An automated workflow for enhancing microbial bioprocess optimization on a novel microbioreactor platform

PubMed Central

2012-01-01

Background High-throughput methods are widely-used for strain screening effectively resulting in binary information regarding high or low productivity. Nevertheless achieving quantitative and scalable parameters for fast bioprocess development is much more challenging, especially for heterologous protein production. Here, the nature of the foreign protein makes it impossible to predict the, e.g. best expression construct, secretion signal peptide, inductor concentration, induction time, temperature and substrate feed rate in fed-batch operation to name only a few. Therefore, a high number of systematic experiments are necessary to elucidate the best conditions for heterologous expression of each new protein of interest. Results To increase the throughput in bioprocess development, we used a microtiter plate based cultivation system (Biolector) which was fully integrated into a liquid-handling platform enclosed in laminar airflow housing. This automated cultivation platform was used for optimization of the secretory production of a cutinase from Fusarium solani pisi with Corynebacterium glutamicum. The online monitoring of biomass, dissolved oxygen and pH in each of the microtiter plate wells enables to trigger sampling or dosing events with the pipetting robot used for a reliable selection of best performing cutinase producers. In addition to this, further automated methods like media optimization and induction profiling were developed and validated. All biological and bioprocess parameters were exclusively optimized at microtiter plate scale and showed perfect scalable results to 1 L and 20 L stirred tank bioreactor scale. Conclusions The optimization of heterologous protein expression in microbial systems currently requires extensive testing of biological and bioprocess engineering parameters. This can be efficiently boosted by using a microtiter plate cultivation setup embedded into a liquid-handling system, providing more throughput by parallelization and automation. Due to improved statistics by replicate cultivations, automated downstream analysis, and scalable process information, this setup has superior performance compared to standard microtiter plate cultivation. PMID:23113930

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.

Scale-up bioprocess development for production of the antibiotic valinomycin in Escherichia coli based on consistent fed-batch cultivations.

PubMed

Li, Jian; Jaitzig, Jennifer; Lu, Ping; Süssmuth, Roderich D; Neubauer, Peter

2015-06-12

Heterologous production of natural products in Escherichia coli has emerged as an attractive strategy to obtain molecules of interest. Although technically feasible most of them are still constrained to laboratory scale production. Therefore, it is necessary to develop reasonable scale-up strategies for bioprocesses aiming at the overproduction of targeted natural products under industrial scale conditions. To this end, we used the production of the antibiotic valinomycin in E. coli as a model system for scalable bioprocess development based on consistent fed-batch cultivations. In this work, the glucose limited fed-batch strategy based on pure mineral salt medium was used throughout all scales for valinomycin production. The optimal glucose feed rate was initially detected by the use of a biocatalytically controlled glucose release (EnBase® technology) in parallel cultivations in 24-well plates with continuous monitoring of pH and dissolved oxygen. These results were confirmed in shake flasks, where the accumulation of valinomycin was highest when the specific growth rate decreased below 0.1 h(-1). This correlation was also observed for high cell density fed-batch cultivations in a lab-scale bioreactor. The bioreactor fermentation produced valinomycin with titers of more than 2 mg L(-1) based on the feeding of a concentrated glucose solution. Valinomycin production was not affected by oscillating conditions (i.e. glucose and oxygen) in a scale-down two-compartment reactor, which could mimic similar situations in industrial bioreactors, suggesting that the process is very robust and a scaling of the process to a larger industrial scale appears a realistic scenario. Valinomycin production was scaled up from mL volumes to 10 L with consistent use of the fed-batch technology. This work presents a robust and reliable approach for scalable bioprocess development and represents an example for the consistent development of a process for a heterologously expressed natural product towards the industrial scale.

Fluorescence Spectroscopy and Chemometric Modeling for Bioprocess Monitoring

PubMed Central

Faassen, Saskia M.; Hitzmann, Bernd

2015-01-01

On-line sensors for the detection of crucial process parameters are desirable for the monitoring, control and automation of processes in the biotechnology, food and pharma industry. Fluorescence spectroscopy as a highly developed and non-invasive technique that enables the on-line measurements of substrate and product concentrations or the identification of characteristic process states. During a cultivation process significant changes occur in the fluorescence spectra. By means of chemometric modeling, prediction models can be calculated and applied for process supervision and control to provide increased quality and the productivity of bioprocesses. A range of applications for different microorganisms and analytes has been proposed during the last years. This contribution provides an overview of different analysis methods for the measured fluorescence spectra and the model-building chemometric methods used for various microbial cultivations. Most of these processes are observed using the BioView® Sensor, thanks to its robustness and insensitivity to adverse process conditions. Beyond that, the PLS-method is the most frequently used chemometric method for the calculation of process models and prediction of process variables. PMID:25942644

Incorporating unnatural amino acids to engineer biocatalysts for industrial bioprocess applications.

PubMed

Ravikumar, Yuvaraj; Nadarajan, Saravanan Prabhu; Hyeon Yoo, Tae; Lee, Chong-Soon; Yun, Hyungdon

2015-12-01

The bioprocess engineering with biocatalysts broadly spans its development and actual application of enzymes in an industrial context. Recently, both the use of bioprocess engineering and the development and employment of enzyme engineering techniques have been increasing rapidly. Importantly, engineering techniques that incorporate unnatural amino acids (UAAs) in vivo has begun to produce enzymes with greater stability and altered catalytic properties. Despite the growth of this technique, its potential value in bioprocess applications remains to be fully exploited. In this review, we explore the methodologies involved in UAA incorporation as well as ways to synthesize these UAAs. In addition, we summarize recent efforts to increase the yield of UAA engineered proteins in Escherichia coli and also the application of this tool in enzyme engineering. Furthermore, this protein engineering tool based on the incorporation of UAA can be used to develop immobilized enzymes that are ideal for bioprocess applications. Considering the potential of this tool and by exploiting these engineered enzymes, we expect the field of bioprocess engineering to open up new opportunities for biocatalysis in the near future. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Teaching Bioprocess Engineering to Undergraduates: Multidisciplinary Hands-On Training in a One-Week Practical Course

ERIC Educational Resources Information Center

Henkel, Marius; Zwick, Michaela; Beuker, Janina; Willenbacher, Judit; Baumann, Sandra; Oswald, Florian; Neumann, Anke; Siemann-Herzberg, Martin; Syldatk, Christoph; Hausmann, Rudolf

2015-01-01

Bioprocess engineering is a highly interdisciplinary field of study which is strongly benefited by practical courses where students can actively experience the interconnection between biology, engineering, and physical sciences. This work describes a lab course developed for 2nd year undergraduate students of bioprocess engineering and related…

Electrochemical Detection of Multiple Bioprocess Analytes

NASA Technical Reports Server (NTRS)

Rauh, R. David

2010-01-01

An apparatus that includes highly miniaturized thin-film electrochemical sensor array has been demonstrated as a prototype of instruments for simultaneous detection of multiple substances of interest (analytes) and measurement of acidity or alkalinity in bioprocess streams. Measurements of pH and of concentrations of nutrients and wastes in cell-culture media, made by use of these instruments, are to be used as feedback for optimizing the growth of cells or the production of desired substances by the cultured cells. The apparatus is designed to utilize samples of minimal volume so as to minimize any perturbation of monitored processes. The apparatus can function in a potentiometric mode (for measuring pH), an amperometric mode (detecting analytes via oxidation/reduction reactions), or both. The sensor array is planar and includes multiple thin-film microelectrodes covered with hydrous iridium oxide. The oxide layer on each electrode serves as both a protective and electrochemical transducing layer. In its transducing role, the oxide provides electrical conductivity for amperometric measurement or pH response for potentiometric measurement. The oxide on an electrode can also serve as a matrix for one or more enzymes that render the electrode sensitive to a specific analyte. In addition to transducing electrodes, the array includes electrodes for potential control. The array can be fabricated by techniques familiar to the microelectronics industry. The sensor array is housed in a thin-film liquid-flow cell that has a total volume of about 100 mL. The flow cell is connected to a computer-controlled subsystem that periodically draws samples from the bioprocess stream to be monitored. Before entering the cell, each 100-mL sample is subjected to tangential-flow filtration to remove particles. In the present version of the apparatus, the electrodes are operated under control by a potentiostat and are used to simultaneously measure the pH and the concentration of glucose. It is anticipated that development of procedures for trapping more enzymes into hydrous iridium oxide (and possibly into other electroactive metal oxides) and of means for imparting long-term stability to the transducer layers should make it possible to monitor concentrations of products of many enzyme reactions for example, such key bioprocess analytes as amino acids, vitamins, lactose, and acetate.

Microscale bioprocess optimisation.

PubMed

Micheletti, Martina; Lye, Gary J

2006-12-01

Microscale processing techniques offer the potential to speed up the delivery of new drugs to the market, reducing development costs and increasing patient benefit. These techniques have application across both the chemical and biopharmaceutical sectors. The approach involves the study of individual bioprocess operations at the microlitre scale using either microwell or microfluidic formats. In both cases the aim is to generate quantitative bioprocess information early on, so as to inform bioprocess design and speed translation to the manufacturing scale. Automation can enhance experimental throughput and will facilitate the parallel evaluation of competing biocatalyst and process options.

Multisensor Instrument for Real-Time Biological Monitoring

NASA Technical Reports Server (NTRS)

Zhang, Sean (Zhanxiang); Xu, Guoda; Qiu, Wei; Lin, Freddie

2004-01-01

The figure schematically depicts an instrumentation system, called a fiber optic-based integration system (FOBIS), that is undergoing development to enable real-time monitoring of fluid cell cultures, bioprocess flows, and the like. The FOBIS design combines a micro flow cytometer (MFC), a microphotometer (MP), and a fluorescence-spectrum- or binding-force-measuring micro-sensor (MS) in a single instrument that is capable of measuring multiple biological parameters simultaneously or sequentially. The fiber-optic-based integration system is so named because the MFC, the MP, and the MS are integrated into a single optical system that is coupled to light sources and photometric equipment via optical fibers. The optical coupling components also include a wavelength-division multiplexer and diffractive optical elements. The FOBIS includes a laserdiode- and fiber-optic-based optical trapping subsystem (optical tweezers ) with microphotometric and micro-sensing capabilities for noninvasive confinement and optical measurement of relevant parameters of a single cell or other particle. Some of the measurement techniques implemented together by the FOBIS have long been used separately to obtain basic understanding of the optical properties of individual cells and other organisms, the optical properties of populations of organisms, and the interrelationships among these properties, physiology of the organisms, and physical processes that govern the media that surround the organisms. For example, flow cytometry yields information on numerical concentrations, cross-sectional areas, and types of cells or other particles. Micro-sensing can be used to measure pH and concentrations of oxygen, carbon dioxide, glucose, metabolites, calcium, and antigens in a cell-culture fluid, thereby providing feedback that can be helpful in improving control over a bioprocess. Microphotometry (including measurements of scattering and fluorescence) can yield further information about optically trapped individual particles. In addition to the multifunctionality not previously available in a single biological monitoring system, the FOBIS offers advantages of low mass, sensitivity, accuracy, portability, low cost, compactness (the overall dimensions of the fully developed FOBIS sensor head are expected to be less than 1 by 1 by 2 cm), and immunity to electromagnetic interference at suboptical frequencies. FOBIS could be useful in a variety of laboratory and field settings in such diverse endeavors as medical, veterinary, and general biological research; medical and veterinary diagnosis monitoring of industrial bioprocesses; and analysis of biological contaminants in air, water, and food.

Bioprocessing automation in cell therapy manufacturing: Outcomes of special interest group automation workshop.

PubMed

Ball, Oliver; Robinson, Sarah; Bure, Kim; Brindley, David A; Mccall, David

2018-04-01

Phacilitate held a Special Interest Group workshop event in Edinburgh, UK, in May 2017. The event brought together leading stakeholders in the cell therapy bioprocessing field to identify present and future challenges and propose potential solutions to automation in cell therapy bioprocessing. Here, we review and summarize discussions from the event. Deep biological understanding of a product, its mechanism of action and indication pathogenesis underpin many factors relating to bioprocessing and automation. To fully exploit the opportunities of bioprocess automation, therapeutics developers must closely consider whether an automation strategy is applicable, how to design an 'automatable' bioprocess and how to implement process modifications with minimal disruption. Major decisions around bioprocess automation strategy should involve all relevant stakeholders; communication between technical and business strategy decision-makers is of particular importance. Developers should leverage automation to implement in-process testing, in turn applicable to process optimization, quality assurance (QA)/ quality control (QC), batch failure control, adaptive manufacturing and regulatory demands, but a lack of precedent and technical opportunities can complicate such efforts. Sparse standardization across product characterization, hardware components and software platforms is perceived to complicate efforts to implement automation. The use of advanced algorithmic approaches such as machine learning may have application to bioprocess and supply chain optimization. Automation can substantially de-risk the wider supply chain, including tracking and traceability, cryopreservation and thawing and logistics. The regulatory implications of automation are currently unclear because few hardware options exist and novel solutions require case-by-case validation, but automation can present attractive regulatory incentives. Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Intelligent control of mixed-culture bioprocesses

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

Stoner, D.L.; Larsen, E.D.; Miller, K.S.

A hierarchical control system is being developed and applied to a mixed culture bioprocess in a continuous stirred tank reactor. A bioreactor, with its inherent complexity and non-linear behavior was an interesting, yet, difficult application for control theory. The bottom level of the hierarchy was implemented as a number of integrated set point controls and data acquisition modules. Within the second level was a diagnostic system that used expert knowledge to determine the operational status of the sensors, actuators, and control modules. A diagnostic program was successfully implemented for the detection of stirrer malfunctions, and to monitor liquid delivery ratesmore » and recalibrate the pumps when deviations from desired flow rates occurred. The highest control level was a supervisory shell that was developed using expert knowledge and the history of the reactor operation to determine the set points required to meet a set of production criteria. At this stage the supervisory shell analyzed the data to determine the state of the system. In future implementations, this shell will determine the set points required to optimize a cost function using expert knowledge and adaptive learning techniques.« less

Generic Raman-based calibration models enabling real-time monitoring of cell culture bioreactors.

PubMed

Mehdizadeh, Hamidreza; Lauri, David; Karry, Krizia M; Moshgbar, Mojgan; Procopio-Melino, Renee; Drapeau, Denis

2015-01-01

Raman-based multivariate calibration models have been developed for real-time in situ monitoring of multiple process parameters within cell culture bioreactors. Developed models are generic, in the sense that they are applicable to various products, media, and cell lines based on Chinese Hamster Ovarian (CHO) host cells, and are scalable to large pilot and manufacturing scales. Several batches using different CHO-based cell lines and corresponding proprietary media and process conditions have been used to generate calibration datasets, and models have been validated using independent datasets from separate batch runs. All models have been validated to be generic and capable of predicting process parameters with acceptable accuracy. The developed models allow monitoring multiple key bioprocess metabolic variables, and hence can be utilized as an important enabling tool for Quality by Design approaches which are strongly supported by the U.S. Food and Drug Administration. © 2015 American Institute of Chemical Engineers.

Quantitative Analysis, Design, and Fabrication of Biosensing and Bioprocessing Devices in Living Cells

DTIC Science & Technology

2015-03-10

AFRL-OSR-VA-TR-2015-0080 Biosensing and Bioprocessing Devices in Living Cells Domitilla Del Vecchio MASSACHUSETTS INSTITUTE OF TECHNOLOGY Final...Of Biosensing And Bioprocessing Devices In Living Cells FA9550-12-1-0129 D. Del Vecchio Massachusetts Institute of Technology -- 77 Massachusetts...research is to develop quantitative techniques for the de novo design and fabrication of biosensing devices in living cells . Such devices will be entirely

From Process Development to Manufacturing: Lab-Intensive Courses in Downstream Bioprocessing

ERIC Educational Resources Information Center

Gilleskie, Gary L.; Reeves, Baley A.

2014-01-01

Most chemical engineering graduates work in industry, a fact that underscores the need for courses to provide experiences that prepare them for industry. The Biomanufacturing Training and Education Center (BTEC) at North Carolina State University has addressed this need by developing and delivering a comprehensive downstream bioprocessing program…

Mining manufacturing data for discovery of high productivity process characteristics.

PubMed

Charaniya, Salim; Le, Huong; Rangwala, Huzefa; Mills, Keri; Johnson, Kevin; Karypis, George; Hu, Wei-Shou

2010-06-01

Modern manufacturing facilities for bioproducts are highly automated with advanced process monitoring and data archiving systems. The time dynamics of hundreds of process parameters and outcome variables over a large number of production runs are archived in the data warehouse. This vast amount of data is a vital resource to comprehend the complex characteristics of bioprocesses and enhance production robustness. Cell culture process data from 108 'trains' comprising production as well as inoculum bioreactors from Genentech's manufacturing facility were investigated. Each run constitutes over one-hundred on-line and off-line temporal parameters. A kernel-based approach combined with a maximum margin-based support vector regression algorithm was used to integrate all the process parameters and develop predictive models for a key cell culture performance parameter. The model was also used to identify and rank process parameters according to their relevance in predicting process outcome. Evaluation of cell culture stage-specific models indicates that production performance can be reliably predicted days prior to harvest. Strong associations between several temporal parameters at various manufacturing stages and final process outcome were uncovered. This model-based data mining represents an important step forward in establishing a process data-driven knowledge discovery in bioprocesses. Implementation of this methodology on the manufacturing floor can facilitate a real-time decision making process and thereby improve the robustness of large scale bioprocesses. 2010 Elsevier B.V. All rights reserved.

Implementation and use of cloud-based electronic lab notebook in a bioprocess engineering teaching laboratory.

PubMed

Riley, Erin M; Hattaway, Holly Z; Felse, P Arthur

2017-01-01

Electronic lab notebooks (ELNs) are better equipped than paper lab notebooks (PLNs) to handle present-day life science and engineering experiments that generate large data sets and require high levels of data integrity. But limited training and a lack of workforce with ELN knowledge have restricted the use of ELN in academic and industry research laboratories which still rely on cumbersome PLNs for recordkeeping. We used LabArchives, a cloud-based ELN in our bioprocess engineering lab course to train students in electronic record keeping, good documentation practices (GDPs), and data integrity. Implementation of ELN in the bioprocess engineering lab course, an analysis of user experiences, and our development actions to improve ELN training are presented here. ELN improved pedagogy and learning outcomes of the lab course through stream lined workflow, quick data recording and archiving, and enhanced data sharing and collaboration. It also enabled superior data integrity, simplified information exchange, and allowed real-time and remote monitoring of experiments. Several attributes related to positive user experiences of ELN improved between the two subsequent years in which ELN was offered. Student responses also indicate that ELN is better than PLN for compliance. We demonstrated that ELN can be successfully implemented in a lab course with significant benefits to pedagogy, GDP training, and data integrity. The methods and processes presented here for ELN implementation can be adapted to many types of laboratory experiments.

Bioprocessing of bio-based chemicals produced from lignocellulosic feedstocks.

PubMed

Kawaguchi, Hideo; Hasunuma, Tomohisa; Ogino, Chiaki; Kondo, Akihiko

2016-12-01

The feedstocks used for the production of bio-based chemicals have recently expanded from edible sugars to inedible and more recalcitrant forms of lignocellulosic biomass. To produce bio-based chemicals from renewable polysaccharides, several bioprocessing approaches have been developed and include separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), and consolidated bioprocessing (CBP). In the last decade, SHF, SSF, and CBP have been used to generate macromolecules and aliphatic and aromatic compounds that are capable of serving as sustainable, drop-in substitutes for petroleum-based chemicals. The present review focuses on recent progress in the bioprocessing of microbially produced chemicals from renewable feedstocks, including starch and lignocellulosic biomass. In particular, the technological feasibility of bio-based chemical production is discussed in terms of the feedstocks and different bioprocessing approaches, including the consolidation of enzyme production, enzymatic hydrolysis of biomass, and fermentation. Copyright © 2016. Published by Elsevier Ltd.

Biological processing in oscillatory baffled reactors: operation, advantages and potential

PubMed Central

Abbott, M. S. R.; Harvey, A. P.; Perez, G. Valente; Theodorou, M. K.

2013-01-01

The development of efficient and commercially viable bioprocesses is essential for reducing the need for fossil-derived products. Increasingly, pharmaceuticals, fuel, health products and precursor compounds for plastics are being synthesized using bioprocessing routes as opposed to more traditional chemical technologies. Production vessels or reactors are required for synthesis of crude product before downstream processing for extraction and purification. Reactors are operated either in discrete batches or, preferably, continuously in order to reduce waste, cost and energy. This review describes the oscillatory baffled reactor (OBR), which, generally, has a niche application in performing ‘long’ processes in plug flow conditions, and so should be suitable for various bioprocesses. We report findings to suggest that OBRs could increase reaction rates for specific bioprocesses owing to low shear, good global mixing and enhanced mass transfer compared with conventional reactors. By maintaining geometrical and dynamic conditions, the technology has been proved to be easily scaled up and operated continuously, allowing laboratory-scale results to be easily transferred to industrial-sized processes. This is the first comprehensive review of bioprocessing using OBRs. The barriers facing industrial adoption of the technology are discussed alongside some suggested strategies to overcome these barriers. OBR technology could prove to be a major aid in the development of commercially viable and sustainable bioprocesses, essential for moving towards a greener future. PMID:24427509

Organic Chemicals from Bioprocesses in China

NASA Astrophysics Data System (ADS)

Huang, Jin; Huang, Lei; Lin, Jianping; Xu, Zhinan; Cen, Peilin

Over the last 20 years, China has successfully established a modern biotechnology industry from almost nothing. Presently, China is a major producer of a vast array of products involving bioprocesses, for some China is even the world's top producer. The ever-increasing list of products includes organic acids, amino acids, antibiotics, solvents, chiral chemicals, biopesticides, and biopolymers. Herein, the research and development of bioprocesses in China will be reviewed briefly. We will concentrate on three categories of products: small molecules produced via fermentation, biopolymers produced via fermentation and small chemicals produced by enzyme-catalyzed reactions. In comparison with the traditional chemical process, in which, nonrenewable mineral resources are generally used, products in the first and second categories noted above can use renewable bioresources as raw materials. The bioprocesses are generally energy saving and environmentally benign. For products developed via the third category, although the raw materials still need to be obtained from mineral resources, the biocatalysts are more effective with higher selectivity and productivity, and the bioprocesses occur under ambient temperature and pressure, therefore, these are "green processes." Most of the products such as citric acid, xanthan and acrylamide etc., discussed in this paper have been in large-scale commercial production in China. Also introduced herein are three scientists, Prof. Shen Yinchu, Prof. Ouyang Pingkai and Prof. Chen Guoqiang, and six enterprises, Anhui Fengyuan Biochemical Co. Ltd., Shandong Hiland Biotechnology Co. Ltd., Shandong Fufeng Fermentation Co. Ltd., Shandong Bausch & Lomb-Freda Pharmaceutical Co. Ltd., Zhejiang Hangzhou Xinfu Pharmaceutical Co. Ltd., and Changzhou Changmao Biochemical Engineering Co. Ltd.; they have all contributed a great deal to research and development in the commercialization of bioprocesses.

