 ± 1.21 g/L) of the mixed culture achieved in the batch culture were significantly higher than that of the mono-cultures (p < 0.05). The fed-batch culture further raised the biomass and lipid yield to 31.45 ± 4.93 g/L and 18.47 ± 3.25 g/L, respectively. The lipids mainly composed of oleic acid and palmitic acid, suggesting the potential applications such as biofuel feedstock, cosmetics, food additives and lubricant. This study provided new insights for the integration of the economical SCO production with agro-industrial waste disposal. Copyright © 2018 Elsevier Ltd. All rights reserved.

Systemic acquired resistance in moss: further evidence for conserved defense mechanisms in plants.

PubMed

Winter, Peter S; Bowman, Collin E; Villani, Philip J; Dolan, Thomas E; Hauck, Nathanael R

2014-01-01

Vascular plants possess multiple mechanisms for defending themselves against pathogens. One well-characterized defense mechanism is systemic acquired resistance (SAR). In SAR, a plant detects the presence of a pathogen and transmits a signal throughout the plant, inducing changes in the expression of various pathogenesis-related (PR) genes. Once SAR is established, the plant is capable of mounting rapid responses to subsequent pathogen attacks. SAR has been characterized in numerous angiosperm and gymnosperm species; however, despite several pieces of evidence suggesting SAR may also exist in non-vascular plants6-8, its presence in non-vascular plants has not been conclusively demonstrated, in part due to the lack of an appropriate culture system. Here, we describe and use a novel culture system to demonstrate that the moss species Amblystegium serpens does initiate a SAR-like reaction upon inoculation with Pythium irregulare, a common soil-borne oomycete. Infection of A. serpens gametophores by P. irregulare is characterized by localized cytoplasmic shrinkage within 34 h and chlorosis and necrosis within 7 d of inoculation. Within 24 h of a primary inoculation (induction), moss gametophores grown in culture became highly resistant to infection following subsequent inoculation (challenge) by the same pathogen. This increased resistance was a response to the pathogen itself and not to physical wounding. Treatment with β-1,3 glucan, a structural component of oomycete cell walls, was equally effective at triggering SAR. Our results demonstrate, for the first time, that this important defense mechanism exists in a non-vascular plant, and, together with previous studies, suggest that SAR arose prior to the divergence of vascular and non-vascular plants. In addition, this novel moss - pathogen culture system will be valuable for future characterization of the mechanism of SAR in moss, which is necessary for a better understanding of the evolutionary history of SAR in plants.

Starter Culture Selection for Making Chinese Sesame-Flavored Liquor Based on Microbial Metabolic Activity in Mixed-Culture Fermentation

PubMed Central

Wu, Qun; Ling, Jie

2014-01-01

Selection of a starter culture with excellent viability and metabolic activity is important for inoculated fermentation of traditional food. To obtain a suitable starter culture for making Chinese sesame-flavored liquor, the yeast and bacterium community structures were investigated during spontaneous and solid-state fermentations of this type of liquor. Five dominant species in spontaneous fermentation were identified: Saccharomyces cerevisiae, Pichia membranaefaciens, Issatchenkia orientalis, Bacillus licheniformis, and Bacillus amyloliquefaciens. The metabolic activity of each species in mixed and inoculated fermentations of liquor was investigated in 14 different cocultures that used different combinations of these species. The relationships between the microbial species and volatile metabolites were analyzed by partial least-squares (PLS) regression analysis. We found that S. cerevisiae was positively correlated to nonanal, and B. licheniformis was positively associated with 2,3-butanediol, isobutyric acid, guaiacol, and 4-vinyl guaiacol, while I. orientalis was positively correlated to butyric acid, isovaleric acid, hexanoic acid, and 2,3-butanediol. These three species are excellent flavor producers for Chinese liquor. Although P. membranaefaciens and B. amyloliquefaciens were not efficient flavor producers, the addition of them alleviated competition among the other three species and altered their growth rates and flavor production. As a result, the coculture of all five dominant species produced the largest amount of flavor compounds. The result indicates that flavor producers and microbial interaction regulators are important for inoculated fermentation of Chinese sesame-flavored liquor. PMID:24814798

Sustained Ocular Delivery of Ciprofloxacin Using Nanospheres and Conventional Contact Lens Materials

PubMed Central

Garhwal, Rahul; Shady, Sally F.; Ellis, Edward J.; Ellis, Jeanne Y.; Leahy, Charles D.; McCarthy, Stephen P.; Crawford, Kathryn S.

2012-01-01

Purpose. To formulate conventional contact lenses that incorporate nanosphere-encapsulated antibiotic and demonstrate that the lenses provide for sustained antibacterial activity. Methods. A copolymer composed of pullulan and polycaprolactone (PCL) was used to synthesize core-shell nanospheres that encapsulated ciprofloxacin. Bactericidal activity of the nanosphere-encapsulated ciprofloxacin (nanosphere/cipro) was tested by using liquid cultures of either Staphylococcus aureus or Pseudomonas aeruginosa. Nanosphere/cipro was then incorporated into HEMA-based contact lenses that were tested for growth inhibition of S. aureus or P. aeruginosa in liquid cultures inoculated daily with fresh bacteria. Lens designs included thin or thick lenses incorporating nanosphere/cipro and ciprofloxacin-HCl-soaked Acuvue lenses (Acuvue; Johnson & Johnson Vision Care, Inc., Jacksonville, FL). Results. Less than 2 μg/mL of nanosphere/cipro effectively inhibited the proliferation of cultures inoculated with 107 or 108 bacteria/mL of S. aureus and P. aeruginosa, respectively. HEMA-based contact lenses polymerized with nanosphere/cipro were transparent, effectively inhibited the proliferation of greater than 107/mL of bacteria added daily over 3 days of culture, and killed up to 5 × 109 total microbes in a single inoculation. A thicker lens design provided additional inhibition of bacterial growth for up to 96 hours. Conclusions. Core-shell nanospheres loaded with an antibiotic can be incorporated into a conventional, transparent contact lens and provide for sustained and effective bactericidal activity and thereby provide a new drug delivery platform for widespread use in treating ocular disorders. PMID:22266514

Systematic evaluation of sericin protein as a substitute for fetal bovine serum in cell culture.

PubMed

Liu, Liyuan; Wang, Jinhuan; Duan, Shengchang; Chen, Lei; Xiang, Hui; Dong, Yang; Wang, Wen

2016-08-17

Fetal bovine serum (FBS) shows obvious deficiencies in cell culture, such as low batch to batch consistency, adventitious biological contaminant risk, and high cost, which severely limit the development of the cell culture industry. Sericin protein derived from the silkworm cocoon has become increasingly popular due to its diverse and beneficial cell culture characteristics. However, systematic evaluation of sericin as a substitute for FBS in cell culture medium remains limited. In this study, we conducted cellular morphological, physiological, and transcriptomic evaluation on three widely used mammalian cells. Compared with cells cultured in the control, those cultured in sericin-substitute medium showed similar cellular morphology, similar or higher cellular overall survival, lower population doubling time (PDT), and a higher percentage of S-phase with similar G2/G1 ratio, indicating comparable or better cell growth and proliferation. At the transcriptomic level, differentially expressed genes between cells in the two media were mainly enriched in function and biological processes related to cell growth and proliferation, reflecting that genes were activated to facilitate cell growth and proliferation. The results of this study suggest that cells cultured in sericin-substituted medium perform as well as, or even better than, those cultured in FBS-containing medium.

Systematic evaluation of sericin protein as a substitute for fetal bovine serum in cell culture

PubMed Central

Liu, Liyuan; Wang, Jinhuan; Duan, Shengchang; Chen, Lei; Xiang, Hui; Dong, Yang; Wang, Wen

2016-01-01

Fetal bovine serum (FBS) shows obvious deficiencies in cell culture, such as low batch to batch consistency, adventitious biological contaminant risk, and high cost, which severely limit the development of the cell culture industry. Sericin protein derived from the silkworm cocoon has become increasingly popular due to its diverse and beneficial cell culture characteristics. However, systematic evaluation of sericin as a substitute for FBS in cell culture medium remains limited. In this study, we conducted cellular morphological, physiological, and transcriptomic evaluation on three widely used mammalian cells. Compared with cells cultured in the control, those cultured in sericin-substitute medium showed similar cellular morphology, similar or higher cellular overall survival, lower population doubling time (PDT), and a higher percentage of S-phase with similar G2/G1 ratio, indicating comparable or better cell growth and proliferation. At the transcriptomic level, differentially expressed genes between cells in the two media were mainly enriched in function and biological processes related to cell growth and proliferation, reflecting that genes were activated to facilitate cell growth and proliferation. The results of this study suggest that cells cultured in sericin-substituted medium perform as well as, or even better than, those cultured in FBS-containing medium. PMID:27531556

Photosynthetic light reactions increase total lipid accumulation in carbon-supplemented batch cultures of Chlorella vulgaris.

PubMed

Woodworth, Benjamin D; Mead, Rebecca L; Nichols, Courtney N; Kolling, Derrick R J

2015-03-01

Microalgae are an attractive biofuel feedstock because of their high lipid to biomass ratios, lipid compositions that are suitable for biodiesel production, and the ability to grow on varied carbon sources. While algae can grow autotrophically, supplying an exogenous carbon source can increase growth rates and allow heterotrophic growth in the absence of light. Time course analyses of dextrose-supplemented Chlorella vulgaris batch cultures demonstrate that light availability directly influences growth rate, chlorophyll production, and total lipid accumulation. Parallel photomixotrophic and heterotrophic cultures grown to stationary phase reached the same amount of biomass, but total lipid content was higher for algae grown in the presence of light (an average of 1.90 mg/mL vs. 0.77 mg/mL over 5 days of stationary phase growth). Copyright © 2014 Elsevier Ltd. All rights reserved.

The mechanism of improved pullulan production by nitrogen limitation in batch culture of Aureobasidium pullulans.

PubMed

Wang, Dahui; Chen, Feifei; Wei, Gongyuan; Jiang, Min; Dong, Mingsheng

2015-08-20

Batch culture of Aureobasidium pullulans CCTCC M 2012259 for pullulan production at different concentrations of ammonium sulfate and yeast extract was investigated. Increased pullulan production was obtained under nitrogen-limiting conditions, as compared to that without nitrogen limitation. The mechanism of nitrogen limitation favoring to pullulan overproduction was revealed by determining the activity as well as gene expression of key enzymes, and energy supply for pullulan biosynthesis. Results indicated that nitrogen limitation increased the activities of α-phosphoglucose mutase and glucosyltransferase, up-regulated the transcriptional levels of pgm1 and fks genes, and supplied more ATP intracellularly, which were propitious to further pullulan biosynthesis. The economic analysis of batch pullulan production indicated that nitrogen limitation could reduce more than one third of the cost of raw materials when glucose was supplemented to a total concentration of 70 g/L. This study also helps to understand the mechanism of other polysaccharide overproduction by nitrogen limitation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Modeling and parameters identification of 2-keto-L-gulonic acid fed-batch fermentation.

PubMed

Wang, Tao; Sun, Jibin; Yuan, Jingqi

2015-04-01

This article presents a modeling approach for industrial 2-keto-L-gulonic acid (2-KGA) fed-batch fermentation by the mixed culture of Ketogulonicigenium vulgare (K. vulgare) and Bacillus megaterium (B. megaterium). A macrokinetic model of K. vulgare is constructed based on the simplified metabolic pathways. The reaction rates obtained from the macrokinetic model are then coupled into a bioreactor model such that the relationship between substrate feeding rates and the main state variables, e.g., the concentrations of the biomass, substrate and product, is constructed. A differential evolution algorithm using the Lozi map as the random number generator is utilized to perform the model parameters identification, with the industrial data of 2-KGA fed-batch fermentation. Validation results demonstrate that the model simulations of substrate and product concentrations are well in coincidence with the measurements. Furthermore, the model simulations of biomass concentrations reflect principally the growth kinetics of the two microbes in the mixed culture.

Mangifera indica L. extract (Vimang) and mangiferin modulate mouse humoral immune responses.

PubMed

García, D; Leiro, J; Delgado, R; Sanmartín, M L; Ubeira, F M

2003-12-01

The present study investigated the effects of orally administered Vimang (an aqueous extract of Mangifera indica) and mangiferin (the major polyphenol present in Vimang) on mouse antibody responses induced by inoculation with spores of microsporidian parasites. Inoculation induced specific antibody production with an exponential timecourse, peaking after about one month. Vimang significantly inhibited this antibody production from about three weeks post-inoculation, and most markedly by four weeks post-inoculation; by contrast, mangiferin had no significant effect. Determination of Ig isotypes showed that the IgM to IgG switch began about four weeks post-inoculation, with IgG2a predominating. Vimang significantly inhibited IgG production, but had no effect on IgM. Mangiferin did no affect either IgM or IgG2a, but significantly enhanced production of IgG1 and IgG2b. Neither Vimang nor mangiferin enhanced specific antibody secretion by splenic plasma cells from mice inoculated with microsporidian spores, whether administered in vivo before serum extraction or in vitro to the culture medium. Inoculation with spores induced splenomegaly, which was significantly reduced by Vimang and significantly enhanced by mangiferin. These results suggest that components of Mangifera indica extracts may be of potential value for modulating the humoral response in different immunopathological disorders. Copyright 2003 John Wiley & Sons, Ltd.

Bioelectrochemical sulphate reduction on batch reactors: Effect of inoculum-type and applied potential on sulphate consumption and pH.

PubMed

Gacitúa, Manuel A; Muñoz, Enyelbert; González, Bernardo

2018-02-01

Microbial electrolysis batch reactor systems were studied employing different conditions, paying attention on the effect that biocathode potential has on pH and system performance, with the overall aim to distinguish sulphate reduction from H 2 evolution. Inocula from pure strains (Desulfovibrio paquesii and Desulfobacter halotolerans) were compared to a natural source conditioned inoculum. The natural inoculum possess the potential for sulphate reduction on serum bottles experiments due to the activity of mutualistic bacteria (Sedimentibacter sp. and Bacteroides sp.) that assist sulphate-reducing bacterial cells (Desulfovibrio sp.) present in the consortium. Electrochemical batch reactors were monitored at two different potentials (graphite-bar cathodes poised at -900 and -400mV versus standard hydrogen electrode) in an attempt to isolate bioelectrochemical sulphate reduction from hydrogen evolution. At -900mV all inocula were able to reduce sulphate with the consortium demonstrating superior performance (SO 4 2- consumption: 25.71gm -2 day -1 ), despite the high alkalinisation of the media. At -400mV only the pure Desulfobacter halotolerans inoculated system was able to reduce sulphate (SO 4 2- consumption: 17.47gm -2 day -1 ) and, in this potential condition, pH elevation was less for all systems, confirming direct (or at least preferential) bioelectrochemical reduction of sulphate over H 2 production. Copyright © 2017 Elsevier B.V. All rights reserved.

High Solid Fed-batch Butanol Fermentation with Simultaneous Product Recovery: Part II - Process Integration.

PubMed

Qureshi, Nasib; Klasson, K Thomas; Saha, Badal C; Liu, Siqing

2018-04-25

In these studies liquid hot water (LHW) pretreated and enzymatically hydrolyzed Sweet Sorghum Bagasse (SSB) hydrolyzates were fermented in a fed-batch reactor. As reported in the preceding paper, the culture was not able to ferment the hydrolyzate I in a batch process due to presence of high level of toxic chemicals, in particular acetic acid released from SSB during the hydrolytic process. To be able to ferment the hydrolyzate I obtained from 250 gL -1 SSB hydrolysis, a fed-batch reactor with in-situ butanol recovery was devised. The process was started with the hydrolyzate II and when good cell growth and vigorous fermentation were observed, the hydrolyzate I was slowly fed to the reactor. In this manner the culture was able to ferment all the sugars present in both the hydrolyzates to acetone butanol ethanol (ABE). In a control batch reactor in which ABE was produced from glucose, ABE productivity and yield of 0.42 gL -1 h -1 and 0.36 were obtained, respectively. In the fed-batch reactor fed with SSB hydrolyzates these productivity and yield values were 0.44 gL -1 h -1 and 0.45, respectively. ABE yield in the integrated system was high due to utilization of acetic acid to convert to ABE. In summary we were able to utilize both the hydrolyzates obtained from LHW pretreated and enzymatically hydrolyzed SSB (250 gL -1 ) and convert them to ABE. Complete fermentation was possible due to simultaneous recovery of ABE by vacuum. This article is protected by copyright. All rights reserved. © 2018 American Institute of Chemical Engineers.

Comparison of batch cultivation strategies for cost-effective biomass production of Micractinium inermum NLP-F014 using a blended wastewater medium.

PubMed

Park, Seonghwan; Kim, Jeongmi; Park, Younghyun; Son, Suyoung; Cho, Sunja; Kim, Changwon; Lee, Taeho

2017-06-01

Two competitive strategies, fed-batch and sequencing-batch cultivation, were compared in cost-effective biomass production of a high lipid microalgae, Micractinium inermum NLP-F014 using a blended wastewater medium. For fed-batch cultivations, additional nutrient was supplemented at day 2 (FB1) or consecutively added at day 2 and 4 (FB2). Through inoculum size test, 1.0g-DCWL -1 was selected for the sequencing-batch cultivation (SB) where about 65% of culture was replaced with fresh medium every 2days. Both fed-batch cultivations showed the maximum biomass productivity of 0.95g-DCWL -1 d -1 , while average biomass productivity in SB was slightly higher as 0.96±0.08g-DCWL -1 d -1 . Furthermore, remained concentrations of organics (426mg-CODL -1 ), total nitrogen (15.4mg-NL -1 ) and phosphorus (0.6mg-PL -1 ) in SB were much lower than those of fed-batch conditions. The results suggested that SB could be a promising strategy to cultivate M. inermum NLP-F014 with the blended wastewater medium. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evaluation of two types of swabs for sampling allograft musculoskeletal tissue.

PubMed

Varettas, Kerry

2015-01-01

Allograft musculoskeletal tissue is commonly sampled by a swab for bioburden screening. To determine if bioburden recovery could be improved at the pre-analytical stage, two swab systems were evaluated: the Amies gel swab and the ESwab. In vitro studies were performed to determine the recovery of each swab system with <100 colony-forming unit of challenge organisms using inoculated swabs and by sampling inoculated femoral heads. The standard culture protocol used in this laboratory was also evaluated after sampling of inoculated femoral heads. A prospective study was performed with both swab systems used in parallel to sample cadaveric allograft musculoskeletal tissue. The challenge organisms could be recovered from the in vitro inoculated studies. The standard culture protocol in this laboratory recovered all challenge organisms from both swab systems. One hundred and six paired Amies and ESwabs were collected from eight cadaveric donors with skin commensals the predominant isolates. The sampling of an inoculated femoral head was included to reflect routine swab sampling practice as was the inclusion of the standard method used in this laboratory. This appears to be the first study to compare Amies gel swabs with ESwabs to sample allograft femoral heads and in a prospective study with cadaveric allograft musculoskeletal tissue. Other comparative studies of swab systems have used a much higher inoculum to mimic an infection; however, sepsis is an exclusion criterion for allograft donors. It was found that the Amies gel swab and ESwab are both suitable sampling devices for bioburden testing of allograft musculoskeletal tissue. © 2014 Royal Australasian College of Surgeons.

Oronasal and intramuscular vaccination of swine with a modified live porcine parvovirus vaccine: multiplication and transmission of the vaccine virus.

PubMed

Paul, P S; Mengeling, W L

1984-12-01

An attenuated strain NADL-2 of porcine parvovirus (PPV) has been used at the 54th cell culture passage as a modified live-virus (MLV) vaccine. The present study was conducted to determine the minimum immunizing dose of MLV, the extent of MLV multiplication in swine tissues, and its transmission from swine administered MLV oronasally or intramuscularly. Immune response to MLV was dose dependent and swine responded to as little as 10(2) median cell-culture infective doses (CCID50). A 10(5) CCID50 of MLV, the largest dose given, induced the best immune response and was used in subsequent experiments. Route of MLV administration also was found to be important. The MLV replicated in tissues of swine after IM inoculation; however, viral antigen in tissues was less, as measured by immunofluorescence, and serum hemagglutination-inhibition titers for PPV were lower in MLV-inoculated swine than we have previously observed in virulent PPV-inoculated swine. In contrast, oronasal inoculation with MLV did not consistently result in infection of pigs; only 5 of 23 swine had virologic and/or serologic evidence of infection. Virus transmission studies indicated that MLV is shed in feces, but shedding occurs later than that in virulent-PPV-inoculated swine and is inconsistent. Delayed transmission of MLV was observed in contact pigs, which were seronegative at 2 weeks, but became seropositive at 4 weeks--indicating that perhaps a virus population capable of infecting pigs by oronasal route was selected by passage through the pig.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular and structural characteristics in toxic algae cultures of Ostreopsis ovata and Ostreopsis spp. evidenced by FTIR and FTNIR spectroscopy

NASA Astrophysics Data System (ADS)

Mecozzi, Mauro; Pietroletti, Marco; Tornambè, Andrea

2011-05-01

In this article we investigated the compositional and structural characteristics of the principal biomolecules such as carbohydrates, proteins, lipids, nucleic acids and chlorophyll pigments present in biofilm cultures of Ostreopsis spp. and in batch cultures of Ostreopsis ovata. Our approach based on the use of infrared (FTIR) and near infrared (FTNIR) spectroscopy showed the marked differences existing between biofilm cultures and batch cultures. FTIR spectroscopy showed the higher contents of polysaccharides and chlorophyll pigments in O. ovata from batch cultures with respect to Ostreopsis spp. Second derivative FTIR spectroscopy showed different features concerning the secondary structure of proteins because in O. ovata samples the beta sheet and beta turn structures were observed whereas in Ostreopsis spp. samples the alpha helix structure was the most evident. FTNIR spectroscopy showed other structural differences observed existing between O. ovata and Ostreopsis spp. mainly related to hydrogen bond interactions determining more packed structures in the nucleus of O. ovata. In addition, the interpretation of FTIR and FTNIR spectral information was also supported by the application of two statistical methods, the independent component analysis (ICA) and the spectral cross correlation analysis (SCCA). ICA was used as spectral deconvolution technique to separate the effects of the interference bicarbonate ion from algal FTIR spectra so to verify the high similar qualitative composition of the three biofilm samples of Ostreopsis spp. At last, SCCA applied to FTIR and FTNIR spectra was useful to evidence some structural differences involving -CH and CH 2 groups of aliphatic chains in O. ovata and Ostreopsis spp. samples. Though preliminary, these results agree with some previous studies suggesting that the presence of different ecophysiological characteristics in O. ovata and Ostreopsis spp. depending on the parameters related to the condition growth.

The Growth Advantage in Stationary-Phase Phenotype Conferred by rpoS Mutations Is Dependent on the pH and Nutrient Environment

PubMed Central

Farrell, Michael J.; Finkel, Steven E.

2003-01-01

Escherichia coli cells that are aged in batch culture display an increased fitness referred to as the growth advantage in stationary phase, or GASP, phenotype. A common early adaptation to this culture environment is a mutant rpoS allele, such as rpoS819, that results in attenuated RpoS activity. However, it is important to note that during long-term batch culture, environmental conditions are in flux. To date, most studies of the GASP phenotype have focused on identifying alleles that render an advantage in a specific environment, Luria-Bertani broth (LB) batch culture. To determine what role environmental conditions play in rendering relative fitness advantages to E. coli cells carrying either the wild-type or rpoS819 alleles, we performed competitions under a variety of culture conditions in which either the available nutrients, the pH, or both were manipulated. In LB medium, we found that while the rpoS819 allele confers a strong competitive fitness advantage at basic pH, it confers a reduced advantage under neutral conditions, and it is disadvantageous under acidic conditions. Similar results were found using other media. rpoS819 conferred its greatest advantage in basic minimal medium in which either glucose or Casamino Acids were the sole source of carbon and energy. In acidic medium supplemented with either Casamino Acids or glucose, the wild-type allele conferred a slight advantage. In addition, populations were dynamic under all pH conditions tested, with neither the wild-type nor mutant rpoS alleles sweeping a culture. We also found that the strength of the fitness advantage gained during a 10-day incubation is pH dependent. PMID:14645263

Development and characterization of an ex-vivo brain slice culture model of chronic wasting disease

USDA-ARS?s Scientific Manuscript database

Prion diseases have long incubation times in vivo, therefore, modeling the diseases ex-vivo will advance the development of rationale-based therapeutic strategies. An organotypic slice culture assay (POSCA) was recently developed for scrapie prions by inoculating mouse cerebellar brain slices with R...

Modified ecometric technique (four-quadrant sequential streak) to evaluate Campylobacter enrichment broth proficiency in suppressing background microflora

USDA-ARS?s Scientific Manuscript database

Ecometric technique is a semi-quantitative scoring method used for quality control of culture media in microbiological laboratories. The technique involves inoculation with defined populations of specific culture onto solid media via a standardized chronological streaking technique, leading to ever-...

Dramatic reduction of culture time of Mycobacterium tuberculosis

NASA Astrophysics Data System (ADS)

Ghodbane, Ramzi; Raoult, Didier; Drancourt, Michel

2014-02-01

Mycobacterium tuberculosis culture, a critical technique for routine diagnosis of tuberculosis, takes more than two weeks. Here, step-by-step improvements in the protocol including a new medium, microaerophlic atmosphere or ascorbic-acid supplement and autofluorescence detection dramatically shortened this delay. In the best case, primary culture and rifampicin susceptibility testing were achieved in 72 hours when specimens were inoculated directly on the medium supplemented by antibiotic at the beginning of the culture.

Possible evidence for contribution of arbuscular mycorrhizal fungi (AMF) in phytoremediation of iron-cyanide (Fe-CN) complexes.