Multi-function microfluidic platform for sensor integration.

PubMed

Fernandes, Ana C; Semenova, Daria; Panjan, Peter; Sesay, Adama M; Gernaey, Krist V; Krühne, Ulrich

2018-03-06

The limited availability of metabolite-specific sensors for continuous sampling and monitoring is one of the main bottlenecks contributing to failures in bioprocess development. Furthermore, only a limited number of approaches exist to connect currently available measurement systems with high throughput reactor units. This is especially relevant in the biocatalyst screening and characterization stage of process development. In this work, a strategy for sensor integration in microfluidic platforms is demonstrated, to address the need for rapid, cost-effective and high-throughput screening in bioprocesses. This platform is compatible with different sensor formats by enabling their replacement and was built in order to be highly flexible and thus suitable for a wide range of applications. Moreover, this re-usable platform can easily be connected to analytical equipment, such as HPLC, laboratory scale reactors or other microfluidic chips through the use of standardized fittings. In addition, the developed platform includes a two-sensor system interspersed with a mixing channel, which allows the detection of samples that might be outside the first sensor's range of detection, through dilution of the sample solution up to 10 times. In order to highlight the features of the proposed platform, inline monitoring of glucose levels is presented and discussed. Glucose was chosen due to its importance in biotechnology as a relevant substrate. The platform demonstrated continuous measurement of substrate solutions for up to 12 h. Furthermore, the influence of the fluid velocity on substrate diffusion was observed, indicating the need for in-flow calibration to achieve a good quantitative output. Copyright © 2018 Elsevier B.V. All rights reserved.

Automatic extraction of angiogenesis bioprocess from text

PubMed Central

Wang, Xinglong; McKendrick, Iain; Barrett, Ian; Dix, Ian; French, Tim; Tsujii, Jun'ichi; Ananiadou, Sophia

2011-01-01

Motivation: Understanding key biological processes (bioprocesses) and their relationships with constituent biological entities and pharmaceutical agents is crucial for drug design and discovery. One way to harvest such information is searching the literature. However, bioprocesses are difficult to capture because they may occur in text in a variety of textual expressions. Moreover, a bioprocess is often composed of a series of bioevents, where a bioevent denotes changes to one or a group of cells involved in the bioprocess. Such bioevents are often used to refer to bioprocesses in text, which current techniques, relying solely on specialized lexicons, struggle to find. Results: This article presents a range of methods for finding bioprocess terms and events. To facilitate the study, we built a gold standard corpus in which terms and events related to angiogenesis, a key biological process of the growth of new blood vessels, were annotated. Statistics of the annotated corpus revealed that over 36% of the text expressions that referred to angiogenesis appeared as events. The proposed methods respectively employed domain-specific vocabularies, a manually annotated corpus and unstructured domain-specific documents. Evaluation results showed that, while a supervised machine-learning model yielded the best precision, recall and F1 scores, the other methods achieved reasonable performance and less cost to develop. Availability: The angiogenesis vocabularies, gold standard corpus, annotation guidelines and software described in this article are available at http://text0.mib.man.ac.uk/~mbassxw2/angiogenesis/ Contact: xinglong.wang@gmail.com PMID:21821664

Guiding bioprocess design by microbial ecology.

PubMed

Volmer, Jan; Schmid, Andreas; Bühler, Bruno

2015-06-01

Industrial bioprocess development is driven by profitability and eco-efficiency. It profits from an early stage definition of process and biocatalyst design objectives. Microbial bioprocess environments can be considered as synthetic technical microbial ecosystems. Natural systems follow Darwinian evolution principles aiming at survival and reproduction. Technical systems objectives are eco-efficiency, productivity, and profitable production. Deciphering technical microbial ecology reveals differences and similarities of natural and technical systems objectives, which are discussed in this review in view of biocatalyst and process design and engineering strategies. Strategies for handling opposing objectives of natural and technical systems and for exploiting and engineering natural properties of microorganisms for technical systems are reviewed based on examples. This illustrates the relevance of considering microbial ecology for bioprocess design and the potential for exploitation by synthetic biology strategies. Copyright © 2015 Elsevier Ltd. All rights reserved.

Continuous downstream processing for high value biological products: A Review.

PubMed

Zydney, Andrew L

2016-03-01

There is growing interest in the possibility of developing truly continuous processes for the large-scale production of high value biological products. Continuous processing has the potential to provide significant reductions in cost and facility size while improving product quality and facilitating the design of flexible multi-product manufacturing facilities. This paper reviews the current state-of-the-art in separations technology suitable for continuous downstream bioprocessing, focusing on unit operations that would be most appropriate for the production of secreted proteins like monoclonal antibodies. This includes cell separation/recycle from the perfusion bioreactor, initial product recovery (capture), product purification (polishing), and formulation. Of particular importance are the available options, and alternatives, for continuous chromatographic separations. Although there are still significant challenges in developing integrated continuous bioprocesses, recent technological advances have provided process developers with a number of attractive options for development of truly continuous bioprocessing operations. © 2015 Wiley Periodicals, Inc.

Raman spectroscopy as a process analytical technology for pharmaceutical manufacturing and bioprocessing.

PubMed

Esmonde-White, Karen A; Cuellar, Maryann; Uerpmann, Carsten; Lenain, Bruno; Lewis, Ian R

2017-01-01

Adoption of Quality by Design (QbD) principles, regulatory support of QbD, process analytical technology (PAT), and continuous manufacturing are major factors effecting new approaches to pharmaceutical manufacturing and bioprocessing. In this review, we highlight new technology developments, data analysis models, and applications of Raman spectroscopy, which have expanded the scope of Raman spectroscopy as a process analytical technology. Emerging technologies such as transmission and enhanced reflection Raman, and new approaches to using available technologies, expand the scope of Raman spectroscopy in pharmaceutical manufacturing, and now Raman spectroscopy is successfully integrated into real-time release testing, continuous manufacturing, and statistical process control. Since the last major review of Raman as a pharmaceutical PAT in 2010, many new Raman applications in bioprocessing have emerged. Exciting reports of in situ Raman spectroscopy in bioprocesses complement a growing scientific field of biological and biomedical Raman spectroscopy. Raman spectroscopy has made a positive impact as a process analytical and control tool for pharmaceutical manufacturing and bioprocessing, with demonstrated scientific and financial benefits throughout a product's lifecycle.

Adaptation of in-situ microscopy for crystallization processes

NASA Astrophysics Data System (ADS)

Bluma, A.; Höpfner, T.; Rudolph, G.; Lindner, P.; Beutel, S.; Hitzmann, B.; Scheper, T.

2009-08-01

In biotechnological and pharmaceutical engineering, the study of crystallization processes gains importance. An efficient analytical inline sensor could help to improve the knowledge about these processes in order to increase efficiency and yields. The in-situ microscope (ISM) is an optical sensor developed for the monitoring of bioprocesses. A new application for this sensor is the monitoring in downstream processes, e.g. the crystallization of proteins and other organic compounds. This contribution shows new aspects of using in-situ microscopy to monitor crystallization processes. Crystals of different chemical compounds were precipitated from supersaturated solutions and the crystal growth was monitored. Exemplified morphological properties and different forms of crystals could be distinguished on the basis of offline experiments. For inline monitoring of crystallization processes, a special 0.5 L stirred tank reactor was developed and equipped with the in-situ microscope. This reactor was utilized to carry out batch experiments for crystallizations of O-acetylsalicyclic acid (ASS) and hen egg white lysozyme (HEWL). During the whole crystallization process, the in-situ microscope system acquired images directly from the crystallization broth. For the data evaluation, an image analysis algorithm was developed and implemented in the microscope analysis software.

Fiber optic chemical sensors: The evolution of high- density fiber-optic DNA microarrays

NASA Astrophysics Data System (ADS)

Ferguson, Jane A.

2001-06-01

Sensors were developed for multianalyte monitoring, fermentation monitoring, lactate analysis, remote oxygen detection for use in bioremediation monitoring and in a fuel spill clean-up project, heavy metal analysis, and high density DNA microarrays. The major focus of this thesis involved creating and improving high-density DNA gene arrays. Fiber optic sensors are created using fluorescent indicators, polymeric supports, and optical fiber substrates. The fluorescent indicator is entrapped in a polymer layer and attached to the tip of the optical fiber. The tip of the fiber bearing the sensing layer (the distal end) is placed in the sample of interest while the other end of the fiber (the proximal end) is connected to an analysis system. Any length of fiber can be used without compromising the integrity or sensitivity of the system. A fiber optic oxygen sensor was designed incorporating an oxygen sensitive fluorescent dye and a gas permeable polymer attached to an optical fiber. The construction simplicity and ruggedness of the sensor enabled its deployment for in situ chemical oxidation and bioremediation studies. Optical fibers were also used as the substrate to detect biomolecules in solution. To monitor bioprocesses, the production of the analyte of interest must be coupled with a species that is optically measurable. For example, oxygen is consumed in many metabolic functions. The fiber optic oxygen sensor is equipped with an additional sensing layer. Upon contact with a specific biochemical in the sample, a reaction occurs in the additional sensing layer that either consumes or produces oxygen. This dual layer system was used to monitor the presence of lactate, an important metabolite for clinical and bioprocess analysis. In many biological and environmental systems, the generation of one species occurs coincidentally with the generation or consumption of another species. A multianalyte sensor was prepared that can monitor the simultaneous activity of pH, CO2 and O2. This sensor is useful for monitoring bioprocesses such as (beer) fermentation and for clinical situations such as blood gas analysis. DNA sensors were created by attaching short single strands of DNA (probes) to the fiber tip. A matching single strand (target) forms a strong interacting pair with the probe upon contact. The target strands in a sample are labeled with a fluorescent dye. When a probe-target pair is formed and excitation light is sent down the fiber, the fiber bearing the pair emits light that is captured and detected. A high density DNA array was created by isolating thousands of discrete DNA sensors on the tip of an imaging optical fiber. This array was made possible by the formation of microwells on the imaging fiber tip. Microspheres functionalized with DNA were placed in the wells of the fiber and each microsphere was independently and simultaneously monitored. (Abstract shortened by UMI.)

Developing a Continuous Bioprocessing Approach to Stromal Cell Manufacture.

PubMed

Miotto, Martina; Gouveia, Ricardo; Abidin, Fadhilah Zainal; Figueiredo, Francisco; Connon, Che J

2017-11-29

To this day, the concept of continuous bioprocessing has been applied mostly to the manufacture of molecular biologics such as proteins, growth factors, and secondary metabolites with biopharmaceutical uses. The present work now sets to explore the potential application of continuous bioprocess methods to source large numbers of human adherent cells with potential therapeutic value. To this purpose, we developed a smart multifunctional surface coating capable of controlling the attachment, proliferation, and subsequent self-detachment of human corneal stromal cells. This system allowed the maintenance of cell cultures under steady-state growth conditions, where self-detaching cells were continuously replenished by the proliferation of those remaining attached. This facilitated a closed, continuous bioprocessing platform with recovery of approximately 1% of the total adherent cells per hour, a yield rate that was maintained for 1 month. Moreover, both attached and self-detached cells were shown to retain their original phenotype. Together, these results represent the proof-of-concept for a new high-throughput, high-standard, and low-cost biomanufacturing strategy with multiple potentials and important downstream applications.

PTR-MS in Italy: A Multipurpose Sensor with Applications in Environmental, Agri-Food and Health Science

PubMed Central

Cappellin, Luca; Loreto, Francesco; Aprea, Eugenio; Romano, Andrea; del Pulgar, José Sánchez; Gasperi, Flavia; Biasioli, Franco

2013-01-01

Proton Transfer Reaction Mass Spectrometry (PTR-MS) has evolved in the last decade as a fast and high sensitivity sensor for the real-time monitoring of volatile compounds. Its applications range from environmental sciences to medical sciences, from food technology to bioprocess monitoring. Italian scientists and institutions participated from the very beginning in fundamental and applied research aiming at exploiting the potentialities of this technique and providing relevant methodological advances and new fundamental indications. In this review we describe this activity on the basis of the available literature. The Italian scientific community has been active mostly in food science and technology, plant physiology and environmental studies and also pioneered the applications of the recently released PTR-ToF-MS (Proton Transfer Reaction-Time of Flight-Mass Spectrometry) in food science and in plant physiology. In the very last years new results related to bioprocess monitoring and health science have been published as well. PTR-MS data analysis, particularly in the case of the ToF based version, and the application of advanced chemometrics and data mining are also aspects characterising the activity of the Italian community. PMID:24021966

Nanobiocatalyst advancements and bioprocessing applications

PubMed Central

Misson, Mailin; Zhang, Hu; Jin, Bo

2015-01-01

The nanobiocatalyst (NBC) is an emerging innovation that synergistically integrates advanced nanotechnology with biotechnology and promises exciting advantages for improving enzyme activity, stability, capability and engineering performances in bioprocessing applications. NBCs are fabricated by immobilizing enzymes with functional nanomaterials as enzyme carriers or containers. In this paper, we review the recent developments of novel nanocarriers/nanocontainers with advanced hierarchical porous structures for retaining enzymes, such as nanofibres (NFs), mesoporous nanocarriers and nanocages. Strategies for immobilizing enzymes onto nanocarriers made from polymers, silicas, carbons and metals by physical adsorption, covalent binding, cross-linking or specific ligand spacers are discussed. The resulting NBCs are critically evaluated in terms of their bioprocessing performances. Excellent performances are demonstrated through enhanced NBC catalytic activity and stability due to conformational changes upon immobilization and localized nanoenvironments, and NBC reutilization by assembling magnetic nanoparticles into NBCs to defray the high operational costs associated with enzyme production and nanocarrier synthesis. We also highlight several challenges associated with the NBC-driven bioprocess applications, including the maturation of large-scale nanocarrier synthesis, design and development of bioreactors to accommodate NBCs, and long-term operations of NBCs. We suggest these challenges are to be addressed through joint collaboration of chemists, engineers and material scientists. Finally, we have demonstrated the great potential of NBCs in manufacturing bioprocesses in the near future through successful laboratory trials of NBCs in carbohydrate hydrolysis, biofuel production and biotransformation. PMID:25392397

Rapid monitoring of glycerol in fermentation growth media: Facilitating crude glycerol bioprocess development.

PubMed

Abad, Sergi; Pérez, Xavier; Planas, Antoni; Turon, Xavier

2014-04-01

Recently, the need for crude glycerol valorisation from the biodiesel industry has generated many studies for practical and economic applications. Amongst them, fermentations based on glycerol media for the production of high value metabolites are prominent applications. This has generated a need to develop analytical techniques which allow fast and simple glycerol monitoring during fermentation. The methodology should be fast and inexpensive to be adopted in research, as well as in industrial applications. In this study three different methods were analysed and compared: two common methodologies based on liquid chromatography and enzymatic kits, and the new method based on a DotBlot assay coupled with image analysis. The new methodology is faster and cheaper than the other conventional methods, with comparable performance. Good linearity, precision and accuracy were achieved in the lower range (10 or 15 g/L to depletion), the most common range of glycerol concentrations to monitor fermentations in terms of growth kinetics. Copyright © 2014 Elsevier B.V. All rights reserved.

Cell bioprocessing in space - Applications of analytical cytology

NASA Technical Reports Server (NTRS)

Todd, P.; Hymer, W. C.; Goolsby, C. L.; Hatfield, J. M.; Morrison, D. R.

1988-01-01

Cell bioprocessing experiments in space are reviewed and the development of on-board cell analytical cytology techniques that can serve such experiments is discussed. Methods and results of experiments involving the cultivation and separation of eukaryotic cells in space are presented. It is suggested that an advanced cytometer should be developed for the quantitative analysis of large numbers of specimens of suspended eukaryotic cells and bioparticles in experiments on the Space Station.

ECUT: Energy Conversion and Utilization Technologies program. Biocatalysis project

NASA Technical Reports Server (NTRS)

1990-01-01

The Biocatalysis Project is a mission-oriented, applied research and exploratory development activity directed toward resolution of the major generic technical barriers that impede the development of biologically catalyzed commercial chemical production. The approach toward achieving project objectives involves an integrated participation of Universities, Industrial Companies and Government Research Laboratories. The Project's technical activities were organized into three work elements: molecular modeling and applied genetics; bioprocess engineering; and bioprocess design and assessment.

Production of polyol oils from soybean oil by bioprocess and Philippines edible medicinal wild mushrooms

USDA-ARS?s Scientific Manuscript database

We have been trying to develop a bioprocess for the production of polyol oils directly from soybean oil. We reported earlier the polyol products produced from soybean oil by Acinetobacter haemolyticus A01-35 (NRRL B-59985) (Hou and Lin, 2013). The objective of this study is to identify the chemical ...

White paper on continuous bioprocessing. May 20-21, 2014 Continuous Manufacturing Symposium.

PubMed

Konstantinov, Konstantin B; Cooney, Charles L

2015-03-01

There is a growing interest in realizing the benefits of continuous processing in biologics manufacturing, which is reflected by the significant number of industrial and academic researchers who are actively involved in the development of continuous bioprocessing systems. These efforts are further encouraged by guidance expressed in recent US FDA conference presentations. The advantages of continuous manufacturing include sustained operation with consistent product quality, reduced equipment size, high-volumetric productivity, streamlined process flow, low-process cycle times, and reduced capital and operating cost. This technology, however, poses challenges, which need to be addressed before routine implementation is considered. This paper, which is based on the available literature and input from a large number of reviewers, is intended to provide a consensus of the opportunities, technical needs, and strategic directions for continuous bioprocessing. The discussion is supported by several examples illustrating various architectures of continuous bioprocessing systems. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Micro-Electromechanical Affinity Sensor for the Monitoring of Glucose in Bioprocess Media

PubMed Central

Theuer, Lorenz; Lehmann, Micha; Junne, Stefan; Neubauer, Peter; Birkholz, Mario

2017-01-01

An affinity-viscometry-based micro-sensor probe for continuous glucose monitoring was investigated with respect to its suitability for bioprocesses. The sensor operates with glucose and dextran competing as binding partner for concanavalin A, while the viscosity of the assay scales with glucose concentration. Changes in viscosity are determined with a micro-electromechanical system (MEMS) in the measurement cavity of the sensor probe. The study aimed to elucidate the interactions between the assay and a typical phosphate buffered bacterial cultivation medium. It turned out that contact with the medium resulted in a significant long-lasting drift of the assay’s viscosity at zero glucose concentration. Adding glucose to the medium lowers the drift by a factor of eight. The cglc values measured off-line with the glucose sensor for monitoring of a bacterial cultivation were similar to the measurements with an enzymatic assay with a difference of less than ±0.15 g·L−1. We propose that lectin agglomeration, the electro-viscous effect, and constitutional changes of concanavalin A due to exchanges of the incorporated metal ions may account for the observed viscosity increase. The study has demonstrated the potential of the MEMS sensor to determine sensitive viscosity changes within very small sample volumes, which could be of interest for various biotechnological applications. PMID:28594350

New perspectives for the design of sustainable bioprocesses for phosphorus recovery from waste.

PubMed

Tarayre, Cédric; De Clercq, Lies; Charlier, Raphaëlle; Michels, Evi; Meers, Erik; Camargo-Valero, Miller; Delvigne, Frank

2016-04-01

Phosphate rock has long been used for the production of phosphorus based chemicals. However, considering the depletion of the reservoirs and the decrease of the quality of phosphate rocks, a potential market is now emerging for the recovery of phosphate from waste and its reuse for different applications. Notably, phosphate recovery from wastewater could be included in a circular economy approach. This review focuses on the use of microbial systems for phosphorus accumulation and recovery, by considering the actual range of analytical techniques available for the monitoring of phosphorus accumulating organisms, as well as the actual biochemical and metabolic engineering toolbox available for the optimization of bioprocesses. In this context, knowledge gathered from process, system and synthetic biology could potentially lead to innovative process design. Copyright © 2016 Elsevier Ltd. All rights reserved.

A biological/chemical process for reduced waste and energy consumption: caprolactam production. Final report

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

NONE

1996-05-01

A biological/chemical process for converting cyclohexane into caprolactam was investigated: microorganisms in a bioreactor would be used to convert cyclohexane into caprolactone followed by chemical synthesis of caprolactam using ammonia. Four microorganisms were isolated from natural soil and water, that can utilize cyclohexane as a sole source of C and energy for growth. They were shown to have the correct metabolic intermediates and enzymes to convert cyclohexane into cyclohexanol, cyclohexanone, and caprolactone. Genetic techniques to create and select for caprolactone hydrolase negative-mutants were developed; those are used to convert cyclohexane into caprolactone but, because of the block, are unable tomore » metabolize the caprolactone further. Because of a new nylon carpet reycle process and the long time frame for a totally new bioprocess, a limited study was done to evaluate whether a simplified bioprocess to convert cyclohexanol into cyclohexanone or caprolactone was feasible; growth rates and key enzyme levels were measured in a collection of microorganisms that metabolize cyclohexanol to determine if the bioactivity is high enough to support an economical cyclohexanol bioprocess. Although these microorganisms had sufficient bioactivity, they could tolerate only low levels (<1%) of cyclohexanol and thus are not suitable for developing a cost effective bioprocess because of the high cost of dilute product recovery.« less

Multiple reaction monitoring targeted LC-MS analysis of potential cell death marker proteins for increased bioprocess control.

PubMed

Albrecht, Simone; Kaisermayer, Christian; Reinhart, David; Ambrose, Monica; Kunert, Renate; Lindeberg, Anna; Bones, Jonathan

2018-05-01

The monitoring of protein biomarkers for the early prediction of cell stress and death is a valuable tool for process characterization and efficient biomanufacturing control. A representative set of six proteins, namely GPDH, PRDX1, LGALS1, CFL1, TAGLN2 and MDH, which were identified in a previous CHO-K1 cell death model using discovery LC-MS E was translated into a targeted liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) platform and verified. The universality of the markers was confirmed in a cell growth model for which three Chinese hamster ovary host cell lines (CHO-K1, CHO-S, CHO-DG44) were grown in batch culture in two different types of basal media. LC-MRM-MS was also applied to spent media (n = 39) from four perfusion biomanufacturing series. Stable isotope-labelled peptide analogues and a stable isotope-labelled monoclonal antibody were used for improved protein quantitation and simultaneous monitoring of the workflow reproducibility. Significant increases in protein concentrations were observed for all viability marker proteins upon increased dead cell numbers and allowed for discrimination of spent media with dead cell densities below and above 1 × 10 6  dead cells/mL which highlights the potential of the selected viability marker proteins in bioprocess control. Graphical abstract Overview of the LC-MRM-MS workflow for the determination of proteomic markers in conditioned media from the bioreactor that correlate with CHO cell death.

Bioprocessing development: Immune/cellular applications: Anti-Ig autoantibody and complement-mediated destruction of neoplastic cells

NASA Technical Reports Server (NTRS)

Twomey, J. J.

1976-01-01

This space bioprocessing contract effort was comprised of four general objectives. These were: (1) the evaluation of current separation processes, (2) the identification of problems relevant to the separation of important biologicals, (3) the identification of ground-based assay methods needed for pre- and postflight analysis of space bioprocessing separation technology; and (4) the establishment of methods to determine the efficiency of space bioprocessing separation procedures. Immunology was deemed advantageous to study the diversity of cells and cell products involved and the extensive interest being given to their separation. Upon recognition of a cellular or molecular agent as foreign to the body, the immune system becomes activated to produce cells whose function is to destroy that agent and cell products whose function is to inactivate the agent and assist in its destruction. Long after the agent is removed from the body, some cells remain in a state of readiness to continue these destructive actions specifically against that agent should further exposure to it occur. This is the basis of acquired immunity to disease.

Microbial biocatalyst developments to upgrade fossil fuels.

PubMed

Kilbane, John J

2006-06-01

Steady increases in the average sulfur content of petroleum and stricter environmental regulations concerning the sulfur content have promoted studies of bioprocessing to upgrade fossil fuels. Bioprocesses can potentially provide a solution to the need for improved and expanded fuel upgrading worldwide, because bioprocesses for fuel upgrading do not require hydrogen and produce far less carbon dioxide than thermochemical processes. Recent advances have demonstrated that biodesulfurization is capable of removing sulfur from hydrotreated diesel to yield a product with an ultra-low sulfur concentration that meets current environmental regulations. However, the technology has not yet progressed beyond laboratory-scale testing, as more efficient biocatalysts are needed. Genetic studies to obtain improved biocatalysts for the selective removal of sulfur and nitrogen from petroleum provide the focus of current research efforts.

1H NMR-based metabolic profiling for evaluating poppy seed rancidity and brewing.

PubMed

Jawień, Ewa; Ząbek, Adam; Deja, Stanisław; Łukaszewicz, Marcin; Młynarz, Piotr

2015-12-01

Poppy seeds are widely used in household and commercial confectionery. The aim of this study was to demonstrate the application of metabolic profiling for industrial monitoring of the molecular changes which occur during minced poppy seed rancidity and brewing processes performed on raw seeds. Both forms of poppy seeds were obtained from a confectionery company. Proton nuclear magnetic resonance (1H NMR) was applied as the analytical method of choice together with multivariate statistical data analysis. Metabolic fingerprinting was applied as a bioprocess control tool to monitor rancidity with the trajectory of change and brewing progressions. Low molecular weight compounds were found to be statistically significant biomarkers of these bioprocesses. Changes in concentrations of chemical compounds were explained relative to the biochemical processes and external conditions. The obtained results provide valuable and comprehensive information to gain a better understanding of the biology of rancidity and brewing processes, while demonstrating the potential for applying NMR spectroscopy combined with multivariate data analysis tools for quality control in food industries involved in the processing of oilseeds. This precious and versatile information gives a better understanding of the biology of these processes.

Review of nonconventional bioreactor technology

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

Turick, C.E.; Mcllwain, M.E.

1993-09-01

Biotechnology will significantly affect many industrial sectors in the future. Industrial sectors that will be affected include pharmaceutical, chemical, fuel, agricultural, and environmental remediation. Future research is needed to improve bioprocessing efficiency and cost-effectiveness in order to compete with traditional technologies. This report describes recent advances in bioprocess technologies and bioreactor designs and relates them to problems encountered in many industrial bioprocessing operations. The primary focus is directed towards increasing gas and vapor transfer for enhanced bioprocess kinetics as well as unproved by-product separation and removal. The advantages and disadvantages of various conceptual designs such as hollow-fiber, gas-phase, hyperbaric/hypobaric, andmore » electrochemical bioreactors are also discussed. Specific applications that are intended for improved bioprocesses include coal desulfurization, coal liquefaction, soil bioremediation, biomass conversion to marketable chemicals, biomining, and biohydrometallurgy as well as bioprocessing of gases and vapors.« less

Hydrodynamic chronoamperometry for probing kinetics of anaerobic microbial metabolism--case study of Faecalibacterium prausnitzii.

PubMed

Prévoteau, Antonin; Geirnaert, Annelies; Arends, Jan B A; Lannebère, Sylvain; Van de Wiele, Tom; Rabaey, Korneel

2015-07-01

Monitoring in vitro the metabolic activity of microorganisms aids bioprocesses and enables better understanding of microbial metabolism. Redox mediators can be used for this purpose via different electrochemical techniques that are either complex or only provide non-continuous data. Hydrodynamic chronoamperometry using a rotating disc electrode (RDE) can alleviate these issues but was seldom used and is poorly characterized. The kinetics of Faecalibacterium prausnitzii A2-165, a beneficial gut microbe, were determined using a RDE with riboflavin as redox probe. This butyrate producer anaerobically ferments glucose and reduces riboflavin whose continuous monitoring on a RDE provided highly accurate kinetic measurements of its metabolism, even at low cell densities. The metabolic reaction rate increased linearly over a broad range of cell concentrations (9 × 10(4) to 5 × 10(7) cells.mL(-1)). Apparent Michaelis-Menten kinetics was observed with respect to riboflavin (KM = 6 μM; kcat = 5.3 × 10(5) s(-1), at 37 °C) and glucose (KM = 6 μM; kcat = 2.4 × 10(5) s(-1)). The short temporal resolution allows continuous monitoring of fast cellular events such as kinetics inhibition with butyrate. Furthermore, we detected for the first time riboflavin reduction by another potential probiotic, Butyricicoccus pullicaecorum. The ability of the RDE for fast, accurate, simple and continuous measurements makes it an ad hoc tool for assessing bioprocesses at high resolution.

Integrating human stem cell expansion and neuronal differentiation in bioreactors

PubMed Central

Serra, Margarida; Brito, Catarina; Costa, Eunice M; Sousa, Marcos FQ; Alves, Paula M

2009-01-01

Background Human stem cells are cellular resources with outstanding potential for cell therapy. However, for the fulfillment of this application, major challenges remain to be met. Of paramount importance is the development of robust systems for in vitro stem cell expansion and differentiation. In this work, we successfully developed an efficient scalable bioprocess for the fast production of human neurons. Results The expansion of undifferentiated human embryonal carcinoma stem cells (NTera2/cl.D1 cell line) as 3D-aggregates was firstly optimized in spinner vessel. The media exchange operation mode with an inoculum concentration of 4 × 105 cell/mL was the most efficient strategy tested, with a 4.6-fold increase in cell concentration achieved in 5 days. These results were validated in a bioreactor where similar profile and metabolic performance were obtained. Furthermore, characterization of the expanded population by immunofluorescence microscopy and flow cytometry showed that NT2 cells maintained their stem cell characteristics along the bioreactor culture time. Finally, the neuronal differentiation step was integrated in the bioreactor process, by addition of retinoic acid when cells were in the middle of the exponential phase. Neurosphere composition was monitored and neuronal differentiation efficiency evaluated along the culture time. The results show that, for bioreactor cultures, we were able to increase significantly the neuronal differentiation efficiency by 10-fold while reducing drastically, by 30%, the time required for the differentiation process. Conclusion The culture systems developed herein are robust and represent one-step-forward towards the development of integrated bioprocesses, bridging stem cell expansion and differentiation in fully controlled bioreactors. PMID:19772662

Single cell oil production by Mortierella isabellina from steam exploded corn stover degraded by three-stage enzymatic hydrolysis in the context of on-site enzyme production.

PubMed

Fang, Hao; Zhao, Chen; Chen, Shaolin

2016-09-01

Single cell oil (SCO), promising as alternative oil source, was produced from steam exploded corn stover (SECS) by Mortierella isabellina. Different bioprocesses from SECS to SCO were compared and the bioprocess C using the three-stage enzymatic hydrolysis was found to be the most efficient one. The bioprocess C used the lowest enzyme input 20FPIU cellulase/g glucan and the shortest time 222h, but produced 44.94g dry cell biomass and 25.77g lipid from 327.63g dry SECS. It had the highest lipid content 57.34%, and its productivities and yields were much higher than those of the bioprocess B and comparable to the bioprocess A, indicating that the three-stage enzymatic hydrolysis could greatly improve the efficiency of the bioprocess from high solid loading SECS to SCO by Mortierella isabellina. This work testified the application value of three-stage enzymatic hydrolysis in lignocellulose-based bioprocesses. Copyright © 2016 Elsevier Ltd. All rights reserved.

Winemaking and Bioprocesses Strongly Shaped the Genetic Diversity of the Ubiquitous Yeast Torulaspora delbrueckii

PubMed Central

Comte, Guillaume; Panfili, Aurélie; Delcamp, Adline; Salin, Franck; Marullo, Philippe; Bely, Marina

2014-01-01

The yeast Torulaspora delbrueckii is associated with several human activities including oenology, bakery, distillery, dairy industry, etc. In addition to its biotechnological applications, T. delbrueckii is frequently isolated in natural environments (plant, soil, insect). T. delbrueckii is thus a remarkable ubiquitous yeast species with both wild and anthropic habitats, and appears to be a perfect yeast model to search for evidence of human domestication. For that purpose, we developed eight microsatellite markers that were used for the genotyping of 110 strains from various substrates and geographical origins. Microsatellite analysis showed four genetic clusters: two groups contained most nature strains from Old World and Americas respectively, and two clusters were associated with winemaking and other bioprocesses. Analysis of molecular variance (AMOVA) confirmed that human activities significantly shaped the genetic variability of T. delbrueckii species. Natural isolates are differentiated on the basis of geographical localisation, as expected for wild population. The domestication of T. delbrueckii probably dates back to the Roman Empire for winemaking (∼1900 years ago), and to the Neolithic era for bioprocesses (∼4000 years ago). Microsatellite analysis also provided valuable data regarding the life-cycle of the species, suggesting a mostly diploid homothallic life. In addition to population genetics and ecological studies, the microsatellite tool will be particularly useful for further biotechnological development of T. delbrueckii strains for winemaking and other bioprocesses. PMID:24718638

Winemaking and bioprocesses strongly shaped the genetic diversity of the ubiquitous yeast Torulaspora delbrueckii.

PubMed

Albertin, Warren; Chasseriaud, Laura; Comte, Guillaume; Panfili, Aurélie; Delcamp, Adline; Salin, Franck; Marullo, Philippe; Bely, Marina

2014-01-01

The yeast Torulaspora delbrueckii is associated with several human activities including oenology, bakery, distillery, dairy industry, etc. In addition to its biotechnological applications, T. delbrueckii is frequently isolated in natural environments (plant, soil, insect). T. delbrueckii is thus a remarkable ubiquitous yeast species with both wild and anthropic habitats, and appears to be a perfect yeast model to search for evidence of human domestication. For that purpose, we developed eight microsatellite markers that were used for the genotyping of 110 strains from various substrates and geographical origins. Microsatellite analysis showed four genetic clusters: two groups contained most nature strains from Old World and Americas respectively, and two clusters were associated with winemaking and other bioprocesses. Analysis of molecular variance (AMOVA) confirmed that human activities significantly shaped the genetic variability of T. delbrueckii species. Natural isolates are differentiated on the basis of geographical localisation, as expected for wild population. The domestication of T. delbrueckii probably dates back to the Roman Empire for winemaking (∼ 1900 years ago), and to the Neolithic era for bioprocesses (∼ 4000 years ago). Microsatellite analysis also provided valuable data regarding the life-cycle of the species, suggesting a mostly diploid homothallic life. In addition to population genetics and ecological studies, the microsatellite tool will be particularly useful for further biotechnological development of T. delbrueckii strains for winemaking and other bioprocesses.

Introduction to Session 5

NASA Astrophysics Data System (ADS)

Zullo, Luca; Snyder, Seth W.

Production of bio-based products that are cost competitive in the market place requires well-developed operations that include innovative processes and separation solutions. Separations costs can make the difference between an interesting laboratory project and a successful commercial process. Bioprocessing and separations research and development addresses some of the most significant cost barriers in production of bioffuels and bio-based chemicals. Models of integrated biorefineries indicate that success will require production of higher volume fuels in conjunction with high margin chemical products. Addressing the bioprocessing and separations cost barriers will be critical to the overall success of the integrated biorefinery.

Immobilized metal affinity cryogel-based high-throughput platform for screening bioprocess and chromatographic parameters of His6-GTPase.

PubMed

Sarkar, Joyita; Kumar, Ashok

2017-04-01

Among various tools of product monitoring, chromatography is of vital importance as it also extends to the purification of product. Immobilized metal affinity cryogel (Cu(II)-iminodiacetic acid- and Ni(II)-nitrilotriacetic acid-polyacrylamide) minicolumns (diameter 8 mm, height 4 mm, void volume 250 μl) were inserted in open-ended 96-well plate and different chromatographic parameters and bioprocess conditions were analysed. The platform was first validated with lysozyme. Optimum binding of lysozyme (∼90%) was achieved when 50 μg of protein in 20 mM Tris, pH 8.0 was applied to the minicolumns with maximum recovery (∼90%) upon elution with 300 mM imidazole. Thereafter, the platform was screened for chromatographic conditions of His 6 -GTPase. Since cryogels have large pore size, they can easily process non-clarified samples containing debris and particulate matters. The bound enzymes on the gel retain its activity and therefore can be assayed on-column by adding substrate and then displacing the product. Highest binding of His 6 -GTPase was achieved when 50 μl of non-clarified cell lysate was applied to the cryogel and subsequently washed with 50 mM Tris, 150 mM NaCl, 5 mM MgCl 2 , 10 mM imidazole, pH 8.0 with dynamic and static binding capacities of ∼1.5 and 3 activity units. Maximum recovery was obtained upon elution with 300 mM imidazole with a purification fold of ∼10; the purity was also analysed by SDS-PAGE. The platform showed reproducible results which were validated by Bland-Altman plot. The minicolumn was also scaled up for chromatographic capture and recovery of His 6 -GTPase. The bioprocess conditions were monitored which displayed that optimum production of His 6 -GTPase was attained by induction with 200 μM isopropyl-β-D-thiogalactoside at 25 °C for 12 h. It was concluded that immobilized metal affinity cryogel-based platform can be successfully used as a high-throughput platform for screening of bioprocess and chromatographic parameters. Graphical abstract Capture and on-column analysis of bound enzyme from non-clarified cell lysate on immobilized metal affinity cryogel minicolumn-based high-throughput platform.

Commercial Generic Bioprocessing Apparatus

NASA Technical Reports Server (NTRS)

1998-01-01

CGBA, a facility developed by BioServe Space Technologies, a NASA Commercial Generic Bioprocessing Space Center, allows a variety of sophisticated bioprocessing research to be performed using a common device. The Fluids Processing Apparatus is essentially a microgravity test tube that allows a variety of complex investigations to be performed in space. This is a glass barrel containing several chambers separated by rubber stoppers. Eight FPAs are placed together in a Group Activation Pack (GAP), which allows all of the research to be started simultaneously by turning a single crank. Eight GAPs, or similar-sized payloads, can be stored in a single CGBA temperature controlled locker, which now uses motor drives to automatically turn the cranks to start and stop experiments. On STS-95, research efforts cover eight major areas that will benefit Earth-based products ranging from the production of pharmaceuticals to fish hatcheries.

Biodecontamination of concrete

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

Rogers, R.D.

1995-12-31

This paper describes the development and results of a demonstration for a continuous bioprocess for mixed waste treatment. A key element of the process is a unique microbial strain, which tolerates high levels of aromatic solvents and surfactants. This microorganism is the biocatalysis of the continuous flow system designed for processing stored liquid scintillation wastes. During the past year, a process demonstration has been conducted on commercial formulation of liquid scintillation cocktails (LSQ). Based on data obtained from this demonstration, the Ohio Environmental Protection Agency granted the Mound Applied Technologies Laboratory a treatability permit allowing the limited processing of actualmore » mixed waste. Since August 1994, the system has been successfully processing stored {open_quotes}hot{close_quotes} LSC waste. This paper discusses the bioprocess, rates of processing, effluent, and implications of bioprocessing for mixed waste management.« less

Growth of near-infrared spectroscopy in pharmaceutical and medical sciences

NASA Astrophysics Data System (ADS)

Ciurczak, Emil W.

2002-06-01

Near-IR Spectroscopy (NIRS) is used extensively in the health services industries: medical research, pharmaceutical production, and bioprocessing. NIRS Is rugged, simple to operate, flexible, and relatively inexpensive. It may be used to monitor the progress biochemical reactions. It is used to control mixing, blending, drying, and coating in pharmaceutical production and is used for imaging and chemical determinations in living patients.

In situ Raman spectroscopy for simultaneous monitoring of multiple process parameters in mammalian cell culture bioreactors.

PubMed

Whelan, Jessica; Craven, Stephen; Glennon, Brian

2012-01-01

In this study, the application of Raman spectroscopy to the simultaneous quantitative determination of glucose, glutamine, lactate, ammonia, glutamate, total cell density (TCD), and viable cell density (VCD) in a CHO fed-batch process was demonstrated in situ in 3 L and 15 L bioreactors. Spectral preprocessing and partial least squares (PLS) regression were used to correlate spectral data with off-line reference data. Separate PLS calibration models were developed for each analyte at the 3 L laboratory bioreactor scale before assessing its transferability to the same bioprocess conducted at the 15 L pilot scale. PLS calibration models were successfully developed for all analytes bar VCD and transferred to the 15 L scale. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

Systems metabolic engineering: the creation of microbial cell factories by rational metabolic design and evolution.

PubMed

Furusawa, Chikara; Horinouchi, Takaaki; Hirasawa, Takashi; Shimizu, Hiroshi

2013-01-01

It is widely acknowledged that in order to establish sustainable societies, production processes should shift from petrochemical-based processes to bioprocesses. Because bioconversion technologies, in which biomass resources are converted to valuable materials, are preferable to processes dependent on fossil resources, the former should be further developed. The following two approaches can be adopted to improve cellular properties and obtain high productivity and production yield of target products: (1) optimization of cellular metabolic pathways involved in various bioprocesses and (2) creation of stress-tolerant cells that can be active even under severe stress conditions in the bioprocesses. Recent progress in omics analyses has facilitated the analysis of microorganisms based on bioinformatics data for molecular breeding and bioprocess development. Systems metabolic engineering is a new area of study, and it has been defined as a methodology in which metabolic engineering and systems biology are integrated to upgrade the designability of industrially useful microorganisms. This chapter discusses multi-omics analyses and rational design methods for molecular breeding. The first is an example of the rational design of metabolic networks for target production by flux balance analysis using genome-scale metabolic models. Recent progress in the development of genome-scale metabolic models and the application of these models to the design of desirable metabolic networks is also described in this example. The second is an example of evolution engineering with omics analyses for the creation of stress-tolerant microorganisms. Long-term culture experiments to obtain the desired phenotypes and omics analyses to identify the phenotypic changes are described here.

Integrated study plan for space bioprocessing (phase 1)

NASA Technical Reports Server (NTRS)

1977-01-01

Current economic evaluation and analytical techniques are applied to decision problems faced by the space bioprocessing program. NASA decision makers are enabled to choose candidate substances, after ranking them according to their potential economic benefit. The determination of appropriate evaluation techniques necessary to obtain measures of potential economic benefits which result from the pursuit of various space bioprocessing endeavors are focused upon. The treatment of each disease is impacted by a successful outcome of space bioprocessing and specify data and other input needs for each candidate substance.

Laser intensity modulated real time monitoring cell growth sensor for bioprocess applications

NASA Astrophysics Data System (ADS)

Kishore, P.; Babu, P. Ravindra; Devi, V. Rama; Maunika, T.; Soujanya, P.; Kishore, P. V. N.; Dinakar, D.

2016-04-01

This article proposes an optical method for monitoring the growth of Escherichia coli in Luria Bertani medium and Saccharomyces cereviciae in YPD. Suitable light is selected which on interaction with the analyte under consideration, gets adsorption / scattered. Required electronic circuitry is designed to drive the laser source and to detect the intensity of light using Photo-detector. All these components are embedded and arranged in a proper way and monitored the growth of the microbs in real time. The sensors results are compared with standard techniques such as colorimeter, Nephelometer and hemocytometer. The experimental results are in good agreement with the existed techniques and well suitable for real time monitoring applications of the growth of the microbs.

Biosensing Technologies for Mycobacterium tuberculosis Detection: Status and New Developments

PubMed Central

Zhou, Lixia; He, Xiaoxiao; He, Dinggeng; Wang, Kemin; Qin, Dilan

2011-01-01

Biosensing technologies promise to improve Mycobacterium tuberculosis (M. tuberculosis) detection and management in clinical diagnosis, food analysis, bioprocess, and environmental monitoring. A variety of portable, rapid, and sensitive biosensors with immediate “on-the-spot” interpretation have been developed for M. tuberculosis detection based on different biological elements recognition systems and basic signal transducer principles. Here, we present a synopsis of current developments of biosensing technologies for M. tuberculosis detection, which are classified on the basis of basic signal transducer principles, including piezoelectric quartz crystal biosensors, electrochemical biosensors, and magnetoelastic biosensors. Special attention is paid to the methods for improving the framework and analytical parameters of the biosensors, including sensitivity and analysis time as well as automation of analysis procedures. Challenges and perspectives of biosensing technologies development for M. tuberculosis detection are also discussed in the final part of this paper. PMID:21437177

The Use of Multidimensional Image-Based Analysis to Accurately Monitor Cell Growth in 3D Bioreactor Culture

PubMed Central

Baradez, Marc-Olivier; Marshall, Damian

2011-01-01

The transition from traditional culture methods towards bioreactor based bioprocessing to produce cells in commercially viable quantities for cell therapy applications requires the development of robust methods to ensure the quality of the cells produced. Standard methods for measuring cell quality parameters such as viability provide only limited information making process monitoring and optimisation difficult. Here we describe a 3D image-based approach to develop cell distribution maps which can be used to simultaneously measure the number, confluency and morphology of cells attached to microcarriers in a stirred tank bioreactor. The accuracy of the cell distribution measurements is validated using in silico modelling of synthetic image datasets and is shown to have an accuracy >90%. Using the cell distribution mapping process and principal component analysis we show how cell growth can be quantitatively monitored over a 13 day bioreactor culture period and how changes to manufacture processes such as initial cell seeding density can significantly influence cell morphology and the rate at which cells are produced. Taken together, these results demonstrate how image-based analysis can be incorporated in cell quality control processes facilitating the transition towards bioreactor based manufacture for clinical grade cells. PMID:22028809

The use of multidimensional image-based analysis to accurately monitor cell growth in 3D bioreactor culture.