PubMed

Sut, Magdalena; Boldt-Burisch, Katja; Raab, Thomas

2016-08-01

Arbuscular mycorrhizal fungi (AMF) are integral functioning parts of plant root systems and are widely recognized for enhancing contaminants uptake and metabolism on severely disturbed sites. However, the patterns of their influence on the phytoremediation of iron-cyanide (Fe-CN) complexes are unknown. Fe-CN complexes are of great common interest, as iron is one of the most abundant element in soil and water. Effect of ryegrass (Lolium perenne L.) roots inoculation, using mycorrhizal fungi (Rhizophagus irregularis and a mixture of R. irregularis, Funneliformis mosseae, Rhizophagus aggregatus, and Claroideoglomus etunicatum), on iron-cyanide sorption was studied. Results indicated significantly higher colonization of R. irregularis than the mixture of AMF species on ryegrass roots. Series of batch experiments using potassium hexacyanoferrate (II) solutions, in varying concentrations revealed significantly higher reduction of total CN and free CN content in the mycorrhizal roots, indicating greater cyanide decrease in the treatment inoculated with R. irregularis. Our study is a first indication of the possible positive contribution of AM fungi on the phytoremediation of iron-cyanide complexes.

Enhanced production and immunological characterization of recombinant West Nile virus envelope domain III protein.

PubMed

Tripathi, Nagesh K; Karothia, Divyanshi; Shrivastava, Ambuj; Banger, Swati; Kumar, Jyoti S

2018-05-13

West Nile virus (WNV) is an emerging mosquito-borne virus which is responsible for severe and fatal encephalitis in humans and for which there is no licensed vaccine or therapeutic available to prevent infection. The envelope domain III protein (EDIII) of WNV was over-expressed in Escherichia coli and purified using a two-step chromatography process which included immobilized metal affinity chromatography and ion exchange chromatography. E. coli cells were grown in a bioreactor to high density using batch and fed-batch cultivation. Wet biomass obtained after batch and fed-batch cultivation processes was 11.2 g and 84 g/L of culture respectively. Protein yield after affinity purification was 5.76 mg and 5.81 mg/g wet cell weight after batch and fed-batch processes respectively. The purified WNV EDIII elicited specific antibodies in rabbits, confirming its immunogenicity. Moreover, the antibodies were able to neutralize WNV in vitro. These results established that the refolded and purified WNV EDIII could be a potential vaccine candidate. Copyright © 2018 Elsevier B.V. All rights reserved.

A rapid chemiluminescent slot blot immunoassay for the detection and quantification of Clostridium botulinum neurotoxin type E, in cultures.

PubMed

Cadieux, Brigitte; Blanchfield, Burke; Smith, James P; Austin, John W

2005-05-01

A simple, rapid, cost-effective in vitro slot blot immunoassay was developed for the detection and quantification of botulinum neurotoxin type E (BoNT/E) in cultures. Culture supernatants of 36 strains of clostridia, including 12 strains of Clostridium botulinum type E, 12 strains of other C. botulinum neurotoxin serotypes, and 12 strains of other clostridial species were tested. Samples containing BoNT/E were detected using affinity-purified polyclonal rabbit antisera prepared against BoNT/E with subsequent detection of secondary antibodies using chemiluminescence. All strains of C. botulinum type E tested positive, while all non C. botulinum type E strains tested negative. The sensitivity of the slot blot immunoassay for detection of BoNT/E was approximately four mouse lethal doses (MLD). The intensity of chemiluminescence was directly correlated with the concentration of BoNT/E up to 128 MLD, allowing quantification of BoNT/E between 4 and 128 MLD. The slot blot immunoassay was compared to the mouse bioassay for detection of BoNT/E using cultures derived from fish samples inoculated with C. botulinum type E, and cultures derived from naturally contaminated environmental samples. A total of 120 primary enrichment cultures derived from fish samples, of which 103 were inoculated with C. botulinum type E, and 17 were uninoculated controls, were assayed. Of the 103 primary enrichment cultures derived from inoculated fish samples, all were positive by mouse bioassay, while 94 were also positive by slot blot immunoassay, resulting in a 7.5% false-negative rate. All 17 primary enrichment cultures derived from the uninoculated fish samples were negative by both mouse bioassay and slot blot immunoassay. A total of twenty-six primary enrichment cultures derived from environmental samples were tested by mouse bioassay and slot blot immunoassay. Of 13 primary enrichment cultures positive by mouse bioassay, 12 were also positive by slot blot immunoassay, resulting in a 3.8% false-negative rate. All 13 primary enrichment cultures that tested negative by mouse bioassay also tested negative by slot blot immunoassay. The slot blot immunoassay could be used routinely as a positive screen for BoNT/E in primary enrichment cultures, and could be used as a replacement for the mouse bioassay for pure cultures.

Contribution of acetate to butyrate formation by human faecal bacteria.

PubMed

Duncan, Sylvia H; Holtrop, Grietje; Lobley, Gerald E; Calder, A Graham; Stewart, Colin S; Flint, Harry J

2004-06-01

Acetate is normally regarded as an endproduct of anaerobic fermentation, but butyrate-producing bacteria found in the human colon can be net utilisers of acetate. The butyrate formed provides a fuel for epithelial cells of the large intestine and influences colonic health. [1-(13)C]Acetate was used to investigate the contribution of exogenous acetate to butyrate formation. Faecalibacterium prausnitzii and Roseburia spp. grown in the presence of 60 mm-acetate and 10 mm-glucose derived 85-90 % butyrate-C from external acetate. This was due to rapid interchange between extracellular acetate and intracellular acetyl-CoA, plus net acetate uptake. In contrast, a Coprococcus-related strain that is a net acetate producer derived only 28 % butyrate-C from external acetate. Different carbohydrate-derived energy sources affected butyrate formation by mixed human faecal bacteria growing in continuous or batch cultures. The ranking order of butyrate production rates was amylopectin > oat xylan > shredded wheat > inulin > pectin (continuous cultures), and inulin > amylopectin > oat xylan > shredded wheat > pectin (batch cultures). The contribution of external acetate to butyrate formation in these experiments ranged from 56 (pectin) to 90 % (xylan) in continuous cultures, and from 72 to 91 % in the batch cultures. This is consistent with a major role for bacteria related to F. prausnitzii and Roseburia spp. in butyrate formation from a range of substrates that are fermented in the large intestine. Variations in the dominant metabolic type of butyrate producer between individuals or with variations in diet are not ruled out, however, and could influence butyrate supply in the large intestine.

Alginate Beads as Synthetic Inoculant Carriers for Slow Release of Bacteria That Affect Plant Growth †‡

PubMed Central

Bashan, Yoav

1986-01-01

Uniform synthetic beads were developed as carriers for the bacterial inoculation of plants. The beads are made of sodium alginate and skim milk and contain a large reservoir of bacterial culture which releases the bacteria at a slow and constant rate. The beads are biodegradable and produce no environmental pollution. The strength of the beads, the rate of bacterial release, and the time of their survival in the soil can be controlled by several hardening treatments. The final product, lyophilized beads, is simple to use and is applied to the seeds concomitantly with sowing. The released bacteria are available for root colonization immediately at seed germination. Dry beads containing bacteria can be stored at ambient temperature over a long period without loss of bacterial content; storage requires a limited space, and the quality control of a number of bacteria in the bead is simple. The level of plant inoculation with beads was similar to that with previously used peat inoculants, but the former method yielded more consistent results, as the frequency of inoculated plants was much higher. The former method provides a different approach for inoculation of plants with beneficial rhizosphere bacteria. Images PMID:16347055

Evidence of Archaeal Methanogens in Brain Abscess.

PubMed

Drancourt, Michel; Nkamga, Vanessa Demonfort; Lakhe, Ndèye Aïssatou; Régis, Jean-Marie; Dufour, Henry; Fournier, Pierre-Edouard; Bechah, Yassina; Scheld, W Michael; Raoult, Didier

2017-07-01

Methanogens are antibiotic-resistant anaerobic archaea that escape routine detection in clinical microbiology. We hypothesized that methanogens are part of the anaerobic community that cause brain abscess. Methanogens were investigated in 1 index sample using specific polymerase chain reaction (PCR) sequencing and culture. The pathogenesis of a methanogen isolate was assessed in a mouse model. Archaea-specific quantitative (q) PCR and metagenomics were used to detect specific archaeal sequences in brain abscess samples and controls. In 1 index sample, routine culture found Porphyromonas endodontalis and Streptococcus intermedius, and specific culture found Methanobrevibacter oralis susceptible to metronidazole and fusidic acid. Archaea-targeted PCR sequencing and metagenomics confirmed M. oralis along with 14 bacteria, including S. intermedius. Archaea-specific qPCR yielded archaea in 8/18 brain abscess specimens and 1/27 controls (P < .003), and metagenomics yielded archaea, mostly methanogens, in 28/32 brain abscess samples, and no archaea in 71 negative controls (P < 10-6). Infection of mice brains yielded no mortality in 14 controls and death in 17/22 M. oralis-inoculated mice (P < 10-6), 32/95 S. intermedius-inoculated mice (P < 10-6), and 75/104 mice inoculated with M. oralis mixed with S. intermedius (P < 10-6) 7 days post-inoculation. Methanogens belong to the anaerobic community responsible for brain abscess, and M. oralis may participate in the pathogenicity of this deadly infection. In mice, a synergy of M. oralis and S. intermedius was observed. Antibiotic treatment of brain abscess should contain anti-archaeal compounds such as imidazole derivatives in most cases. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Using common mycorrhizal networks for controlled inoculation of Quercus spp. with Tuber melanosporum: the nurse plant method.

PubMed

Pereira, Guillermo; Palfner, Götz; Chávez, Daniel; Suz, Laura M; Machuca, Angela; Honrubia, Mario

2013-07-01

The high cost and restricted availability of black truffle spore inoculum for controlled mycorrhiza formation of host trees produced for truffle orchards worldwide encourage the search for more efficient and sustainable inoculation methods that can be applied globally. In this study, we evaluated the potential of the nurse plant method for the controlled inoculation of Quercus cerris and Quercus robur with Tuber melanosporum by mycorrhizal networks in pot cultures. Pine bark compost, adjusted to pH 7.8 by liming, was used as substrate for all assays. Initially, Q. robur seedlings were inoculated with truffle spores and cultured for 12 months. After this period, the plants presenting 74 % mycorrhizal fine roots were transferred to larger containers. Nurse plants were used for two treatments of two different nursling species: five sterilized acorns or five 45-day-old, axenically grown Q. robur or Q. cerris seedlings, planted in containers around the nurse plant. After 6 months, colonized nursling plant root tips showed that mycorrhiza formation by T. melanosporum was higher than 45 % in the seedlings tested, with the most successful nursling combination being Q. cerris seedlings, reaching 81 % colonization. Bulk identification of T. melanosporum mycorrhizae was based on morphological and anatomical features and confirmed by sequencing of the internal transcribed spacer region of the ribosomal DNA of selected root tips. Our results show that the nurse plant method yields attractive rates of mycorrhiza formation by the Périgord black truffle and suggest that establishing and maintaining common mycorrhizal networks in pot cultures enables sustained use of the initial spore inoculum.

Growth kinetics of Bacillus stearothermophilus BR219

DOE Office of Scientific and Technical Information (OSTI.GOV)

Worden, R.M.; Subramanian, R.; Bly, M.J.

1991-12-31

Bacillus stearothermophilus BR219, a phenol-resistant thermophile, can convert phenol to the specialty chemical catechol. The growth kinetics of this organism were studied in batch, continuous, and immobilized-cell culture. Batch growth was insensitive to pH between 6.0 and 8.0, but little growth occurred at 5.5. In continuous culture on a dilute medium supplemented with 10 mM phenol, several steady states were achieved between dilution rates of 0.25 and 1.3 h{sup -1}. Phenol degradation was found to be uncoupled from growth. Immobilized cells grew rapidly in a rich medium, but cell viability plummeted following a switch to a dilute medium supplemented withmore » 5 mM phenol.« less

Effect of dissolved aromatic hydrocarbons on the growth of marine bacteria in batch culture.

PubMed Central

Calder, J A; Lader, J H

1976-01-01

Dissolved aromatic hydrocarbons were found to decrease growth rate and maximum cell density of marine bacteria in batch cultures. The magnitude of the decrement was observed to be a function of concentration of the hydrocarbon and inherent toxicity. The inherent toxicity was observed to increase inversely with solubility such that naphthalene at 100 muM concentration demonstrated a toxic effect similar to benzopyrene at 0.02 muM. A partial oxidation product of naphthalene was found to be more effective in decreasing growth parameters than naphthalene at equivalent concentrations and to cause complete cessation of growth at the higher concentrations permitted by its polar structure. PMID:970939

Study of methods for the improvement of bacterial transport media

NASA Technical Reports Server (NTRS)

Gardner, R. L.; Beakley, J. W.

1973-01-01

A series of 500 transport media recipes was tested for ability to hold pure cultures of Streptococcus equisimilus, Corynebacterium equi, Neisseria perflava, and Haemophilus parainfluenzae for 21 days. Stuart Medium Base with 0.4% agar was used as the control medium for this and the other experiments in the investigation. At the end of the holding period inoculated transport media were quantitatively assayed, and the control media were assayed immediately after inoculation. Three vials of each medium were inoculated with an organism, and each vial's medium was diluted and spread on duplicate plates. Assay media for this experiment included Brain Heart Infusion,(BHIA) Tryptic Soy Agar, and BHIA with 1% Isovitalex enrichment.

Dynamics of yeast immobilized-cell fluidized-bed bioreactors systems in ethanol fermentation from lactose-hydrolyzed whey and whey permeate.

PubMed

Gabardo, Sabrina; Pereira, Gabriela Feix; Klein, Manuela P; Rech, Rosane; Hertz, Plinho F; Ayub, Marco Antônio Záchia

2016-01-01

We studied the dynamics of ethanol production on lactose-hydrolyzed whey (LHW) and lactose-hydrolyzed whey permeate (LHWP) in batch fluidized-bed bioreactors using single and co-cultures of immobilized cells of industrial strains of Saccharomyces cerevisiae and non-industrial strains of Kluyveromyces marxianus. Although the co-culture of S. cerevisiae CAT-1 and K. marxianus CCT 4086 produced two- to fourfold the ethanol productivity of single cultures of S. cerevisiae, the single cultures of the K. marxianus CCT 4086 produced the best results in both media (Y EtOH/S = 0.47-0.49 g g(-1) and Q P = 1.39-1.68 g L(-1) h(-1), in LHW and LHWP, respectively). Ethanol production on concentrated LHWP (180 g L(-1)) reached 79.1 g L(-1), with yields of 0.46 g g(-1) for K. marxianus CCT 4086 cultures. Repeated batches of fluidized-bed bioreactor on concentrated LHWP led to increased ethanol productivity, reaching 2.8 g L(-1) h(-1).

Enhancement of Biohydrogen Production via pH Variation using Molasses as Feedstock in an Attached Growth System

NASA Astrophysics Data System (ADS)

Che Zuhar, C. N. S.; Lutpi, N. A.; Idris, N.; Wong, Y. S.; Tengku Izhar, T. N.

2018-03-01

In this study, mesophilic biohydrogen production by a mixed culture, obtained from a continuous anaerobic reactor treating molasses effluent from sugarcane bagasse, was improved by using granular activated carbon (GAC) as the carrier material. A series of batch fermentation were performed at 37°C by feeding the anaerobic sludge bacteria with molasses to determine the effect of initial pH in the range of 5.5 to 7.5, and the effect of repeated batch cultivation on biohydrogen production. The enrichment of granular activated carbon (GAC) immobilised cells from the repeated batch cultivation were used as immobilised seed culture to obtain the optimal initial pH. The cumulative hydrogen production results from the optimal pH were fitted into modified Gompertz equation in order to obtained the batch profile of biohydrogen production. The optimal hydrogen production was obtained at an initial pH of 5.5 with the maximum hydrogen production (Hm) was found to be 84.14 ml, and maximum hydrogen production rate (Rm) was 3.63 mL/h with hydrogen concentration of 759 ppm. The results showed that the granular activated carbon was successfully enhanced the biohydrogen production by stabilizing the pH and therefore could be used as a carrier material for fermentative hydrogen production using industrial effluent.

Biotechnological enhancement of capsaicin biosynthesis in cell suspension cultures of Naga King Chili (Capsicum chinense Jacq.).

PubMed

Kehie, Mechuselie; Kumaria, Suman; Tandon, Pramod

2016-01-01

Cell suspension cultures were initiated from hypocotyl derived callus to induce capsaicin biosynthesis in suspension cultures of Naga King Chili (Capsicum chinense Jacq.). Efficient capsaicin production with high growth index (GI) was obtained by exposing cells to salicylic acid (SA) and calcium channel modulators in suspension cultures. The time course of capsaicin formation is related to the cell growth profile in a batch culture. Cells cultivated in the standard medium (SM) initially showed low level of capsaicin yield during active growth. When the cells approached stationary phase, cell growth and cell viability decreased whereas capsaicin production increased continuously. In the fed-batch cultures, the highest capsaicin yield (567.4 ± 8.1 μgg(1) fresh weight) (f.wt) was obtained by feeding the cells with 1 mM SA. However, SA feeding during cultivation repressed the cell growth. Enhanced cell growth (3.1 ± 0.1 GI/culture) and capsaicin yield (534 ± 7.8 μgg(-1)f.wt) were obtained when the cells were fed with calcium ionophore A23187 (0.5 mM) on day 25 as compared to the control. Addition of the calcium channel blocker verapamil hydrochloride (100 mM) inhibited cell growth and capsaicin production in Naga King Chili suspension cell cultures.

Establishment and assessment of an integrated citric acid-methane production process.

PubMed

Xu, Jian; Chen, Yang-Qiu; Zhang, Hong-Jian; Bao, Jia-Wei; Tang, Lei; Wang, Ke; Zhang, Jian-Hua; Chen, Xu-Sheng; Mao, Zhong-Gui

2015-01-01

To solve the problem of extraction wastewater in citric acid industrial production, an improved integrated citric acid-methane production process was established in this study. Extraction wastewater was treated by anaerobic digestion and then the anaerobic digestion effluent (ADE) was stripped by air to remove ammonia. Followed by solid-liquid separation to remove metal ion precipitation, the supernatant was recycled for the next batch of citric acid fermentation, thus eliminating wastewater discharge and reducing water consumption. 130U/g glucoamylase was added to medium after inoculation and the recycling process performed for 10 batches. Fermentation time decreased by 20% in recycling and the average citric acid production (2nd-10th) was 145.9±3.4g/L, only 2.5% lower than that with tap water (149.6g/L). The average methane production was 292.3±25.1mL/g CODremoved and stable in operation. Excessive Na(+) concentration in ADE was confirmed to be the major challenge for the proposed process. Copyright © 2014 Elsevier Ltd. All rights reserved.

Impact of 2,4-Diacetylphloroglucinol-Producing Biocontrol Strain Pseudomonas fluorescens F113 on Intraspecific Diversity of Resident Culturable Fluorescent Pseudomonads Associated with the Roots of Field-Grown Sugar Beet Seedlings

PubMed Central

Moënne-Loccoz, Yvan; Tichy, Hans-Volker; O'Donnell, Anne; Simon, Reinhard; O'Gara, Fergal

2001-01-01

The impact of the 2,4-diacetylphloroglucinol-producing biocontrol agent Pseudomonas fluorescens F113Rif on the diversity of the resident community of culturable fluorescent pseudomonads associated with the roots of field-grown sugar beet seedlings was evaluated. At 19 days after sowing, the seed inoculant F113Rif had replaced some of the resident culturable fluorescent pseudomonads at the rhizoplane but had no effect on the number of these bacteria in the rhizosphere. A total of 498 isolates of resident fluorescent pseudomonads were obtained and characterized by molecular means at the level of broad phylogenetic groups (by amplified ribosomal DNA restriction analysis) and at the strain level (with random amplified polymorphic DNA markers) as well as phenotypically (55 physiological tests). The introduced pseudomonad induced a major shift in the composition of the resident culturable fluorescent Pseudomonas community, as the percentage of rhizoplane isolates capable of growing on three carbon substrates (erythritol, adonitol, and l-tryptophan) not assimilated by the inoculant was increased from less than 10% to more than 40%. However, the pseudomonads selected did not display enhanced resistance to 2,4-diacetylphloroglucinol. The shift in the resident populations, which was spatially limited to the surface of the root (i.e., the rhizoplane), took place without affecting the relative proportions of phylogenetic groups or the high level of strain diversity of the resident culturable fluorescent Pseudomonas community. These results suggest that the root-associated Pseudomonas community of sugar beet seedlings is resilient to the perturbation that may be caused by a taxonomically related inoculant. PMID:11472913

Mitigation of soil N2O emission by inoculation with a mixed culture of indigenous Bradyrhizobium diazoefficiens

NASA Astrophysics Data System (ADS)

Akiyama, Hiroko; Hoshino, Yuko Takada; Itakura, Manabu; Shimomura, Yumi; Wang, Yong; Yamamoto, Akinori; Tago, Kanako; Nakajima, Yasuhiro; Minamisawa, Kiwamu; Hayatsu, Masahito

2016-09-01

Agricultural soil is the largest source of nitrous oxide (N2O), a greenhouse gas. Soybean is an important leguminous crop worldwide. Soybean hosts symbiotic nitrogen-fixing soil bacteria (rhizobia) in root nodules. In soybean ecosystems, N2O emissions often increase during decomposition of the root nodules. Our previous study showed that N2O reductase can be used to mitigate N2O emission from soybean fields during nodule decomposition by inoculation with nosZ++ strains [mutants with increased N2O reductase (N2OR) activity] of Bradyrhizobium diazoefficiens. Here, we show that N2O emission can be reduced at the field scale by inoculation with a mixed culture of indigenous nosZ+ strains of B. diazoefficiens USDA110 group isolated from Japanese agricultural fields. Our results also suggested that nodule nitrogen is the main source of N2O production during nodule decomposition. Isolating nosZ+ strains from local soybean fields would be more applicable and feasible for many soybean-producing countries than generating mutants.

Mitigation of soil N2O emission by inoculation with a mixed culture of indigenous Bradyrhizobium diazoefficiens

PubMed Central

Akiyama, Hiroko; Hoshino, Yuko Takada; Itakura, Manabu; Shimomura, Yumi; Wang, Yong; Yamamoto, Akinori; Tago, Kanako; Nakajima, Yasuhiro; Minamisawa, Kiwamu; Hayatsu, Masahito

2016-01-01

Agricultural soil is the largest source of nitrous oxide (N2O), a greenhouse gas. Soybean is an important leguminous crop worldwide. Soybean hosts symbiotic nitrogen-fixing soil bacteria (rhizobia) in root nodules. In soybean ecosystems, N2O emissions often increase during decomposition of the root nodules. Our previous study showed that N2O reductase can be used to mitigate N2O emission from soybean fields during nodule decomposition by inoculation with nosZ++ strains [mutants with increased N2O reductase (N2OR) activity] of Bradyrhizobium diazoefficiens. Here, we show that N2O emission can be reduced at the field scale by inoculation with a mixed culture of indigenous nosZ+ strains of B. diazoefficiens USDA110 group isolated from Japanese agricultural fields. Our results also suggested that nodule nitrogen is the main source of N2O production during nodule decomposition. Isolating nosZ+ strains from local soybean fields would be more applicable and feasible for many soybean-producing countries than generating mutants. PMID:27633524

Experimental root canal infections in conventional and germ-free mice.

PubMed

Sobrinho, A P; Barros, M H; Nicoli, J R; Carvalho, M A; Farias, L M; Bambirra, E A; Bahia, M G; Vieira, E C

1998-06-01

A small animal model was evaluated to study the interrelationships between microorganisms after their implantation in root canals (inferior central incisors) using germ-free (GF) and conventional (CV) mice. The selected microorganisms were: Porphyromonas endodontalis (ATCC 35406), Eubacterium lentum (ATCC 25559), Peptostreptococcus anaerobius (ATCC 27337), Fusobacterium nucleatum (ATCC 10953), Escherichia coli (ATCC 25922), and Enterococcus faecalis (ATCC 4083). Only P. anaerobius, E. coli, and E. faecalis, respectively, were able to colonize when inoculated alone into the root canal of both CV and GF mice. E. lentum, when inoculated alone colonized only in CV animals. P. endodontalis and F. nucleatum were unable to colonize in CV and GF animals after single inoculation. It is concluded that the experimental animal model presented herein is valuable for ecological studies of root canal infections and that only some strict anaerobic bacteria are able to colonize mice root canals when inoculated by themselves alone in pure culture.

Cultured High-Fidelity Three-Dimensional Human Urogenital Tract Carcinomas and Process

NASA Technical Reports Server (NTRS)

Goodwin, Thomas J. (Inventor); Prewett, Tacey L. (Inventor); Spaulding, Glenn F. (Inventor); Wolf, David A. (Inventor)

1998-01-01

Artificial high-fidelity three-dimensional human urogenital tract carcinomas are propagated under in vitro-microgravity conditions from carcinoma cells. Artificial high-fidelity three-dimensional human urogenital tract carcinomas are also propagated from a coculture of normal urogenital tract cells inoculated with carcinoma cells. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

Bacterial adaptation to the gut environment favors successful colonization: microbial and metabonomic characterization of a simplified microbiota mouse model.

PubMed

Rezzonico, Enea; Mestdagh, Renaud; Delley, Michèle; Combremont, Séverine; Dumas, Marc-Emmanuel; Holmes, Elaine; Nicholson, Jeremy; Bibiloni, Rodrigo

2011-01-01

Rodent models harboring a simple yet functional human intestinal microbiota provide a valuable tool to study the relationships between mammals and their bacterial inhabitants. In this study, we aimed to develop a simplified gnotobiotic mouse model containing 10 easy-to-grow bacteria, readily available from culture repositories, and of known genome sequence, that overall reflect the dominant commensal bacterial makeup found in adult human feces. We observed that merely inoculating a mix of fresh bacterial cultures into ex-germ free mice did not guarantee a successful intestinal colonization of the entire bacterial set, as mice inoculated simultaneously with all strains only harbored 3 after 21 d. Therefore, several inoculation procedures were tested and levels of individual strains were quantified using molecular tools. Best results were obtained by inoculating single bacterial strains into individual animals followed by an interval of two weeks before allowing the animals to socialize to exchange their commensal microbes. Through this procedure, animals were colonized with almost the complete bacterial set (9/10). Differences in the intestinal composition were also reflected in the urine and plasma metabolic profiles, where changes in lipids, SCFA, and amino acids were observed. We conclude that adaptation of bacterial strains to the host's gut environment (mono-colonization) may predict a successful establishment of a more complex microbiota in rodents.

Growth rates of Dekkera/Brettanomyces yeasts hinder their ability to compete with Saccharomyces cerevisiae in batch corn mash fermentations.

PubMed

Abbott, D A; Hynes, S H; Ingledew, W M

2005-03-01

Growth rates determined by linear regression analysis revealed that Saccharomyces cerevisiae consistently grew more rapidly than Brettanomyces yeasts under a wide array of batch fermentative conditions, including acetic acid stress, in normal gravity (ca. 20 degrees Plato) mashes made from ground corn. Brettanomyces yeasts only grew more rapidly than S. cerevisiae when acetic acid concentrations were elevated to industrially irrelevant levels (>0.45%, w/v). Furthermore, the three Brettanomyces isolates used in this study failed to produce significant quantities of acetic acid under pure culture fermentative conditions. In fact, the small amounts of acetic acid which accumulated in pure culture fermentations of whole corn mash were below the concentration required to inhibit the growth and metabolism of S. cerevisiae. Acetic acid concentrations in pure culture Brettanomyces fermentations exceeded 0.05% (w/v) only in media containing low levels of glucose (<4%, w/v) or when aeration rates were elevated to at least 0.03 vol. air vol.-1 mash min-1. Consequently, it was concluded that Brettanomyces yeasts would not be capable of competing with S. cerevisiae in industrial batch fermentations of whole corn mash based solely on growth rates, nor would they be capable of producing inhibitory concentrations of acetic acid in such fermentations.

Inferring mixed-culture growth from total biomass data in a wavelet approach

NASA Astrophysics Data System (ADS)

Ibarra-Junquera, V.; Escalante-Minakata, P.; Murguía, J. S.; Rosu, H. C.

2006-10-01

It is shown that the presence of mixed-culture growth in batch fermentation processes can be very accurately inferred from total biomass data by means of the wavelet analysis for singularity detection. This is accomplished by considering simple phenomenological models for the mixed growth and the more complicated case of mixed growth on a mixture of substrates. The main quantity provided by the wavelet analysis is the Hölder exponent of the singularity that we determine for our illustrative examples. The numerical results point to the possibility that Hölder exponents can be used to characterize the nature of the mixed-culture growth in batch fermentation processes with potential industrial applications. Moreover, the analysis of the same data affected by the common additive Gaussian noise still lead to the wavelet detection of the singularities although the Hölder exponent is no longer a useful parameter.

Enhanced production and purification of recombinant surface array protein (Sap) for use in detection of Bacillus anthracis.

PubMed

Puranik, Nidhi; Tripathi, N K; Pal, V; Goel, Ajay Kumar

2018-05-01

Surface array protein (Sap) can be an important biomarker for specific detection of Bacillus anthracis , which is released by the bacterium during its growth in culture broth. In the present work, we have cloned and expressed Sap in Escherichia coli . The culture conditions and cultivation media were optimized and used in batch fermentation process for scale up of Sap in soluble form. The recombinant Sap was purified employing affinity chromatography followed by diafiltration. The final yield of purified protein was 20 and 46 mg/l of culture during shake flasks and batch fermentation, respectively. The protein purity and its reactivity were confirmed employing SDS-PAGE and Western blot, respectively. The antibodies raised against purified Sap were evaluated by Western blotting for detection of Sap released by B. anthracis . Our results showed that the Sap could be a novel marker for detection and confirmation of B. anthracis .

Dynamic changes in the initial colonization of Actinomyces naeslundii and Streptococcus gordonii using a new animal model.

PubMed

Zhang, Xi; Senpuku, Hidenobu

2013-01-01

Actinomyces naeslundii and Streptococcus gordonii are the predominant bacteria and initial colonizers of oral microflora. The binding of A. naeslundii and S. gordonii and the interaction between them on the salivary pellicle-coated tooth surface play an important role in the biofilm development. Recently, we reported that NOD/SCID.e2f1(-) mice are a useful model for studying oral biofilm formation by Streptococcus mutans on the tooth surface. In this study, we aimed to determine whether NOD/SCID.e2f1(-) mice can be used for studying oral colonization of A. naeslundii and S. gordonii. Colonization of A. naeslundii in mice fed with 1% sucrose water for 24 h before inoculation was higher than that among mice fed with sucrose water for 1 h. A. naeslundii colonization using mixed species-inoculation was lower than that using single-species inoculation 30-90 min after inoculation; however, the colonization was higher 120-180 min after inoculation. The mixed inoculation induced better colonization of S. gordonii than single-species inoculation 60-180 min after inoculation. Polyclonal and fluorescein isothiocyanate-labeled antibody stained bacteria showed better colonization of S. gordonii when a mixed culture is used in vivo. NOD/SCID.e2f1(-) mice were useful for studying the initial colonization of A. naeslundii and S. gordonii. Long-term supply of sucrose water creates a favorable environment for the initial colonization of A. naeslundii that, in turn, supports the colonization of S. gordonii.

Saccharomyces and non-Saccharomyces Competition during Microvinification under Different Sugar and Nitrogen Conditions

PubMed Central

Lleixà, Jessica; Manzano, Maria; Mas, Albert; Portillo, María del C.

2016-01-01

The inoculation of wines with autochthonous yeast allows obtaining complex wines with a peculiar microbial footprint characteristic from a wine region. Mixed inoculation of non-Saccharomyces yeasts and S. cerevisiae is of interest for the wine industry for technological and sensory reasons. However, the interactions between these yeasts are not well understood, especially those regarding the availability of nutrients. The aim of the present study was to analyze the effect of nitrogen and sugar concentration on the evolution of mixed yeast populations on controlled laboratory-scale fermentations monitored by density, plate culturing, PCR-DGGE and sugar and nitrogen consumption. Furthermore, the effect of the time of inoculation of Saccharomyces cerevisiae respect the initial co-inoculation of three non-Saccharomyces yeasts was evaluated over the evolution of fermentation. Our results have shown that S. cerevisiae inoculation during the first 48 h conferred a stabilizing effect over the fermentations with non-Saccharomyces strains tested and, generally, reduced yeast diversity at the end of the fermentation. On the other hand, nitrogen limitation increased the time of fermentation and also the proportion of non-Saccharomyces yeasts at mid and final fermentation. High sugar concentration resulted in different proportions of the inoculated yeast depending on the time of S. cerevisiae inoculation. This work emphasizes the importance of the concentration of nutrients on the evolution of mixed fermentations and points to the optimal conditions for a stable fermentation in which the inoculated yeasts survived until the end. PMID:27994585

Decontamination methods for samples preserved in cetylpyridinium chloride and cultured on thin-layer agar.

PubMed

Ardizzoni, E; Mulders, W; Sanchez-Padilla, E; Varaine, F; de Jong, B C; Rigouts, L

2014-08-01

Long transportation times of samples to culture laboratories can lead to higher contamination rates and significant loss of viability, resulting in lower culture positivity rates. Thin-layer agar (TLA) is a sensitive culture method for the isolation of Mycobacterium tuberculosis that has been optimised with N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) decontaminated samples. The combination of the TLA culture method and other decontamination procedures has not been extensively validated. Among 390 smear-positive samples, we compared the culture positivity of samples decontaminated using the Petroff method vs. NALC-NaOH neutralised with phosphate buffer (PBS), applied to samples preserved with cetylpyridinium chloride (CPC) or CPC-free, and then of CPC-preserved samples decontaminated with NALC-NaOH neutralised using Difco neutralising buffer. The sediments were inoculated on TLA, and then on MGIT 960 or Löwenstein-Jensen (LJ) as gold standards. Decontamination with NALC-NaOH yielded higher culture positivity in TLA than in the Petroff method, which was further enhanced by neutralising CPC with the Difco buffer. Surprisingly, culture positivity on LJ also increased after using Difco buffer, suggesting that CPC may not be completely neutralised in egg-based medium. After transportation in CPC, decontamination using NALC-NaOH followed by neutralisation using Difco buffer resulted in the best recovery rates for samples inoculated on TLA and on LJ.

[Effects of arbuscular mycorrhizal fungi on root system morphology and sucrose and glucose contents of Poncirus trifoliata].

PubMed

Zou, Ying-Ning; Wu, Qiang-Sheng; Li, Yan; Huang, Yong-Ming

2014-04-01

The effects of inoculation with Glomus mosseae, G. versiforme, and their mixture on plant growth, root system morphology, and sucrose and glucose contents of trifoliate orange (Poncirus trifoliata L.) were studied by pot culture. The results showed that all the inoculated treatments significantly increased the plant height, stem diameter, leaf number, and shoot and root biomass. In addition, the mycorrhizal treatments significantly increased the number of 1st, 2nd, and 3rd lateral roots. Inoculation with arbuscular mycorrhizal fungi significantly increased the root projected area, surface area, volume, and total root length (mainly 0-1 cm root length), but decreased the root average diameter. Meanwhile, G. versiforme showed the best effects. Mycorrhizal inoculation significantly increased the leaf sucrose and root glucose contents, but decreased the leaf glucose and root sucrose contents. Owing to the 'mycorrhizal carbon pool' in roots, inoculation with arbuscular mycorrhizal fungi resulted in high glucose content and low sucrose content of roots, which would facilitate the root growth and development, thereby the establishment of better root system morphology of host plants.

The widely distributed hard tick, Haemaphysalis longicornis, can retain canine parvovirus, but not be infected in laboratory condition

PubMed Central

MORI, Hiroyuki; TANAKA, Tetsuya; MOCHIZUKI, Masami

2014-01-01

ABSTRACT. Ticks are known to transmit various pathogens, radically threatening humans and animals. Despite the close contact between ticks and viruses, our understanding on their interaction and biology is still lacking. The aim of this study was to experimentally assess the interaction between canine parvovirus (CPV) and a widely distributed hard tick, Haemaphysalis longicornis, in laboratory condition. After inoculation of CPV into the hemocoel of the ticks, polymerase chain reaction assay revealed that CPV persisted in inoculated unfed adult female ticks for 28 days. Canine parvovirus was recovered from the inoculated ticks using a cell culture, indicating that the virus retained intact in the ticks after inoculation, but significant positive reaction indicating virus infection was not detected in the tick organs by immunofluorescence antibody test using a monoclonal antibody. In the case of ticks inoculated with feline leukemia virus, the virus had shorter persistence in the ticks compared to CPV. These findings provide significant important information on the characteristic interaction of tick with non-tick-borne virus. PMID:25650060

Survival of Campylobacter jejuni inoculated into ground beef.

PubMed Central

Stern, N J; Kotula, A W

1982-01-01

Ground beef was inoculated with mixed cultures of Campylobacter jejuni, and the samples were subjected to various cooking and cold-storage temperatures. When samples were heated in an oven at either 190 or 218 degrees C, approximately 10(7) cells of C. jejuni per g were inactivated (less than 30 cells per g) in less than 10 min after the ground beef reached an internal temperature of 70 degrees C. When the samples were held at -15 degrees C over 14 days of storage, the numbers of C. jejuni declined by 3 log10. When inoculated samples were stored with an equal amount of Cary-Blair diluent at 4 degrees C, no changes in viability were observed over 14 days of storage. Twenty-five times as much C. jejuni was recovered from inoculated ground beef when either 10% glycerol or 10% dimethyl sulfoxide was added to an equal amount of ground beef before freezing as was recovered from peptone-diluted ground beef. Twice as much inoculated C. jejuni was recovered from ground beef plus Cary-Blair diluent as was recovered from ground beef plus peptone diluent. PMID:7181502

Isolation and characterization of a plant growth-promoting rhizobacterium, Serratia sp. SY5.

PubMed

Koo, So-Yeon; Cho, Kyung-Suk

2009-11-01

The role of plant growth-promoting rhizobacteria (PGPR) in the phytoremediation of heavy-metal-contaminated soils is important in overcoming its limitations for field application. A plant growth-promoting rhizobacterium, Serratia sp. SY5, was isolated from the rhizoplane of barnyard grass (Echinochloa crus-galli) grown in petroleum and heavy-metal-contaminated soil. This isolate has shown capacities for indole acetic acid production and siderophores synthesis. Compared with a non-inoculated control, the radicular root growth of Zea mays seedlings inoculated with SY5 can be increased by 27- or 15.4-fold in the presence of 15 mg-Cd/l or 15 mg-Cu/l, respectively. The results from hydroponic cultures showed that inoculation of Serratia sp. SY5 had a favorable influence on the initial shoot growth and biomass of Zea mays under noncontaminated conditions. However, under Cd-contaminated conditions, the inoculation of SY5 significantly increased the root biomass of Zea mays. These results indicate that Serratia sp. SY5 can serve as a promising microbial inoculant for increased plant growth in heavy-metal-contaminated soils to improve the phytoremediation efficiency.

Effect of inoculation with Penicillium expansum on the microbial community and maturity of compost.

PubMed

Wang, Hong-yuan; Fan, Bing-quan; Hu, Qing-xiu; Yin, Zhong-wei

2011-12-01

Compost prepared from wheat straw and cattle/chicken mature was inoculated with the lignocellulolytic fungus, Penicillium expansum. Compared to uninoculated compost, the inoculated compost exhibited a 150% higher germination index, more than 1.2 g kg(-1)-dw of changes in NH(4)(+)-N concentrations, a ca. 12.0% higher humus content and a lignocellulose degradation that proceeded 57.5% faster. Culture-based determinations of microbial populations demonstrated that aerobic heterotrophic bacteria and fungi were about 1-2 orders of magnitude higher in inoculated than in uninoculated compost. The number of ammonifying, ammonium-oxidizing, nitrite-oxidizing, denitrifying bacteria and cellulose-decomposing bacteria was 6.1-9.0 log(10) CFU g(-1)-dw, 1.2-4.3 log(10) MPN g(-1)-dw, 3.5-6.8 log(10) MPN g(-1)-dw, 3.58-4.34 log(10) MPN g(-1)-dw, 1.4-3.8 log(10)MPN g(-1)-dw, and 4.2-8.8 log(10) CFU g(-1)-dw higher in the compost inoculated with P. expansum. Copyright © 2011 Elsevier Ltd. All rights reserved.

A poliomyelitis model through mucosal infection in transgenic mice bearing human poliovirus receptor, TgPVR21.

PubMed

Nagata, Noriyo; Iwasaki, Takuya; Ami, Yasushi; Sato, Yuko; Hatano, Ikuyoshi; Harashima, Ayako; Suzaki, Yuriko; Yoshii, Takao; Hashikawa, Tsutomu; Sata, Tetsutaro; Horiuchi, Yoshinobu; Koike, Satoshi; Kurata, Takeshi; Nomoto, Akio

2004-03-30

Transgenic mice bearing the human poliovirus receptor (TgPVR) are less susceptible to oral inoculation, although they are susceptible to parenteral inoculation. We investigated the susceptibility of TgPVR 21 line [Arch. Virol. 130 (1994) 351] to poliovirus through various mucosal routes. Intranasal inoculation of a neurovirulent Mahoney strain (OM1) caused flaccid paralysis with viral replication in the central nervous system at a dose of 10(6) cell culture infectious dose (CCID50), in contrast, no paralysis following oral or intragastric inoculation of the same dose. Intranasal inoculation of a vaccine strain, Sabin 1, at 10(6) CCID50, resulted in no paralysis. Initial replication of poliovirus in the nasal cavity was confirmed by virus isolation and detection of negative-stranded replicative intermediates by RT-PCR and viral antigens using a high-sensitive immunohistochemistry and genome/transcripts by in situ hybridization. Poliovirus-specific IgG antibodies were elevated in the sera of surviving TgPVR21. This model can be used as a mucosal infection model and for differentiation of neurovirulent and attenuated poliovirus strains.

Detection of shigella in lettuce by the use of a rapid molecular assay with increased sensitivity

PubMed Central

Jiménez, Kenia Barrantes; McCoy², Clyde B.; Achí, Rosario

2010-01-01

A Multiplex Polymerase Chain Reaction (PCR) assay to be used as an alternative to the conventional culture method in detecting Shigella and enteroinvasive Escherichia coli (EIEC) virulence genes ipaH and ial in lettuce was developed. Efficacy and rapidity of the molecular method were determined as compared to the conventional culture. Lettuce samples were inoculated with different Shigella flexneri concentrations (from 10 CFU/ml to 107 CFU/ml). DNA was extracted directly from lettuce after inoculation (direct-PCR) and after an enrichment step (enrichment PCR). Multiplex PCR detection limit was 104CFU/ml, diagnostic sensitivity and specificity were 100% accurate. An internal amplification control (IAC) of 100 bp was used in order to avoid false negative results. This method produced results in 1 to 2 days while the conventional culture method required 5 to 6 days. Also, the culture method detection limit was 106 CFU/ml, diagnostic sensitivity was 53% and diagnostic specificity was 100%. In this study a Multiplex PCR method for detection of virulence genes in Shigella and EIEC was shown to be effective in terms of diagnostic sensitivity, detection limit and amount of time as compared to Shigella conventional culture. PMID:24031579

Influence of Infection Court, Host Vigor, and Culture Filtrates on Canker Production by Botryodiplodia Theobromae Conidia in Sycamore

Treesearch

R. Lewis

1978-01-01

Some of the factors that influence canker development in American sycamores inoculated with Botryodiplodia theobromae conidia were determined. A combination of B. theobromae culture filtrates and conidia resulted in 100% canker production when introduced into stem wounds; however, a combination of Cephalosporium diospyri...

Preparation of a Lactobacillus plantarum starter culture for cucumber fermentations that can meet kosher guidelines

USDA-ARS?s Scientific Manuscript database

A method is described for growth of a Lactobacillus plantarum starter culture in jars of commercially available pasteurized fresh-pack kosher dill cucumbers so that jars can be used to inoculate commercial scale cucumber fermentation tanks. A procedure is also described to transfer lactic acid bacte...

A four-quadrant sequential streak technique to evaluate Campylobacter selective broths for suppressing background flora in broiler carcass rinses

USDA-ARS?s Scientific Manuscript database

The ecometric technique is a semi-quantitative scoring method used in the quality control of culture media in microbiology laboratories. This technique involves inoculation with defined populations of a specific culture onto solid media via a standardized chronological streaking technique, leading ...

Effect of red cyst cell inoculation and iron(II) supplementation on autotrophic astaxanthin production by Haematococcus pluvialis under outdoor summer conditions.

PubMed

Hong, Min-Eui; Choi, Yoon Young; Sim, Sang Jun

2016-01-20

The negative effect of heat stress on the autotrophic astaxanthin production by Haematococcus pluvialis has been observed during outdoor culture in summer. Under the summer conditions, the proliferation of vegetative cells was highly halted in the green stage and the inducibility in the biosynthesis of astaxanthin was partly hindered in the red stage. Herein, under outdoor summer conditions in which variations of the diurnal temperature occur, heat-stress-driven inefficient vegetative growth of H. pluvialis was highly improved by inoculating the red cyst cells; thereby, maintaining relatively moderate intracellular carotenoid levels in the green stage. Subsequently, a remarkably enhanced astaxanthin titer was successfully obtained by supplementing 50 μM iron(II) to induce the heat stress-driven Haber-Weiss reaction in the red stage. As a result, the productivity of astaxanthin in the cells cultured under summer temperature conditions (23.4-33.5 °C) using the two methods of red cell (cyst) inoculation and the iron(Fe(2+)) supplementation was increased by 147% up to 5.53 mg/L day compared with that of the cells cultured under spring temperature conditions (17.5-27.3 °C). Our technical solutions will definitely improve the annual natural astaxanthin productivity in H. pluvialis in locations confronted by hot summer weather, particularly in large-scale closed photobioreactor systems. Copyright © 2015 Elsevier B.V. All rights reserved.

Yeast Monitoring of Wine Mixed or Sequential Fermentations Made by Native Strains from D.O. "Vinos de Madrid" Using Real-Time Quantitative PCR.

PubMed

García, Margarita; Esteve-Zarzoso, Braulio; Crespo, Julia; Cabellos, Juan M; Arroyo, Teresa

2017-01-01

There is an increasing trend toward understanding the impact of non- Saccharomyces yeasts on the winemaking process. Although Saccharomyces cerevisiae is the predominant species at the end of fermentation, it has been recognized that the presence of non- Saccharomyces species during alcoholic fermentation can produce an improvement in the quality and complexity of the final wines. A previous work was developed for selecting the best combinations between S. cerevisiae and five non- Saccharomyces ( Torulaspora delbrueckii, Schizosaccharomyces pombe, Candida stellata, Metschnikowia pulcherrima , and Lachancea thermotolorans ) native yeast strains from D.O. "Vinos de Madrid" at the laboratory scale. The best inoculation strategies between S. cerevisiae and non- Saccharomyces strains were chosen to analyze, by real-time quantitative PCR (qPCR) combined with the use of specific primers, the dynamics of inoculated populations throughout the fermentation process at the pilot scale using the Malvar white grape variety. The efficiency of the qPCR system was verified independently of the samples matrix, founding the inoculated yeast species throughout alcoholic fermentation. Finally, we can validate the positive effect of selected co-cultures in the Malvar wine quality, highlighting the sequential cultures of T. delbrueckii CLI 918/ S. cerevisiae CLI 889 and C. stellata CLI 920/ S. cerevisiae CLI 889 and, mixed and sequential cultures of L. thermotolerans 9-6C combined with S. cerevisiae CLI 889.

Influence of l-amino acids on aggregation and biofilm formation in Azotobacter chroococcum and Trichoderma viride.

PubMed

Velmourougane, K; Prasanna, R

2017-10-01

The effects of l-amino acids on growth and biofilm formation in Azotobacter chroococcum (Az) and Trichoderma viride (Tv) as single (Az, Tv) and staggered inoculated cultures (Az-Tv, Tv-Az) were investigated. A preliminary study using a set of 20 l-amino acids, identified 6 amino acids (l-Glu, l-Gln, l-His, l-Ser, l-Thr and l-Trp) which significantly enhanced growth and biofilm formation. Supplementation of these amino acids at different concentrations revealed that 40 mmol l -1 was most effective. l-Glu and l-Gln favoured planktonic growth in both single and in staggered inoculated cultures, while l-Trp and l-Thr, enhanced aggregation and biofilm formation. Addition of l-Glu or l-Gln increased carbohydrate content and planktonic population. Principal component analysis revealed the significant role of proteins in growth and biofilm formation, particularly with supplementation of l-Trp, l-Thr and l-Ser. Azotobacter was found to function better as biofilm under staggered inoculated culture with Trichoderma. The results illustrate that amino acids play crucial roles in microbial biofilm formation, by influencing growth, aggregation and carbohydrates synthesized. The differential and specific roles of amino acids on biofilm formation are of significance for agriculturally important micro-organisms that grow as biofilms, colonize and benefit the plants more effectively. © 2017 The Society for Applied Microbiology.

Ferrous Iron Oxidation by Thiobacillus ferrooxidans: Inhibition with Benzoic Acid, Sorbic Acid, and Sodium Lauryl Sulfate

PubMed Central

Onysko, Steven J.; Kleinmann, Robert L. P.; Erickson, Patricia M.

1984-01-01

Benzoic acid, sorbic acid, and sodium lauryl sulfate at low concentrations (5 to 10 mg/liter) each effectively inhibited bacterial oxidation of ferrous iron in batch cultures of Thiobacillus ferrooxidans. The rate of chemical oxidation of ferrous iron in low-pH, sterile batch reactors was not substantially affected at the tested concentrations (5 to 50 mg/liter) of any of the compounds. PMID:16346592

Modelling and properties of a nonlinear autonomous switching system in fed-batch culture of glycerol

NASA Astrophysics Data System (ADS)

Wang, Juan; Sun, Qingying; Feng, Enmin

2012-11-01

A nonlinear autonomous switching system is proposed to describe the coupled fed-batch fermentation with the pH as the feedback parameter. We prove the non-Zeno behaviors of the switching system and some basic properties of its solution, including the existence, uniqueness, boundedness and regularity. Numerical simulation is also carried out, which reveals that the proposed system can describe the factual fermentation process properly.

Transmission of vesicular stomatitis New Jersey virus to cattle by the biting midge Culicoides sonorensis (Diptera: Ceratopogonidae).

PubMed

Perez de Leon, Adalberto A; Tabachnick, Walter J

2006-03-01

Laboratory-reared Culicoides sonorensis Wirth & Jones were infected with vesicular stomatitis virus serotype New Jersey (family Rhabdoviridae, genus Vesiculovirus, VSNJV) through intrathoracic inoculation. After 10-d incubation at 25 degrees C, these insects were allowed to blood feed on four steers. Two other steers were exposed to VSNJV through intralingual inoculation with 10(8) tissue culture infective dose50 VSNJV. All six steers became seropositive for VSNJV. The results demonstrate the ability of C. sonorensis to transmit VSNJV to livestock. Only the animals intralingually inoculated with VSNJV showed clinical signs in the form of vesicles at the site of inoculation. Uninfected C. sonorensis allowed to feed on the exposed animals did not become infected with VSNJV. Animals infected by C. sonorensis showed a slower antibody response compared with intralingually inoculated animals. This is probably because of different amounts of virus received via insect transmission and syringe inoculation. A significant difference was found in the serum acute-phase protein alpha-1-acid glycoprotein in animals that received VSNJV through C. sonorensis transmission. These animals had previously been exposed to insect attack in the field compared with intralingually inoculated animals and C. sonorensis-infected animals that had been protected from insect attack. The failure to observe clinical signs of vesicular stomatitis through transmission of VSNJV by C. sonorensis may explain widespread subclinical infections during vesicular stomatitis epidemics.