PubMed

Baradez, Marc-Olivier; Marshall, Damian

2011-01-01

The transition from traditional culture methods towards bioreactor based bioprocessing to produce cells in commercially viable quantities for cell therapy applications requires the development of robust methods to ensure the quality of the cells produced. Standard methods for measuring cell quality parameters such as viability provide only limited information making process monitoring and optimisation difficult. Here we describe a 3D image-based approach to develop cell distribution maps which can be used to simultaneously measure the number, confluency and morphology of cells attached to microcarriers in a stirred tank bioreactor. The accuracy of the cell distribution measurements is validated using in silico modelling of synthetic image datasets and is shown to have an accuracy >90%. Using the cell distribution mapping process and principal component analysis we show how cell growth can be quantitatively monitored over a 13 day bioreactor culture period and how changes to manufacture processes such as initial cell seeding density can significantly influence cell morphology and the rate at which cells are produced. Taken together, these results demonstrate how image-based analysis can be incorporated in cell quality control processes facilitating the transition towards bioreactor based manufacture for clinical grade cells.

Stem cell bioprocessing: fundamentals and principles

PubMed Central

Placzek, Mark R.; Chung, I-Ming; Macedo, Hugo M.; Ismail, Siti; Mortera Blanco, Teresa; Lim, Mayasari; Min Cha, Jae; Fauzi, Iliana; Kang, Yunyi; Yeo, David C.L.; Yip Joan Ma, Chi; Polak, Julia M.; Panoskaltsis, Nicki; Mantalaris, Athanasios

2008-01-01

In recent years, the potential of stem cell research for tissue engineering-based therapies and regenerative medicine clinical applications has become well established. In 2006, Chung pioneered the first entire organ transplant using adult stem cells and a scaffold for clinical evaluation. With this a new milestone was achieved, with seven patients with myelomeningocele receiving stem cell-derived bladder transplants resulting in substantial improvements in their quality of life. While a bladder is a relatively simple organ, the breakthrough highlights the incredible benefits that can be gained from the cross-disciplinary nature of tissue engineering and regenerative medicine (TERM) that encompasses stem cell research and stem cell bioprocessing. Unquestionably, the development of bioprocess technologies for the transfer of the current laboratory-based practice of stem cell tissue culture to the clinic as therapeutics necessitates the application of engineering principles and practices to achieve control, reproducibility, automation, validation and safety of the process and the product. The successful translation will require contributions from fundamental research (from developmental biology to the ‘omics’ technologies and advances in immunology) and from existing industrial practice (biologics), especially on automation, quality assurance and regulation. The timely development, integration and execution of various components will be critical—failures of the past (such as in the commercialization of skin equivalents) on marketing, pricing, production and advertising should not be repeated. This review aims to address the principles required for successful stem cell bioprocessing so that they can be applied deftly to clinical applications. PMID:19033137

Stem cell bioprocessing: fundamentals and principles.

PubMed

Placzek, Mark R; Chung, I-Ming; Macedo, Hugo M; Ismail, Siti; Mortera Blanco, Teresa; Lim, Mayasari; Cha, Jae Min; Fauzi, Iliana; Kang, Yunyi; Yeo, David C L; Ma, Chi Yip Joan; Polak, Julia M; Panoskaltsis, Nicki; Mantalaris, Athanasios

2009-03-06

In recent years, the potential of stem cell research for tissue engineering-based therapies and regenerative medicine clinical applications has become well established. In 2006, Chung pioneered the first entire organ transplant using adult stem cells and a scaffold for clinical evaluation. With this a new milestone was achieved, with seven patients with myelomeningocele receiving stem cell-derived bladder transplants resulting in substantial improvements in their quality of life. While a bladder is a relatively simple organ, the breakthrough highlights the incredible benefits that can be gained from the cross-disciplinary nature of tissue engineering and regenerative medicine (TERM) that encompasses stem cell research and stem cell bioprocessing. Unquestionably, the development of bioprocess technologies for the transfer of the current laboratory-based practice of stem cell tissue culture to the clinic as therapeutics necessitates the application of engineering principles and practices to achieve control, reproducibility, automation, validation and safety of the process and the product. The successful translation will require contributions from fundamental research (from developmental biology to the 'omics' technologies and advances in immunology) and from existing industrial practice (biologics), especially on automation, quality assurance and regulation. The timely development, integration and execution of various components will be critical-failures of the past (such as in the commercialization of skin equivalents) on marketing, pricing, production and advertising should not be repeated. This review aims to address the principles required for successful stem cell bioprocessing so that they can be applied deftly to clinical applications.

[Advances of consolidated bioprocessing based on recombinant strategy].

PubMed

Zheng, Zongbao; Zhao, Meina; Chen, Tao; Zhao, Xueming

2013-10-01

Lignocellulosic biomass represents an abundant, low-cost and renewable source of potentially fermentable sugars. It is acandidate besides petroleum as feedstock for fuel and chemical production. Recent researches on utilizing lignocellulosicsas feedstock boost development of numerous-promising processes for a variety of fuels and chemicals, such as biodiesel, biohydrogen and ethanol. However, high cost in depolymerization is a primary obstacle preventing the use of lignocellulosic biomass as feedstock. Consolidated bioprocessing (CBP), refers to the bioprocess without any exogenous cellulolyotic enzymes added, converting the lignocellulosic material into biochemicals directly, which could potentially avoid the cost of the dedicated enzyme generation step by incorporating enzyme-generating, biomass-degrading and bioproduct-producing capabilities into a single organism through genetic engineering. There are two CBP strategies, native strategy and recombinant strategy. We mainly introduce the recombinant strategy, including its principle, the two responding styles, the contributions of synthetic biology and metabolic engineering and the future challenges.

Continuous bioprocessing: The real thing this time?

PubMed Central

Farid, Suzanne S; Thompson, Bill; Davidson, Andrew

2014-01-01

The Annual bioProcessUK Conference has acted as the key networking event for bioprocess scientists and engineers in the UK for the past 10 years. The following article is a report from the sessions that focused on continuous bioprocessing during the 10th Annual bioProcessUK Conference (London, December 2013). These sessions were organized by the ‘EPSRC Centre for Innovative Manufacturing in Emergent Macromolecular Therapies’ hosted at University College London. A plenary lecture and workshop provided a forum for participants to debate topical issues in roundtable discussions with industry and academic experts from institutions such as Genzyme, Janssen, Novo Nordisk, Pfizer, Merck, GE Healthcare and University College London. The aim of these particular sessions was to understand better the challenges and opportunities for continuous bioprocessing in the bioprocessing sector. PMID:25484060

Therapeutic antibodies: market considerations, disease targets and bioprocessing.

PubMed

Elvin, John G; Couston, Ruairidh G; van der Walle, Christopher F

2013-01-02

Antibodies are well established in mainstream clinical practice and present an exciting area for collaborative research and development in industry and academia alike. In this review, we will provide an overview of the current market and an outlook to 2015, focussing on whole antibody molecules while acknowledging the next generation scaffolds containing variable fragments. The market will be discussed in the context of disease targets, particularly in the areas of oncology and immune disorders which generate the greatest revenue by a wide margin. Emerging targets include central nervous system disorders which will also stimulate new delivery strategies. It is becoming increasingly apparent that a better understanding of bioprocessing is required in order to optimize the steps involved in the preparation of a protein prior to formulation. The latter is outside the scope of this review and nor is it our intention to discuss protein delivery and pharmacokinetics. The challenges that lie ahead include the discovery of new disease targets and the development of robust bioprocessing operations. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

Generic HPLC platform for automated enzyme reaction monitoring: Advancing the assay toolbox for transaminases and other PLP-dependent enzymes.

PubMed

Börner, Tim; Grey, Carl; Adlercreutz, Patrick

2016-08-01

Methods for rapid and direct quantification of enzyme kinetics independent of the substrate stand in high demand for both fundamental research and bioprocess development. This study addresses the need for a generic method by developing an automated, standardizable HPLC platform monitoring reaction progress in near real-time. The method was applied to amine transaminase (ATA) catalyzed reactions intensifying process development for chiral amine synthesis. Autosampler-assisted pipetting facilitates integrated mixing and sampling under controlled temperature. Crude enzyme formulations in high and low substrate concentrations can be employed. Sequential, small (1 µL) sample injections and immediate detection after separation permits fast reaction monitoring with excellent sensitivity, accuracy and reproducibility. Due to its modular design, different chromatographic techniques, e.g. reverse phase and size exclusion chromatography (SEC) can be employed. A novel assay for pyridoxal 5'-phosphate-dependent enzymes is presented using SEC for direct monitoring of enzyme-bound and free reaction intermediates. Time-resolved changes of the different cofactor states, e.g. pyridoxal 5'-phosphate, pyridoxamine 5'-phosphate and the internal aldimine were traced in both half reactions. The combination of the automated HPLC platform with SEC offers a method for substrate-independent screening, which renders a missing piece in the assay and screening toolbox for ATAs and other PLP-dependent enzymes. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hydrodynamic chronoamperometry for probing kinetics of anaerobic microbial metabolism - case study of Faecalibacterium prausnitzii

NASA Astrophysics Data System (ADS)

Prévoteau, Antonin; Geirnaert, Annelies; Arends, Jan B. A.; Lannebère, Sylvain; van de Wiele, Tom; Rabaey, Korneel

2015-07-01

Monitoring in vitro the metabolic activity of microorganisms aids bioprocesses and enables better understanding of microbial metabolism. Redox mediators can be used for this purpose via different electrochemical techniques that are either complex or only provide non-continuous data. Hydrodynamic chronoamperometry using a rotating disc electrode (RDE) can alleviate these issues but was seldom used and is poorly characterized. The kinetics of Faecalibacterium prausnitzii A2-165, a beneficial gut microbe, were determined using a RDE with riboflavin as redox probe. This butyrate producer anaerobically ferments glucose and reduces riboflavin whose continuous monitoring on a RDE provided highly accurate kinetic measurements of its metabolism, even at low cell densities. The metabolic reaction rate increased linearly over a broad range of cell concentrations (9 × 104 to 5 × 107 cells.mL-1). Apparent Michaelis-Menten kinetics was observed with respect to riboflavin (KM = 6 μM kcat = 5.3×105 s-1, at 37 °C) and glucose (KM = 6 μM kcat = 2.4 × 105 s-1). The short temporal resolution allows continuous monitoring of fast cellular events such as kinetics inhibition with butyrate. Furthermore, we detected for the first time riboflavin reduction by another potential probiotic, Butyricicoccus pullicaecorum. The ability of the RDE for fast, accurate, simple and continuous measurements makes it an ad hoc tool for assessing bioprocesses at high resolution.

Does Nonlinear Modeling Play a Role in Plasmid Bioprocess Monitoring Using Fourier Transform Infrared Spectra?

PubMed

Lopes, Marta B; Calado, Cecília R C; Figueiredo, Mário A T; Bioucas-Dias, José M

2017-06-01

The monitoring of biopharmaceutical products using Fourier transform infrared (FT-IR) spectroscopy relies on calibration techniques involving the acquisition of spectra of bioprocess samples along the process. The most commonly used method for that purpose is partial least squares (PLS) regression, under the assumption that a linear model is valid. Despite being successful in the presence of small nonlinearities, linear methods may fail in the presence of strong nonlinearities. This paper studies the potential usefulness of nonlinear regression methods for predicting, from in situ near-infrared (NIR) and mid-infrared (MIR) spectra acquired in high-throughput mode, biomass and plasmid concentrations in Escherichia coli DH5-α cultures producing the plasmid model pVAX-LacZ. The linear methods PLS and ridge regression (RR) are compared with their kernel (nonlinear) versions, kPLS and kRR, as well as with the (also nonlinear) relevance vector machine (RVM) and Gaussian process regression (GPR). For the systems studied, RR provided better predictive performances compared to the remaining methods. Moreover, the results point to further investigation based on larger data sets whenever differences in predictive accuracy between a linear method and its kernelized version could not be found. The use of nonlinear methods, however, shall be judged regarding the additional computational cost required to tune their additional parameters, especially when the less computationally demanding linear methods herein studied are able to successfully monitor the variables under study.

FTIR microspectroscopy for rapid screening and monitoring of polyunsaturated fatty acid production in commercially valuable marine yeasts and protists.

PubMed

Vongsvivut, Jitraporn; Heraud, Philip; Gupta, Adarsha; Puri, Munish; McNaughton, Don; Barrow, Colin J

2013-10-21

The increase in polyunsaturated fatty acid (PUFA) consumption has prompted research into alternative resources other than fish oil. In this study, a new approach based on focal-plane-array Fourier transform infrared (FPA-FTIR) microspectroscopy and multivariate data analysis was developed for the characterisation of some marine microorganisms. Cell and lipid compositions in lipid-rich marine yeasts collected from the Australian coast were characterised in comparison to a commercially available PUFA-producing marine fungoid protist, thraustochytrid. Multivariate classification methods provided good discriminative accuracy evidenced from (i) separation of the yeasts from thraustochytrids and distinct spectral clusters among the yeasts that conformed well to their biological identities, and (ii) correct classification of yeasts from a totally independent set using cross-validation testing. The findings further indicated additional capability of the developed FPA-FTIR methodology, when combined with partial least squares regression (PLSR) analysis, for rapid monitoring of lipid production in one of the yeasts during the growth period, which was achieved at a high accuracy compared to the results obtained from the traditional lipid analysis based on gas chromatography. The developed FTIR-based approach when coupled to programmable withdrawal devices and a cytocentrifugation module would have strong potential as a novel online monitoring technology suited for bioprocessing applications and large-scale production.

Continuous bioprocessing: the real thing this time? 10(th) Annual bioProcessUK Conference, December 3-4, 2013, London, UK.

PubMed

Farid, Suzanne S; Thompson, Bill; Davidson, Andrew

2014-01-01

The Annual bioProcessUK Conference has acted as the key networking event for bioprocess scientists and engineers in the UK for the past 10 years. The following article is a report from the sessions that focused on continuous bioprocessing during the 10(th) Annual bioProcessUK Conference (London, December 2013). These sessions were organized by the 'EPSRC Centre for Innovative Manufacturing in Emergent Macromolecular Therapies' hosted at University College London. A plenary lecture and workshop provided a forum for participants to debate topical issues in roundtable discussions with industry and academic experts from institutions such as Genzyme, Janssen, Novo Nordisk, Pfizer, Merck, GE Healthcare and University College London. The aim of these particular sessions was to understand better the challenges and opportunities for continuous bioprocessing in the bioprocessing sector.

Bioprocessing of a stored mixed liquid waste

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

Wolfram, J.H.; Rogers, R.D.; Finney, R.

1995-12-31

This paper describes the development and results of a demonstration for a continuous bioprocess for mixed waste treatment. A key element of the process is an unique microbial strain which tolerates high levels of aromatic solvents and surfactants. This microorganism is the biocatalysis of the continuous flow system designed for the processing of stored liquid scintillation wastes. During the past year a process demonstration has been conducted on commercial formulation of liquid scintillation cocktails (LSC). Based on data obtained from this demonstration, the Ohio EPA granted the Mound Applied Technologies Lab a treatability permit allowing the limited processing of actualmore » mixed waste. Since August 1994, the system has been successfully processing stored, {open_quotes}hot{close_quotes} LSC waste. The initial LSC waste fed into the system contained 11% pseudocumene and detectable quantities of plutonium. Another treated waste stream contained pseudocumene and tritium. Data from this initial work shows that the hazardous organic solvent, and pseudocumene have been removed due to processing, leaving the aqueous low level radioactive waste. Results to date have shown that living cells are not affected by the dissolved plutonium and that 95% of the plutonium was sorbed to the biomass. This paper discusses the bioprocess, rates of processing, effluent, and the implications of bioprocessing for mixed waste management.« less

Allogeneic cell therapy bioprocess economics and optimization: downstream processing decisions.

PubMed

Hassan, Sally; Simaria, Ana S; Varadaraju, Hemanthram; Gupta, Siddharth; Warren, Kim; Farid, Suzanne S

2015-01-01

To develop a decisional tool to identify the most cost effective process flowsheets for allogeneic cell therapies across a range of production scales. A bioprocess economics and optimization tool was built to assess competing cell expansion and downstream processing (DSP) technologies. Tangential flow filtration was generally more cost-effective for the lower cells/lot achieved in planar technologies and fluidized bed centrifugation became the only feasible option for handling large bioreactor outputs. DSP bottlenecks were observed at large commercial lot sizes requiring multiple large bioreactors. The DSP contribution to the cost of goods/dose ranged between 20-55%, and 50-80% for planar and bioreactor flowsheets, respectively. This analysis can facilitate early decision-making during process development.

Optimal bioprocess design through a gene regulatory network - growth kinetic hybrid model: Towards Replacing Monod kinetics.

PubMed

Tsipa, Argyro; Koutinas, Michalis; Usaku, Chonlatep; Mantalaris, Athanasios

2018-05-02

Currently, design and optimisation of biotechnological bioprocesses is performed either through exhaustive experimentation and/or with the use of empirical, unstructured growth kinetics models. Whereas, elaborate systems biology approaches have been recently explored, mixed-substrate utilisation is predominantly ignored despite its significance in enhancing bioprocess performance. Herein, bioprocess optimisation for an industrially-relevant bioremediation process involving a mixture of highly toxic substrates, m-xylene and toluene, was achieved through application of a novel experimental-modelling gene regulatory network - growth kinetic (GRN-GK) hybrid framework. The GRN model described the TOL and ortho-cleavage pathways in Pseudomonas putida mt-2 and captured the transcriptional kinetics expression patterns of the promoters. The GRN model informed the formulation of the growth kinetics model replacing the empirical and unstructured Monod kinetics. The GRN-GK framework's predictive capability and potential as a systematic optimal bioprocess design tool, was demonstrated by effectively predicting bioprocess performance, which was in agreement with experimental values, when compared to four commonly used models that deviated significantly from the experimental values. Significantly, a fed-batch biodegradation process was designed and optimised through the model-based control of TOL Pr promoter expression resulting in 61% and 60% enhanced pollutant removal and biomass formation, respectively, compared to the batch process. This provides strong evidence of model-based bioprocess optimisation at the gene level, rendering the GRN-GK framework as a novel and applicable approach to optimal bioprocess design. Finally, model analysis using global sensitivity analysis (GSA) suggests an alternative, systematic approach for model-driven strain modification for synthetic biology and metabolic engineering applications. Copyright © 2018. Published by Elsevier Inc.

A rational design for hepatitis B virus X protein refolding and bioprocess development guided by second virial coefficient studies.

PubMed

Basu, Anindya; Chen, Wei Ning; Leong, Susanna Su Jan

2011-04-01

The hepatitis B virus X (HBx) protein is well known for its role in hepatitis B virus infection that often leads to hepatocellular carcinoma. Despite the clinical importance of HBx, there is little progress in anti-HBx drug development strategies due to shortage of HBx from native sources. Consistent expression of HBx as insoluble inclusion bodies within various expression systems has largely hindered HBx manufacturing via economical biosynthesis routes. Confronted by this roadblock, this study aims to quantitatively understand HBx protein behaviour in solution that will guide the rational development of a refolding-based bioprocess for HBx production. Second virial coefficient (SVC) measurements were employed to study the effects of varying physicochemical parameters on HBx intermolecular protein interaction. The SVC results suggest that covalent HBx aggregates play a key role in protein destabilisation during refolding. The use of an SVC-optimised refolding environment yielded bioactive and soluble HBx proteins from the denatured-reduced inclusion body state. This study provides new knowledge on HBx solubility behaviour in vitro, which is important in structure-function elucidation behaviour of this hydrophobic protein. Importantly, a rational refolding-based Escherichia coli bioprocess that can deliver purified and soluble HBx at large scale is successfully developed, which opens the way for rapid preparation of soluble HBx for further clinical and characterisation studies.

Recent Advances and Challenges towards Sustainable Polyhydroxyalkanoate (PHA) Production.

PubMed

Kourmentza, Constantina; Plácido, Jersson; Venetsaneas, Nikolaos; Burniol-Figols, Anna; Varrone, Cristiano; Gavala, Hariklia N; Reis, Maria A M

2017-06-11

Sustainable biofuels, biomaterials, and fine chemicals production is a critical matter that research teams around the globe are focusing on nowadays. Polyhydroxyalkanoates represent one of the biomaterials of the future due to their physicochemical properties, biodegradability, and biocompatibility. Designing efficient and economic bioprocesses, combined with the respective social and environmental benefits, has brought together scientists from different backgrounds highlighting the multidisciplinary character of such a venture. In the current review, challenges and opportunities regarding polyhydroxyalkanoate production are presented and discussed, covering key steps of their overall production process by applying pure and mixed culture biotechnology, from raw bioprocess development to downstream processing.

Teaching bioprocess engineering to undergraduates: Multidisciplinary hands-on training in a one-week practical course.

PubMed

Henkel, Marius; Zwick, Michaela; Beuker, Janina; Willenbacher, Judit; Baumann, Sandra; Oswald, Florian; Neumann, Anke; Siemann-Herzberg, Martin; Syldatk, Christoph; Hausmann, Rudolf

2015-01-01

Bioprocess engineering is a highly interdisciplinary field of study which is strongly benefited by practical courses where students can actively experience the interconnection between biology, engineering, and physical sciences. This work describes a lab course developed for 2nd year undergraduate students of bioprocess engineering and related disciplines, where students are challenged with a real-life bioprocess-engineering application, the production of recombinant protein in a fed-batch process. The lab course was designed to introduce students to the subject of operating and supervising an experiment in a bioreactor, along with the analysis of collected data and a final critical evaluation of the experiment. To provide visual feedback of the experimental outcome, the organism used during class was Escherichia coli which carried a plasmid to recombinantly produce enhanced green fluorescent protein (eGFP) upon induction. This can easily be visualized in both the bioreactor and samples by using ultraviolet light. The lab course is performed with bioreactors of the simplest design, and is therefore highly flexible, robust and easy to reproduce. As part of this work the implementation and framework, the results, the evaluation and assessment of student learning combined with opinion surveys are presented, which provides a basis for instructors intending to implement a similar lab course at their respective institution. © 2015 by the International Union of Biochemistry and Molecular Biology.