Bacterial community changes in an industrial algae production system.

PubMed

Fulbright, Scott P; Robbins-Pianka, Adam; Berg-Lyons, Donna; Knight, Rob; Reardon, Kenneth F; Chisholm, Stephen T

2018-04-01

While microalgae are a promising feedstock for production of fuels and other chemicals, a challenge for the algal bioproducts industry is obtaining consistent, robust algae growth. Algal cultures include complex bacterial communities and can be difficult to manage because specific bacteria can promote or reduce algae growth. To overcome bacterial contamination, algae growers may use closed photobioreactors designed to reduce the number of contaminant organisms. Even with closed systems, bacteria are known to enter and cohabitate, but little is known about these communities. Therefore, the richness, structure, and composition of bacterial communities were characterized in closed photobioreactor cultivations of Nannochloropsis salina in F/2 medium at different scales, across nine months spanning late summer-early spring, and during a sequence of serially inoculated cultivations. Using 16S rRNA sequence data from 275 samples, bacterial communities in small, medium, and large cultures were shown to be significantly different. Larger systems contained richer bacterial communities compared to smaller systems. Relationships between bacterial communities and algae growth were complex. On one hand, blooms of a specific bacterial type were observed in three abnormal, poorly performing replicate cultivations, while on the other, notable changes in the bacterial community structures were observed in a series of serial large-scale batch cultivations that had similar growth rates. Bacteria common to the majority of samples were identified, including a single OTU within the class Saprospirae that was found in all samples. This study contributes important information for crop protection in algae systems, and demonstrates the complex ecosystems that need to be understood for consistent, successful industrial algae cultivation. This is the first study to profile bacterial communities during the scale-up process of industrial algae systems.

Ultraviolet Light (UV) Inactivation of Porcine Parvovirus in Liquid Plasma and Effect of UV Irradiated Spray Dried Porcine Plasma on Performance of Weaned Pigs

PubMed Central

Polo, Javier; Rodríguez, Carmen; Ródenas, Jesús; Russell, Louis E.; Campbell, Joy M.; Crenshaw, Joe D.; Torrallardona, David; Pujols, Joan

2015-01-01

A novel ultraviolet light irradiation (UV-C, 254 nm) process was designed as an additional safety feature for manufacturing of spray dried porcine plasma (SDPP). In Exp. 1, three 10-L batches of bovine plasma were inoculated with 105.2±0.12 tissue culture infectious dose 50 (TCID50) of porcine parvovirus (PPV) per mL of plasma and subjected to UV-C ranging from 0 to 9180 J/L. No viable PPV was detected in bovine plasma by micro-titer assay in SK6 cell culture after UV-C at 2295 J/L. In Exp. 2, porcine plasma was subjected to UV-C (3672 J/L), then spray dried and mixed in complete mash diets. Diets were a control without SDPP (Control), UV-C SDPP either at 3% (UVSDPP3) or 6% (UVSDPP6) and non-UV-C SDPP at 3% (SDPP3) or 6% (SDPP6). Diets were fed ad libitum to 320 weaned pigs (26 d of age; 16 pens/diet; 4 pigs/pen) for 14 d after weaning and a common diet was fed d 15 to 28. During d 0 to 14, pigs fed UVSDPP3, UVSDPP6, or SDPP6 had higher (P < 0.05) weight gain and feed intake than control. During d 0 to 28, pigs fed UVSDPP3 and UVSDPP6 had higher (P < 0.05) weight gain and feed intake than control and SDPP3, and SDPP6 had higher (P < 0.05) feed intake than control. Also, pigs fed UVSDPP had higher (P < 0.05) weight gain than pigs fed SDPP. In conclusion, UV-C inactivated PPV in liquid plasma and UVSDPP used in pig feed had no detrimental effects on pig performance. PMID:26171968

Effect of culture density on biomass production and light utilization efficiency of Synechocystis sp. PCC 6803.

PubMed

Straka, Levi; Rittmann, Bruce E

2018-02-01

The viability of large-scale microalgae cultivation depends on providing optimal growth conditions, for which a key operational parameter is culture density. Using Synechocystis sp. PCC 6803, we conducted a series of fixed-density, steady-state experiments and one batch-growth experiment to investigate the role of culture density on biomass production and light utilization efficiency. In all cases, the fixed-density, steady-state experiments and batch-growth experiment showed good agreement. The highest biomass production rates (260 mg L -1  d -1 ) and efficiency for converting light energy to biomass (0.80 μg (μmol photons) -1 ) occurred together at a culture density near 760 mg L -1 , which approximately corresponded to the lowest culture density where almost all incident light was absorbed. The ratio of OD 680 /OD 735 increased with culture density up to the point of maximum productivity, where it plateaued (at a value of 2.4) for higher culture densities. This change in OD 680 /OD 735 indicates a photoacclimation effect that depended on culture density. Very high culture densities led to a sharp decline in efficiency of biomass production per photons absorbed, likely due to a combination of increased decay relative to growth, metabolic changes due to cell-cell interactions, and photodamage due to mixing between regions with high light intensity and zero light intensity. © 2017 Wiley Periodicals, Inc.

First evaluation of automated specimen inoculation for wound swab samples by use of the Previ Isola system compared to manual inoculation in a routine laboratory: finding a cost-effective and accurate approach.

PubMed

Mischnik, Alexander; Mieth, Markus; Busch, Cornelius J; Hofer, Stefan; Zimmermann, Stefan

2012-08-01

Automation of plate streaking is ongoing in clinical microbiological laboratories, but evaluation for routine use is mostly open. In the present study, the recovery of microorganisms from the Previ Isola system plated polyurethane (PU) swab samples is compared to manually plated control viscose swab samples from wounds according to the CLSI procedure M40-A (quality control of microbiological transport systems). One hundred twelve paired samples (224 swabs) were analyzed. In 80/112 samples (71%), concordant culture results were obtained with the two methods. In 32/112 samples (29%), CFU recovery of microorganisms from the two methods was discordant. In 24 (75%) of the 32 paired samples with a discordant result, Previ Isola plated PU swabs were superior. In 8 (25%) of the 32 paired samples with a discordant result, control viscose swabs were superior. The quality of colony growth on culture media for further investigations was superior with Previ Isola inoculated plates compared to manual plating techniques. Gram stain results were concordant between the two methods in 62/112 samples (55%). In 50/112 samples (45%), the results of Gram staining were discordant between the two methods. In 34 (68%) of the 50 paired samples with discordant results, Gram staining of PU swabs was superior to that of control viscose swabs. In 16 (32%) of the 50 paired samples, Gram staining of control viscose swabs was superior to that of PU swabs. We report the first clinical evaluation of Previ Isola automated specimen inoculation for wound swab samples. This study suggests that use of an automated specimen inoculation system has good results with regard to CFU recovery, quality of Gram staining, and accuracy of diagnosis.

First Evaluation of Automated Specimen Inoculation for Wound Swab Samples by Use of the Previ Isola System Compared to Manual Inoculation in a Routine Laboratory: Finding a Cost-Effective and Accurate Approach

PubMed Central

Mieth, Markus; Busch, Cornelius J.; Hofer, Stefan; Zimmermann, Stefan

2012-01-01

Automation of plate streaking is ongoing in clinical microbiological laboratories, but evaluation for routine use is mostly open. In the present study, the recovery of microorganisms from the Previ Isola system plated polyurethane (PU) swab samples is compared to manually plated control viscose swab samples from wounds according to the CLSI procedure M40-A (quality control of microbiological transport systems). One hundred twelve paired samples (224 swabs) were analyzed. In 80/112 samples (71%), concordant culture results were obtained with the two methods. In 32/112 samples (29%), CFU recovery of microorganisms from the two methods was discordant. In 24 (75%) of the 32 paired samples with a discordant result, Previ Isola plated PU swabs were superior. In 8 (25%) of the 32 paired samples with a discordant result, control viscose swabs were superior. The quality of colony growth on culture media for further investigations was superior with Previ Isola inoculated plates compared to manual plating techniques. Gram stain results were concordant between the two methods in 62/112 samples (55%). In 50/112 samples (45%), the results of Gram staining were discordant between the two methods. In 34 (68%) of the 50 paired samples with discordant results, Gram staining of PU swabs was superior to that of control viscose swabs. In 16 (32%) of the 50 paired samples, Gram staining of control viscose swabs was superior to that of PU swabs. We report the first clinical evaluation of Previ Isola automated specimen inoculation for wound swab samples. This study suggests that use of an automated specimen inoculation system has good results with regard to CFU recovery, quality of Gram staining, and accuracy of diagnosis. PMID:22692745

Enhancement of a culture-based bacterial detection system (eBDS) for platelet products based on measurement of oxygen consumption.

PubMed

Holme, Stein; McAlister, Morven B; Ortolano, Girolamo A; Chong, Chiyong; Cortus, Mary Anne; Jacobs, Michael R; Yomtovian, Roslyn; Freundlich, Lawrence F; Wenz, Barry

2005-06-01

An enhanced bacterial detection system (Pall eBDS) was developed that distinguishes itself from its predecessor (Pall BDS) by removal of the platelet (PLT)-retaining filter allowing for optimal bacterial transfer, modification of the culture tablet to reduce the confounding effects of respiring PLTs while enhancing bacterial growth, and facilitation of nutrients and gas exchange by agitating the sample pouch during incubation at 35 degrees C. The objective was to evaluate the performance of the new eBDS. Leukoreduced whole blood-derived PLT concentrates (LR-PCs) and LR single-donor PLTs (LR-SDPs) were inoculated with 1 to 15 colony-forming units (CFUs) of bacteria per mL in studies of each of 10 bacterial species associated with fatal transfusion-transmitted bacterial infection. Immediately after inoculation and after 24 hours of storage at 22 degrees C, samples of inoculated LR-PCs were aseptically transferred into the eBDS pouches. Pouches were then incubated for 24 hours at 35 degrees C with agitation and oxygen concentration was then measured. Median inoculation levels ranged from 5 to 13 CFUs per mL for each species studied. No significant differences in oxygen concentration were found when comparing LR-PCs with LR-SDPs. When sampling occurred from the PLTs 24 hours after inoculation, all 280 cases (24-33 replicates of each species) were detected as contaminated by the device (100% sensitivity). No false-positives were obtained with 713 uninoculated PLT units. The eBDS demonstrated improved detection sensitivity in the range of 1 to 15 CFUs per mL with no observed false-positives compared to the original BDS (detection range 100 to 500 CFUs/mL) with no false-positives.

Effect of starter culture and turmeric on physico-chemical quality of carabeef pastirma.

PubMed

Maurya, P; Borpuzari, R N; Nath, D R; Nath, N C

2010-01-01

Carabeef samples were sliced, pressed, cured and divided into 6 groups. Starter cultures (Micrococcus varians M483 (MV), Staphylococcus carnosus (SC), Lactobacillus sakei (LS), M. varians M483+ Lb. sakei and Staph. carnosus + Lb. sakei) were inoculated at the dose of 10(6)-0(7)cfu/g and stored at 10 ± 1°C for 7 days. Uninoculated samples were maintained as control. Samples were then divided into 2 treatment groups. Samples of treatment 1 (T1) were smeared with a paste of turmeric followed by application of a thick layer of the paste of garlic, cumin, black pepper and red pepper whereas, samples of treatment 2 (T2) were applied with a thick layer of spices as above without turmeric. With the gradual fall in pH there was a reduction in water-holding capacity (WHC) of samples. The WHC of samples treated with SC+LS of T1 reduced to 6.3 ± 0.03 cm(2) and those inoculated with MV+LS of T2 to 6.2 ± 0.03 cm(2). The extract release volume (ERV) increased in all samples during storage. The least ERV of 11.7 and 11.6 ml were recorded in samples inoculated with MV of T1 and T2, respectively. The tyrosine (TV) and thiobarbituric acid (TBA) number of turmeric treated samples were significantly lower than non turmeric treated samples. The samples inoculated with LS had the least TV of 30.9 mg tyrosine/100 g of meat and TBA number of 0.06 mg manoladehyde/kg of meat. Samples inoculated with MV and LS of both T1 and T2 were better in physico-chemical qualities.

Interaction between mycobacteria and mucus on a human respiratory tissue organ culture model with an air interface.

PubMed

Middleton, A M; Chadwick, M V; Nicholson, A G; Wilson, R; Thornton, D J; Kirkham, S; Sheehan, J K

2004-01-01

Mycobacteria adhere specifically to extracellular matrix (ECM) and mucus with a fibrous, but not globular, appearance, in organ cultures of human respiratory mucosa examined by scanning electron microscopy. Previously, light microscopy sections made of tissue infected for 7 days demonstrated mycobacteria associated with mucus on the organ culture surface, and within submucosal glands in areas of damaged epithelium. The authors have now investigated the interactions between Mycobacterium avium complex (MAC), Mycobacterium tuberculosis (MTB), and Mycobacterium smegmatis (MS) and mucus by preincubating bacteria with purified mucins MUC5AC and MUC5B prior to inoculation onto the organ culture mucosal surface. They have also measured mucin production by the organ culture after mycobacterial infection. Mucus did not cause clumping of mycobacteria. There was a significant (P=.03) increase in the amount of fibrous mucus, but not globular mucus, observed on tissue inoculated with mucins compared to controls. The number of bacteria adhering to ECM was markedly reduced after incubation with mucins, which could indicate a protective effect. Mycobacterial infection did not increase mucin production by the organ culture. Mycobacterial adherence to mucins may play a role in the pathogenicity of mycobacteria in diseases such as cystic fibrosis, bronchiectasis, and chronic obstructive pulmonary disease (COPD), in which there are changes in mucus composition and clearance.

Detection of Infectious Bovine Rhinotracheitis and Bovine Viral Diarrhea Viruses in the Nasal Epithelial Cells by the Direct Immunofluorescence Technique

PubMed Central

Silim, A.; Elazhary, M.A.S.Y.

1983-01-01

Nasal epithelial cells were collected by cotton swabs for the diagnosis in experimental and field cases of infectious bovine rhinotracheitis and field cases of bovine viral diarrhea in calves. A portion of the cells was washed twice in phosphate buffered saline and a 25 µL drop was placed on microscope slides. The cells were dried, fixed and stained according to the direct fluorescent antibody technique. Another portion of the same specimen was inoculated onto primary bovine skin cell cultures for virus isolation. In the experimental studies for infectious bovine rhinotracheitis, 29/35 specimens were positive by fluorescent antibody technique and 32/35 by cell culture and in the field cases, 22/119 were positive by fluorescent antibody technique and 19/119 by cell culture. In the field cases of bovine viral diarrhea, 28/69 samples were positive by fluorescent antibody technique and 14/69 by cell culture. When fluorescent antibody technique was performed on inoculated cell cultures a total of 24/69 specimens were positive for bovine viral diarrhea. The sensitivity of fluorescent antibody technique was thus comparable to that of cell culture method for infectious bovine rhinotracheitis and bovine viral diarrhea. ImagesFig. 1.Fig. 2.Fig. 3. PMID:6299484

AN ATTEMPT TO ISOLATE A FILTER-PASSING VIRUS IN EPIDEMIC INFLUENZA

PubMed Central

Detweiler, Herbert K.; Hodge, W. Ray

1924-01-01

1. Six cases of epidemic influenza in various stages of disease were investigated. 2. Suspicious cultures of bodies resembling Bacterium pneumosintes were obtained in three cases, twice from the lung filtrates of inoculated animals and once from the filtrate of nasopharyngeal washings direct. 3. In these instances material was obtained within 36 hours of the acute onset, thus confirming the observations of Olitsky and Gates. 4. Macroscopic and microscopic findings in the lungs of inoculated animals were uniformly negative. PMID:19868833

C3 fixed in vivo to cornea from horses inoculated with Leptospira interrogans.

PubMed

Parma, A E; Cerone, S I; Sansinanea, S A; Ghezzi, M

1992-10-01

C3 was detected bound in vivo to the opaque cornea of horses inoculated with killed Leptospira interrogans. Employing epithelial corneal cells isolated from a monolayer in tissue culture, we proved that C3 is fixed in vitro to the intact cell surface after incubation with a fresh equine anti-Leptospira serum. These findings, in addition to the infiltration of cornea with neutrophils and lymphocytes, may explain the mechanisms of tissue damage in recurrent uveitis of horses with leptospirosis.

Capacity planning for batch and perfusion bioprocesses across multiple biopharmaceutical facilities.

PubMed

Siganporia, Cyrus C; Ghosh, Soumitra; Daszkowski, Thomas; Papageorgiou, Lazaros G; Farid, Suzanne S

2014-01-01

Production planning for biopharmaceutical portfolios becomes more complex when products switch between fed-batch and continuous perfusion culture processes. This article describes the development of a discrete-time mixed integer linear programming (MILP) model to optimize capacity plans for multiple biopharmaceutical products, with either batch or perfusion bioprocesses, across multiple facilities to meet quarterly demands. The model comprised specific features to account for products with fed-batch or perfusion culture processes such as sequence-dependent changeover times, continuous culture constraints, and decoupled upstream and downstream operations that permit independent scheduling of each. Strategic inventory levels were accounted for by applying cost penalties when they were not met. A rolling time horizon methodology was utilized in conjunction with the MILP model and was shown to obtain solutions with greater optimality in less computational time than the full-scale model. The model was applied to an industrial case study to illustrate how the framework aids decisions regarding outsourcing capacity to third party manufacturers or building new facilities. The impact of variations on key parameters such as demand or titres on the optimal production plans and costs was captured. The analysis identified the critical ratio of in-house to contract manufacturing organization (CMO) manufacturing costs that led the optimization results to favor building a future facility over using a CMO. The tool predicted that if titres were higher than expected then the optimal solution would allocate more production to in-house facilities, where manufacturing costs were lower. Utilization graphs indicated when capacity expansion should be considered. © 2014 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers.

Capacity Planning for Batch and Perfusion Bioprocesses Across Multiple Biopharmaceutical Facilities

PubMed Central

Siganporia, Cyrus C; Ghosh, Soumitra; Daszkowski, Thomas; Papageorgiou, Lazaros G; Farid, Suzanne S

2014-01-01

Production planning for biopharmaceutical portfolios becomes more complex when products switch between fed-batch and continuous perfusion culture processes. This article describes the development of a discrete-time mixed integer linear programming (MILP) model to optimize capacity plans for multiple biopharmaceutical products, with either batch or perfusion bioprocesses, across multiple facilities to meet quarterly demands. The model comprised specific features to account for products with fed-batch or perfusion culture processes such as sequence-dependent changeover times, continuous culture constraints, and decoupled upstream and downstream operations that permit independent scheduling of each. Strategic inventory levels were accounted for by applying cost penalties when they were not met. A rolling time horizon methodology was utilized in conjunction with the MILP model and was shown to obtain solutions with greater optimality in less computational time than the full-scale model. The model was applied to an industrial case study to illustrate how the framework aids decisions regarding outsourcing capacity to third party manufacturers or building new facilities. The impact of variations on key parameters such as demand or titres on the optimal production plans and costs was captured. The analysis identified the critical ratio of in-house to contract manufacturing organization (CMO) manufacturing costs that led the optimization results to favor building a future facility over using a CMO. The tool predicted that if titres were higher than expected then the optimal solution would allocate more production to in-house facilities, where manufacturing costs were lower. Utilization graphs indicated when capacity expansion should be considered. © 2013 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 30:594–606, 2014 PMID:24376262

Bioreactor Performance Parameters for an Industrially-Promising Methanotroph Methylomicrobium buryatense 5GB1

DOE PAGES

Gilman, Alexey; Laurens, Lieve M.; Puri, Aaron W.; ...

2015-11-16

Methane is a feedstock of interest for the future, both from natural gas and from renewable biogas sources. Methanotrophic bacteria have the potential to enable commercial methane bioconversion to value-added products such as fuels and chemicals. A strain of interest for such applications is Methylomicrobium buryatense 5GB1, due to its robust growth characteristics. But, to take advantage of the potential of this methanotroph, it is important to generate comprehensive bioreactor-based datasets for different growth conditions to compare bioprocess parameters. The datasets of growth parameters, gas utilization rates, and products (total biomass, extracted fatty acids, glycogen, excreted acids) were obtained formore » cultures of M. buryatense 5GB1 grown in continuous culture under methane limitation and O2 limitation conditions. Additionally, experiments were performed involving unrestricted batch growth conditions with both methane and methanol as substrate. All four growth conditions show significant differences. The most notable changes are the high glycogen content and high formate excretion for cells grown on methanol (batch), and high O2:CH4 utilization ratio for cells grown under methane limitation. The results presented here represent the most comprehensive published bioreactor datasets for a gamma-proteobacterial methanotroph. This information shows that metabolism by M. buryatense 5GB1 differs significantly for each of the four conditions tested. O2 limitation resulted in the lowest relative O2 demand and fed-batch growth on methane the highest. Future studies are needed to understand the metabolic basis of these differences. However, these results suggest that both batch and continuous culture conditions have specific advantages, depending on the product of interest.« less

Microimaging FT-IR of oral cavity tumours. Part III: Cells, inoculated tissues and human tissues

NASA Astrophysics Data System (ADS)

Conti, C.; Ferraris, P.; Giorgini, E.; Pieramici, T.; Possati, L.; Rocchetti, R.; Rubini, C.; Sabbatini, S.; Tosi, G.; Mariggiò, M. A.; Lo Muzio, L.

2007-05-01

The biochemistry of healthy and tumour cell cultures, inoculated tissues and oral cavity tissues have been studied by FT-IR Microscopy with the aim to relate spectral patterns with microbiological and histopathological findings. 'Supervised' and 'unsupervised' procedures of data handling afforded a satisfactory degree of accordance between spectroscopic and the other two techniques. In particular, changes in frequency and intensity of proteins, connective and nucleic acids vibrational modes as well as the visualization of biochemical single wave number or band ratio images, allowed an evaluation of the pathological changes. The spectroscopic patterns of inoculated tissues resulted quite similar to human tissues; differences of both types of sections with cellular lines could be explained by the influence of the environment.

Mimicking lichens: incorporation of yeast strains together with sucrose-secreting cyanobacteria improves survival, growth, ROS removal, and lipid production in a stable mutualistic co-culture production platform

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Tingting; Li, Chien-Ting; Butler, Kirk

The feasibility of heterotrophic-phototrophic symbioses was tested via pairing of yeast strains Cryptococcus curvatus, Rhodotorula glutinis, or Saccharomyces cerevisiae with a sucrose-secreting cyanobacterium Synechococcus elongatus. The phototroph S. elongatus showed no growth in standard BG-11 medium with yeast extract, but grew well in BG-11 medium alone or supplemented with yeast nitrogen base without amino acids (YNB w/o aa). Among three yeast species, C. curvatus and R. glutinis adapted well to the BG-11 medium supplemented with YNB w/o aa, sucrose, and various concentrations of NaCl needed to maintain sucrose secretion from S. elongatus, while growth of S. cerevisiae was highly dependentmore » on sucrose levels. R. glutinis and C. curvatus grew efficiently and utilized sucrose produced by the partner in co-culture. Co-cultures of S. elongatus and R. glutinis were sustained over 1 month in both batch and in semi-continuous culture, with the final biomass and overall lipid yields in the batch co-culture 40 to 60% higher compared to batch mono-cultures of S. elongatus. The co-cultures showed enhanced levels of palmitoleic and linoleic acids. Furthermore, cyanobacterial growth in co-culture with R. glutinis was significantly superior to axenic growth, as S. elongatus was unable to grow in the absence of the yeast partner when cultivated at lower densities in liquid medium. Accumulated reactive oxygen species was observed to severely inhibit axenic growth of cyanobacteria, which was efficiently alleviated through catalase supply and even more effectively with co-cultures of R. glutinis. In conclusion, the pairing of a cyanobacterium and eukaryotic heterotroph in the artificial lichen of this study demonstrates the importance of mutual interactions between phototrophs and heterotrophs, e.g., phototrophs provide a carbon source to heterotrophs, and heterotrophs assist phototrophic growth and survival by removing/eliminating oxidative stress. Our results establish a potential stable production platform that combines the metabolic capability of photoautotrophs to capture inorganic carbon with the channeling of the resulting organic carbon directly to a robust heterotroph partner for producing biofuel and other chemical precursors.« less

Mimicking lichens: incorporation of yeast strains together with sucrose-secreting cyanobacteria improves survival, growth, ROS removal, and lipid production in a stable mutualistic co-culture production platform

DOE PAGES

Li, Tingting; Li, Chien-Ting; Butler, Kirk; ...

2017-03-21

The feasibility of heterotrophic-phototrophic symbioses was tested via pairing of yeast strains Cryptococcus curvatus, Rhodotorula glutinis, or Saccharomyces cerevisiae with a sucrose-secreting cyanobacterium Synechococcus elongatus. The phototroph S. elongatus showed no growth in standard BG-11 medium with yeast extract, but grew well in BG-11 medium alone or supplemented with yeast nitrogen base without amino acids (YNB w/o aa). Among three yeast species, C. curvatus and R. glutinis adapted well to the BG-11 medium supplemented with YNB w/o aa, sucrose, and various concentrations of NaCl needed to maintain sucrose secretion from S. elongatus, while growth of S. cerevisiae was highly dependentmore » on sucrose levels. R. glutinis and C. curvatus grew efficiently and utilized sucrose produced by the partner in co-culture. Co-cultures of S. elongatus and R. glutinis were sustained over 1 month in both batch and in semi-continuous culture, with the final biomass and overall lipid yields in the batch co-culture 40 to 60% higher compared to batch mono-cultures of S. elongatus. The co-cultures showed enhanced levels of palmitoleic and linoleic acids. Furthermore, cyanobacterial growth in co-culture with R. glutinis was significantly superior to axenic growth, as S. elongatus was unable to grow in the absence of the yeast partner when cultivated at lower densities in liquid medium. Accumulated reactive oxygen species was observed to severely inhibit axenic growth of cyanobacteria, which was efficiently alleviated through catalase supply and even more effectively with co-cultures of R. glutinis. In conclusion, the pairing of a cyanobacterium and eukaryotic heterotroph in the artificial lichen of this study demonstrates the importance of mutual interactions between phototrophs and heterotrophs, e.g., phototrophs provide a carbon source to heterotrophs, and heterotrophs assist phototrophic growth and survival by removing/eliminating oxidative stress. Our results establish a potential stable production platform that combines the metabolic capability of photoautotrophs to capture inorganic carbon with the channeling of the resulting organic carbon directly to a robust heterotroph partner for producing biofuel and other chemical precursors.« less

High-cell-density fed-batch cultivation of the docosahexaenoic acid producing marine alga Crypthecodinium cohnii.

PubMed

De Swaaf, Martin E; Sijtsma, Lolke; Pronk, Jack T

2003-03-20

The heterotrophic marine alga Crypthecodinium cohnii is known to produce docosahexaenoic acid (DHA), a polyunsaturated fatty acid with food and pharmaceutical applications, during batch cultivation on complex media containing sea salt, yeast extract, and glucose. In the present study, fed-batch cultivation was studied as an alternative fermentation strategy for DHA production. Glucose and acetic acid were compared as carbon sources. For both substrates, the feed rate was adapted to the maximum specific consumption rate of C. cohnii. In glucose-grown cultures, this was done by maintaining a significant glucose concentration (between 5 and 20 g/L) throughout fermentation. In acetic acid-grown cultures, the medium feed was automatically controlled via the culture pH. A feed consisting of acetic acid (50% w/w) resulted in a higher overall volumetric productivity of DHA (r(DHA)) than a feed consisting of 50% (w/v) glucose (38 and 14 mg/L/h, respectively). The r(DHA) was further increased to 48 mg/L/h using a feed consisting of pure acetic acid. The latter fermentation strategy resulted in final concentrations of 109 g/L dry biomass, 61 g/L lipid, and 19 g/L DHA. These are the highest biomass, lipid, and DHA concentrations reported to date for a heterotrophic alga. Vigorous mixing was required to sustain aerobic conditions during high-cell-density cultivation. This was complicated by culture viscosity, which resulted from the production of viscous extracellular polysaccharides. These may present a problem for large-scale industrial production of DHA. Addition of a commercial polysaccharide-hydrolase preparation could decrease the viscosity of the culture and the required stirring. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 666-672, 2003.

Solar Disinfection of MODS Mycobacterial Cultures in Resource-Poor Settings

PubMed Central

Nathavitharana, Ruvandhi; Coronel, Jorge; Moore, David A. J.

2007-01-01

Introduction Safe disposal of TB culture material in which the infectious burden of clinical samples has been greatly amplified is an important challenge in resource-limited settings. The bactericidal capacity of solar cookers has been demonstrated previously for conventional bacteria and contaminated clinical waste. We investigated the use of a simple solar cooker for the sterilization of mycobacterial broth cultures from the microscopic observation drug susceptibility assay (MODS). Methods Simulated TB culture materials were prepared by inoculating 24-well MODS plates with 500 µL of a known concentration of Mycobacterium bovis BCG. In a series of experiments, samples were simultaneously placed inside a box-type solar cooker and control box and removed at timepoints between 15 minutes and 6 hours. Quantitative cultures were performed using retrieved samples to determine sterilization effect. Results All cultures from the control box were positive at or within 1–4 logs of inoculation concentration. Simulated culture plates at concentrations from 103colony-forming-units (CFU)/ml to 107 CFU/ml were completely sterilized after only one hour of cooker exposure, at temperatures between 50–102°C. At 109 CFU/ml (far in excess of diagnostic cultures), it was only possible to recover mycobacterial growth in plates removed after 15 minutes. By 30 minutes all plates were effectively sterilized. Discussion Solar disinfection provides a very effective, safe and low-cost alternative to conventional equipment used for disposal of mycobacterial culture material. Effect of climatic conditions and optimal operating procedure remain to be defined. PMID:17971863

In vitro fermentation and prebiotic potential of novel low molecular weight polysaccharides derived from agar and alginate seaweeds.

PubMed

Ramnani, Priya; Chitarrari, Roberto; Tuohy, Kieran; Grant, John; Hotchkiss, Sarah; Philp, Kevin; Campbell, Ross; Gill, Chris; Rowland, Ian

2012-02-01

Fermentation properties and prebiotic potential of novel low molecular weight polysaccharides (LMWPs) derived from agar and alginate bearing seaweeds was investigated. Ten LMWPs were supplemented to pH, temperature controlled anaerobic batch cultures inoculated with human feces from three donors, in triplicate. Microbiota changes were monitored using Fluorescent in-situ hybridization and short chain fatty acids, the fermentation end products were analysed using gas chromatography. Of the ten LMWPs tested, Gelidium seaweed CC2253 of molecular weight 64.64 KDa showed a significant increase in bifidobacterial populations from log(10) 8.06 at 0 h to log(10) 8.55 at 24 h (p = 0.018). For total bacterial populations, alginate powder CC2238 produced a significant increase from log(10) 9.01 at 0 h to log(10) 9.58 at 24 h (p = 0.032). No changes were observed in the other bacterial groups tested viz. Bacteroides, Lactobacilli/Enterococci, Eubacterium rectale/Clostridium coccoides and Clostridium histolyticum. The polysaccharides also showed significant increases in total SCFA production, particularly acetic and propionic acids, indicating that they were readily fermented. In conclusion, some LMWPs derived from agar and alginate bearing seaweeds were fermented by gut bacteria and exhibited potential to be used a novel source of prebiotics. Copyright © 2011 Elsevier Ltd. All rights reserved.

Analysis of the Anti-Cancer Effects of Cincau Extract (Premna oblongifolia Merr) and Other Types of Non-Digestible Fibre Using Faecal Fermentation Supernatants and Caco-2 Cells as a Model of the Human Colon

PubMed Central

Nurdin, Samsu U.; Le Leu, Richard K.; Young, Graeme P.; Stangoulis, James C. R.; Christophersen, Claus T.; Abbott, Catherine A.

2017-01-01

Green cincau (Premna oblongifolia Merr) is an Indonesian food plant with a high dietary fibre content. Research has shown that dietary fibre mixtures may be more beneficial for colorectal cancer prevention than a single dietary fibre type. The aim of this study was to investigate the effects of green cincau extract on short chain fatty acid (SCFA) production in anaerobic batch cultures inoculated with human faecal slurries and to compare these to results obtained using different dietary fibre types (pectin, inulin, and cellulose), singly and in combination. Furthermore, fermentation supernatants (FSs) were evaluated in Caco-2 cells for their effect on cell viability, differentiation, and apoptosis. Cincau increased total SCFA concentration by increasing acetate and propionate, but not butyrate concentration. FSs from all dietary fibre sources, including cincau, reduced Caco-2 cell viability. However, the effects of all FSs on cell viability, cell differentiation, and apoptosis were not simply explainable by their butyrate content. In conclusion, products of fermentation of cincau extracts induced cell death, but further work is required to understand the mechanism of action. This study demonstrates for the first time that this Indonesian traditional source of dietary fibre may be protective against colorectal cancer. PMID:28368356

Kinetics of sugars consumption and ethanol inhibition in carob pulp fermentation by Saccharomyces cerevisiae in batch and fed-batch cultures.

PubMed

Lima-Costa, Maria Emília; Tavares, Catarina; Raposo, Sara; Rodrigues, Brígida; Peinado, José M

2012-05-01

The waste materials from the carob processing industry are a potential resource for second-generation bioethanol production. These by-products are small carob kibbles with a high content of soluble sugars (45-50%). Batch and fed-batch Saccharomyces cerevisiae fermentations of high density sugar from carob pods were analyzed in terms of the kinetics of sugars consumption and ethanol inhibition. In all the batch runs, 90-95% of the total sugar was consumed and transformed into ethanol with a yield close to the theoretical maximum (0.47-0.50 g/g), and a final ethanol concentration of 100-110 g/l. In fed-batch runs, fresh carob extract was added when glucose had been consumed. This addition and the subsequent decrease of ethanol concentrations by dilution increased the final ethanol production up to 130 g/l. It seems that invertase activity and yeast tolerance to ethanol are the main factors to be controlled in carob fermentations. The efficiency of highly concentrated carob fermentation makes it a very promising process for use in a second-generation ethanol biorefinery.

Dynamic model of temperature impact on cell viability and major product formation during fed-batch and continuous ethanolic fermentation in Saccharomyces cerevisiae.

PubMed

Amillastre, Emilie; Aceves-Lara, César-Arturo; Uribelarrea, Jean-Louis; Alfenore, Sandrine; Guillouet, Stéphane E

2012-08-01

The impact of the temperature on an industrial yeast strain was investigated in very high ethanol performance fermentation fed-batch process within the range of 30-47 °C. As previously observed with a lab strain, decoupling between growth and glycerol formation occurred at temperature of 36 °C and higher. A dynamic model was proposed to describe the impact of the temperature on the total and viable biomass, ethanol and glycerol production. The model validation was implemented with experimental data sets from independent cultures under different temperatures, temperature variation profiles and cultivation modes. The proposed model fitted accurately the dynamic evolutions for products and biomass concentrations over a wide range of temperature profiles. R2 values were above 0.96 for ethanol and glycerol in most experiments. The best results were obtained at 37 °C in fed-batch and chemostat cultures. This dynamic model could be further used for optimizing and monitoring the ethanol fermentation at larger scale. Copyright © 2012 Elsevier Ltd. All rights reserved.

A triply cloned strain of xylella fastidiosa multiplies and induces symptoms of citrus variegated chlorosis in sweet orange

PubMed

Li; Zreik; Fernandes; Miranda; Teixeira; Ayres; Garnier; Bov

1999-08-01

Xylella fastidiosa isolate 8.1.b obtained from a sweet orange tree affected by citrus variegated chlorosis in the state of Sao Paulo, Brazil, and shown in 1993 to be the causal agent of the disease, was cloned by repeated culture in liquid and on solid PW medium, yielding triply cloned strain 9a5c. The eighth and the 16th passages of strain 9a5c were mechanically inoculated into sweet orange plants. Presence of X. fastidiosa in sweet orange leaves of shoots having grown after inoculation (first-flush shoots) was detected by DAS-ELISA and PCR. Thirty-eight days after inoculation, 70% of the 20 inoculated plants tested positive, and all plants gave strong positive reactions 90 days after inoculation. Symptoms first appeared after 3 months and were conspicuous after 5 months. X. fastidiosa was reisolated from sweet orange leaves, 44 days after inoculation. These results indicate that X. fastidiosa strain 9a5c, derived from pathogenic isolate 8.1.b by triply cloning, is also pathogenic. Strain 9a5c is now used for the X. fastidiosa genome sequencing project undertaken on a large scale in Brazil.http://link. springer-ny.com/link/service/journals/00284/bibs/39n2p106.html

STUDIES ON VARIANTS OF POLIOMYELITIS VIRUS

PubMed Central

Sabin, Albert B.; Hennessen, Walter A.; Winsser, Johan

1954-01-01

Attempts were made to "convert" highly virulent strains of the 3 immunologic types of poliomyelitis virus (Mahoney, Y-SK, and Leon) into avirulent variants. Tests involving intracerebral, intramuscular, or oral administration of virus to cynomolgus monkeys indicated that mere propagation in cultures of kidney tissue of cynomolgus monkeys had no effect on virulence when single or small numbers of virus particles were used as seed, and harvests were delayed for 24 hours or more after the appearance of cytopathogenic change. On the other hand, passages at 24 hour intervals with large inocula (105 to 106 TCD60) produced culture fluids with diminished virulence and unusual patterns of response in cynomolgus monkeys. Purification of such culture fluids by the terminal dilution technique yielded modified strains which proved to be avirulent after administration by the intracerebral, intramuscular, or oral routes in cynomolgus monkeys. Neither paralysis nor CNS lesions were found in any of more than 80 monkeys inoculated intracerebrally with various amounts of virus. However, focal neuronal lesions were found in the spinal cord of 3 of 48 monkeys inoculated intramuscularly with various amounts of the Mahoney variant, in 2 of 20 receiving the Y-SK variant, though in none of 40 inoculated with various amounts of the Leon variant. Virus recovered from the spinal cord of one of the monkeys in the Mahoney group produced no paralysis on intracerebral passage in monkeys. It is assumed that all 3 modified viruses possess a limited capacity to affect lower motor neurones of cynomolgus monkeys when these are directly exposed to them by accidental intraneural or traumatic intracerebral injection. On propagation in cynomolgus kidney cultures the modified viruses reached titers of approximately 107 TCD50 per ml., as measured by cytopathogenic activity on renal epithelial cells in vitro, yet produced no perceptible pathologic changes in the muscles, kidneys, testes, ovaries, heart, pancreas, adrenals, liver, or spleen of cynomolgus monkeys inoculated intramuscularly. The modified viruses were immunogenic after intramuscular injection, but a large proportion of cynomolgus monkeys failed to develop antibody after small doses, indicating that in this host the experimentally produced variants multiplied less readily in non-nervous tissue than the virulent parent strains. Tests with the Type 1 virus showed that the orally administered avirulent variant can induce the formation of antibody and bring about resistance to the occurrence of paralysis such as results from ingestion of the virulent, parent strain. The Types 1 and 2 modified viruses are paralytogenic in mice after direct spinal inoculation whereas the Type 3 virus is not. The Type 1 virus became paralytogenic for mice when it lost its virulence for cynomolgus monkeys by the indicated routes. The Type 2 virus lost its virulence for mice by the intracerebral but not intraspinal routes when it was still fully virulent for cynomolgus monkeys, and retained its paralytogenic activity in intraspinally inoculated mice after it had lost its virulence for cynomolgus monkeys by the indicated routes. The parent Type 3 virus was paralytogenic in intraspinally inoculated mice when it was still fully virulent for cynomolgus monkeys, but this property disappeared in the modified virus when it became avirulent for monkeys. PMID:13163327

Optimization of adventitious root culture for production of biomass and secondary metabolites in Prunella vulgaris L.

PubMed

Fazal, Hina; Abbasi, Bilal Haider; Ahmad, Nisar

2014-11-01

Adventitious root cultures of Prunella vulgaris L. were established in shaking flask system for the production of biomass and secondary metabolites. Adventitious root cultures were induced from callus cultures obtained from leaf explants on solid Murashige and Skoog (MS) medium containing combination of 6-benzyladenine (BA; 1.0 mg l(-1)) and naphthalene acetic acid (NAA; 1.5 mg l(-1)). Thereafter, 0.49 g inoculum was transferred to liquid MS medium supplemented with different concentrations of NAA (0.5-2.0 mg l(-1)). Growth kinetics of adventitious roots was recorded with an interval of 7 days for 49 days period. Highest biomass accumulation (2.13 g/l) was observed in liquid medium containing 1.0 mg l(-1) NAA after 21 days of inoculation. However, other concentrations of NAA also showed similar accumulation pattern but the biomass gradually decreases after 49 days of inoculation. Adventitious roots were collected and dried for investigation of total phenolics (TP), total flavonoids (TF), and antioxidant activities. Higher TPC (0.995 GAE mg/g-DRB) and TFC (6.615 RE mg/g-DRB) were observed in 0.5 mg l(-1) NAA treated cultures. In contrast, higher antioxidant activity (83.53 %) was observed 1.5 mg l(-1) NAA treated cultures. These results are helpful in up scaling of root cultures into bioreactor for secondary metabolites production.

Synergistic degradation of crude oil by indigenous bacterial consortium and exogenous fungus Scedosporium boydii.

PubMed

Yuan, Xiaoyu; Zhang, Xinying; Chen, Xueping; Kong, Dewen; Liu, Xiaoyan; Shen, Siyuan

2018-05-19

The purpose of this study was to investigate the potential of defined co-culture of indigenous bacterial consortium and exogenous fungus Scedosporium boydii for biodegradation of crude oil. After 7 days of incubation, residual oil, n-alkanes and aromatic fraction were analyzed. The degradation rate of crude oil was increased from 61.06% to 81.45% by the defined co-culture according to the 3:1 inoculation ratio of bacteria to fungi. The microbial activity was enhanced markedly and the formation of biofilms was accelerated after suitable inoculation of Scedosporium boydii. High throughput analysis showed that bacterial evenness and diversity were increased and the relative abundance of Paraburkholderia tropica was increased observably from 7.67% to 56.13% in the defined co-culture. These results indicated that synergistic degradation of crude oil in the bacteria-fungi consortium may be advantageous for bioremediation of petroleum-contaminated site. Copyright © 2018 Elsevier Ltd. All rights reserved.

Biofumigation on Post-Harvest Diseases of Fruits Using a New Volatile-Producing Fungus of Ceratocystis fimbriata

PubMed Central

Li, Qian; Wu, Lei; Hao, Jianjun; Luo, Laixin; Cao, Yongsong; Li, Jianqiang

2015-01-01

A variety of volatile organic compounds (VOCs) produced by Ceratocystis fimbriata have strong bioactivity against a wide range of fungi, bacteria and oomycetes. Mycelial growth, conidial production, and spore germination of fungi and oomycetes were significantly inhibited after exposure to cultures of C. fimbriata, and colony formation of bacteria was also inhibited. Two post-harvest diseases, peach brown rot caused by Monilinia fructicola and citrus green mold caused by Penicillium digitatum, were controlled during a 4-day storage by enclosing wound-inoculated fruits with 10 standard diameter Petri plate cultures of C. fimbriata in a 15 L box. The fruits were freshly inoculated at onset of storage and the cultures of C. fimbriata were 6 days old. Percentage of control was 92 and 97%, respectively. After exposure to C. fimbriata VOCs, severely misshapen hyphae and conidia of these two post-harvest pathogens were observed by scanning electron microscopy, and their pathogenicity was lost or greatly reduced. PMID:26147922

Effect of selected strains of Debaryomyces hansenii on the volatile compound production of dry fermented sausage "salchichón".

PubMed

Andrade, M A Jesús; Córdoba, Juan José; Casado, Eva M A; Córdoba, María G; Rodríguez, Mar

2010-06-01

Different biotypes of Debaryomyces hansenii, characterized by mitochondrial DNA (mtDNA) restriction analysis, were inoculated in dry fermented sausages to evaluate their influence as single starter culture on volatile compound generation throughout the ripening process. Similar evolution of physicochemical parameters and microbial population was observed in both uninoculated and inoculated sausages. The tested biotypes modified the volatile compound profile of sausages specially in esters, branched alcohols and aldehydes. The biotype of D. hansenii with the E mtDNA restriction pattern is the most suitable to be used as starter culture since it produced volatile compounds involved in flavour development of dry-cured meat products such as 3-methylbutanol, 3-methylbutanal and 2-propanone. Moreover, the use of D. hansenii strains with the B, C2 and E mtDNA restriction patterns, as a mixed starter culture, should be also considered to generate low amount of sulphur compounds in dry-cured meat products. Copyright 2010 Elsevier Ltd. All rights reserved.

Isolation of Neospora caninum from dairy zero grazing cattle in Israel.

PubMed

Fish, L; Mazuz, M; Molad, T; Savitsky, I; Shkap, V

2007-11-10

First Israeli Neospora caninum isolates were obtained from brain tissues of aborted fetuses (NcIs491 and NcIs580) from dairy farms endemic for neosporosis and maintaining cattle on zero grazing. Tissues from different parts of the fetus brains were used to infect Vero cells. Tachyzoites of N. caninum were first observed in cultures from days 30 and 32 after infection. To confirm the identity of the isolated parasites, DNA extracts from brains and cultures were tested by PCR with specific primers based on the Nc5 gene. Specific fragments were amplified by PCR from infected cultures of both fetuses on day 25. Susceptible seronegative gerbils (Meriones tristrami) were inoculated intraperitoneally with 10(3) to 10(5) tenfold dilutions of subculture tachyzoites. The inoculated gerbils developed specific antibodies to N. caninum, with end-point serum dilution of 1:4096 in the IFA assay, whereas no neurological signs or deaths were seen during 4 months of observation.

Characterization of two diesel fuel degrading microbial consortia enriched from a non acclimated, complex source of microorganisms

PubMed Central

2010-01-01

Background The bioremediation of soils impacted by diesel fuels is very often limited by the lack of indigenous microflora with the required broad substrate specificity. In such cases, the soil inoculation with cultures with the desired catabolic capabilities (bioaugmentation) is an essential option. The use of consortia of microorganisms obtained from rich sources of microbes (e.g., sludges, composts, manure) via enrichment (i.e., serial growth transfers) on the polluting hydrocarbons would provide bioremediation enhancements more robust and reproducible than those achieved with specialized pure cultures or tailored combinations (co-cultures) of them, together with none or minor risks of soil loading with unrelated or pathogenic allocthonous microorganisms. Results In this work, two microbial consortia, i.e., ENZ-G1 and ENZ-G2, were enriched from ENZYVEBA (a complex commercial source of microorganisms) on Diesel (G1) and HiQ Diesel (G2), respectively, and characterized in terms of microbial composition and hydrocarbon biodegradation capability and specificity. ENZ-G1 and ENZ-G2 exhibited a comparable and remarkable biodegradation capability and specificity towards n-C10 to n-C24 linear paraffins by removing about 90% of 1 g l-1 of diesel fuel applied after 10 days of aerobic shaken flask batch culture incubation at 30°C. Cultivation dependent and independent approaches evidenced that both consortia consist of bacteria belonging to the genera Chryseobacterium, Acinetobacter, Psudomonas, Stenotrophomonas, Alcaligenes and Gordonia along with the fungus Trametes gibbosa. However, only the fungus was found to grow and remarkably biodegrade G1 and G2 hydrocarbons under the same conditions. The biodegradation activity and specificity and the microbial composition of ENZ-G1 and ENZ-G2 did not significantly change after cryopreservation and storage at -20°C for several months. Conclusions ENZ-G1 and ENZ-G2 are very similar highly enriched consortia of bacteria and a fungus capable of extensively degrading a broad range of the hydrocarbons mainly composing diesel fuels. Given their remarkable biodegradation potential, stability and resistance to cryopreservation, both consortia appear very interesting candidates for bioaugmentation operations on Diesel fuel impacted soils and sites. PMID:20158909

Characterization of two diesel fuel degrading microbial consortia enriched from a non acclimated, complex source of microorganisms.

PubMed

Zanaroli, Giulio; Di Toro, Sara; Todaro, Daniela; Varese, Giovanna C; Bertolotto, Antonio; Fava, Fabio

2010-02-16

The bioremediation of soils impacted by diesel fuels is very often limited by the lack of indigenous microflora with the required broad substrate specificity. In such cases, the soil inoculation with cultures with the desired catabolic capabilities (bioaugmentation) is an essential option. The use of consortia of microorganisms obtained from rich sources of microbes (e.g., sludges, composts, manure) via enrichment (i.e., serial growth transfers) on the polluting hydrocarbons would provide bioremediation enhancements more robust and reproducible than those achieved with specialized pure cultures or tailored combinations (co-cultures) of them, together with none or minor risks of soil loading with unrelated or pathogenic allocthonous microorganisms. In this work, two microbial consortia, i.e., ENZ-G1 and ENZ-G2, were enriched from ENZYVEBA (a complex commercial source of microorganisms) on Diesel (G1) and HiQ Diesel (G2), respectively, and characterized in terms of microbial composition and hydrocarbon biodegradation capability and specificity. ENZ-G1 and ENZ-G2 exhibited a comparable and remarkable biodegradation capability and specificity towards n-C10 to n-C24 linear paraffins by removing about 90% of 1 g l-1 of diesel fuel applied after 10 days of aerobic shaken flask batch culture incubation at 30 degrees C. Cultivation dependent and independent approaches evidenced that both consortia consist of bacteria belonging to the genera Chryseobacterium, Acinetobacter, Psudomonas, Stenotrophomonas, Alcaligenes and Gordonia along with the fungus Trametes gibbosa. However, only the fungus was found to grow and remarkably biodegrade G1 and G2 hydrocarbons under the same conditions. The biodegradation activity and specificity and the microbial composition of ENZ-G1 and ENZ-G2 did not significantly change after cryopreservation and storage at -20 degrees C for several months. ENZ-G1 and ENZ-G2 are very similar highly enriched consortia of bacteria and a fungus capable of extensively degrading a broad range of the hydrocarbons mainly composing diesel fuels. Given their remarkable biodegradation potential, stability and resistance to cryopreservation, both consortia appear very interesting candidates for bioaugmentation operations on Diesel fuel impacted soils and sites.

Feedback between environment and traits under selection in a seasonal environment: consequences for experimental evolution.

PubMed

Collot, Dorian; Nidelet, Thibault; Ramsayer, Johan; Martin, Olivier C; Méléard, Sylvie; Dillmann, Christine; Sicard, Delphine; Legrand, Judith

2018-04-11

Batch cultures are frequently used in experimental evolution to study the dynamics of adaptation. Although they are generally considered to simply drive a growth rate increase, other fitness components can also be selected for. Indeed, recurrent batches form a seasonal environment where different phases repeat periodically and different traits can be under selection in the different seasons. Moreover, the system being closed, organisms may have a strong impact on the environment. Thus, the study of adaptation should take into account the environment and eco-evolutionary feedbacks. Using data from an experimental evolution on yeast Saccharomyces cerevisiae , we developed a mathematical model to understand which traits are under selection, and what is the impact of the environment for selection in a batch culture. We showed that two kinds of traits are under selection in seasonal environments: life-history traits, related to growth and mortality, but also transition traits, related to the ability to react to environmental changes. The impact of environmental conditions can be summarized by the length of the different seasons which weight selection on each trait: the longer a season is, the higher the selection on associated traits. Since phenotypes drive season length, eco-evolutionary feedbacks emerge. Our results show how evolution in successive batches can affect season lengths and strength of selection on different traits. © 2018 The Author(s).