Development of a circulation direct sampling and monitoring system for O2 and CO2 concentrations in the gas-liquid phases of shake-flask systems during microbial cell culture.

PubMed

Takahashi, Masato; Sawada, Yoshisuke; Aoyagi, Hideki

2017-08-23

Monitoring the environmental factors during shake-flask culture of microorganisms can help to optimise the initial steps of bioprocess development. Herein, we developed a circulation direct monitoring and sampling system (CDMSS) that can monitor the behaviour of CO 2 and O 2 in the gas-liquid phases and obtain a sample without interrupting the shaking of the culture in Erlenmeyer flasks capped with breathable culture plugs. Shake-flask culturing of Escherichia coli using this set-up indicated that a high concentration of CO 2 accumulated not only in the headspace (maximum ~100 mg/L) but also in the culture broth (maximum ~85 mg/L) during the logarithmic phase (4.5-9.0 h). By packing a CO 2 absorbent in the gas circulation unit of CDMSS, a specialised shake-flask culture was developed to remove CO 2 from the headspace. It was posited that removing CO 2 from the headspace would suppress increases in the dissolved CO 2 concentration in the culture broth (maximum ~15 mg/L). Furthermore, the logarithmic growth phase (4.5-12.0 h) was extended, the U.O.D. 580 and pH value increased, and acetic acid concentration was reduced, compared with the control. To our knowledge, this is the first report of a method aimed at improving the growth of E. coli cells without changing the composition of the medium, temperature, and shaking conditions.

Design-for-Six-Sigma To Develop a Bioprocess Knowledge Management Framework.

PubMed

Junker, Beth; Maheshwari, Gargi; Ranheim, Todd; Altaras, Nedim; Stankevicz, Michael; Harmon, Lori; Rios, Sandra; D'anjou, Marc

2011-01-01

Owing to the high costs associated with biopharmaceutical development, considerable pressure has developed for the biopharmaceutical industry to increase productivity by becoming more lean and flexible. The ability to reuse knowledge was identified as one key advantage to streamline productivity, efficiently use resources, and ultimately perform better than the competition. A knowledge management (KM) strategy was assembled for bioprocess-related information using the technique of Design-for-Six-Sigma (DFSS). This strategy supported quality-by-design and process validation efforts for pipeline as well as licensed products. The DFSS technique was selected because it was both streamlined and efficient. These characteristics permitted development of a KM strategy with minimized team leader and team member resources. DFSS also placed a high emphasis on the voice of the customer, information considered crucial to the selection of solutions most appropriate for the current knowledge-based challenges of the organization. The KM strategy developed was comprised of nine workstreams, constructed from related solution buckets which in turn were assembled from the individual solution tasks that were identified. Each workstream's detailed design was evaluated against published and established best practices, as well as the KM strategy project charter and design inputs. Gaps and risks were identified and mitigated as necessary to improve the robustness of the proposed strategy. Aggregated resources (specifically expense/capital funds and staff) and timing were estimated to obtain vital management sponsorship for implementation. Where possible, existing governance and divisional/corporate information technology efforts were leveraged to minimize the additional bioprocess resources required for implementation. Finally, leading and lagging indicator metrics were selected to track the success of pilots and eventual implementation. A knowledge management framework was assembled for bioprocess-related information using a streamlined and efficient technique that minimized team leader and member resources. The technique also highly emphasized input from the staff, who generated and used the knowledge, information considered crucial to selection of solutions most appropriate for the current knowledge-based challenges in the organization. The framework developed was comprised of nine workstreams, constructed from related solution buckets which were assembled from individual solution tasks that were identified. Each workstream's detailed design was evaluated against published and established best practices, as well as the project charter and design inputs. Gaps and risks were identified and mitigated to improve robustness of the proposed framework. Aggregated resources (specifically expense/capital funds and staff) and timing were estimated to obtain vital management sponsorship for implementation. Where possible, existing governance and information technology efforts were leveraged to minimize additional bioprocess resources required for implementation. Finally, metrics were selected to track the success of pilots and eventual implementation.

The use of 'Omics technology to rationally improve industrial mammalian cell line performance.

PubMed

Lewis, Amanda M; Abu-Absi, Nicholas R; Borys, Michael C; Li, Zheng Jian

2016-01-01

Biologics represent an increasingly important class of therapeutics, with 7 of the 10 top selling drugs from 2013 being in this class. Furthermore, health authority approval of biologics in the immuno-oncology space is expected to transform treatment of patients with debilitating and deadly diseases. The growing importance of biologics in the healthcare field has also resulted in the recent approvals of several biosimilars. These recent developments, combined with pressure to provide treatments at lower costs to payers, are resulting in increasing need for the industry to quickly and efficiently develop high yielding, robust processes for the manufacture of biologics with the ability to control quality attributes within narrow distributions. Achieving this level of manufacturing efficiency and the ability to design processes capable of regulating growth, death and other cellular pathways through manipulation of media, feeding strategies, and other process parameters will undoubtedly be facilitated through systems biology tools generated in academic and public research communities. Here we discuss the intersection of systems biology, 'Omics technologies, and mammalian bioprocess sciences. Specifically, we address how these methods in conjunction with traditional monitoring techniques represent a unique opportunity to better characterize and understand host cell culture state, shift from an empirical to rational approach to process development and optimization of bioreactor cultivation processes. We summarize the following six key areas: (i) research applied to parental, non-recombinant cell lines; (ii) systems level datasets generated with recombinant cell lines; (iii) datasets linking phenotypic traits to relevant biomarkers; (iv) data depositories and bioinformatics tools; (v) in silico model development, and (vi) examples where these approaches have been used to rationally improve cellular processes. We critically assess relevant and state of the art research being conducted in academic, government and industrial laboratories. Furthermore, we apply our expertise in bioprocess to define a potential model for integration of these systems biology approaches into biologics development. © 2015 Wiley Periodicals, Inc.

Study of Material Used in Nanotechnology for the Recycling of Industrial Waste Water

NASA Astrophysics Data System (ADS)

Larbi, L.; Fertikh, N.; Toubal, A.

The objective of our study is to recycle the industrial waste water of a industrial Complex after treatment by the bioprocess MBR (membrane bioreactor). In order to apply this bioprocess, the water quality in question was first of all studied. To characterize this industrial waste water, a series of physicochemical analysis was carried out according to standardized directives and methods. Following-up the water quality to meet the regulatory requirements with rejection of this industrial waste water, a study was done thanks to the permanently monitoring of the following relevant parameters(P): the flow, the potential of hydrogen (pH), the total suspended solids(TSS), the turbidity (Turb), the chemical oxygen demand (COD),the biochemical oxygen demand (BOD), the Kjeldahl total nitrogen (KTN) and ammonia (NH4+), the total phosphorus (Ptot), the fluorine (F), the oils (O), the fats (F) and the phenols (Ph). According to collected information, it was established the sampling rates to which the quality control was done, the selected analytical methods were validated by the control charts and the analysis test number was determined by the Cochran test. The results of the quality control show that some rejected water contents are not in the Algerian standards, but, in our case, the objective is the preoccupation for a standard setting of these industrial water parameters so as to recycle it. The process adopted by MBR for waste water treatment is being studied, first in the development of the experimental characterizing of the reactor and the selected membrane.

Bioprocess automation on a Mini Pilot Plant enables fast quantitative microbial phenotyping.

PubMed

Unthan, Simon; Radek, Andreas; Wiechert, Wolfgang; Oldiges, Marco; Noack, Stephan

2015-03-11

The throughput of cultivation experiments in bioprocess development has drastically increased in recent years due to the availability of sophisticated microliter scale cultivation devices. However, as these devices still require time-consuming manual work, the bottleneck was merely shifted to media preparation, inoculation and finally the analyses of cultivation samples. A first step towards solving these issues was undertaken in our former study by embedding a BioLector in a robotic workstation. This workstation already allowed for the optimization of heterologous protein production processes, but remained limited when aiming for the characterization of small molecule producer strains. In this work, we extended our workstation to a versatile Mini Pilot Plant (MPP) by integrating further robotic workflows and microtiter plate assays that now enable a fast and accurate phenotyping of a broad range of microbial production hosts. A fully automated harvest procedure was established, which repeatedly samples up to 48 wells from BioLector cultivations in response to individually defined trigger conditions. The samples are automatically clarified by centrifugation and finally frozen for subsequent analyses. Sensitive metabolite assays in 384-well plate scale were integrated on the MPP for the direct determination of substrate uptake (specifically D-glucose and D-xylose) and product formation (specifically amino acids). In a first application, we characterized a set of Corynebacterium glutamicum L-lysine producer strains and could rapidly identify a unique strain showing increased L-lysine titers, which was subsequently confirmed in lab-scale bioreactor experiments. In a second study, we analyzed the substrate uptake kinetics of a previously constructed D-xylose-converting C. glutamicum strain during cultivation on mixed carbon sources in a fully automated experiment. The presented MPP is designed to face the challenges typically encountered during early-stage bioprocess development. Especially the bottleneck of sample analyses from fast and parallelized microtiter plate cultivations can be solved using cutting-edge robotic automation. As robotic workstations become increasingly attractive for biotechnological research, we expect our setup to become a template for future bioprocess development.

A framework for accelerated phototrophic bioprocess development: integration of parallelized microscale cultivation, laboratory automation and Kriging-assisted experimental design.

PubMed

Morschett, Holger; Freier, Lars; Rohde, Jannis; Wiechert, Wolfgang; von Lieres, Eric; Oldiges, Marco

2017-01-01

Even though microalgae-derived biodiesel has regained interest within the last decade, industrial production is still challenging for economic reasons. Besides reactor design, as well as value chain and strain engineering, laborious and slow early-stage parameter optimization represents a major drawback. The present study introduces a framework for the accelerated development of phototrophic bioprocesses. A state-of-the-art micro-photobioreactor supported by a liquid-handling robot for automated medium preparation and product quantification was used. To take full advantage of the technology's experimental capacity, Kriging-assisted experimental design was integrated to enable highly efficient execution of screening applications. The resulting platform was used for medium optimization of a lipid production process using Chlorella vulgaris toward maximum volumetric productivity. Within only four experimental rounds, lipid production was increased approximately threefold to 212 ± 11 mg L -1  d -1 . Besides nitrogen availability as a key parameter, magnesium, calcium and various trace elements were shown to be of crucial importance. Here, synergistic multi-parameter interactions as revealed by the experimental design introduced significant further optimization potential. The integration of parallelized microscale cultivation, laboratory automation and Kriging-assisted experimental design proved to be a fruitful tool for the accelerated development of phototrophic bioprocesses. By means of the proposed technology, the targeted optimization task was conducted in a very timely and material-efficient manner.

Advancing biopharmaceutical process development by system-level data analysis and integration of omics data.

PubMed

Schaub, Jochen; Clemens, Christoph; Kaufmann, Hitto; Schulz, Torsten W

2012-01-01

Development of efficient bioprocesses is essential for cost-effective manufacturing of recombinant therapeutic proteins. To achieve further process improvement and process rationalization comprehensive data analysis of both process data and phenotypic cell-level data is essential. Here, we present a framework for advanced bioprocess data analysis consisting of multivariate data analysis (MVDA), metabolic flux analysis (MFA), and pathway analysis for mapping of large-scale gene expression data sets. This data analysis platform was applied in a process development project with an IgG-producing Chinese hamster ovary (CHO) cell line in which the maximal product titer could be increased from about 5 to 8 g/L.Principal component analysis (PCA), k-means clustering, and partial least-squares (PLS) models were applied to analyze the macroscopic bioprocess data. MFA and gene expression analysis revealed intracellular information on the characteristics of high-performance cell cultivations. By MVDA, for example, correlations between several essential amino acids and the product concentration were observed. Also, a grouping into rather cell specific productivity-driven and process control-driven processes could be unraveled. By MFA, phenotypic characteristics in glycolysis, glutaminolysis, pentose phosphate pathway, citrate cycle, coupling of amino acid metabolism to citrate cycle, and in the energy yield could be identified. By gene expression analysis 247 deregulated metabolic genes were identified which are involved, inter alia, in amino acid metabolism, transport, and protein synthesis.

Recent Advances and Challenges towards Sustainable Polyhydroxyalkanoate (PHA) Production

PubMed Central

Kourmentza, Constantina; Plácido, Jersson; Venetsaneas, Nikolaos; Burniol-Figols, Anna; Varrone, Cristiano; Gavala, Hariklia N.; Reis, Maria A. M.

2017-01-01

Sustainable biofuels, biomaterials, and fine chemicals production is a critical matter that research teams around the globe are focusing on nowadays. Polyhydroxyalkanoates represent one of the biomaterials of the future due to their physicochemical properties, biodegradability, and biocompatibility. Designing efficient and economic bioprocesses, combined with the respective social and environmental benefits, has brought together scientists from different backgrounds highlighting the multidisciplinary character of such a venture. In the current review, challenges and opportunities regarding polyhydroxyalkanoate production are presented and discussed, covering key steps of their overall production process by applying pure and mixed culture biotechnology, from raw bioprocess development to downstream processing. PMID:28952534

Conversion of bioprocess ethanol to industrial chemical products - Applications of process models for energy-economic assessments

NASA Technical Reports Server (NTRS)

Rohatgi, Naresh K.; Ingham, John D.

1992-01-01

An assessment approach for accurate evaluation of bioprocesses for large-scale production of industrial chemicals is presented. Detailed energy-economic assessments of a potential esterification process were performed, where ethanol vapor in the presence of water from a bioreactor is catalytically converted to ethyl acetate. Results show that such processes are likely to become more competitive as the cost of substrates decreases relative to petrolium costs. A commercial ASPEN process simulation provided a reasonably consistent comparison with energy economics calculated using JPL developed software. Detailed evaluations of the sensitivity of production cost to material costs and annual production rates are discussed.

ISS Flight 2A.2B (STS-106): Commercial Generic Bioprocessing Apparatus (CGBA) Payload BioServe Space Technologies

NASA Technical Reports Server (NTRS)

Stodieck, Louis; Klaus, David

2001-01-01

The two experiments housed in the Commercial Generic Bioprocessing Apparatus (CGBA) during STS-106 were designed to explore how biological processes are affected by microgravity. The first was a developmental study into the effects of microgravity on motor-neuronal growth in the fruit fly species Drosophila melanogaster and the second study was designed to characterize changes in kidney cell gene expression. The objective of the primary experiment, called NIH-B1, was to determine how gravity affects neuronal development of the D. melanogaster embryo and larvae in microgravity, specifically observing the neural connections to muscle fibers.

Biopharmaceuticals from microorganisms: from production to purification.

PubMed

Jozala, Angela Faustino; Geraldes, Danilo Costa; Tundisi, Louise Lacalendola; Feitosa, Valker de Araújo; Breyer, Carlos Alexandre; Cardoso, Samuel Leite; Mazzola, Priscila Gava; Oliveira-Nascimento, Laura de; Rangel-Yagui, Carlota de Oliveira; Magalhães, Pérola de Oliveira; Oliveira, Marcos Antonio de; Pessoa, Adalberto

2016-12-01

The use of biopharmaceuticals dates from the 19th century and within 5-10 years, up to 50% of all drugs in development will be biopharmaceuticals. In the 1980s, the biopharmaceutical industry experienced a significant growth in the production and approval of recombinant proteins such as interferons (IFN α, β, and γ) and growth hormones. The production of biopharmaceuticals, known as bioprocess, involves a wide range of techniques. In this review, we discuss the technology involved in the bioprocess and describe the available strategies and main advances in microbial fermentation and purification process to obtain biopharmaceuticals. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Monitoring lipase/esterase activity by stopped flow in a sequential injection analysis system using p-nitrophenyl butyrate.

PubMed

Pliego, Jorge; Mateos, Juan Carlos; Rodriguez, Jorge; Valero, Francisco; Baeza, Mireia; Femat, Ricardo; Camacho, Rosa; Sandoval, Georgina; Herrera-López, Enrique J

2015-01-27

Lipases and esterases are biocatalysts used at the laboratory and industrial level. To obtain the maximum yield in a bioprocess, it is important to measure key variables, such as enzymatic activity. The conventional method for monitoring hydrolytic activity is to take out a sample from the bioreactor to be analyzed off-line at the laboratory. The disadvantage of this approach is the long time required to recover the information from the process, hindering the possibility to develop control systems. New strategies to monitor lipase/esterase activity are necessary. In this context and in the first approach, we proposed a lab-made sequential injection analysis system to analyze off-line samples from shake flasks. Lipase/esterase activity was determined using p-nitrophenyl butyrate as the substrate. The sequential injection analysis allowed us to measure the hydrolytic activity from a sample without dilution in a linear range from 0.05-1.60 U/mL, with the capability to reach sample dilutions up to 1000 times, a sampling frequency of five samples/h, with a kinetic reaction of 5 min and a relative standard deviation of 8.75%. The results are promising to monitor lipase/esterase activity in real time, in which optimization and control strategies can be designed.

Monitoring Lipase/Esterase Activity by Stopped Flow in a Sequential Injection Analysis System Using p-Nitrophenyl Butyrate

PubMed Central

Pliego, Jorge; Mateos, Juan Carlos; Rodriguez, Jorge; Valero, Francisco; Baeza, Mireia; Femat, Ricardo; Camacho, Rosa; Sandoval, Georgina; Herrera-López, Enrique J.

2015-01-01

Lipases and esterases are biocatalysts used at the laboratory and industrial level. To obtain the maximum yield in a bioprocess, it is important to measure key variables, such as enzymatic activity. The conventional method for monitoring hydrolytic activity is to take out a sample from the bioreactor to be analyzed off-line at the laboratory. The disadvantage of this approach is the long time required to recover the information from the process, hindering the possibility to develop control systems. New strategies to monitor lipase/esterase activity are necessary. In this context and in the first approach, we proposed a lab-made sequential injection analysis system to analyze off-line samples from shake flasks. Lipase/esterase activity was determined using p-nitrophenyl butyrate as the substrate. The sequential injection analysis allowed us to measure the hydrolytic activity from a sample without dilution in a linear range from 0.05–1.60 U/mL, with the capability to reach sample dilutions up to 1000 times, a sampling frequency of five samples/h, with a kinetic reaction of 5 min and a relative standard deviation of 8.75%. The results are promising to monitor lipase/esterase activity in real time, in which optimization and control strategies can be designed. PMID:25633600

Multiplexed chemostat system for quantification of biodiversity and ecosystem functioning in anaerobic digestion

PubMed Central

Plouchart, Diane; Guizard, Guillaume; Latrille, Eric

2018-01-01

Continuous cultures in chemostats have proven their value in microbiology, microbial ecology, systems biology and bioprocess engineering, among others. In these systems, microbial growth and ecosystem performance can be quantified under stable and defined environmental conditions. This is essential when linking microbial diversity to ecosystem function. Here, a new system to test this link in anaerobic, methanogenic microbial communities is introduced. Rigorously replicated experiments or a suitable experimental design typically require operating several chemostats in parallel. However, this is labor intensive, especially when measuring biogas production. Commercial solutions for multiplying reactors performing continuous anaerobic digestion exist but are expensive and use comparably large reactor volumes, requiring the preparation of substantial amounts of media. Here, a flexible system of Lab-scale Automated and Multiplexed Anaerobic Chemostat system (LAMACs) with a working volume of 200 mL is introduced. Sterile feeding, biomass wasting and pressure monitoring are automated. One module containing six reactors fits the typical dimensions of a lab bench. Thanks to automation, time required for reactor operation and maintenance are reduced compared to traditional lab-scale systems. Several modules can be used together, and so far the parallel operation of 30 reactors was demonstrated. The chemostats are autoclavable. Parameters like reactor volume, flow rates and operating temperature can be freely set. The robustness of the system was tested in a two-month long experiment in which three inocula in four replicates, i.e., twelve continuous digesters were monitored. Statistically significant differences in the biogas production between inocula were observed. In anaerobic digestion, biogas production and consequently pressure development in a closed environment is a proxy for ecosystem performance. The precision of the pressure measurement is thus crucial. The measured maximum and minimum rates of gas production could be determined at the same precision. The LAMACs is a tool that enables us to put in practice the often-demanded need for replication and rigorous testing in microbial ecology as well as bioprocess engineering. PMID:29518106

Design of experiments applications in bioprocessing: concepts and approach.

PubMed

Kumar, Vijesh; Bhalla, Akriti; Rathore, Anurag S

2014-01-01

Most biotechnology unit operations are complex in nature with numerous process variables, feed material attributes, and raw material attributes that can have significant impact on the performance of the process. Design of experiments (DOE)-based approach offers a solution to this conundrum and allows for an efficient estimation of the main effects and the interactions with minimal number of experiments. Numerous publications illustrate application of DOE towards development of different bioprocessing unit operations. However, a systematic approach for evaluation of the different DOE designs and for choosing the optimal design for a given application has not been published yet. Through this work we have compared the I-optimal and D-optimal designs to the commonly used central composite and Box-Behnken designs for bioprocess applications. A systematic methodology is proposed for construction of the model and for precise prediction of the responses for the three case studies involving some of the commonly used unit operations in downstream processing. Use of Akaike information criterion for model selection has been examined and found to be suitable for the applications under consideration. © 2013 American Institute of Chemical Engineers.

Bioprocessing strategies for the large-scale production of human mesenchymal stem cells: a review.

PubMed

Panchalingam, Krishna M; Jung, Sunghoon; Rosenberg, Lawrence; Behie, Leo A

2015-11-23

Human mesenchymal stem cells (hMSCs), also called mesenchymal stromal cells, have been of great interest in regenerative medicine applications because of not only their differentiation potential but also their ability to secrete bioactive factors that can modulate the immune system and promote tissue repair. This potential has initiated many early-phase clinical studies for the treatment of various diseases, disorders, and injuries by using either hMSCs themselves or their secreted products. Currently, hMSCs for clinical use are generated through conventional static adherent cultures in the presence of fetal bovine serum or human-sourced supplements. However, these methods suffer from variable culture conditions (i.e., ill-defined medium components and heterogeneous culture environment) and thus are not ideal procedures to meet the expected future demand of quality-assured hMSCs for human therapeutic use. Optimizing a bioprocess to generate hMSCs or their secreted products (or both) promises to improve the efficacy as well as safety of this stem cell therapy. In this review, current media and methods for hMSC culture are outlined and bioprocess development strategies discussed.