Gentiana dinarica Beck hairy root cultures and evaluation of factors affecting growth and xanthone production

USDA-ARS?s Scientific Manuscript database

The induction and establishment of hairy root cultures of Gentiana dinarica using two strains of Agrobacterium rhizogenes (A4M70GUS and 15834/PI) is reported for the first time. Hairy roots were formed from the shoots 25 days after inoculation, and strain 15834/PI had higher induction rate of hairy ...

[Effects of fungus on the growth of Dendrobium candidum and D. nobile in vitro culture].

PubMed

Song, J Y; Guo, S X

2001-12-01

To examine effects of fungus AR-18 (Epulorhiza sp.) on the growth of Dendrobium candidum and D. nobile in vitro culture. Effects of fungus AR-18 on fresh weight and dry weight of D. candidum and D. nobile were studied in vitro culture, when agar was used as rest, and effects of fungus AR-18 and nutrients on fresh weight and dry weight of D. candidum and D. nobile were studied in vitro culture, when vermiculite was used as rest. In agar medium, effects of fungus AR-18 on fresh weight and dry weight of D. candidum and D. nobile were not significant (P > 0.05). However, in vermiculite medium, the effect of fungus AR-18 on fresh weight of D. nobile was significant (P < 0.05). Fresh weight of D. nobile inoculated with fungus AR-18 was increased by 16% compared with the non-inoculated ones (control). The effect of fungus AR-18 on dry weight of D. nobile was very significant (P < 0.01). Dry weight of D. nobile inoculated with fungus AR-18 was increased by 21% compared with the control. In vermiculite medium, the effect of combination treatment (Fungus AR-18 + Distilled H2O) on fresh weight of D. nobile was very significant (P < 0.01). Fresh weight of D. nobile inoculated with fungus AR-18 was increased by 47% compared with the control, while the effect of combination treatment (Fungus AR-18 + Nutrients) on fresh weight of D. nobile was not significant (P > 0.05). In addition, the effect of combinations treatment (Fungus AR-18 + Nutrients) and (Fungus AR-18 + Distilled H2O) on dry weight of D. nobile was not significant (P > 0.05). So the combination treatment (Fungus AR-18 + Distilled H2O) was the best for the growth of D. nobile. In vermiculite medium, effects of fungus AR-18 and nutrients on fresh weight and dry weight of D. candidum were not significant (P > 0.05). It is important in vitro culture to select a proper rest for the control of the growth of fungus to establish a beneficial symbiotic relationship between plants and fungi. If the fungus AR-18 is utilized as biological manure in got-up planting of D. nobile, a good result may be obtained.

Expression of Haemophilus ducreyi Collagen Binding Outer Membrane Protein NcaA Is Required for Virulence in Swine and Human Challenge Models of Chancroid

PubMed Central

Fulcher, Robert A.; Cole, Leah E.; Janowicz, Diane M.; Toffer, Kristen L.; Fortney, Kate R.; Katz, Barry P.; Orndorff, Paul E.; Spinola, Stanley M.; Kawula, Thomas H.

2006-01-01

Haemophilus ducreyi, the etiologic agent of the sexually transmitted genital ulcer disease chancroid, has been shown to associate with dermal collagen fibers within infected skin lesions. Here we describe NcaA, a previously uncharacterized outer membrane protein that is important for H. ducreyi collagen binding and host colonization. An H. ducreyi strain lacking the ncaA gene was impaired in adherence to type I collagen but not fibronectin (plasma or cellular form) or heparin. The mutation had no effect on serum resistance or binding to HaCaT keratinocytes or human foreskin fibroblasts in vitro. Escherichia coli expressing H. ducreyi NcaA bound to type I collagen, demonstrating that NcaA is sufficient to confer collagen attachment. The importance of NcaA in H. ducreyi pathogenesis was assessed using both swine and human experimental models of chancroid. In the swine model, 20% of lesions from sites inoculated with the ncaA mutant were culture positive for H. ducreyi 7 days after inoculation, compared to 73% of wild-type-inoculated sites. The average number of CFU recovered from mutant-inoculated lesions was also significantly reduced compared to that recovered from wild-type-inoculated sites at both 2 and 7 days after inoculation. In the human challenge model, 8 of 30 sites inoculated with wild-type H. ducreyi progressed to the pustular stage, compared to 0 of 30 sites inoculated with the ncaA mutant. Together these results demonstrate that the collagen binding protein NcaA is required for H. ducreyi infection. PMID:16622201

Expression of Haemophilus ducreyi collagen binding outer membrane protein NcaA is required for virulence in swine and human challenge models of chancroid.

PubMed

Fulcher, Robert A; Cole, Leah E; Janowicz, Diane M; Toffer, Kristen L; Fortney, Kate R; Katz, Barry P; Orndorff, Paul E; Spinola, Stanley M; Kawula, Thomas H

2006-05-01

Haemophilus ducreyi, the etiologic agent of the sexually transmitted genital ulcer disease chancroid, has been shown to associate with dermal collagen fibers within infected skin lesions. Here we describe NcaA, a previously uncharacterized outer membrane protein that is important for H. ducreyi collagen binding and host colonization. An H. ducreyi strain lacking the ncaA gene was impaired in adherence to type I collagen but not fibronectin (plasma or cellular form) or heparin. The mutation had no effect on serum resistance or binding to HaCaT keratinocytes or human foreskin fibroblasts in vitro. Escherichia coli expressing H. ducreyi NcaA bound to type I collagen, demonstrating that NcaA is sufficient to confer collagen attachment. The importance of NcaA in H. ducreyi pathogenesis was assessed using both swine and human experimental models of chancroid. In the swine model, 20% of lesions from sites inoculated with the ncaA mutant were culture positive for H. ducreyi 7 days after inoculation, compared to 73% of wild-type-inoculated sites. The average number of CFU recovered from mutant-inoculated lesions was also significantly reduced compared to that recovered from wild-type-inoculated sites at both 2 and 7 days after inoculation. In the human challenge model, 8 of 30 sites inoculated with wild-type H. ducreyi progressed to the pustular stage, compared to 0 of 30 sites inoculated with the ncaA mutant. Together these results demonstrate that the collagen binding protein NcaA is required for H. ducreyi infection.

Monochloramine Cometabolism by Mixed-Culture Nitrifiers ...

EPA Pesticide Factsheets

The current research investigated monochloramine cometabolism by nitrifying mixed cultures grown under drinking water relevant conditions and harvested from sand-packed reactors before conducting suspended growth batch kinetic experiments. Three batch reactors were used in each experiment: (1) a positive control to estimate ammonia kinetic parameters, (2) a negative control to account for abiotic reactions, and (3) a cometabolism reactor to estimate cometabolism kinetic constants. Kinetic parameters were estimated in AQUASIM with a simultaneous fit to all experimental data. Cometabolism kinetics were best described by a first order model. Monochloramine cometabolism kinetics were similar to those of ammonia metabolism, and monochloramine cometabolism was a significant loss mechanism (30% of the observed monochloramine loss). These results demonstrated that monochloramine cometabolism occurred in mixed cultures similar to those found in drinking water distribution systems; thus, cometabolism may be a significant contribution to monochloramine loss during nitrification episodes in drinking water distribution systems. The results demonstrated that monochloramine cometabolism occurred in mixed cultures similar to those found in drinking water distribution systems; thus, cometabolism may be a significant contribution to monochloramine loss during nitrification episodes in drinking water distribution systems.

Comparative study on major bioactive components in natural, artificial and in-vitro cultured Calculus Bovis.

PubMed

Yan, Shi-Kai; Wu, Yan-Wen; Liu, Run-Hui; Zhang, Wei-Dong

2007-01-01

Major bioactive components in various Calculus Bovis, including natural, artificial and in-vitro cultured Calculus Bovis, were comparatively studied. An approach of high-performance liquid chromatography coupled with ultraviolet and evaporative light scattering detections (HPLC/UV/ELSD) was established to simultaneously determinate six bioactive components thereof, including five bile acids (cholic acid, deoxycholic acid, ursodeoxycholic, chenodeoxycholic acid, hyodeoxycholic acid) and bilirubin. ELSD and UV detector were applied to detect bile acids and bilirubin respectively. The assay was performed on a C(18) column with water-acetonitrile gradient elution and the investigated constituents were authenticated by comparing retention times and mass spectra with those of reference compounds. The proposed method was applied to analyze twenty-one Calculus Bovis extraction samples, and produced data with acceptable linearity, precision, repeatability and accuracy. The result indicated the variations among Calculus Bovis samples under different developmental conditions. Artificial and in-vitro cultured Calculus Bovis, especially in-vitro cultured ones, which contain total bioactive constituents no less than natural products and have the best batch-to-batch uniformity, suffice to be used as substitutes of natural Calculus Bovis.

Degradation pathway of 2-chloroethanol in Pseudomonas stutzeri strain JJ under denitrifying conditions.

PubMed

Dijk, John A; Gerritse, Jan; Schraa, Gosse; Stams, Alfons J M

2004-12-01

The pathway of 2-chloroethanol degradation in the denitrifying Pseudomonas stutzeri strain JJ was investigated. In cell-free extracts, activities of a phenazine methosulfate (PMS)-dependent chloroethanol dehydrogenase, an NAD-dependent chloroacetaldehyde dehydrogenase, and a chloroacetate dehalogenase were detected. This suggested that the 2-chloroethanol degradation pathway in this denitrifying strain is the same as found in aerobic bacteria that degrade chloroethanol. Activity towards primary alcohols, secondary alcohols, diols, and other chlorinated alcohols could be measured in cell-free extracts with chloroethanol dehydrogenase (CE-DH) activity. PMS and phenazine ethosulfate (PES) were used as primary electron acceptors, but not NAD, NADP or ferricyanide. Cells of strain JJ cultured in a continuous culture under nitrate limitation exhibited chloroethanol dehydrogenase activity that was a 12 times higher than in cells grown in batch culture. However, under chloroethanol-limiting conditions, CE-DH activity was in the same range as in batch culture. Cells grown on ethanol did not exhibit CE-DH activity. Instead, NAD-dependent ethanol dehydrogenase (E-DH) activity and PMS-dependent E-DH activity were detected.

Biosynthesis of highly enriched 13C-lycopene for human metabolic studies using repeated batch tomato cell culturing with 13C-glucose

PubMed Central

Moran, Nancy E.; Rogers, Randy B.; Lu, Chi-Hua; Conlon, Lauren E.; Lila, Mary Ann; Clinton, Steven K.; Erdman, John W.

2013-01-01

While putative disease-preventing lycopene metabolites are found in both tomato (Solanum lycopersicum) products and in their consumers, mammalian lycopene metabolism is poorly understood. Advances in tomato cell culturing techniques offer an economical tool for generation of highly-enriched 13C-lycopene for human bioavailability and metabolism studies. To enhance the 13C-enrichment and yields of labeled lycopene from the hp-1 tomato cell line, cultures were first grown in 13C-glucose media for three serial batches and produced increasing proportions of uniformly labeled lycopene (14.3 +/− 1.2 %, 39.6 +/− 0.5 %, and 48.9 +/− 1.5% with consistent yields (from 5.8 to 9 mg/L). An optimized 9-day-long 13C-loading and 18-day-long labeling strategy developed based on glucose utilization and lycopene yields, yielded 13C-lycopene with 93% 13C isotopic purity, and 55% of isotopomers were uniformly labeled. Furthermore, an optimized acetone and hexane extraction led to a four-fold increase in lycopene recovery from cultures compared to a standard extraction. PMID:23561155

Genetically different isolates of Trypanosoma cruzi elicit different infection dynamics in raccoons (Procyon lotor) and Virginia opossums (Didelphis virginiana)

PubMed Central

Roellig, Dawn M.; Ellis, Angela E.; Yabsley, Michael J.

2009-01-01

Trypanosoma cruzi is a genetically and biologically diverse species. In the current study we determined T. cruzi infection dynamics in two common North American reservoirs, Virginia opossums (Didelphis virginiana) and raccoons (Procyon lotor). Based on previous molecular and culture data from naturally-exposed animals, we hypothesized that raccoons would have a longer patent period than opossums, and raccoons would be competent reservoirs for both genotypes T. cruzi I (TcI) and TcIIa, while opossums would only serve as hosts for TcI. Individuals (n = 2 or 3) of each species were inoculated with 1 × 106 culture-derived T. cruzi trypomastigotes of TcIIa (North American (NA) - raccoon), TcI (NA - opossum), TcIIb (South American - human), or both TcI and TcIIa. Parasitemias in opossums gradually increased and declined rapidly, whereas parasitemias peaked sooner in raccoons and they maintained relatively high parasitemia for 5 weeks. Raccoons became infected with all three T. cruzi strains, while opossums only became infected with TcI and TcIIb. Although opossums were susceptible to TcIIb, infection dynamics were dramatically different compared with TcI. Opossums inoculated with TcIIb seroconverted, but parasitemia duration was short and only detectable by PCR. In addition, raccoons seroconverted sooner (3–7 days post inoculation) than opossums (10 days post inoculation). These data suggest that infection dynamics of various T. cruzi strains can differ considerably in different wildlife hosts. PMID:19607833

Cell culture for three-dimensional modeling in rotating-wall vessels: an application of simulated microgravity

NASA Technical Reports Server (NTRS)

Schwarz, R. P.; Goodwin, T. J.; Wolf, D. A.

1992-01-01

High-density, three-dimensional cell cultures are difficult to grow in vitro. The rotating-wall vessel (RWV) described here has cultured BHK-21 cells to a density of 1.1 X 10(7) cells/ml. Cells on microcarriers were observed to grow with enhanced bridging in this batch culture system. The RWV is a horizontally rotated tissue culture vessel with silicon membrane oxygenation. This design results in a low-turbulence, low-shear cell culture environment with abundant oxygenation. The RWV has the potential to culture a wide variety of normal and neoplastic cells.

A new species of Burkholderia isolated from sugarcane roots promotes plant growth

PubMed Central

Paungfoo-Lonhienne, Chanyarat; Lonhienne, Thierry G A; Yeoh, Yun Kit; Webb, Richard I; Lakshmanan, Prakash; Chan, Cheong Xin; Lim, Phaik-Eem; Ragan, Mark A; Schmidt, Susanne; Hugenholtz, Philip

2014-01-01

Sugarcane is a globally important food, biofuel and biomaterials crop. High nitrogen (N) fertilizer rates aimed at increasing yield often result in environmental damage because of excess and inefficient application. Inoculation with diazotrophic bacteria is an attractive option for reducing N fertilizer needs. However, the efficacy of bacterial inoculants is variable, and their effective formulation remains a knowledge frontier. Here, we take a new approach to investigating diazotrophic bacteria associated with roots using culture-independent microbial community profiling of a commercial sugarcane variety (Q208A) in a field setting. We first identified bacteria that were markedly enriched in the rhizosphere to guide isolation and then tested putative diazotrophs for the ability to colonize axenic sugarcane plantlets (Q208A) and promote growth in suboptimal N supply. One isolate readily colonized roots, fixed N2 and stimulated growth of plantlets, and was classified as a new species, Burkholderia australis sp. nov. Draft genome sequencing of the isolate confirmed the presence of nitrogen fixation. We propose that culture-independent identification and isolation of bacteria that are enriched in rhizosphere and roots, followed by systematic testing and confirming their growth-promoting capacity, is a necessary step towards designing effective microbial inoculants. PMID:24350979

Alleviation of salt stress by halotolerant and halophilic plant growth-promoting bacteria in wheat (Triticum aestivum).

PubMed

Orhan, Furkan

2016-01-01

In the current study, 18 halotolerant and halophilic bacteria have been investigated for their plant growth promoting abilities in vitro and in a hydroponic culture. The bacterial strains have been investigated for ammonia, indole-3-acetic acid and 1-aminocyclopropane-1-carboxylate-deaminase production, phosphate solubilisation and nitrogen fixation activities. Of the tested bacteria, eight were inoculated with Triticum aestivum in a hydroponic culture. The investigated bacterial strains were found to have different plant-growth promoting activities in vitro. Under salt stress (200mM NaCl), the investigated bacterial strains significantly increased the root and shoot length and total fresh weight of the plants. The growth rates of the plants inoculated with bacterial strains ranged from 62.2% to 78.1%. Identifying of novel halophilic and halotolerant bacteria that promote plant growth can be used as alternatives for salt sensitive plants. Extensive research has been conducted on several halophilic and halotolerant bacterial strains to investigate their plant growth promoting activities. However, to the best of my knowledge, this is the first study to inoculate these bacterial strains with wheat. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Acclimation of Saccharomyces cerevisiae to low temperature: a chemostat-based transcriptome analysis.

PubMed

Tai, Siew Leng; Daran-Lapujade, Pascale; Walsh, Michael C; Pronk, Jack T; Daran, Jean-Marc

2007-12-01

Effects of suboptimal temperatures on transcriptional regulation in yeast have been extensively studied in batch cultures. To eliminate indirect effects of specific growth rates that are inherent to batch-cultivation studies, genome-wide transcriptional responses to low temperatures were analyzed in steady-state chemostats, grown at a fixed specific growth rate (0.03 h(-1)). Although in vivo metabolic fluxes were essentially the same in cultures grown at 12 and at 30 degrees C, concentrations of the growth-limiting nutrients (glucose or ammonia) were higher at 12 degrees C. This difference was reflected by transcript levels of genes that encode transporters for the growth-limiting nutrients. Several transcriptional responses to low temperature occurred under both nutrient-limitation regimes. Increased transcription of ribosome-biogenesis genes emphasized the importance of adapting protein-synthesis capacity to low temperature. In contrast to observations in cold-shock and batch-culture studies, transcript levels of environmental stress response genes were reduced at 12 degrees C. Transcription of trehalose-biosynthesis genes and intracellular trehalose levels indicated that, in contrast to its role in cold-shock adaptation, trehalose is not involved in steady-state low-temperature adaptation. Comparison of the chemostat-based transcriptome data with literature data revealed large differences between transcriptional reprogramming during long-term low-temperature acclimation and the transcriptional responses to a rapid transition to low temperature.

Performance of high intensity fed-batch mammalian cell cultures in disposable bioreactor systems.

PubMed

Smelko, John Paul; Wiltberger, Kelly Rae; Hickman, Eric Francis; Morris, Beverly Janey; Blackburn, Tobias James; Ryll, Thomas

2011-01-01

The adoption of disposable bioreactor technology as an alternate to traditional nondisposable technology is gaining momentum in the biotechnology industry. Evaluation of current disposable bioreactors systems to sustain high intensity fed-batch mammalian cell culture processes needs to be explored. In this study, an assessment was performed comparing single-use bioreactors (SUBs) systems of 50-, 250-, and 1,000-L operating scales with traditional stainless steel (SS) and glass vessels using four distinct mammalian cell culture processes. This comparison focuses on expansion and production stage performance. The SUB performance was evaluated based on three main areas: operability, process scalability, and process performance. The process performance and operability aspects were assessed over time and product quality performance was compared at the day of harvest. Expansion stage results showed disposable bioreactors mirror traditional bioreactors in terms of cellular growth and metabolism. Set-up and disposal times were dramatically reduced using the SUB systems when compared with traditional systems. Production stage runs for both Chinese hamster ovary and NS0 cell lines in the SUB system were able to model SS bioreactors runs at 100-, 200-, 2,000-, and 15,000-L scales. A single 1,000-L SUB run applying a high intensity fed-batch process was able to generate 7.5 kg of antibody with comparable product quality. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

An In Vitro Approach to Study Effects of Prebiotics and Probiotics on the Faecal Microbiota and Selected Immune Parameters Relevant to the Elderly

PubMed Central

Liu, Yue; Gibson, Glenn R.; Walton, Gemma E.

2016-01-01

The aging process leads to alterations of gut microbiota and modifications to the immune response, such changes may be associated with increased disease risk. Prebiotics and probiotics can modulate microbiome changes induced by aging; however, their effects have not been directly compared. The aim of this study was to use anaerobic batch culture fermenters to assess the impact of various fermentable carbohydrates and microorganisms on the gut microbiota and selected immune markers. Elderly volunteers were used as donors for these experiments to enable relevance to an aging population. The impact of fermentation supernatants on immune markers relevant to the elderly were assessed in vitro. Levels of IL-1β, IL-6, IL-8, IL-10 and TNF-α in peripheral blood mononuclear cell culture supernatants were measured using flow cytometry. Trans-galactooligosaccharides (B-GOS) and inulin both stimulated bifidobacteria compared to other treatments (p<0.05). Fermentation supernatants taken from faecal batch cultures supplemented with B-GOS, inulin, B. bifidum, L. acidophilus and Ba. coagulans inhibited LPS induced TNF-α (p<0.05). IL-10 production, induced by LPS, was enhanced by fermentation supernatants from faecal batch cultures supplemented with B-GOS, inulin, B. bifidum, L. acidophilus, Ba. coagulans and Bac. thetaiotaomicron (p<0.05). To conclude, prebiotics and probiotics could lead to potentially beneficial effects to host health by targeting specific bacterial groups, increasing saccharolytic fermentation and decreasing inflammation associated with aging. Compared to probiotics, prebiotics led to greater microbiota modulation at the genus level within the fermenters. PMID:27612304

An In Vitro Approach to Study Effects of Prebiotics and Probiotics on the Faecal Microbiota and Selected Immune Parameters Relevant to the Elderly.

PubMed

Liu, Yue; Gibson, Glenn R; Walton, Gemma E

2016-01-01

The aging process leads to alterations of gut microbiota and modifications to the immune response, such changes may be associated with increased disease risk. Prebiotics and probiotics can modulate microbiome changes induced by aging; however, their effects have not been directly compared. The aim of this study was to use anaerobic batch culture fermenters to assess the impact of various fermentable carbohydrates and microorganisms on the gut microbiota and selected immune markers. Elderly volunteers were used as donors for these experiments to enable relevance to an aging population. The impact of fermentation supernatants on immune markers relevant to the elderly were assessed in vitro. Levels of IL-1β, IL-6, IL-8, IL-10 and TNF-α in peripheral blood mononuclear cell culture supernatants were measured using flow cytometry. Trans-galactooligosaccharides (B-GOS) and inulin both stimulated bifidobacteria compared to other treatments (p<0.05). Fermentation supernatants taken from faecal batch cultures supplemented with B-GOS, inulin, B. bifidum, L. acidophilus and Ba. coagulans inhibited LPS induced TNF-α (p<0.05). IL-10 production, induced by LPS, was enhanced by fermentation supernatants from faecal batch cultures supplemented with B-GOS, inulin, B. bifidum, L. acidophilus, Ba. coagulans and Bac. thetaiotaomicron (p<0.05). To conclude, prebiotics and probiotics could lead to potentially beneficial effects to host health by targeting specific bacterial groups, increasing saccharolytic fermentation and decreasing inflammation associated with aging. Compared to probiotics, prebiotics led to greater microbiota modulation at the genus level within the fermenters.

FT-IR microspectroscopy in rapid identification of bacteria in pure and mixed culture

NASA Astrophysics Data System (ADS)

Fontoura, Inglid; Belo, Ricardo; Sakane, Kumiko; Cardoso, Maria Angélica Gargione; Khouri, Sônia; Uehara, Mituo; Raniero, Leandro; Martin, Airton A.

2010-02-01

In recent years FT-IR microspectroscopy has been developed for microbiology analysis and applied successfully in pure cultures of microorganisms to rapidly identify strains of bacteria, yeasts and fungi. The investigation and characterization of microorganism mixed cultures is also of growing importance, especially in hospitals where it is common to poly-microbial infections. In this work, the rapid identification of bacteria in pure and mixed cultures was studied. The bacteria were obtained from the Institute Oswaldo Cruz culture collection at Brazil. Escherichia coli ATCC 10799 and Staphylococcus aureus ATCC 14456 were analyzed, 3 inoculations were examined in triplicate: Escherichia coli, Staphylococcus aureus and a mixed culture of them. The inoculations were prepared according to McFarland 0.5, incubated at 37 ° C for 6 hours, diluted in saline, placed in the CaF2 window and store for one hour at 50°C to obtain thin film. The measurement was performed by Spectrum Spotlight 400 (Perkin-Elmer) equipment in the range of 4000-900 cm-1, with 32 scans using a transmittance technique with point and image modes. The data were processed (baseline, normalization, calculation of first derivate followed by smoothing with 9 point using a Savitzky-Golay algorithm) and a cluster analysis were done by Ward's algorithm and an excellent discrimination between pure and mixed culture was obtained. Our preliminary results indicate that the FT-IR microspectroscopy associated with cluster analysis can be used to discriminate between pure and mixed culture.

Managing Your Wine Fermentation to Reduce the Risk of Biogenic Amine Formation

PubMed Central

Smit, Anita Yolandi; Engelbrecht, Lynn; du Toit, Maret

2012-01-01

Biogenic amines are nitrogenous organic compounds produced in wine from amino acid precursors mainly by microbial decarboxylation. The concentration of biogenic amines that can potentially be produced is dependent on the amount of amino acid precursors in the medium, the presence of decarboxylase positive microorganisms and conditions that enable microbial or biochemical activity such as the addition of nutrients to support the inoculated starter cultures for alcoholic and malolactic fermentation (MLF). MLF can be conducted using co-inoculation or an inoculation after the completion of alcoholic fermentation that may also affect the level of biogenic amines in wine. This study focused on the impact of the addition of complex commercial yeast and bacterial nutrients and the use of different MLF inoculation scenarios on the production of biogenic amines in wine. Results showed that the addition of complex nutrients to real grape must could potentially increase histamine concentrations in wine. The same experiment in synthetic grape must showed a similar trend for putrescine and cadaverine. The effect of different MLF inoculation scenarios was examined in two cultivars, Pinotage and Shiraz. Conflicting results was obtained. In the Shiraz, co-inoculation resulted in lower biogenic amine concentrations after MLF compared to before MLF, while the concentration was higher in the Pinotage. However, the production of biogenic amines was affected more by the presence of decarboxylase positive lactic acid bacteria than by the addition of complex nutrients or the inoculation scenario. PMID:22419915

Use of whey lactose from dairy industry for economical kefiran production by Lactobacillus kefiranofaciens in mixed cultures with yeasts.

PubMed

Cheirsilp, Benjamas; Radchabut, Sirilaor

2011-10-01

To evaluate the feasibility of producing kefiran industrially, whey lactose, a by-product from dairy industry, was used as a low cost carbon source. Because the accumulation of lactic acid as a by-product of Lactobacillus kefiranofaciens inhibited cell growth and kefiran production, the kefir grain derived and non-derived yeasts were screened for their abilities to reduce lactic acid and promote kefiran production in a mixed culture. Six species of yeasts were examined: Torulaspora delbrueckii IFO 1626; Saccharomyces cerevisiae IFO 0216; Debaryomyces hansenii TISTR 5155; Saccharomyces exiguus TISTR 5081; Zygosaccharomyces rouxii TISTR 5044; and Saccharomyces carlsbergensis TISTR 5018. The mixed culture of L. kefiranofaciens with S. cerevisiae IFO 0216 enhanced the kefiran production best from 568 mg/L in the pure culture up to 807 and 938 mg/L in the mixed cultures under anaerobic and microaerobic conditions, respectively. The optimal conditions for kefiran production by the mixed culture were: whey lactose 4%; yeast extract 4%; initial pH of 5.5; and initial amounts of L. kefiranofaciens and S. cerevisiae IFO 0216 of 2.1×10(7) and 4.0×10(6)CFU/mL, respectively. Scaling up the mixed culture in a 2L bioreactor with dissolved oxygen control at 5% and pH control at 5.5 gave the maximum kefiran production of 2,580 mg/L in batch culture and 3,250 mg/L in fed-batch culture. Copyright © 2011 Elsevier B.V. All rights reserved.

Approaches to Optimizing Animal Cell Culture Process: Substrate Metabolism Regulation and Protein Expression Improvement

NASA Astrophysics Data System (ADS)

Zhang, Yuanxing

Some high value proteins and vaccines for medical and veterinary applications by animal cell culture have an increasing market in China. In order to meet the demands of large-scale productions of proteins and vaccines, animal cell culture technology has been widely developed. In general, an animal cell culture process can be divided into two stages in a batch culture. In cell growth stage a high specific growth rate is expected to achieve a high cell density. In production stage a high specific production rate is stressed for the expression and secretion of qualified protein or replication of virus. It is always critical to maintain high cell viability in fed-batch and perfusion cultures. More concern has been focused on two points by the researchers in China. First, the cell metabolism of substrates is analyzed and the accumulation of toxic by-products is decreased through regulating cell metabolism in the culture process. Second, some important factors effecting protein expression are understood at the molecular level and the production ability of protein is improved. In pace with the rapid development of large-scale cell culture for the production of vaccines, antibodies and other recombinant proteins in China, the medium design and process optimization based on cell metabolism regulation and protein expression improvement will play an important role. The chapter outlines the main advances in metabolic regulation of cell and expression improvement of protein in animal cell culture in recent years.

Comparison of Reductive Dechlorination of Chlorinated Ethylene in Batch and Continuous-Flow Reactor

NASA Astrophysics Data System (ADS)

Park, S.; Jonghwan, L.; Hong, U.; Kim, N.; Ahn, H.; Lee, S.; Kim, Y.

2010-12-01

A 1.28 L-Batch reactor and continuous-flow stirred tank reactor (CFSTR) fed with formate and trichloriethene (TCE) were operated for 120 days and 72 days, respectively, to study the effect of formate as electron donor on reductive dechlorination of TCE to cis-1,2-dichloroethylene (c-DCE), vinyl chloride (VC), and ethylene (ETH). In batch reactor, injected 60 μmol TCE was completely degraded in presence of 20% hydrogen gas (H2) in less than 8 days by Evanite culture (300 mg-soluble protein) with ability to completely degrade tetrachloroethene (PCE) and TCE to ETH under anaerobic conditions. To determine the effect of formate as electron donor instead of H2, about 3 or 11 mmol of formate injected into batch-reactor every 15 days was enough to support H2 for dechlorination of c-DCE to VC and ETH. Soluble protein concentration of Evanite culture during the batch test increased from 300 mg to 688 mg for 120 days. In CFSTR test, TCE was fed continuously at 9.9 ppm (75.38 μmol/L) and the influent formate feed concentration increased stepwise from 1.3 mmol/L to 14.3 mmol/L. Injected TCE was accumulated at HRT 18 days for 13 days, but TCE was completed degraded at HRT 36 days without accumulation during left of experiment period, getting H2 from fermentative hydrogen production of injected formate. Although c-DCE was also accumulated for 23 days after CFSTR operation, it reached steady-state without accumulation in presence of excessive formate. However, since c-DCE in CFSTR was not completely dechlorinated, we will determine the transcriptional level of enzyme involved in reductive dechlorination of TCE, c-DCE, and VC in our future work.

Hydrogenase activity in aged, nonviable Desulfovibrio vulgaris cultures and its significance in anaerobic biocorrosion.

PubMed Central

Chatelus, C; Carrier, P; Saignes, P; Libert, M F; Berlier, Y; Lespinat, P A; Fauque, G; Legall, J

1987-01-01

Batch cultures of Desulfovibrio vulgaris stored at 32 degrees C for 10 months have been found to retain 50% of the hydrogenase activity of a 1-day culture. The hydrogenase found in old cultures needs reducing conditions for its activation. Viable cell counts are negative after 6 months, showing that the hydrogenase activity does not depend on the presence of viable cells. These observations are of importance in the understanding of anaerobic biocorrosion of metals caused by depolarization phenomena. PMID:3310883

Isolation of Salmonellae from Foods Samples

PubMed Central

Taylor, Welton I.; Silliker, John H.

1961-01-01

A comparison of various methods of enhancing frequency of Salmonella isolations revealed that inoculation of a second enrichment broth, with culture from the first, was no improvement over the single direct enrichment method. It was inferior to centrifugation. Selenite was observed to produce more positive isolations at 48 hr than at 24. No change occurred in tetrathionate. Reconstitution of dried albumen with water produced a significant increase in isolations over direct inoculation of enrichment broth in the case of tetrathionate but not selenite broth. Pre-enrichment in lactose broth before inoculation of enrichment media was vastly superior to reconstitution in water for both enrichment broths. A comparison of results obtained using dulcitol, mannitol, lactose and carbohydrate-free purple broths in pre-enrichment indicated that the carbohydrate added was immaterial. PMID:13920002

The Microbial Diversity of Traditional Spontaneously Fermented Lambic Beer

PubMed Central

Spitaels, Freek; Wieme, Anneleen D.; Janssens, Maarten; Aerts, Maarten; Daniel, Heide-Marie; Van Landschoot, Anita; De Vuyst, Luc; Vandamme, Peter

2014-01-01

Lambic sour beers are the products of a spontaneous fermentation that lasts for one to three years before bottling. The present study determined the microbiota involved in the fermentation of lambic beers by sampling two fermentation batches during two years in the most traditional lambic brewery of Belgium, using culture-dependent and culture-independent methods. From 14 samples per fermentation, over 2000 bacterial and yeast isolates were obtained and identified. Although minor variations in the microbiota between casks and batches and a considerable species diversity were found, a characteristic microbial succession was identified. This succession started with a dominance of Enterobacteriaceae in the first month, which were replaced at 2 months by Pediococcus damnosus and Saccharomyces spp., the latter being replaced by Dekkera bruxellensis at 6 months fermentation duration. PMID:24748344

The microbial diversity of traditional spontaneously fermented lambic beer.

PubMed

Spitaels, Freek; Wieme, Anneleen D; Janssens, Maarten; Aerts, Maarten; Daniel, Heide-Marie; Van Landschoot, Anita; De Vuyst, Luc; Vandamme, Peter

2014-01-01

Lambic sour beers are the products of a spontaneous fermentation that lasts for one to three years before bottling. The present study determined the microbiota involved in the fermentation of lambic beers by sampling two fermentation batches during two years in the most traditional lambic brewery of Belgium, using culture-dependent and culture-independent methods. From 14 samples per fermentation, over 2000 bacterial and yeast isolates were obtained and identified. Although minor variations in the microbiota between casks and batches and a considerable species diversity were found, a characteristic microbial succession was identified. This succession started with a dominance of Enterobacteriaceae in the first month, which were replaced at 2 months by Pediococcus damnosus and Saccharomyces spp., the latter being replaced by Dekkera bruxellensis at 6 months fermentation duration.

Unconditioned commercial embryo culture media contain a large variety of non-declared proteins: a comprehensive proteomics analysis.

PubMed

Dyrlund, Thomas F; Kirkegaard, Kirstine; Poulsen, Ebbe Toftgaard; Sanggaard, Kristian W; Hindkjær, Johnny J; Kjems, Jørgen; Enghild, Jan J; Ingerslev, Hans Jakob

2014-11-01

Which non-declared proteins (proteins not listed on the composition list of the product data sheet) are present in unconditioned commercial embryo culture media? A total of 110 non-declared proteins were identified in unconditioned media and between 6 and 8 of these were quantifiable and therefore represent the majority of the total protein in the media samples. There are no data in the literature on what non-declared proteins are present in unconditioned (fresh media in which no embryos have been cultured) commercial embryo media. The following eight commercial embryo culture media were included in this study: G-1 PLUS and G-2 PLUS G5 Series from Vitrolife, Sydney IVF Cleavage Medium and Sydney IVF Blastocyst Medium from Cook Medical and EmbryoAssist, BlastAssist, Sequential Cleav and Sequential Blast from ORIGIO. Two batches were analyzed from each of the Sydney IVF media and one batch from each of the other media. All embryo culture media are supplemented by the manufacturers with purified human serum albumin (HSA 5 mg/ml). The purified HSA (HSA-solution from Vitrolife) and the recombinant human albumin supplement (G-MM from Vitrolife) were also analyzed. For protein quantification, media samples were in-solution digested with trypsin and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). For in-depth protein identification, media were albumin depleted, dialyzed and concentrated before sodium dodecyl sulfate polyacrylamide gel electrophoresis. The gel was cut into 14 slices followed by in-gel trypsin digestion, and analysis by LC-MS/MS. Proteins were further investigated using gene ontology (GO) terms analysis. Using advanced mass spectrometry and high confidence criteria for accepting proteins (P < 0.01), a total of 110 proteins other than HSA were identified. The average HSA content was found to be 94% (92-97%) of total protein. Other individual proteins accounted for up to 4.7% of the total protein. Analysis of purified HSA strongly suggests that these non-declared proteins are introduced to the media when the albumin is added. GO analysis showed that many of these proteins have roles in defence pathways, for example 18 were associated with the innate immune response and 17 with inflammatory responses. Eight proteins have been reported previously as secreted embryo proteins. For six of the commercial embryo culture media only one batch was analyzed. However, this does not affect the overall conclusions. The results showed that the HSA added to IVF media contained many other proteins and that the amount varies from batch to batch. These variations in protein profiles are problematic when attempting to identify proteins derived from the embryos. Therefore, when studying the embryo secretome and analyzing conditioned media with the aim of finding potential biomarkers that can distinguish normal and abnormal embryo development, it is important that the medium used in the experimental and control groups is from the same batch. Furthermore, the proteins present in unconditioned media could potentially influence embryonic development, gestation age, birthweight and perhaps have subsequent effects on health of the offspring. The study was supported by the Danish Agency for Science, Technology and Innovation. Research at the Fertility Clinic, Aarhus University Hospital is supported by an unrestricted grant from Merck Sharp & Dohme Corp and Ferring. The authors declare no conflicts of interest. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Biological control of fusarium seedling blight disease of wheat and barley.

PubMed

Khan, Mojibur R; Fischer, Sven; Egan, Damian; Doohan, Fiona M

2006-04-01

ABSTRACT Fusarium fungi, including F. culmorum, cause seedling blight, foot rot, and head blight diseases of cereals, resulting in yield loss. In a screen for potential disease control organisms and agents, Pseudomonas fluorescens strains MKB 100 and MKB 249, P. frederiksbergensis strain 202, Pseudomonas sp. strain MKB 158, and chitosan all significantly reduced the extent of both wheat coleoptile growth retardation and wheat and barley seedling blight caused by F. culmorum (by 53 to 91%). Trichodiene synthase is a Fusarium enzyme necessary for trichothecene mycotoxin biosynthesis; expression of the gene encoding this enzyme in wheat was 33% lower in stem base tissue coinoculated with Pseudomonas sp. strain MKB 158 and F. culmorum than in wheat treated with bacterial culture medium and F. culmorum. When wheat and barley were grown in soil amended with either chitosan, P. fluorescens strain MKB 249, Pseudomonas sp. strain MKB 158, or culture filtrates of these bacteria, the level of disease symptoms on F. culmorum-inoculated stem base tissue (at 12 days post- F. culmorum inoculation) was >/=31% less than the level on F. culmorum-inoculated plants grown in culture medium-amended soil. It seems likely that at least part of the biocontrol activity of these bacteria and chitosan may be due to the induction of systemic disease resistance in host plants. Also, in coinoculation studies, Pseudomonas sp. strain MKB 158 induced the expression of a wheat class III plant peroxidase gene (a pathogenesis-related gene).

Yeast Monitoring of Wine Mixed or Sequential Fermentations Made by Native Strains from D.O. “Vinos de Madrid” Using Real-Time Quantitative PCR

PubMed Central

García, Margarita; Esteve-Zarzoso, Braulio; Crespo, Julia; Cabellos, Juan M.; Arroyo, Teresa

2017-01-01

There is an increasing trend toward understanding the impact of non-Saccharomyces yeasts on the winemaking process. Although Saccharomyces cerevisiae is the predominant species at the end of fermentation, it has been recognized that the presence of non-Saccharomyces species during alcoholic fermentation can produce an improvement in the quality and complexity of the final wines. A previous work was developed for selecting the best combinations between S. cerevisiae and five non-Saccharomyces (Torulaspora delbrueckii, Schizosaccharomyces pombe, Candida stellata, Metschnikowia pulcherrima, and Lachancea thermotolorans) native yeast strains from D.O. “Vinos de Madrid” at the laboratory scale. The best inoculation strategies between S. cerevisiae and non-Saccharomyces strains were chosen to analyze, by real-time quantitative PCR (qPCR) combined with the use of specific primers, the dynamics of inoculated populations throughout the fermentation process at the pilot scale using the Malvar white grape variety. The efficiency of the qPCR system was verified independently of the samples matrix, founding the inoculated yeast species throughout alcoholic fermentation. Finally, we can validate the positive effect of selected co-cultures in the Malvar wine quality, highlighting the sequential cultures of T. delbrueckii CLI 918/S. cerevisiae CLI 889 and C. stellata CLI 920/S. cerevisiae CLI 889 and, mixed and sequential cultures of L. thermotolerans 9-6C combined with S. cerevisiae CLI 889. PMID:29326669

Comparison of Assays for Sensitive and Reproducible Detection of Cell Culture-Infectious Cryptosporidium parvum and Cryptosporidium hominis in Drinking Water

PubMed Central

Di Giovanni, George D.; Rochelle, Paul A.

2012-01-01

This study compared the three most commonly used assays for detecting Cryptosporidium sp. infections in cell culture: immunofluorescent antibody and microscopy assay (IFA), PCR targeting Cryptosporidium sp.-specific DNA, and reverse transcriptase PCR (RT-PCR) targeting Cryptosporidium sp.-specific mRNA. Monolayers of HCT-8 cells, grown in 8-well chamber slides or 96-well plates, were inoculated with a variety of viable and inactivated oocysts to assess assay performance. All assays detected infection with low doses of flow cytometry-enumerated Cryptosporidium parvum oocysts, including infection with one oocyst and three oocysts. All methods also detected infection with Cryptosporidium hominis. The RT-PCR assay, IFA, and PCR assay detected infection in 23%, 25%, and 51% of monolayers inoculated with three C. parvum oocysts and 10%, 9%, and 16% of monolayers inoculated with one oocyst, respectively. The PCR assay was the most sensitive, but it had the highest frequency of false positives with mock-infected cells and inactivated oocysts. IFA was the only infection detection assay that did not produce false positives with mock-infected monolayers. IFA was also the only assay that detected infections in all experiments with spiked oocysts recovered from Envirochek capsules following filtration of 1,000 liters of treated water. Consequently, cell culture with IFA detection is the most appropriate method for routine and sensitive detection of infectious Cryptosporidium parvum and Cryptosporidium hominis in drinking water. PMID:22038611

Biodegradation of diesel oil by a novel microbial consortium: comparison between co-inoculation with biosurfactant-producing strain and exogenously added biosurfactants.

PubMed

Mnif, Inès; Mnif, Sami; Sahnoun, Rihab; Maktouf, Sameh; Ayedi, Younes; Ellouze-Chaabouni, Semia; Ghribi, Dhouha

2015-10-01

Bioremediation, involving the use of microorganisms to detoxify or remove pollutants, is the most interesting strategy for hydrocarbon remediation. In this aim, four hydrocarbon-degrading bacteria were isolated from oil-contaminated soil in Tunisia. They were identified by the 16S rDNA sequence analysis, as Lysinibacillus bronitolerans RI18 (KF964487), Bacillus thuringiensis RI16 (KM111604), Bacillus weihenstephanensis RI12 (KM094930), and Acinetobacter radioresistens RI7 (KJ829530). Moreover, a lipopeptide biosurfactant produced by Bacillus subtilis SPB1, confirmed to increase diesel solubility, was tested to increase diesel biodegradation along with co-inoculation with two biosurfactant-producing strains. Culture studies revealed the enhancement of diesel biodegradation by the selected consortium with the addition of SPB1 lipopeptide and in the cases of co-inoculation by biosurfactant-producing strain. In fact, an improvement of about 38.42 and 49.65 % of diesel degradation was registered in the presence of 0.1 % lipopeptide biosurfactant and when culturing B. subtilis SPB1 strain with the isolated consortium, respectively. Furthermore, the best improvement, evaluated to about 55.4 %, was recorded when using the consortium cultured with B. subtilis SPB1 and A. radioresistens RI7 strains. Gas chromatography analyses were correlated with the gravimetric evaluation of the residual hydrocarbons. Results suggested the potential applicability of the selected consortium along with the ex situ- and in situ-added biosurfactant for the effective bioremediation of diesel-contaminated water and soil.

Evaluation of a kinetic model for computer simulation of growth and fermentation by Scheffersomyces (Pichia) stipitis fed D-xylose.

PubMed

Slininger, P J; Dien, B S; Lomont, J M; Bothast, R J; Ladisch, M R; Okos, M R

2014-08-01

Scheffersomyces (formerly Pichia) stipitis is a potential biocatalyst for converting lignocelluloses to ethanol because the yeast natively ferments xylose. An unstructured kinetic model based upon a system of linear differential equations has been formulated that describes growth and ethanol production as functions of ethanol, oxygen, and xylose concentrations for both growth and fermentation stages. The model was validated for various growth conditions including batch, cell recycle, batch with in situ ethanol removal and fed-batch. The model provides a summary of basic physiological yeast properties and is an important tool for simulating and optimizing various culture conditions and evaluating various bioreactor designs for ethanol production. © 2014 Wiley Periodicals, Inc.

[Assessment of two DNA extraction methods to amplify the pneumolysin gene (PLY) from blood culture samples of Streptococcus pneumoniae].

PubMed

Hernández, Carolina; Durán, Claudia; Ulloa, María Teresa; Prado, Valeria

2004-05-01

Streptococcus pneumoniae is a common etiologic agent of invasive respiratory infections among children under 5 years of age and older adults. Isolation rates of S. pneumoniae by traditional culture techniques are low. To study the sensitivity and specificity of two different DNA extraction methods to amplify the ply gene, applied to three different types of blood culture broths, experimentally inoculated with S. pneumoniae. DNA was extracted from the cultures using an organic method or a technique that consists in dilution, washing with NaOH and concentration of the sample. This was followed by PCR amplification of a 355 pb fragment of the pneumolysin gene (ply). The organic DNA extraction method inhibited the PCR reaction at all concentrations studied (0.6 to 10(6) colony forming units/mL). Using the NaOH extraction, ply gene amplification was positive in all three blood culture broths, but only at concentrations of 10(3) colony forming units/mL, or higher. Using the same DNA extraction method, PCR was negative when the broths were inoculated with seven other related bacterial species, which results in a 100% specificity. Detection of S. pneumoniae by amplification of ply gene from blood cultures using the protocol of NaOH for DNA extraction is specific and provides results in a short lapse. However, the diagnostic sensitivity is not optimal, which limits its clinical use.

Synthesis and high-throughput processing of polymeric hydrogels for 3D cell culture.

PubMed

Lowe, Stuart B; Tan, Vincent T G; Soeriyadi, Alexander H; Davis, Thomas P; Gooding, J Justin

2014-09-17

3D cell cultures have drawn a large amount of interest in the scientific community with their ability to closely mimic physiological conditions. Hydrogels have been used extensively in the development of extracellular matrix (ECM) mimics for 3D cell culture. Compounds such as collagen and fibrin are commonly used to synthesize natural ECM mimics; however they suffer from batch-to-batch variation. In this Review we explore the synthesis route of hydrogels; how they can be altered to give different chemical and physical properties; how different biomolecules such as arginylglycylaspartic acid (RGD) or vascular endothelial growth factor (VEGF) can be incorporated to give different biological cues; and how to create concentration gradients with UV light. There will also be emphasis on the types of techniques available in high-throughput processing such as nozzle and droplet-based biofabrication, photoenabled biofabrication, and microfluidics. The combination of these approaches and techniques allow the preparation of hydrogels which are capable of mimicking the ECM.

Pseudo-outbreak of Brevundimonas diminuta Attributed to Contamination of Culture Medium Supplement.

PubMed

Lee, Rachael A; Moser, Stephen A; Long, Martha; Butler, Susan L; Whiddon, Jennifer F; Camins, Bernard C

2017-05-01

We report an epidemiological investigation of a cluster of Brevundimonas diminuta isolates cultured from sterile sites. Inoculation of supplement medium yielded growth of B. diminuta. Molecular typing indicated likely contamination of the lot. No B. diminuta was further isolated after replacement of the supplement with a new lot number. Infect Control Hosp Epidemiol 2017;38:598-601.

Radiation sensitivity of poliovirus, a model for norovirus, inoculated in oyster ( Crassostrea gigas) and culture broth under different conditions

NASA Astrophysics Data System (ADS)

Jung, Pil-Mun; Park, Jae Seok; Park, Jin-Gyu; Park, Jae-Nam; Han, In-Jun; Song, Beom-Seok; Choi, Jong-il; Kim, Jae-Hun; Byun, Myung-Woo; Baek, Min; Chung, Young-Jin; Lee, Ju-Woon

2009-07-01

Poliovirus is a recognized surrogate for norovirus, pathogen in water and food, due to the structural and genetic similarity. Although radiation sensitivity of poliovirus in water or media had been reported, there has been no research in food model such as shellfish. In this study, oyster ( Crassostrea gigas) was incubated in artificial seawater contaminated with poliovirus, and thus radiation sensitivity of poliovirus was determined in inoculated oyster. The effects of ionizing radiation on the sensitivity of poliovirus were also evaluated under different conditions such as pH (4-7) and salt concentration (1-15%) in culture broth, and temperature during irradiation. The D10 value of poliovirus in PBS buffer, virus culture broth and oyster was determined to 0.46, 2.84 and 2.94 kGy, respectively. The initial plaque forming unit (PFU) of poliovirus in culture broth was slightly decreased as the decrease of pH and the increase of salt concentration, but radiation sensitivity was not affected by pH and salt contents. However, radiation resistance of poliovirus was increased at frozen state. These results provide the basic information for the inactivation of pathogenic virus in foods by using irradiation.

Performance of two potential probiotic Lactobacillus strains from the olive microbiota as starters in the fermentation of heat shocked green olives.

PubMed

Argyri, Anthoula A; Nisiotou, Aspasia A; Mallouchos, Athanasios; Panagou, Efstathios Z; Tassou, Chrysoula C

2014-02-03

The performance of two potential probiotic Lactobacillus strains from olive microbiota, namely L. pentosus B281 and L. plantarum B282 was assessed as starter cultures in Spanish-style fermentation of heat shocked green olives cv. Halkidiki. Two different initial salt levels were studied, 10% (w/v) and 8% (w/v) NaCl, and the brines were inoculated with (a) L. pentosus B281, (b) L. plantarum B282, and (c) a mixture of both strains. A spontaneous fermentation was also taken into account as control treatment. Prior to brining, olives were heat shocked at 80 °C for 10 min to reduce the level of the indigenous microbiota on olive drupes and facilitate the dominance of the inoculated cultures. Microbiological, physicochemical and sensory analyses were conducted throughout fermentation. The composition of LAB population and the evolution of added inocula were assessed by Pulsed Field Gel Electrophoresis (PFGE). The final population of LAB was maintained above 6 log cycles in olive flesh. Both L. pentosus B281 and L. plantarum B282 were able to dominate over indigenous LAB, albeit strain B281 exhibited higher recovery percentages (100 or 94.7% for B281 and 58.8% or 55.0% for B282 in 10% or 8% NaCl, respectively). L. pentosus B281 also dominated over L. plantarum B282, when the two strains were co-inoculated in olive fermentations. The sensory assessment showed higher preference for inoculated fermentations of L. pentosus and L. plantarum separately in 8% NaCl, followed by the L. plantarum in 10% NaCl. The present study showed that probiotic strains L. pentosus B281 and L. plantarum B282, may offer a great potential for use as functional starter cultures in olive fermentation and deliver a promising probiotic food to the consumer. Copyright © 2013 Elsevier B.V. All rights reserved.

Evaluation of a new generation of plastic culture bottles with an automated microbial detection system for nine common contaminating organisms found in PLT components.