Hyper-production of butyric acid from delignified rice straw by a novel consolidated bioprocess.

PubMed

Chi, Xue; Li, Jianzheng; Wang, Xin; Zhang, Yafei; Antwi, Philip

2018-04-01

A novel consolidated bioprocess for hyper-production of butyric acid from delignified rice straw without exogenous enzymes involved was developed by co-fermentation of Clostridium thermocellum ATCC 27405 and C. thermobutyricum ATCC 49875. Feasibility of the consolidated bioprocess was approved by batch fermentations, with the optimum pH of 6.5. Fed-batch fermentation with a constant pH of 6.5 at 55 °C could enhance the butyric acid yield to a remarkable 33.9 g/L with a selectivity as high as 78%. Metabolic analysis of the co-culture indicated that sugars liberated by C. thermocellum ATCC 27405 were effectively converted to butyric acid by C. thermobutyricum ATCC 49875. Secondary metabolism of C. thermobutyricum ATCC 49875 also contributed to the hyper-production of butyric acid, resulting in the re-assimilation of by-products such as acetic acid and ethanol. This work provides a more effective fermentation process for butyric acid production from lignocellulosic biomass for future applications. Copyright © 2018 Elsevier Ltd. All rights reserved.

Continuous pH monitoring in a perfused bioreactor system using an optical pH sensor

NASA Technical Reports Server (NTRS)

Jeevarajan, Antony S.; Vani, Sundeep; Taylor, Thomas D.; Anderson, Melody M.

2002-01-01

Monitoring and regulating the pH of the solution in a bioprocess is one of the key steps in the success of bioreactor operation. An in-line optical pH sensor, based on the optical absorption properties of phenol red present in the medium, was developed and tested in this work for use in NASA space bioreactors based on a rotating wall-perfused vessel system supporting a baby hamster kidney (BHK-21) cell culture. The sensor was tested over three 30-day and one 124-day cell runs. The pH sensor initially was calibrated and then used during the entire cell culture interval. The pH reported by the sensor was compared to that measured by a fiber optically coupled Shimadzu spectrophotometer and a blood gas analyzer. The maximum standard error of prediction for all the four cell runs for development pH sensor against BGA was +/-0.06 pH unit and for the fiber optically coupled Shimadzu spectrophotometer against the blood gas analyzer was +/-0.05 pH unit. The pH sensor system performed well without need of recalibration for 124 days. Copyright 2002 Wiley Periodicals, Inc.

Biohydrogen production from space crew's waste simulants using thermophilic consolidated bioprocessing.

PubMed

Wang, Jia; Bibra, Mohit; Venkateswaran, Kasthuri; Salem, David R; Rathinam, Navanietha Krishnaraj; Gadhamshetty, Venkataraman; Sani, Rajesh K

2018-05-01

Human waste simulants were for the first time converted into biohydrogen by a newly developed anaerobic microbial consortium via thermophilic consolidated bioprocessing. Four different BioH 2 -producing consortia (denoted as C1, C2, C3 and C4) were isolated, and developed using human waste simulants as substrate. The thermophilic consortium C3, which contained Thermoanaerobacterium, Caloribacterium, and Caldanaerobius species as the main constituents, showed the highest BioH 2 production (3.999 mmol/g) from human waste simulants under optimized conditions (pH 7.0 and 60 °C). The consortium C3 also produced significant amounts of BioH 2 (5.732 mmol/g and 2.186 mmol/g) using wastewater and activated sludge, respectively. The developed consortium in this study is a promising candidate for H 2 production in space applications as in situ resource utilization. Copyright © 2018 Elsevier Ltd. All rights reserved.

Nano metal-organic framework (NMOF)-based strategies for multiplexed microRNA detection in solution and living cancer cells

NASA Astrophysics Data System (ADS)

Wu, Yafeng; Han, Jianyu; Xue, Peng; Xu, Rong; Kang, Yuejun

2015-01-01

MiRNAs are an emerging type of biomarker for diagnostics and prognostics. A reliable sensing strategy that can monitor miRNA expression in living cancer cells would be critical in view of its extensive advantages for fundamental research related to miRNA-associated bioprocesses and biomedical applications. Conventional miRNA sensing methods include northern blot, microarrays and real-time quantitative PCR. However, none of them is able to monitor miRNA levels expressed in living cancer cells in a real-time fashion. Some fluorescennt biosensors developed recently from carbon nanomaterials, such as single-walled carbon nanotubes (SWNTs), graphene oxide (GO), and carbon nanoparticles, have been successfully used for assaying miRNA in vitro; however the preparation processes are often expensive, complicated and time-consuming, which have motivated the research on other substitute and novel materials. Herein we present a novel sensing strategy based on peptide nucleic acid (PNA) probes labeled with fluorophores and conjugated with an NMOF vehicle to monitor multiplexed miRNAs in living cancer cells. The NMOF works as a fluorescence quencher of the labelled PNA that is firmly bound with the metal center. In the presence of a target miRNA, PNA is hybridized and released from the NMOF leading to the recovery of fluorescence. This miRNA sensor not only enables the quantitative and highly specific detection of multiplexed miRNAs in living cancer cells, but it also allows the precise and in situ monitoring of the spatiotemporal changes of miRNA expression.MiRNAs are an emerging type of biomarker for diagnostics and prognostics. A reliable sensing strategy that can monitor miRNA expression in living cancer cells would be critical in view of its extensive advantages for fundamental research related to miRNA-associated bioprocesses and biomedical applications. Conventional miRNA sensing methods include northern blot, microarrays and real-time quantitative PCR. However, none of them is able to monitor miRNA levels expressed in living cancer cells in a real-time fashion. Some fluorescennt biosensors developed recently from carbon nanomaterials, such as single-walled carbon nanotubes (SWNTs), graphene oxide (GO), and carbon nanoparticles, have been successfully used for assaying miRNA in vitro; however the preparation processes are often expensive, complicated and time-consuming, which have motivated the research on other substitute and novel materials. Herein we present a novel sensing strategy based on peptide nucleic acid (PNA) probes labeled with fluorophores and conjugated with an NMOF vehicle to monitor multiplexed miRNAs in living cancer cells. The NMOF works as a fluorescence quencher of the labelled PNA that is firmly bound with the metal center. In the presence of a target miRNA, PNA is hybridized and released from the NMOF leading to the recovery of fluorescence. This miRNA sensor not only enables the quantitative and highly specific detection of multiplexed miRNAs in living cancer cells, but it also allows the precise and in situ monitoring of the spatiotemporal changes of miRNA expression. Electronic supplementary information (ESI) available: Extra figures and tables. See DOI: 10.1039/c4nr05447d

Origin and analysis of microbial population heterogeneity in bioprocesses.

PubMed

Müller, Susann; Harms, Hauke; Bley, Thomas

2010-02-01

Heterogeneity of industrial production cultures is accepted to a certain degree; however, the underlying mechanisms are seldom perceived or included in the development of new bioprocess control strategies. Population heterogeneity and its basics, perceptible in the diverse proficiency of cells, begins with asymmetric birth and is found to recess during the life cycle. Since inefficient subpopulations have significant impact on the productivity of industrial cultures, cellular heterogeneity needs to be detected and quantified by using high speed detection tools like flow cytometry. Possible origins of population heterogeneity, sophisticated fluorescent techniques for detection of individual cell states, and cutting-edge Omics-technologies for extended information beyond the resolution of fluorescent labelling are highlighted.

Comments on "Bioprocessing in space"

PubMed

Volk, T

1993-10-01

An analysis developed by Westgate et al. for the digestible energy of edible and inedible biomass, including hydrolysis and fermentation, is reexamined with state-of-the-art values for the harvest index of hydroponic crops.

Anaerobic microplate assay for direct microbial conversion of switchgrass and Avicel using Clostridium thermocellum.

PubMed

Oguntimein, Gbekeloluwa B; Rodriguez, Miguel; Dumitrache, Alexandru; Shollenberger, Todd; Decker, Stephen R; Davison, Brian H; Brown, Steven D

2018-02-01

To develop and prototype a high-throughput microplate assay to assess anaerobic microorganisms and lignocellulosic biomasses in a rapid, cost-effective screen for consolidated bioprocessing potential. Clostridium thermocellum parent Δhpt strain deconstructed Avicel to cellobiose, glucose, and generated lactic acid, formic acid, acetic acid and ethanol as fermentation products in titers and ratios similar to larger scale fermentations confirming the suitability of a plate-based method for C. thermocellum growth studies. C. thermocellum strain LL1210, with gene deletions in the key central metabolic pathways, produced higher ethanol titers in the Consolidated Bioprocessing (CBP) plate assay for both Avicel and switchgrass fermentations when compared to the Δhpt strain. A prototype microplate assay system is developed that will facilitate high-throughput bioprospecting for new lignocellulosic biomass types, genetic variants and new microbial strains for bioethanol production.

Development of a simple and low cost microbioreactor for high-throughput bioprocessing.

PubMed

Rahman, Pattanathu K S M; Pasirayi, Godfrey; Auger, Vincent; Ali, Zulfiqur

2009-02-01

A simple microbioreactor for high-throughput bioprocessing made from low cost polymer polytetrafluoroethylene (PTFE) tubes with a working volume of 1.5 ml is described. We have developed a microfluidic system that handles a small population of cells of a model microorganism, Pseudomonas aeruginosa DS10-129. Under the conditions of the microbioreactor, the organism produced extracellular secondary metabolites by using nutrient broth modified with glycerol. Pyocyanins were isolated from the fermented medium as a metabolite of interest. Antibiotic properties of pyocyanin were effective against a number of microorganisms such as Staphylococcus aureus, S. epidermis, Bacillus subtilis, Micrococcus luteus and Saccharomyces cerevisiae. Batch fermentation of the model organism in the microbioreactor was compared to shake-flask and conventional bench fermenter methods. Results obtained from the microbioreactor compared favourably with the conventional processes.

High productivity chromatography refolding process for Hepatitis B Virus X (HBx) protein guided by statistical design of experiment studies.

PubMed

Basu, Anindya; Leong, Susanna Su Jan

2012-02-03

The Hepatitis B Virus X (HBx) protein is a potential therapeutic target for the treatment of hepatocellular carcinoma. However, consistent expression of the protein as insoluble inclusion bodies in bacteria host systems has largely hindered HBx manufacturing via economical biosynthesis routes, thereby impeding the development of anti-HBx therapeutic strategies. To eliminate this roadblock, this work reports the development of the first 'chromatography refolding'-based bioprocess for HBx using immobilised metal affinity chromatography (IMAC). This process enabled production of HBx at quantities and purity that facilitate their direct use in structural and molecular characterization studies. In line with the principles of quality by design (QbD), we used a statistical design of experiments (DoE) methodology to design the optimum process which delivered bioactive HBx at a productivity of 0.21 mg/ml/h at a refolding yield of 54% (at 10 mg/ml refolding concentration), which was 4.4-fold higher than that achieved in dilution refolding. The systematic DoE methodology adopted for this study enabled us to obtain important insights into the effect of different bioprocess parameters like the effect of buffer exchange gradients on HBx productivity and quality. Such a bioprocess design approach can play a pivotal role in developing intensified processes for other novel proteins, and hence helping to resolve validation and speed-to-market challenges faced by the biopharmaceutical industry today. Copyright © 2011 Elsevier B.V. All rights reserved.

Nano-Tubular Cellulose for Bioprocess Technology Development

PubMed Central

Koutinas, Athanasios A.; Sypsas, Vasilios; Kandylis, Panagiotis; Michelis, Andreas; Bekatorou, Argyro; Kourkoutas, Yiannis; Kordulis, Christos; Lycourghiotis, Alexis; Banat, Ibrahim M.; Nigam, Poonam; Marchant, Roger; Giannouli, Myrsini; Yianoulis, Panagiotis

2012-01-01

Delignified cellulosic material has shown a significant promotional effect on the alcoholic fermentation as yeast immobilization support. However, its potential for further biotechnological development is unexploited. This study reports the characterization of this tubular/porous cellulosic material, which was done by SEM, porosimetry and X-ray powder diffractometry. The results showed that the structure of nano-tubular cellulose (NC) justifies its suitability for use in “cold pasteurization” processes and its promoting activity in bioprocessing (fermentation). The last was explained by a glucose pump theory. Also, it was demonstrated that crystallization of viscous invert sugar solutions during freeze drying could not be otherwise achieved unless NC was present. This effect as well as the feasibility of extremely low temperature fermentation are due to reduction of the activation energy, and have facilitated the development of technologies such as wine fermentations at home scale (in a domestic refrigerator). Moreover, NC may lead to new perspectives in research such as the development of new composites, templates for cylindrical nano-particles, etc. PMID:22496794

Nano-tubular cellulose for bioprocess technology development.

PubMed

Koutinas, Athanasios A; Sypsas, Vasilios; Kandylis, Panagiotis; Michelis, Andreas; Bekatorou, Argyro; Kourkoutas, Yiannis; Kordulis, Christos; Lycourghiotis, Alexis; Banat, Ibrahim M; Nigam, Poonam; Marchant, Roger; Giannouli, Myrsini; Yianoulis, Panagiotis

2012-01-01

Delignified cellulosic material has shown a significant promotional effect on the alcoholic fermentation as yeast immobilization support. However, its potential for further biotechnological development is unexploited. This study reports the characterization of this tubular/porous cellulosic material, which was done by SEM, porosimetry and X-ray powder diffractometry. The results showed that the structure of nano-tubular cellulose (NC) justifies its suitability for use in "cold pasteurization" processes and its promoting activity in bioprocessing (fermentation). The last was explained by a glucose pump theory. Also, it was demonstrated that crystallization of viscous invert sugar solutions during freeze drying could not be otherwise achieved unless NC was present. This effect as well as the feasibility of extremely low temperature fermentation are due to reduction of the activation energy, and have facilitated the development of technologies such as wine fermentations at home scale (in a domestic refrigerator). Moreover, NC may lead to new perspectives in research such as the development of new composites, templates for cylindrical nano-particles, etc.

Effect of Bioprocessing on the In Vitro Colonic Microbial Metabolism of Phenolic Acids from Rye Bran Fortified Breads.

PubMed

Koistinen, Ville M; Nordlund, Emilia; Katina, Kati; Mattila, Ismo; Poutanen, Kaisa; Hanhineva, Kati; Aura, Anna-Marja

2017-03-08

Cereal bran is an important source of dietary fiber and bioactive compounds, such as phenolic acids. We aimed to study the phenolic acid metabolism of native and bioprocessed rye bran fortified refined wheat bread and to elucidate the microbial metabolic route of phenolic acids. After incubation in an in vitro colon model, the metabolites were analyzed using two different methods applying mass spectrometry. While phenolic acids were released more extensively from the bioprocessed bran bread and ferulic acid had consistently higher concentrations in the bread type during fermentation, there were only minor differences in the appearance of microbial metabolites, including the diminished levels of certain phenylacetic acids in the bioprocessed bran. This may be due to rye matrix properties, saturation of ferulic acid metabolism, or a rapid formation of intermediary metabolites left undetected. In addition, we provide expansion to the known metabolic pathways of phenolic acids.

Bioprocess Forces and Their Impact on Cell Behavior: Implications for Bone Regeneration Therapy

PubMed Central

Brindley, David; Moorthy, Kishaani; Lee, Jae-Ho; Mason, Chris; Kim, Hae-Won; Wall, Ivan

2011-01-01

Bioprocess forces such as shear stress experienced during routine cell culture are considered to be harmful to cells. However, the impact of physical forces on cell behavior is an area of growing interest within the tissue engineering community, and it is widely acknowledged that mechanical stimulation including shear stress can enhance osteogenic differentiation. This paper considers the effects of bioprocess shear stress on cell responses such as survival and proliferation in several contexts, including suspension-adapted cells used for recombinant protein and monoclonal antibody manufacture, adherent cells for therapy in suspension, and adherent cells attached to their growth substrates. The enhanced osteogenic differentiation that fluid flow shear stress is widely found to induce is discussed, along with the tissue engineering of mineralized tissue using perfusion bioreactors. Recent evidence that bioprocess forces produced during capillary transfer or pipetting of cell suspensions can enhance osteogenic responses is also discussed. PMID:21904661

Commercial investigation results for the generic bioprocessing apparatus flown on United States Microgravity Laboratory-1

NASA Technical Reports Server (NTRS)

Stodieck, Louis S.; Robinson, M. C.; Luttges, M. W.

1994-01-01

The Generic Bioprocessing Apparatus (BPA) payload was developed by BioServe to support the commercial flight development needs of our specialized consortia comprised of business, academic, and government entities. The consortia have commitments to explore commercial opportunities in bioprocessing, biomedical models, and closed agricultural systems. In addition, some members of BioServe have interests in the development and/or qualification of enabling flight hardware used in life sciences space flight testing. Some business and academic entities have interests in more than one of these consortia. To aid in payload development, flight, and analysis, each consortium member contributes resources ranging from proprietary expertise and materials, to hardware and cash. Professionals from business, academia, and government often interact with each other via graduate research assistants who do much of the 'hands-on' payload preparation and subsequent data analyses. The GBA supported research, testing, and development activities for each different BioServe consortium. It produced an environment in which professionals from diverse backgrounds came together with a single focus. And, it provided a truly novel learning environment for a youthful new cadre of space professionals committed to the exploration of commercial opportunities presented by space. Since the GBA supported a large number of different experiments, this paper briefly describes the payload characteristics and the essential operations of the payload. A summary of the experiments is presented. Finally, a few of the experiments are described in detail highlighting some novel effects of space flight on life science systems. Portions of the reported work have or will appear in appropriate archival journals as cited in the bibliography. In several instances, data collected from USML-1 have been supplemented with related data collected on more recent STS missions.

Process model comparison and transferability across bioreactor scales and modes of operation for a mammalian cell bioprocess.

PubMed

Craven, Stephen; Shirsat, Nishikant; Whelan, Jessica; Glennon, Brian

2013-01-01

A Monod kinetic model, logistic equation model, and statistical regression model were developed for a Chinese hamster ovary cell bioprocess operated under three different modes of operation (batch, bolus fed-batch, and continuous fed-batch) and grown on two different bioreactor scales (3 L bench-top and 15 L pilot-scale). The Monod kinetic model was developed for all modes of operation under study and predicted cell density, glucose glutamine, lactate, and ammonia concentrations well for the bioprocess. However, it was computationally demanding due to the large number of parameters necessary to produce a good model fit. The transferability of the Monod kinetic model structure and parameter set across bioreactor scales and modes of operation was investigated and a parameter sensitivity analysis performed. The experimentally determined parameters had the greatest influence on model performance. They changed with scale and mode of operation, but were easily calculated. The remaining parameters, which were fitted using a differential evolutionary algorithm, were not as crucial. Logistic equation and statistical regression models were investigated as alternatives to the Monod kinetic model. They were less computationally intensive to develop due to the absence of a large parameter set. However, modeling of the nutrient and metabolite concentrations proved to be troublesome due to the logistic equation model structure and the inability of both models to incorporate a feed. The complexity, computational load, and effort required for model development has to be balanced with the necessary level of model sophistication when choosing which model type to develop for a particular application. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

Nancy Dowe | NREL

Science.gov Websites

Center Bioprocess Development Group. The group is largely made up of fermentation scientists and engineers whose focus is on fermentation process development and pilot plant scale-up. Dowe has nearly 30 years of experience working with a wide variety of fermentation processes and microorganisms for the

Consolidated Bioprocessing for Butyric Acid Production from Rice Straw with Undefined Mixed Culture

PubMed Central

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

2016-01-01

Lignocellulosic biomass is a renewable source with great potential for biofuels and bioproducts. However, the cost of cellulolytic enzymes limits the utilization of the low-cost bioresource. This study aimed to develop a consolidated bioprocessing without the need of supplementary cellulase for butyric acid production from lignocellulosic biomass. A stirred-tank reactor with a working volume of 21 L was constructed and operated in batch and semi-continuous fermentation modes with a cellulolytic butyrate-producing microbial community. The semi-continuous fermentation with intermittent discharging of the culture broth and replenishment with fresh medium achieved the highest butyric acid productivity of 2.69 g/(L· d). In semi-continuous operation mode, the butyric acid and total carboxylic acid concentrations of 16.2 and 28.9 g/L, respectively, were achieved. Over the 21-day fermentation period, their cumulative yields reached 1189 and 2048 g, respectively, corresponding to 41 and 74% of the maximum theoretical yields based on the amount of NaOH pretreated rice straw fed in. This study demonstrated that an undefined mixed culture-based consolidated bioprocessing for butyric acid production can completely eliminate the cost of supplementary cellulolytic enzymes. PMID:27822203

Consolidated Bioprocessing for Butyric Acid Production from Rice Straw with Undefined Mixed Culture.

PubMed

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

2016-01-01

Lignocellulosic biomass is a renewable source with great potential for biofuels and bioproducts. However, the cost of cellulolytic enzymes limits the utilization of the low-cost bioresource. This study aimed to develop a consolidated bioprocessing without the need of supplementary cellulase for butyric acid production from lignocellulosic biomass. A stirred-tank reactor with a working volume of 21 L was constructed and operated in batch and semi-continuous fermentation modes with a cellulolytic butyrate-producing microbial community. The semi-continuous fermentation with intermittent discharging of the culture broth and replenishment with fresh medium achieved the highest butyric acid productivity of 2.69 g/(L· d). In semi-continuous operation mode, the butyric acid and total carboxylic acid concentrations of 16.2 and 28.9 g/L, respectively, were achieved. Over the 21-day fermentation period, their cumulative yields reached 1189 and 2048 g, respectively, corresponding to 41 and 74% of the maximum theoretical yields based on the amount of NaOH pretreated rice straw fed in. This study demonstrated that an undefined mixed culture-based consolidated bioprocessing for butyric acid production can completely eliminate the cost of supplementary cellulolytic enzymes.

Isolated Polynucleotides and Methods of Promoting a Morphology in a Fungus

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

Lasure, Linda L; Dai, Ziyu

2008-10-21

The invention includes isolated polynucleotide molecules that are differentially expressed in a native fungus exhibiting a first morphology relative to the native fungus exhibiting a second morphology. The invention includes a method of enhancing a bioprocess utilizing a fungus. A transformed fungus is produced by transforming a fungus with a recombinant polynucleotide molecule. The recombinant polynucleotide molecule contains an isolated polynucleotide sequence linked operably to a promoter. The polynucleotide sequence is expressed to promote a first morphology. The first morphology of the transformed fungus enhances a bioprocess relative to the bioprocess utilizing a second morphology.