PubMed

Brecher, M E; Hay, S N; Rothenberg, S J

2004-03-01

A microbial detection system (BacT/ALERT 3D, bioMérieux [formerly Organon Teknika]) has previously been validated with a variety of bacterial contaminants in PLTs. The recovery of nine organisms seeded into PLTs with new plastic culture bottles was studied in comparison to the current glass bottles. The use of plastic instead of glass would be expected to reduce the risk of injury. Isolates of Bacillus cereus, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Serratia marcescens, Streptococcus viridans, and Propionibacterium acnes were inoculated into Day 2 (>24 hr <48 hr) apheresis PLT units to 10 and 100 CFUs per mL. Replicate samples (4 mL) were inoculated into both current- and new-generation standard aerobic and anaerobic bottles. All organisms (with the exception of P. acnes) were detected in a mean time of 9.3 to 18.9 hours (10 CFUs/mL) or 8.7 to 18.2 hours (100 CFUs/mL). In aggregate (with the exception of P. acnes), the plastic and glass aerobic bottles had a mean difference in detection of 1.2 hours (p < 0.0001), and the plastic and glass anaerobic bottles had a mean difference of 3.3 hours (p < 0.0001). In all cases, the mean detection time was superior or clinically comparable (within 0.1 hr) with the new plastic bottles. P. acnes (an anaerobic organism) was detected with the new and current anaerobic bottles in a mean of 72.8 and 90.4 hours (10 CFUs/mL) or 64.0 and 80.8 hours (100 CFUs/mL), respectively. The narrower bottle neck and smaller inoculation septum present with the new-generation plastic bottles were inoculated with comparable ease to that of the glass bottles. These data demonstrate that the new plastic bottles are clinically comparable or superior to the current glass standard aerobic and anaerobic culture bottles.

The yield of different pleural fluid volumes for Mycobacterium tuberculosis culture.

PubMed

von Groote-Bidlingmaier, Florian; Koegelenberg, Coenraad Frederik; Bolliger, Chris T; Chung, Pui Khi; Rautenbach, Cornelia; Wasserman, Elizabeth; Bernasconi, Maurizio; Friedrich, Sven Olaf; Diacon, Andreas Henri

2013-03-01

We prospectively compared the culture yields of two pleural fluid volumes (5 and 100 ml) inoculated in liquid culture medium in 77 patients of whom 58 (75.3%) were diagnosed with pleural tuberculosis. The overall fluid culture yield was high (60.3% of cases with pleural tuberculosis). The larger volume had a faster time to positivity (329 vs 376 h, p=0.055) but its yield was not significantly higher (53.5% vs 50%; p=0.75). HIV-positive patients were more likely to have positive cultures (78.9% vs 51.5%; p=0.002).

Removal of arsenic from aqueous solutions using waste iron columns inoculated with iron bacteria.

PubMed

Azhdarpoor, Abooalfazl; Nikmanesh, Roya; Samaei, Mohammad Reza

2015-01-01

Arsenic contamination of water resources is one of the serious risks threatening natural ecosystems and human health. This study investigates arsenic removal using a waste iron column with and without iron bacteria in continuous and batch phases. In batch experiments, the effects of pH, contact time, initial concentration of arsenic and adsorbent dose were investigated. Results indicated that the highest arsenate removal efficiency occurred at pH 7 (96.76%). On increasing the amount of waste iron from 0.25 to 1 g, the removal rate changed from about 42.37%-96.70%. The results of continuous experiments on the column containing waste iron showed that as the empty bed contact time increased from 5 to 60 min, the secondary arsenate concentration changed from 23 to 6 µg/l. In experiments involving a waste iron column with iron bacteria, an increase in residence time from 5 to 60 min decreased the secondary arsenate concentration from 14.97 to 4.86 µg/l. The results of this study showed that waste iron containing iron bacteria is a good adsorbent for removal of arsenic from contaminated water.

Influence of feedstock-to-inoculum ratio on performance and microbial community succession during solid-state thermophilic anaerobic co-digestion of pig urine and rice straw.

PubMed

Meng, Lingyu; Xie, Li; Kinh, Co Thi; Suenaga, Toshikazu; Hori, Tomoyuki; Riya, Shohei; Terada, Akihiko; Hosomi, Masaaki

2018-03-01

This study investigated the effect of the feedstock-to-inoculum (F/I) ratio on performance of the solid-state anaerobic co-digestion of pig urine and rice straw inoculated with a solid digestate, and clarified the microbial community succession. A 44-day biochemical methane potential test at F/I ratios of 0.5, 1, 2 and 3 at 55 °C and a 35-day large-scale batch test at F/I ratios of 0.5 and 3 at 55 °C were conducted to investigate the effects of F/I ratio on anaerobic digestibility and analyze microbial community succession, respectively. The highest cumulative methane yield was 353.7 m 3 /t VS in the large-scale batch test. Volatile fatty acids did not accumulate at any F/I ratios. The volatile solids reduction rate was highest at a F/I ratio of 0.5. Microbial community structures were similar between F/I ratios of 3 and 0.5, despite differences in digestion performance, suggesting that stable operation can be achieved at these ratios. Copyright © 2017 Elsevier Ltd. All rights reserved.

A new recycling technique for the waste tires reuse.

PubMed

Derakhshan, Zahra; Ghaneian, Mohammad Taghi; Mahvi, Amir Hossein; Oliveri Conti, Gea; Faramarzian, Mohammad; Dehghani, Mansooreh; Ferrante, Margherita

2017-10-01

In this series of laboratory experiments, the feasibility of using fixed bed biofilm carriers (FBBC) manufactured from existing reclaimed waste tires (RWTs) for wastewater treatment was evaluated. To assess polyamide yarn waste tires as a media, the fixed bed sequence batch reactor (FBSBR) was evaluated under different organic loading rate (OLRs). An experimental model was used to study the kinetics of substrate consumption in biofilm. Removal efficiency of soluble chemical oxygen demand (SCOD) ranged by 76-98% for the FBSBR compared to 71-96% in a sequencing batch reactor (SBR). Removal efficiency of FBBC was significantly increased by inoculating these RWTs carriers. The results revealed that the sludge production yield (Y obs ) was significantly less in the FBSBR compared to the SBR (p < 0.01). It also produced less sludge and recorded a lower stabilization ratio (VSS/TSS). The findings show that the Stover-Kincannon model was the best fit (R 2 > 99%) in a FBSBR. Results from this study suggest that RWTs to support biological activity for a variety of wastewater treatment applications as a biofilm carrier have high potential that better performance as COD and TSS removal and sludge settling properties and effluent quality supported these findings. Copyright © 2017. Published by Elsevier Inc.

Non conventional biological treatment based on Trametes versicolor for the elimination of recalcitrant anticancer drugs in hospital wastewater.

PubMed

Ferrando-Climent, Laura; Cruz-Morató, Carles; Marco-Urrea, Ernest; Vicent, Teresa; Sarrà, Montserrat; Rodriguez-Mozaz, Sara; Barceló, Damià

2015-10-01

This work presents a study about the elimination of anticancer drugs, a group of pollutants considered recalcitrant during conventional activated sludge wastewater treatment, using a biological treatment based on the fungus Trametes versicolor. A 10-L fluidized bed bioreactor inoculated with this fungus was set up in order to evaluate the removal of 10 selected anticancer drugs in real hospital wastewater. Almost all the tested anticancer drugs were completely removed from the wastewater at the end of the batch experiment (8 days) with the exception of Ifosfamide and Tamoxifen. These two recalcitrant compounds, together with Cyclophosphamide, were selected for further studies to test their degradability by T. versicolor under optimal growth conditions. Cyclophosphamide and Ifosfamide were inalterable during batch experiments both at high and low concentration, whereas Tamoxifen exhibited a decrease in its concentration along the treatment. Two positional isomers of a hydroxylated form of Tamoxifen were identified during this experiment using a high resolution mass spectrometry based on ultra-high performance chromatography coupled to an Orbitrap detector (LTQ-Velos Orbitrap). Finally the identified transformation products of Tamoxifen were monitored in the bioreactor run with real hospital wastewater. Copyright © 2015 Elsevier Ltd. All rights reserved.

In vitro propagation of Stevia rebaudina plants using multiple shoot culture.

PubMed

Nepovím, A; Vanek, T

1998-12-01

A multiple shoot culture was induced from nodal segments on MS medium containing half concentration of macroelements, 1% sucrose, and supplemented with NAA (0.01 mg/l). A bioreactor with hormone-free MS medium (300 ml) was inoculated with 1.5 g of the multiple shoot culture and cultivated for a month. The cultivating process of the multiple shoot culture in the bioreactor and the transfer into ex vitro conditions took about 8-9 weeks and produced approx. 600 new seedlings, that could be transferred from greenhouse to field conditions.

CHANGES IN CHLOROPHYLL A FLUORENSCENCE AND PIGMENT RATIOS DURING DIFFERENT GROWTH PHASES OF A UNICELLULAR MARINE CHEATOSEROS (BACILLARIOPHYCEAE) IN BATCH CULTURE

EPA Science Inventory

Photosystem II reaction centers per cell decreased as the cultures began to decline. The degree of inactivation increased daily as the cell numbers continued to decrease. The concentration of chlorophyll a per cell and the ratio of the major accessory pigments to chlorophyll a (e...

Continuous xylose fermentation by Candida shehatae in a two-stage reactor

Treesearch

M. A. Alexander; T. W. Chapman; T. W. Jeffries

1988-01-01

Recent work has identified ethanol toxicity as a major factor preventing continuous production of ethanol at the concentrations obtainable in batch culture. In this paper we investigate the use of a continuous two-stage bioreactor design to circumvent toxic effects of ethanol. Biomass is produced via continuous culture in the first stage reactor in which ethanol...

Improved Diagnosis of Prosthetic Joint Infection by Culturing Periprosthetic Tissue Specimens in Blood Culture Bottles

PubMed Central

Peel, Trisha N.; Dylla, Brenda L.; Hughes, John G.; Lynch, David T.; Greenwood-Quaintance, Kerryl E.; Cheng, Allen C.; Mandrekar, Jayawant N.

2016-01-01

ABSTRACT Despite known low sensitivity, culture of periprosthetic tissue specimens on agars and in broths is routine. Culture of periprosthetic tissue samples in blood culture bottles (BCBs) is potentially more convenient, but it has been evaluated in a limited way and has not been widely adopted. The aim of this study was to compare the sensitivity and specificity of inoculation of periprosthetic tissue specimens into blood culture bottles with standard agar and thioglycolate broth culture, applying Bayesian latent class modeling (LCM) in addition to applying the Infectious Diseases Society of America (IDSA) criteria for prosthetic joint infection. This prospective cohort study was conducted over a 9-month period (August 2013 to April 2014) at the Mayo Clinic, Rochester, MN, and included all consecutive patients undergoing revision arthroplasty. Overall, 369 subjects were studied; 117 (32%) met IDSA criteria for prosthetic joint infection, and 82% had late chronic infection. Applying LCM, inoculation of tissues into BCBs was associated with a 47% improvement in sensitivity compared to the sensitivity of conventional agar and broth cultures (92.1 versus 62.6%, respectively); this magnitude of change was similar when IDSA criteria were applied (60.7 versus 44.4%, respectively; P = 0.003). The time to microorganism detection was shorter with BCBs than with standard media (P < 0.0001), with aerobic and anaerobic BCBs yielding positive results within a median of 21 and 23 h, respectively. Results of our study demonstrate that the semiautomated method of periprosthetic tissue culture in blood culture bottles is more sensitive than and as specific as agar and thioglycolate broth cultures and yields results faster. PMID:26733067

Alleviation of heavy metals toxicity by the application of plant growth promoting rhizobacteria and effects on wheat grown in saline sodic field.

PubMed

Hassan, Tamoor Ul; Bano, Asghari; Naz, Irum

2017-06-03

The aim of the study was to determine tolerance of plant growth promoting rhizobacteria (PGPR) in different concentrations of Cu, Cr, Co, Cd, Ni, Mn, and Pb and to evaluate the PGPR-modulated bioavailability of different heavy metals in the rhizosphere soil and wheat tissues, grown in saline sodic soil. Bacillus cereus and Pseudomonas moraviensis were isolated from Cenchrus ciliaris L. growing in the Khewra salt range. Seven-day-old cultures of PGPR were applied on wheat as single inoculum, co-inoculation and carrier-based biofertilizer (using maize straw and sugarcane husk as carrier). At 100 ppm of Cr and Cu, the survival rates of rhizobacteria were decreased by 40%. Single inoculation of PGPR decreased 50% of Co, Ni, Cr and Mn concentrations in the rhizosphere soil. Co-inoculation of PGPR and biofertilizer treatment further augmented the decreases by 15% in Co, Ni, Cr and Mn over single inoculation except Pb and Co where decreases were 40% and 77%, respectively. The maximum decrease in biological concentration factor (BCF) was observed for Cd, Co, Cr, and Mn. P. moraviensis inoculation decreases the biological accumulation coefficient (BAC) as well as translocation factor (TF) for Cd, Cr, Cu Mn, and Ni. The PGPR inoculation minimized the deleterious effects of heavy metals, and the addition of carriers further assisted the PGPR.

[Quantitative study of the antibacterial effect of cefotaxime and ceftriaxone during experimental Escherichia coli K1 bacteremia in chickens].

PubMed

Labarthe, J C; Guillot, J F; Mouline, C; Bree, A

1989-06-01

In order to assess the in vivo antibacterial activity of two cephalosporins of third generation, cefotaxime and ceftriaxone, we used the model of experimental bacteremia in chickens we had developed for a few years. 93 chickens were inoculated with 10(7) E. coli K1 coming from a meningitis in a new-born baby. 19 chickens were used as a control group; 29 were given ceftriaxone (50 mg/kg); 28 cefotaxime (50 mg/kg) and 17 cefotaxime (100 mg/kg). The antibiotics were injected 4 hours after the inoculation. The bacterial concentrations found in capillaries by using quantitative blood cultures, were significantly lower in the 3 groups of chickens which were given antibiotics than in the control group, at 24, 48 and 72 hours after inoculation. At 24 hours after the inoculation, the bacterial concentration in the chickens treated by ceftriaxone (50 mg/kg) was significantly lower than that found in chickens treated by cefotaxime (50 mg/kg). At 48 or 72 hours the differences of bacterial concentration in the three groups of chickens were not significant. Over 72 hours following inoculation, 4 control and only one treated chickens died. The efficient clearance of E. coli K1 by a single dose of ceftriaxone, found at 24 hours after inoculation, confirms the possibility of using ceftriaxone once daily for serious infections.

Early activation of wheat polyamine biosynthesis during Fusarium head blight implicates putrescine as an inducer of trichothecene mycotoxin production

PubMed Central

2010-01-01

Background The fungal pathogen Fusarium graminearum causes Fusarium Head Blight (FHB) disease on wheat which can lead to trichothecene mycotoxin (e.g. deoxynivalenol, DON) contamination of grain, harmful to mammalian health. DON is produced at low levels under standard culture conditions when compared to plant infection but specific polyamines (e.g. putrescine and agmatine) and amino acids (e.g. arginine and ornithine) are potent inducers of DON by F. graminearum in axenic culture. Currently, host factors that promote mycotoxin synthesis during FHB are unknown, but plant derived polyamines could contribute to DON induction in infected heads. However, the temporal and spatial accumulation of polyamines and amino acids in relation to that of DON has not been studied. Results Following inoculation of susceptible wheat heads by F. graminearum, DON accumulation was detected at two days after inoculation. The accumulation of putrescine was detected as early as one day following inoculation while arginine and cadaverine were also produced at three and four days post-inoculation. Transcripts of ornithine decarboxylase (ODC) and arginine decarboxylase (ADC), two key biosynthetic enzymes for putrescine biosynthesis, were also strongly induced in heads at two days after inoculation. These results indicated that elicitation of the polyamine biosynthetic pathway is an early response to FHB. Transcripts for genes encoding enzymes acting upstream in the polyamine biosynthetic pathway as well as those of ODC and ADC, and putrescine levels were also induced in the rachis, a flower organ supporting DON production and an important route for pathogen colonisation during FHB. A survey of 24 wheat genotypes with varying responses to FHB showed putrescine induction is a general response to inoculation and no correlation was observed between the accumulation of putrescine and infection or DON accumulation. Conclusions The activation of the polyamine biosynthetic pathway and putrescine in infected heads prior to detectable DON accumulation is consistent with a model where the pathogen exploits the generic host stress response of polyamine synthesis as a cue for production of trichothecene mycotoxins during FHB disease. However, it is likely that this mechanism is complicated by other factors contributing to resistance and susceptibility in diverse wheat genetic backgrounds. PMID:21192794

Influences of incubation temperature and various saccharides on the production of organic acids and gases by gut microbes of rainbow trout Oncorhynchus mykiss in a micro-scale batch culture.

PubMed

Kihara, M; Sakata, T

2001-08-01

We studied the influence of incubation temperature and additional saccharides on the metabolism of hindgut microbes of the rainbow trout Oncorhynchus mykiss in a 50 microl-scale batch culture system. Intestinal contents of rainbow trout reared at 15 degrees C were incubated with glucose, lactosucrose, sodium alginate or colloidal chitin (each 10 g/l) at 15 degrees C or 25 degrees C for 12 h. Levels of organic acids at 0 h and 12 h of incubation were quantified with HPLC. We also monitored gas release from these cultures during incubation. The main product was iso-butyric acid, except for the cultures with colloidal chitin where no net production of organic acids was observed. We detected higher levels of iso-butyric acid in cultures with lactosucrose than in the other cultures. Net production of this acid was less in cultures with colloidal chitin than in blank cultures. The volume of released gas was larger when incubated at 25 degrees C than at 15 degrees C. Cultures with colloidal chitin released more gas than blank cultures when they were incubated at 15 degrees C. Cultures with sodium alginate released less gas than blank cultures irrespective of incubation temperature. These results indicate that the hindgut microbes of this carnivorous fish mainly produce branched-chain fatty acids, very likely by microbial digestion of nitrogenous materials rather than saccharides. However, additional saccharides affected production of branched-chain fatty acids. The influence of incubation temperature in the present study also suggested that the environmental temperature of host fish should affect microbial digestion in the fish gut.

Orally Administered Bioadherent Sustained Release Microencapsulated Vaccines

DTIC Science & Technology

1996-09-01

ml flask culture (MMY media) was inoculated with a recombinant Pichia yeast clone containing the vpB gene integrated at the alcohol oxidase (AOX...measured. 6 DAMD17-95-C-5099 2. Materials and Methods 2.1. Materials vpB is expressed in Pichia yeast and isolated and purified as described below. SEA...locus in the Pichia genome. The culture was grown at 30"C for approximately 48 hours until an optical density ( OD 650) of 3.0 was reached. The culture

Factorial experimental design for the culture of human embryonic stem cells as aggregates in stirred suspension bioreactors reveals the potential for interaction effects between bioprocess parameters.

PubMed

Hunt, Megan M; Meng, Guoliang; Rancourt, Derrick E; Gates, Ian D; Kallos, Michael S

2014-01-01

Traditional optimization of culture parameters for the large-scale culture of human embryonic stem cells (ESCs) as aggregates is carried out in a stepwise manner whereby the effect of varying each culture parameter is investigated individually. However, as evidenced by the wide range of published protocols and culture performance indicators (growth rates, pluripotency marker expression, etc.), there is a lack of systematic investigation into the true effect of varying culture parameters especially with respect to potential interactions between culture variables. Here we describe the design and execution of a two-parameter, three-level (3(2)) factorial experiment resulting in nine conditions that were run in duplicate 125-mL stirred suspension bioreactors. The two parameters investigated here were inoculation density and agitation rate, which are easily controlled, but currently, poorly characterized. Cell readouts analyzed included fold expansion, maximum density, and exponential growth rate. Our results reveal that the choice of best case culture parameters was dependent on which cell property was chosen as the primary output variable. Subsequent statistical analyses via two-way analysis of variance indicated significant interaction effects between inoculation density and agitation rate specifically in the case of exponential growth rates. Results indicate that stepwise optimization has the potential to miss out on the true optimal case. In addition, choosing an optimum condition for a culture output of interest from the factorial design yielded similar results when repeated with the same cell line indicating reproducibility. We finally validated that human ESCs remain pluripotent in suspension culture as aggregates under our optimal conditions and maintain their differentiation capabilities as well as a stable karyotype and strong expression levels of specific human ESC markers over several passages in suspension bioreactors.

Recombinant production of biologically active giant grouper (Epinephelus lanceolatus) growth hormone from inclusion bodies of Escherichia coli by fed-batch culture.

PubMed

Chung, Wen-Jen; Huang, Chi-Lung; Gong, Hong-Yi; Ou, Tsung-Yin; Hsu, Jue-Liang; Hu, Shao-Yang

2015-06-01

Growth hormone (GH) performs important roles in regulating somatic growth, reproduction, osmoregulation, metabolism and immunity in teleosts, and thus, it has attracted substantial attention in the field of aquaculture application. Herein, giant grouper GH (ggGH) cDNA was cloned into the pET28a vector and expressed in Shuffle® T7 Competent Escherichia coli. Recombinant N-terminal 6× His-tagged ggGH was produced mainly in insoluble inclusion bodies; the recombinant ggGH content reached 20% of total protein. For large-scale ggGH production, high-cell density E. coli culture was achieved via fed-batch culture with pH-stat. After 30h of cultivation, a cell concentration of 41.1g/l dry cell weight with over 95% plasmid stability was reached. Maximal ggGH production (4.0g/l; 22% total protein) was achieved via mid-log phase induction. Various centrifugal forces, buffer pHs and urea concentrations were optimized for isolation and solubilization of ggGH from inclusion bodies. Hydrophobic interactions and ionic interactions were the major forces in ggGH inclusion body formation. Complete ggGH inclusion body solubilization was obtained in PBS buffer at pH 12 containing 3M urea. Through a simple purification process including Ni-NTA affinity chromatography and refolding, 5.7mg of ggGH was obtained from 10ml of fed-batch culture (45% recovery). The sequence and secondary structure of the purified ggGH were confirmed by LC-MS/MS mass spectrometry and circular dichroism analysis. The cell proliferation-promoting activity was confirmed in HepG2, ZFL and GF-1 cells with the WST-1 colorimetric bioassay. Copyright © 2015 Elsevier Inc. All rights reserved.

Optimization of the yield of dark microaerobic production of hydrogen from lactate by Rhodopseudomonas palustris.

PubMed

Lazaro, Carolina Zampol; Hitit, Zeynep Yilmazer; Hallenbeck, Patrick C

2017-12-01

Hydrogen yields of dark fermentation are limited due to the need to also produce reduced side products, and photofermentation, an alternative, is limited by the need for light. A relatively new strategy, dark microaerobic fermentation, could potentially overcome both these constraints. Here, application of this strategy demonstrated for the first time significant hydrogen production from lactate by a single organism in the dark. Response surface methodology (RSM) was used to optimize substrate and oxygen concentration as well as inoculum using both (1) regular batch and (2) O 2 fed batch cultures. The highest hydrogen yield (HY) was observed under regular batch (1.4±0.1molH 2 /mollactate) and the highest hydrogen production (HP) (173.5µmolH 2 ) was achieved using O 2 fed batch. This study has provided proof of principal for the ability of microaerobic fermentation to drive thermodynamically difficult reactions, such as the conversion of lactate to hydrogen. Copyright © 2017 Elsevier Ltd. All rights reserved.

Production of infectious ferret hepatitis E virus in a human hepatocarcinoma cell line PLC/PRF/5.

PubMed

Li, Tian-Cheng; Yoshizaki, Sayaka; Yang, Tingting; Kataoka, Michiyo; Nakamura, Tomofumi; Ami, Yasushi; Yuriko, Suzaki; Takeda, Naokazu; Wakita, Takaji

2016-02-02

A strain of ferret hepatitis E virus (HEV), sF4370, isolated from an imported ferret was used to inoculate a human hepatocarcinoma cell line, PLC/PRF/5. The virus genome and capsid protein were detected in the cell culture supernatant. Immunofluorescence microscopy indicated that the capsid protein was located in the cytoplasm. The virus particles were purified from the culture supernatant by sucrose gradient ultracentrifugation. The capsid protein with molecular mass of ∼72 kDa was detected in fractions with density of 1.150-1.162 g/cm(3), and particles of ferret HEV was associated with cell membrane. The virus recovered from the supernatant was serially passaged with PLC/PRF/5 cells and had the ability to infect ferrets by oral inoculation, indicating that the ferret HEV grown in PLC/PRF/5 was infectious. The establishment of ferret HEV cell culture system might be useful to understand the life cycle, mechanism of infection and replication of ferret HEV. Copyright © 2015 Elsevier B.V. All rights reserved.

Zinc and glycerol enhance the production of nematicidal compounds in vitro and improve the biocontrol of Meloidogyne javanica in tomato by fluorescent pseudomonads.

PubMed

Siddiqui, I A; Shaukat, S S

2002-01-01

To assess the effects of various carbon and mineral sources on the nematicidal potential of biocontrol inoculants of Pseudomonas aeruginosa IE-6S+ and Ps. fluorescens CHA0 under laboratory and glasshouse conditions. Culture filtrates of strains IE-6S+ and CHA0, cultured in nutrient yeast extract broth, caused substantial mortality of the juveniles of Meloidogyne javanica. The nematicidal activities of the culture filtrates were altered after amendment with various carbon and mineral sources. Soil amendment with zinc alone or in combination with glycerol improved the biocontrol efficacy against root-knot nematode, promoted tomato plant growth and enhanced bacterial rhizosphere and endophytic colonization. Appropriate quantities of glycerol and zinc alone or in combination enhance the nematicidal activity of Ps. aeruginosa and Ps. fluorescens. Glucose reduces the activity of these bacteria against nematodes. Minerals and carbon sources are appealing because they are easy and economical to provide during liquid fermentation of inoculants or as fertilizer amendments to improve the biocontrol activity of indigenous and introduced bacteria.