On-line coupling of a miniaturized bioreactor with capillary electrophoresis, via a membrane interface, for monitoring the production of organic acids by microorganisms.

PubMed

Ehala, S; Vassiljeva, I; Kuldvee, R; Vilu, R; Kaljurand, M

2001-09-01

Capillary electrophoresis (CE) can be a valuable tool for on-line monitoring of bioprocesses. Production of organic acids by phosphorus-solubilizing bacteria and fermentation of UHT milk were monitored and controlled by use of a membrane-interfaced dialysis device and a home-made microsampler for a capillary electrophoresis unit. Use of this specially designed sampling device enabled rapid consecutive injections without interruption of the high voltage. No additional sample preparation was required. The time resolution of monitoring in this particular work was approximately 2 h, but could be reduced to 2 min. Analytes were detected at low microg mL(-1) levels with a reproducibility of approximately 10%. To demonstrate the potential of CE in processes of biotechnological interest, results from monitoring phosphate solubilization by bacteria were submitted to qualitative and quantitative analysis. Fermentation experiments on UHT milk showed that monitoring of the processes by CE can provide good resolution of complex mixtures, although for more specific, detailed characterization the identification of individual substances is needed.

Anaerobic microplate assay for direct microbial conversion of switchgrass and Avicel using Clostridium thermocellum

DOE PAGES

Oguntimein, Gbekeloluwa B.; Rodriguez, Jr., Miguel; Dumitrache, Alexandru; ...

2017-11-09

Here, to develop and prototype a high-throughput microplate assay to assess anaerobic microorganisms and lignocellulosic biomasses in a rapid, cost-effective screen for consolidated bioprocessing potential. Clostridium thermocellum parent Δ hpt strain deconstructed Avicel to cellobiose, glucose, and generated lactic acid, formic acid, acetic acid and ethanol as fermentation products in titers and ratios similar to larger scale fermentations confirming the suitability of a plate-based method for C. thermocellum growth studies. C. thermocellum strain LL1210, with gene deletions in the key central metabolic pathways, produced higher ethanol titers in the Consolidated Bioprocessing (CBP) plate assay for both Avicel and switchgrass fermentationsmore » when compared to the Δ hpt strain. A prototype microplate assay system is developed that will facilitate high-throughput bioprospecting for new lignocellulosic biomass types, genetic variants and new microbial strains for bioethanol production.« less

Anaerobic microplate assay for direct microbial conversion of switchgrass and Avicel using Clostridium thermocellum

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

Oguntimein, Gbekeloluwa B.; Rodriguez, Jr., Miguel; Dumitrache, Alexandru

Here, to develop and prototype a high-throughput microplate assay to assess anaerobic microorganisms and lignocellulosic biomasses in a rapid, cost-effective screen for consolidated bioprocessing potential. Clostridium thermocellum parent Δ hpt strain deconstructed Avicel to cellobiose, glucose, and generated lactic acid, formic acid, acetic acid and ethanol as fermentation products in titers and ratios similar to larger scale fermentations confirming the suitability of a plate-based method for C. thermocellum growth studies. C. thermocellum strain LL1210, with gene deletions in the key central metabolic pathways, produced higher ethanol titers in the Consolidated Bioprocessing (CBP) plate assay for both Avicel and switchgrass fermentationsmore » when compared to the Δ hpt strain. A prototype microplate assay system is developed that will facilitate high-throughput bioprospecting for new lignocellulosic biomass types, genetic variants and new microbial strains for bioethanol production.« less

In situ microscopy for online monitoring of cell concentration in Pichia pastoris cultivations.

PubMed

Marquard, D; Enders, A; Roth, G; Rinas, U; Scheper, T; Lindner, P

2016-09-20

In situ Microscopy (ISM) is an optical non-invasive technique to monitor cells in bioprocesses in real-time. Pichia pastoris is one of the most promising protein expression systems. This yeast combines fast growth on simple media and important eukaryotic features such as glycosylation. In this work, the ISM technology was applied to Pichia pastoris cultivations for online monitoring of the cell concentration during cultivation. Different ISM settings were tested. The acquired images were analyzed with two image processing algorithms. In seven cultivations the cell concentration was monitored by the applied algorithms and offline samples were taken to determine optical density (OD) and dry cell mass (DCM). Cell concentrations up to 74g/L dry cell mass could be analyzed via the ISM. Depending on the algorithm and the ISM settings, an accuracy between 0.3 % and 12 % was achieved. The overall results show that for a robust measurement a combination of the two described algorithms is required. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantification of a bacterial secondary metabolite by SERS combined with SLM extraction for bioprocess monitoring.

PubMed

Morelli, Lidia; Andreasen, Sune Zoëga; Jendresen, Christian Bille; Nielsen, Alex Toftgaard; Emnéus, Jenny; Zór, Kinga; Boisen, Anja

2017-11-20

During the last few decades, great advances have been reached in high-throughput design and building of genetically engineered microbial strains, leading to a need for fast and reliable screening methods. We developed and optimized a microfluidic supported liquid membrane (SLM) extraction device and combined it with surface enhanced Raman scattering (SERS) sensing for the screening of a biological process, namely for the quantification of a bacterial secondary metabolite, p-coumaric acid (pHCA), produced by Escherichia coli. The microfluidic device proved to be robust and reusable, enabling efficient removal of interfering compounds from the real samples, reaching more than 13-fold up-concentration of the donor at 10 μL min -1 flow rate. With this method, we quantified pHCA directly from the bacterial supernatant, distinguishing between various culture conditions based on the pHCA production yield. The obtained data showed good correlation with HPLC analysis.

Simplex-based optimization of numerical and categorical inputs in early bioprocess development: Case studies in HT chromatography.

PubMed

Konstantinidis, Spyridon; Titchener-Hooker, Nigel; Velayudhan, Ajoy

2017-08-01

Bioprocess development studies often involve the investigation of numerical and categorical inputs via the adoption of Design of Experiments (DoE) techniques. An attractive alternative is the deployment of a grid compatible Simplex variant which has been shown to yield optima rapidly and consistently. In this work, the method is combined with dummy variables and it is deployed in three case studies wherein spaces are comprised of both categorical and numerical inputs, a situation intractable by traditional Simplex methods. The first study employs in silico data and lays out the dummy variable methodology. The latter two employ experimental data from chromatography based studies performed with the filter-plate and miniature column High Throughput (HT) techniques. The solute of interest in the former case study was a monoclonal antibody whereas the latter dealt with the separation of a binary system of model proteins. The implemented approach prevented the stranding of the Simplex method at local optima, due to the arbitrary handling of the categorical inputs, and allowed for the concurrent optimization of numerical and categorical, multilevel and/or dichotomous, inputs. The deployment of the Simplex method, combined with dummy variables, was therefore entirely successful in identifying and characterizing global optima in all three case studies. The Simplex-based method was further shown to be of equivalent efficiency to a DoE-based approach, represented here by D-Optimal designs. Such an approach failed, however, to both capture trends and identify optima, and led to poor operating conditions. It is suggested that the Simplex-variant is suited to development activities involving numerical and categorical inputs in early bioprocess development. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Next generation industrial biotechnology based on extremophilic bacteria.

PubMed

Chen, Guo-Qiang; Jiang, Xiao-Ran

2018-04-01

Industrial biotechnology aims to produce bulk chemicals including polymeric materials and biofuels based on bioprocessing sustainable agriculture products such as starch, fatty acids and/or cellulose. However, traditional bioprocesses require bioreactors made of stainless steel, complicated sterilization, difficult and expensive separation procedures as well as well-trained engineers that are able to conduct bioprocessing under sterile conditions, reducing the competitiveness of the bio-products. Amid the continuous low petroleum price, next generation industrial biotechnology (NGIB) allows bioprocessing to be conducted under unsterile (open) conditions using ceramic, cement or plastic bioreactors in a continuous way, it should be an energy, water and substrate saving technology with convenient operation procedure. NGIB also requires less capital investment and reduces demand on highly trained engineers. The foundation for the simplified NGIB is microorganisms that resist contaminations by other microbes, one of the examples is rapid growing halophilic bacteria inoculated under high salt concentration and alkali pH. They have been engineered to produce multiple products in various scales. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evaluating the toxicity of bDtBPP on CHO-K1 cells for testing of single-use bioprocessing systems considering media selection, cell culture volume, mixing, and exposure duration.

PubMed

Shah, Rhythm R; Linville, Taylor W; Whynot, Andrew D; Brazel, Christopher S

2016-09-01

Single-use bioprocessing bags are gaining popularity due to ease of use, lower risk of contamination, and ease of process scale-up. Bis(2,4-di-tert-butylphenyl)phosphate (bDtBPP), a degradant of tris(2,4-di-tert-butylphenyl)phosphite, marketed as Irgafos 168®, which is an antioxidant stabilizer added to resins, has been identified as a potentially toxic leachate which may impact the performance of single-use, multilayer bioprocessing bags. In this study, the toxicity of bDtBPP was tested on CHO-K1 cells grown as adherent or suspended cells. The EC50 (effective concentration to cause 50% cell death) for adherent cells was found to be one order of magnitude higher than that for suspended CHO-K1 cells. While CHO-K1 cells had good cell viability when exposed to moderate concentrations of bDtBPP, the degradant was shown to impact the viable cell density (VCD) at much lower concentrations. Hence, in developing an industry-standard assay for testing the cytotoxicity of leachates, suspended cells (as commonly used in the bioprocessing industry) would likely be most sensitive, particularly when reporting EC50 values based on VCD. The effects of mixing, cell culture volume, and exposure duration were also evaluated for suspended CHO-K1 cells. It was found that the sensitivity of cell culture to leachates from single-use plastic bags was enhanced for suspended cells cultured for longer exposure times and when the cells were subjected to continuous agitation, both of which are important considerations in the production of biopharmaceuticals. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1318-1323, 2016. © 2016 American Institute of Chemical Engineers.

A dual near-infrared and dielectric spectroscopies strategy to monitor populations of Chinese hamster ovary cells in bioreactor.

PubMed

Courtès, Franck; Ebel, Bruno; Guédon, Emmanuel; Marc, Annie

2016-05-01

to develop a new strategy combining near-infrared (NIR) and dielectric spectroscopies for real-time monitoring and in-depth characterizing populations of Chinese hamster ovary cells throughout cultures performed in bioreactors. Spectral data processing was based on off-line analyses of the cells, including trypan blue exclusion method, and lactate dehydrogenase activity (LDH). Viable cell density showed a linear correlation with permittivity up to 6 × 10(6) cells ml(-1), while a logarithmic correlation was found between non-lysed dead cell density and conductivity up to 10(7) cells ml(-1). Additionally, partial least square technique was used to develop a calibration model of the supernatant LDH activity based on online NIR spectra with a RMSEC of 55 U l(-1). Considering the LDH content of viable cells measured to be 110 U per 10(9) cells, the lysed dead cell density could be then estimated. These calibration models provided real-time prediction accuracy (R(2) ≥ 0.95) for the three types of cell populations. The high potential of a dual spectroscopy strategy to enhance the online bioprocesses characterization is demonstrated since it allows the simultaneous determination of viable, dead and lysed cell populations in real time.

Bioprocessing Data for the Production of Marine Enzymes

PubMed Central

Sarkar, Sreyashi; Pramanik, Arnab; Mitra, Anindita; Mukherjee, Joydeep

2010-01-01

This review is a synopsis of different bioprocess engineering approaches adopted for the production of marine enzymes. Three major modes of operation: batch, fed-batch and continuous have been used for production of enzymes (such as protease, chitinase, agarase, peroxidase) mainly from marine bacteria and fungi on a laboratory bioreactor and pilot plant scales. Submerged, immobilized and solid-state processes in batch mode were widely employed. The fed-batch process was also applied in several bioprocesses. Continuous processes with suspended cells as well as with immobilized cells have been used. Investigations in shake flasks were conducted with the prospect of large-scale processing in reactors. PMID:20479981

Turmeric bioprocessed with mycelia from the shiitake culinary-medicinal mushroom lentinus edodes (agaricomycetes) protects mice against salmonellosis

USDA-ARS?s Scientific Manuscript database

Extracts of the shiitake mushroom Lentinus edodes and the spice tumeric (Curcuma longa) have both been reported to have health-promoting properties. The present study investigated the suppressive mechanisms of a bioprocessed Lentinus edodes liquid mushroom mycelia culture supplemented with turmeric ...

Low-level, uniform ultrasound field effects on enzymatic bioprocessing of greige cotton using three fabric weights

USDA-ARS?s Scientific Manuscript database

Ultrasound-assisted enzymatic bio-processing of greige cotton offers an environmentally friendly alternative approach to conventional alkaline scouring. Our research has found that the introduction of a low energy, uniform ultrasound field into enzyme processing solutions greatly improved enzyme ef...

Application of a low level, uniform ultrasound field for the acceleration of enzymatic bio-processing of cotton

USDA-ARS?s Scientific Manuscript database

Enzymatic bio-processing of cotton generates significantly less hazardous wastewater effluents, which are readily biodegradable, but it also has several critical shortcomings that impede its acceptance by industries: expensive processing costs and slow reaction rates. Our research has found that th...

Application of Low Level, Uniform Ultrasound Field for Acceleration of Enzymatic Bio-processing of Cotton

USDA-ARS?s Scientific Manuscript database

Enzymatic bio-processing of cotton generates significantly less hazardous wastewater effluents, which are readily biodegradable, but it also has several critical shortcomings that impede its acceptance by industries: expensive processing costs and slow reaction rates. Our research has found that th...

Integrated Bioprocess Design: A Case Study for Undergraduates.

ERIC Educational Resources Information Center

Titchener-Hooker, Nigel; Zhou, Yu-Hong

2000-01-01

Presents a case study for use in the teaching of bioprocess design. Taking the production and isolation of the intracellular protein s. cerevisae, demonstrates how undergraduates can use a range of data to construct and then investigate the range of processes flowsheet options available for a process duty. (Author/SAH)

Streptomyces sp. TEM 33 possesses high lipolytic activity in solid-state fermentation in comparison with submerged fermentation.

PubMed

Cadirci, Bilge Hilal; Yasa, Ihsan; Kocyigit, Ali

2016-01-01

Solid-state fermentation (SSF) is a bioprocess that doesn't need an excess of free water, and it offers potential benefits for microbial cultivation for bioprocesses and product development. In comparing the antibiotic production, few detailed reports could be found with lipolytic enzyme production by Streptomycetes in SSF. Taking this knowledge into consideration, we prefer to purify Actinomycetes species as a new source for lipase production. The lipase-producing strain Streptomyces sp. TEM 33 was isolated from soil and lipase production was managed by solid-state fermentation (SSF) in comparison with submerged fermentation (SmF). Bioprocess-affecting factors like initial moisture content, incubation time, and various carbon and nitrogen additives and the other enzymes secreted into the media were optimized. Lipase activity was measured as 1.74 ± 0.0005 U/g dry substrate (gds) by the p-nitrophenylpalmitate (pNPP) method on day 6 of fermentation with 71.43% final substrate moisture content. In order to understand the metabolic priority in SSF, cellulase and xylanase activity of Streptomyces sp. TEM33 was also measured. The microorganism degrades the wheat bran to its usable form by excreting cellulases and xylanases; then it secretes the lipase that is necessary for degrading the oil in the medium.

HTRF: A technology tailored for drug discovery - a review of theoretical aspects and recent applications.

PubMed

Degorce, François; Card, Amy; Soh, Sharon; Trinquet, Eric; Knapik, Glenn P; Xie, Bing

2009-05-28

HTRF (Homogeneous Time Resolved Fluorescence) is the most frequently used generic assay technology to measure analytes in a homogenous format, which is the ideal platform used for drug target studies in high-throughput screening (HTS). This technology combines fluorescence resonance energy transfer technology (FRET) with time-resolved measurement (TR). In TR-FRET assays, a signal is generated through fluorescent resonance energy transfer between a donor and an acceptor molecule when in close proximity to each other. Buffer and media interference is dramatically reduced by dual-wavelength detection, and the final signal is proportional to the extent of product formation. The HTRF assay is usually sensitive and robust that can be miniaturized into the 384 and 1536-well plate formats. This assay technology has been applied to many antibody-based assays including GPCR signaling (cAMP and IP-One), kinases, cytokines and biomarkers, bioprocess (antibody and protein production), as well as the assays for protein-protein, proteinpeptide, and protein-DNA/RNA interactions.Since its introduction to the drug-screening world over ten years ago, researchers have used HTRF to expedite the study of GPCRs, kinases, new biomarkers, protein-protein interactions, and other targets of interest. HTRF has also been utilized as an alternative method for bioprocess monitoring. The first-generation HTRF technology, which uses Europium cryptate as a fluorescence donor to monitor reactions between biomolecules, was extended in 2008 through the introduction of a second-generation donor, Terbium cryptate (Tb), enhancing screening performance. Terbium cryptate possesses different photophysical properties compared to Europium, including increased quantum yield and a higher molar extinction coefficient. In addition to being compatible with the same acceptor fluorophors used with Europium, it can serve as a donor fluorophore to green-emitting fluors because it has multiple emission peaks including one at 490 nm. Moreover, all Terbium HTRF assays can be read on the same HTRF-compatible instruments as Europium HTRF assays.Overall, HTRF is a highly sensitive, robust technology for the detection of molecular interactions in vitro and is widely used for primary and secondary screening phases of drug development. This review addresses the general principles of HTRF and its current applications in drug discovery.

Marykate O'Brien | NREL

Science.gov Websites

industry partners and NREL programmatic R&D. Sustainable energy/fuels research and development Catalyst Biological Engineering, University of Colorado, 2009 Professional Experience Bio-Process Engineer, NREL, 2013 Professional Research Assistant, University of Colorado, 2007-2012 Engineering Intern, Baxter Healthcare, 2007

Philip T. Pienkos | NREL

Science.gov Websites

Valorization: Development of a Methane-to-Adipic Acid Bioprocess (consultant) Areas of Expertise Molecular engineering Enzyme and protein engineering Techno-economic analysis Education Ph.D., Molecular Biology Principal Research Supervisor, NREL, Aug. 2007-May 2011 Research Director, Molecular Logix, Inc., The

Biocatalysis: applications and potentials for the chemical industry.

PubMed

Thomas, Stuart M; DiCosimo, Robert; Nagarajan, Vasantha

2002-06-01

The chemical industry is exploring the use of renewable feed stocks to improve sustainability, prompting the exploration of bioprocesses for the production of chemicals. Attractive features of biological systems include versatility, substrate selectivity, regioselectivity, chemoselectivity, enantioselectivity and catalysis at ambient temperatures and pressures. However, a challenge facing bioprocesses is cost competitiveness with chemical processes because capital assets associated with the existing commercial processes are high. The chemical industry will probably use biotechnology with existing feed stocks and processes to extract higher values from feed stocks, process by-products and waste streams. In this decade, bioprocesses that offer either a process or a product advantage over traditional chemical routes will become more widely used.

Technical Aspects of Use of Ultrasound for Intensification of Enzymatic Bio-Processing: New Path to "Green Chemistry"

USDA-ARS?s Scientific Manuscript database

Use of enzymatic processing in the food, textile, and bio-fuel applications is becoming increasingly popular, primarily because of rapid introduction of a new variety of highly efficient enzymes. In general, an enzymatic bio-processing generates less toxic and readily biodegradable wastewater efflue...

Production of polyol oils from soybean oil by bioprocess: results of microbial screening and identification of positive cultures

USDA-ARS?s Scientific Manuscript database

Recently we reported methods for microbial screening and production of polyol oils from soybean oil through bioprocessing (Hou and Lin, 2013). Soy-polyol oils (oxygenated acylglycerols) are important starting materials for the manufacture of polymers such as polyurethane. Currently, they are produce...

Lessons From the Cow: What the Ruminant Animal Can Teach Us About Consolidated Bioprocessing of Cellulosic Biomass

USDA-ARS?s Scientific Manuscript database

Consolidated bioprocessing (CBP), in which anaerobic bacteria produce their own cellulolytic enzymes and ferment the products of cellulose hydrolysis to ethanol in a single reactor, is regarded as a promising future route to cellulosic ethanol. Some of the current limitations to practical use of thi...

The Effects of a Low-Level Uniform Ultrasound Field on Enzymatic Bio-Processing of Cotton: An Investigation of Three Fabric Weights

USDA-ARS?s Scientific Manuscript database

Enzymatic bio-processing of cotton generates significantly less hazardous wastewater effluents, which are readily biodegradable, but it also has several critical shortcomings that impede its acceptance by industries: expensive processing costs and slow reaction rates. Our research has found that t...

Proteomics in biomanufacturing control: Protein dynamics of CHO-K1 cells and conditioned media during apoptosis and necrosis.

PubMed

Albrecht, Simone; Kaisermayer, Christian; Gallagher, Clair; Farrell, Amy; Lindeberg, Anna; Bones, Jonathan

2018-06-01

Cell viability has a critical impact on product quantity and quality during the biomanufacturing of therapeutic proteins. An advanced understanding of changes in the cellular and conditioned media proteomes upon cell stress and death is therefore needed for improved bioprocess control. Here, a high pH/low pH reversed phase data independent 2D-LC-MS E discovery proteomics platform was applied to study the cellular and conditioned media proteomes of CHO-K1 apoptosis and necrosis models where cell death was induced by staurosporine exposure or aeration shear in a benchtop bioreactor, respectively. Functional classification of gene ontology terms related to molecular functions, biological processes, and cellular components revealed both cell death independent and specific features. In addition, label free quantitation using the Hi3 approach resulted in a comprehensive shortlist of 23 potential cell viability marker proteins with highest abundance and a significant increase in the conditioned media upon induction of cell death, including proteins related to cellular stress response, signal mediation, cytoskeletal organization, cell differentiation, cell interaction as well as metabolic and proteolytic enzymes which are interesting candidates for translating into targeted analysis platforms for monitoring bioprocessing response and increasing process control. © 2018 Wiley Periodicals, Inc.

Quality by control: Towards model predictive control of mammalian cell culture bioprocesses.

PubMed

Sommeregger, Wolfgang; Sissolak, Bernhard; Kandra, Kulwant; von Stosch, Moritz; Mayer, Martin; Striedner, Gerald

2017-07-01

The industrial production of complex biopharmaceuticals using recombinant mammalian cell lines is still mainly built on a quality by testing approach, which is represented by fixed process conditions and extensive testing of the end-product. In 2004 the FDA launched the process analytical technology initiative, aiming to guide the industry towards advanced process monitoring and better understanding of how critical process parameters affect the critical quality attributes. Implementation of process analytical technology into the bio-production process enables moving from the quality by testing to a more flexible quality by design approach. The application of advanced sensor systems in combination with mathematical modelling techniques offers enhanced process understanding, allows on-line prediction of critical quality attributes and subsequently real-time product quality control. In this review opportunities and unsolved issues on the road to a successful quality by design and dynamic control implementation are discussed. A major focus is directed on the preconditions for the application of model predictive control for mammalian cell culture bioprocesses. Design of experiments providing information about the process dynamics upon parameter change, dynamic process models, on-line process state predictions and powerful software environments seem to be a prerequisite for quality by control realization. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Versatile common instrumentation for optical detection of pH and dissolved oxygen

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

Sardesai, Neha; Rao, Govind; Kostov, Yordan, E-mail: kostov@umbc.edu

2015-07-15

The recent trend toward use of disposable and miniature bioreactors requires the use of appropriate sensors. pH and dissolved oxygen (DO) are often measured using optical chemical sensors due to their small form factor and convenience in use. These sensors are often interrogated using a specialized opto-electronic transducer that is designed around the optical sensor. In this contribution, we are presenting a new class of opto-electronic transducers that are usable with several different chemical sensors without the need to switch the optics or hardware when changing the type of the chemical sensor. This allows flexibility closer to the lab-grade devicesmore » while the size is closer to a dedicated sensor. This versatile instrumentation is capable of seamlessly switching between the pH and DO measurement modes and is capable of auto recognition of the sensor type. The principle of ratiometric fluorescence is used for pH measurements, and that of fluorescence lifetime for DO measurements. An approach to obtain identical calibrations between several devices is also presented. The described hardware constitutes common instrumentation for measuring either pH or DO and has been tested in actual bioprocesses. It has been found adequate for continuous bioprocess monitoring.« less

Integrated Graduate and Continuing Education in Protein Chromatography for Bioprocess Development and Scale-Up

ERIC Educational Resources Information Center

Carta, Jungbauer

2011-01-01

We describe an intensive course that integrates graduate and continuing education focused on the development and scale-up of chromatography processes used for the recovery and purification of proteins with special emphasis on biotherapeutics. The course includes lectures, laboratories, teamwork, and a design exercise and offers a complete view of…

Bioprocess systems engineering: transferring traditional process engineering principles to industrial biotechnology.

PubMed

Koutinas, Michalis; Kiparissides, Alexandros; Pistikopoulos, Efstratios N; Mantalaris, Athanasios

2012-01-01

The complexity of the regulatory network and the interactions that occur in the intracellular environment of microorganisms highlight the importance in developing tractable mechanistic models of cellular functions and systematic approaches for modelling biological systems. To this end, the existing process systems engineering approaches can serve as a vehicle for understanding, integrating and designing biological systems and processes. Here, we review the application of a holistic approach for the development of mathematical models of biological systems, from the initial conception of the model to its final application in model-based control and optimisation. We also discuss the use of mechanistic models that account for gene regulation, in an attempt to advance the empirical expressions traditionally used to describe micro-organism growth kinetics, and we highlight current and future challenges in mathematical biology. The modelling research framework discussed herein could prove beneficial for the design of optimal bioprocesses, employing rational and feasible approaches towards the efficient production of chemicals and pharmaceuticals.

Bioprocess systems engineering: transferring traditional process engineering principles to industrial biotechnology

PubMed Central

Koutinas, Michalis; Kiparissides, Alexandros; Pistikopoulos, Efstratios N.; Mantalaris, Athanasios

2013-01-01

The complexity of the regulatory network and the interactions that occur in the intracellular environment of microorganisms highlight the importance in developing tractable mechanistic models of cellular functions and systematic approaches for modelling biological systems. To this end, the existing process systems engineering approaches can serve as a vehicle for understanding, integrating and designing biological systems and processes. Here, we review the application of a holistic approach for the development of mathematical models of biological systems, from the initial conception of the model to its final application in model-based control and optimisation. We also discuss the use of mechanistic models that account for gene regulation, in an attempt to advance the empirical expressions traditionally used to describe micro-organism growth kinetics, and we highlight current and future challenges in mathematical biology. The modelling research framework discussed herein could prove beneficial for the design of optimal bioprocesses, employing rational and feasible approaches towards the efficient production of chemicals and pharmaceuticals. PMID:24688682

Bioprocess development for hexavalent chromium reduction

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

Turick, C.E.; Apel, W.A.

1996-10-01

Hexavalent chromium (Cr(VI)) exists in the environment from anthropogenic activity and is regarded as a highly mobile and toxic pollutant. There is considerable interest in developing effective and efficient methods for the remediation of contaminated media. Many bacterial isolates have been demonstrated to metabolically reduce Cr(VI) to Cr(III), a much less toxic, more easily recoverable form of chromium. Aerobic and anaerobic cultures of Cr(VI) reducing bacteria were analyzed for their ability to reduce Cr(VI) prior to bioreactor design and scale up. Batch studies demonstrated Cr(VI) reduction rates with aerobic bacteria of up to 3 mg/hr/g dry cells, while anaerobic culturesmore » exhibited Cr(VI) reduction rates up to 22 mg/hr/g dry cells. An aerobic mixed culture of Cr(VI) reducing bacteria was chosen for bioreactor studies due to better rates of Cr(VI) reduction as well as the robust nature of the culture. These properties will allow for ease in bioprocess operation in the field.« less

Halophiles, coming stars for industrial biotechnology.

PubMed

Yin, Jin; Chen, Jin-Chun; Wu, Qiong; Chen, Guo-Qiang

2015-11-15

Industrial biotechnology aims to produce chemicals, materials and biofuels to ease the challenges of shortage on petroleum. However, due to the disadvantages of bioprocesses including energy consuming sterilization, high fresh water consumption, discontinuous fermentation to avoid microbial contamination, highly expensive stainless steel fermentation facilities and competing substrates for human consumption, industrial biotechnology is less competitive compared with chemical processes. Recently, halophiles have shown promises to overcome these shortcomings. Due to their unique halophilic properties, some halophiles are able to grow in high pH and high NaCl containing medium under higher temperature, allowing fermentation processes to run contamination free under unsterile conditions and continuous way. At the same time, genetic manipulation methods have been developed for halophiles. So far, halophiles have been used to produce bioplastics polyhydroxyalkanoates (PHA), ectoines, enzymes, and bio-surfactants. Increasing effects have been made to develop halophiles into a low cost platform for bioprocessing with advantages of low energy, less fresh water consumption, low fixed capital investment, and continuous production. Copyright © 2014 Elsevier Inc. All rights reserved.

Integration of systems biology with bioprocess engineering: L: -threonine production by systems metabolic engineering of Escherichia coli.

PubMed

Lee, Sang Yup; Park, Jin Hwan

2010-01-01

Random mutation and selection or targeted metabolic engineering without consideration of its impact on the entire metabolic and regulatory networks can unintentionally cause genetic alterations in the region, which is not directly related to the target metabolite. This is one of the reasons why strategies for developing industrial strains are now shifted towards targeted metabolic engineering based on systems biology, which is termed systems metabolic engineering. Using systems metabolic engineering strategies, all the metabolic engineering works are conducted in systems biology framework, whereby entire metabolic and regulatory networks are thoroughly considered in an integrated manner. The targets for purposeful engineering are selected after all possible effects on the entire metabolic and regulatory networks are thoroughly considered. Finally, the strain, which is capable of producing the target metabolite to a high level close to the theoretical maximum value, can be constructed. Here we review strategies and applications of systems biology successfully implemented on bioprocess engineering, with particular focus on developing L: -threonine production strains of Escherichia coli.

Gene and process level modulation to overcome the bottlenecks of recombinant proteins expression in Pichia pastoris.

PubMed

Prabhu, Ashish A; Boro, Bibari; Bharali, Biju; Chakraborty, Shuchishloka; Dasu, Veeranki V

2017-01-01

Process development involving system metabolic engineering and bioprocess engineering has become one of the major thrust for the development of therapeutic proteins or enzymes. Pichia pastoris has emerged as a prominent host for the production of therapeutic protein or enzymes. Regardless of producing high protein titers, various cellular and process level bottlenecks restrict the expression of recombinant proteins in P. pastoris. In the present review, we have summarized the recent developments in the expression of foreign proteins in P. pastoris. Further, we have discussed various cellular engineering strategies which include codon optimization, pathway engineering, signal peptide processing, development of protease deficient strain and glyco-engineered strains for the high yield protein secretion of recombinant protein. Bioprocess development of recombinant proteins in large-scale bioreactor including medium optimization, optimum feeding strategy and co-substrate feeding in fed-batch as well as continuous cultivation have been described. The recent advances in system and synthetic biology studies including metabolic flux analysis in understanding the phenotypic characteristics of recombinant Pichia and genome editing with CRISPR-CAS system have also been summarized. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

ECUT (Energy Conversion and Utilization Technologies Program). Biocatalysis Project

NASA Technical Reports Server (NTRS)

1986-01-01

Presented are the FY 1985 accomplishments, activities, and planned research efforts of the Biocatalysis Project of the U.S. Department of Energy, Energy Conversion and Utilization Technologies (ECUT) Program. The Project's technical activities were organized as follows: In the Molecular Modeling and Applied Genetics work element, research focused on (1) modeling and simulation studies to establish the physiological basis of high temperature tolerance in a selected enzyme and the catalytic mechanisms of three species of another enzyme, and (2) determining the degree of plasmid amplification and stability of several DNA bacterial strains. In the Bioprocess Engineering work element, research focused on (1) studies of plasmid propagation and the generation of models, (2) developing methods for preparing immobilized biocatalyst beads, and (3) developing an enzyme encapsulation method. In the Process Design and Analysis work element, research focused on (1) further refinement of a test case simulation of the economics and energy efficiency of alternative biocatalyzed production processes, (2) developing a candidate bioprocess to determine the potential for reduced energy consumption and facility/operating costs, and (3) a techno-economic assessment of potential advancements in microbial ammonia production.

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

Microgravity

NASA Image and Video Library

1998-10-01

CGBA, a facility developed by BioServe Space Technologies, a NASA Commercial Generic Bioprocessing Space Center, allows a variety of sophisticated bioprocessing research to be performed using a common device. The Fluids Processing Apparatus is essentially a microgravity test tube that allows a variety of complex investigations to be performed in space. This is a glass barrel containing several chambers separated by rubber stoppers. Eight FPAs are placed together in a Group Activation Pack (GAP), which allows all of the research to be started simultaneously by turning a single crank. Eight GAPs, or similar-sized payloads, can be stored in a single CGBA temperature controlled locker, which now uses motor drives to automatically turn the cranks to start and stop experiments. On STS-95, research efforts cover eight major areas that will benefit Earth-based products ranging from the production of pharmaceuticals to fish hatcheries.

Virtual parameter-estimation experiments in Bioprocess-Engineering education.

PubMed

Sessink, Olivier D T; Beeftink, Hendrik H; Hartog, Rob J M; Tramper, Johannes

2006-05-01

Cell growth kinetics and reactor concepts constitute essential knowledge for Bioprocess-Engineering students. Traditional learning of these concepts is supported by lectures, tutorials, and practicals: ICT offers opportunities for improvement. A virtual-experiment environment was developed that supports both model-related and experimenting-related learning objectives. Students have to design experiments to estimate model parameters: they choose initial conditions and 'measure' output variables. The results contain experimental error, which is an important constraint for experimental design. Students learn from these results and use the new knowledge to re-design their experiment. Within a couple of hours, students design and run many experiments that would take weeks in reality. Usage was evaluated in two courses with questionnaires and in the final exam. The faculties involved in the two courses are convinced that the experiment environment supports essential learning objectives well.

ECUT (Energy Conversion and Utilization Technologies) program: Biocatalysis Project

NASA Technical Reports Server (NTRS)

1988-01-01

Fiscal year 1987 research activities and accomplishments for the Biocatalysis Project of the U.S. Department of Energy, Energy Conversion and Utilization Technologies (ECUT) Division are presented. The project's technical activities were organized into three work elements. The Molecular Modeling and Applied Genetics work element includes modeling and simulation studies to verify a dynamic model of the enzyme carboxypeptidase; plasmid stabilization by chromosomal integration; growth and stability characteristics of plasmid-containing cells; and determination of optional production parameters for hyper-production of polyphenol oxidase. The Bioprocess Engineering work element supports efforts in novel bioreactor concepts that are likely to lead to substantially higher levels of reactor productivity, product yields, and lower separation energetics. The Bioprocess Design and Assessment work element attempts to develop procedures (via user-friendly computer software) for assessing the economics and energetics of a given biocatalyst process.

Early-branching Gut Fungi Possess A Large, And Comprehensive Array Of Biomass-Degrading Enzymes

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

Solomon, Kevin V.; Haitjema, Charles; Henske, John K.

The fungal kingdom is the source of almost all industrial enzymes in use for lignocellulose bioprocessing. Its more primitive members, however, remain relatively unexploited. We developed a systems-level approach that integrates RNA-Seq, proteomics, phenotype and biochemical studies of relatively unexplored early-branching free-living fungi. Anaerobic gut fungi isolated from herbivores produce a large array of biomass-degrading enzymes that synergistically degrade crude, unpretreated plant biomass, and are competitive with optimized commercial preparations from Aspergillus and Trichoderma. Compared to these model platforms, gut fungal enzymes are unbiased in substrate preference due to a wealth of xylan-degrading enzymes. These enzymes are universally catabolite repressed,more » and are further regulated by a rich landscape of noncoding regulatory RNAs. Furthermore, we identified several promising sequence divergent enzyme candidates for lignocellulosic bioprocessing.« less

Crewmember in the middeck beside the Commercial Generic Bioprocessing exp.

NASA Image and Video Library

1993-01-19

STS054-07-003 (13-19 Jan 1993) --- Astronaut John H. Casper, mission commander, floats near the Commercial Generic Bioprocessing Apparatus (CGBA) station on Endeavour's middeck. A friction car and its accompanying loop -- part of the Toys in Space package onboard -- can be seen just above Casper's head. The photograph was taken with a 35mm camera.

Bioprocessing preservative-treated waste wood

Treesearch

Barbara L. Illman; Vina W. Yang; Les Ferge

2000-01-01

Disposal of preservative-treated waste wood is a growing problem worldwide. Bioprocessing the treated wood offers one approach to waste management under certain conditions. One goal is to use wood decay fungi to reduce the volume of waste with an easily managed system in a cost-effective manner. Wood decay fungi were obtained from culture collections in the Mycology...

Monitoring cell culture media degradation using surface enhanced Raman scattering (SERS) spectroscopy.

PubMed

Calvet, Amandine; Ryder, Alan G

2014-08-20

The quality of the cell culture media used in biopharmaceutical manufacturing is a crucial factor affecting bioprocess performance and the quality of the final product. Due to their complex composition these media are inherently unstable, and significant compositional variations can occur particularly when in the prepared liquid state. For example photo-degradation of cell culture media can have adverse effects on cell viability and thus process performance. There is therefore, from quality control, quality assurance and process management view points, an urgent demand for the development of rapid and inexpensive tools for the stability monitoring of these complex mixtures. Spectroscopic methods, based on fluorescence or Raman measurements, have now become viable alternatives to more time-consuming and expensive (on a unit analysis cost) chromatographic and/or mass spectrometry based methods for routine analysis of media. Here we demonstrate the application of surface enhanced Raman scattering (SERS) spectroscopy for the simple, fast, analysis of cell culture media degradation. Once stringent reproducibility controls are implemented, chemometric data analysis methods can then be used to rapidly monitor the compositional changes in chemically defined media. SERS shows clearly that even when media are stored at low temperature (2-8°C) and in the dark, significant chemical changes occur, particularly with regard to cysteine/cystine concentration. Copyright © 2014 Elsevier B.V. All rights reserved.

Microalgal process-monitoring based on high-selectivity spectroscopy tools: status and future perspectives.

PubMed

Podevin, Michael; Fotidis, Ioannis A; Angelidaki, Irini

2018-08-01

Microalgae are well known for their ability to accumulate lipids intracellularly, which can be used for biofuels and mitigate CO 2 emissions. However, due to economic challenges, microalgae bioprocesses have maneuvered towards the simultaneous production of food, feed, fuel, and various high-value chemicals in a biorefinery concept. On-line and in-line monitoring of macromolecules such as lipids, proteins, carbohydrates, and high-value pigments will be more critical to maintain product quality and consistency for downstream processing in a biorefinery to maintain and valorize these markets. The main contribution of this review is to present current and prospective advances of on-line and in-line process analytical technology (PAT), with high-selectivity - the capability of monitoring several analytes simultaneously - in the interest of improving product quality, productivity, and process automation of a microalgal biorefinery. The high-selectivity PAT under consideration are mid-infrared (MIR), near-infrared (NIR), and Raman vibrational spectroscopies. The current review contains a critical assessment of these technologies in the context of recent advances in software and hardware in order to move microalgae production towards process automation through multivariate process control (MVPC) and software sensors trained on "big data". The paper will also include a comprehensive overview of off-line implementations of vibrational spectroscopy in microalgal research as it pertains to spectral interpretation and process automation to aid and motivate development.

Artificial neural network associated to UV/Vis spectroscopy for monitoring bioreactions in biopharmaceutical processes.

PubMed

Takahashi, Maria Beatriz; Leme, Jaci; Caricati, Celso Pereira; Tonso, Aldo; Fernández Núñez, Eutimio Gustavo; Rocha, José Celso

2015-06-01

Currently, mammalian cells are the most utilized hosts for biopharmaceutical production. The culture media for these cell lines include commonly in their composition a pH indicator. Spectroscopic techniques are used for biopharmaceutical process monitoring, among them, UV-Vis spectroscopy has found scarce applications. This work aimed to define artificial neural networks architecture and fit its parameters to predict some nutrients and metabolites, as well as viable cell concentration based on UV-Vis spectral data of mammalian cell bioprocess using phenol red in culture medium. The BHK-21 cell line was used as a mammalian cell model. Off-line spectra of supernatant samples taken from batches performed at different dissolved oxygen concentrations in two bioreactor configurations and with two pH control strategies were used to define two artificial neural networks. According to absolute errors, glutamine (0.13 ± 0.14 mM), glutamate (0.02 ± 0.02 mM), glucose (1.11 ± 1.70 mM), lactate (0.84 ± 0.68 mM) and viable cell concentrations (1.89 10(5) ± 1.90 10(5) cell/mL) were suitably predicted. The prediction error averages for monitored variables were lower than those previously reported using different spectroscopic techniques in combination with partial least squares or artificial neural network. The present work allows for UV-VIS sensor development, and decreases cost related to nutrients and metabolite quantifications.

Fueling the future with fungal genomics

Treesearch

Igor.V. Grigoriev; Daniel Cullen; Stephen B. Goodwin; David Hibbett; Thomas W. Jeffries; Christian P. Kubicek; Cheryl Kuske; Jon K. Magnuson; Francis Martin; Joseph W. Spatafora; Adrian Tsang; Scott E. Baker

2011-01-01

Fungi play important roles across the range of current and future biofuel production processes. From crop/feedstock health to plant biomass saccharification, enzyme production to bioprocesses for producing ethanol, higher alcohols, or future hydrocarbon biofuels, fungi are involved. Research and development are underway to understand the underlying biological processes...

Quantitative monitoring of an activated sludge reactor using on-line UV-visible and near-infrared spectroscopy.

PubMed

Sarraguça, Mafalda C; Paulo, Ana; Alves, Madalena M; Dias, Ana M A; Lopes, João A; Ferreira, Eugénio C

2009-10-01

The performance of an activated sludge reactor can be significantly enhanced through use of continuous and real-time process-state monitoring, which avoids the need to sample for off-line analysis and to use chemicals. Despite the complexity associated with wastewater treatment systems, spectroscopic methods coupled with chemometric tools have been shown to be powerful tools for bioprocess monitoring and control. Once implemented and optimized, these methods are fast, nondestructive, user friendly, and most importantly, they can be implemented in situ, permitting rapid inference of the process state at any moment. In this work, UV-visible and NIR spectroscopy were used to monitor an activated sludge reactor using in situ immersion probes connected to the respective analyzers by optical fibers. During the monitoring period, disturbances to the biological system were induced to test the ability of each spectroscopic method to detect the changes in the system. Calibration models based on partial least squares (PLS) regression were developed for three key process parameters, namely chemical oxygen demand (COD), nitrate concentration (N-NO(3)(-)), and total suspended solids (TSS). For NIR, the best results were achieved for TSS, with a relative error of 14.1% and a correlation coefficient of 0.91. The UV-visible technique gave similar results for the three parameters: an error of approximately 25% and correlation coefficients of approximately 0.82 for COD and TSS and 0.87 for N-NO(3)(-) . The results obtained demonstrate that both techniques are suitable for consideration as alternative methods for monitoring and controlling wastewater treatment processes, presenting clear advantages when compared with the reference methods for wastewater treatment process qualification.

HEK293 cell culture media study towards bioprocess optimization: Animal derived component free and animal derived component containing platforms.

PubMed

Liste-Calleja, Leticia; Lecina, Martí; Cairó, Jordi Joan

2014-04-01

The increasing demand for biopharmaceuticals produced in mammalian cells has lead industries to enhance bioprocess volumetric productivity through different strategies. Among those strategies, cell culture media development is of major interest. In the present work, several commercially available culture media for Human Embryonic Kidney cells (HEK293) were evaluated in terms of maximal specific growth rate and maximal viable cell concentration supported. The main objective was to provide different cell culture platforms which are suitable for a wide range of applications depending on the type and the final use of the product obtained. Performing simple media supplementations with and without animal derived components, an enhancement of cell concentration from 2 × 10(6) cell/mL to 17 × 10(6) cell/mL was achieved in batch mode operation. Additionally, the media were evaluated for adenovirus production as a specific application case of HEK293 cells. None of the supplements interfered significantly with the adenovirus infection although some differences were encountered in viral productivity. To the best of our knowledge, the high cell density achieved in the work presented has never been reported before in HEK293 batch cell cultures and thus, our results are greatly promising to further study cell culture strategies in bioreactor towards bioprocess optimization. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Mesoderm Lineage 3D Tissue Constructs Are Produced at Large-Scale in a 3D Stem Cell Bioprocess.

PubMed

Cha, Jae Min; Mantalaris, Athanasios; Jung, Sunyoung; Ji, Yurim; Bang, Oh Young; Bae, Hojae

2017-09-01

Various studies have presented different approaches to direct pluripotent stem cell differentiation such as applying defined sets of exogenous biochemical signals and genetic/epigenetic modifications. Although differentiation to target lineages can be successfully regulated, such conventional methods are often complicated, laborious, and not cost-effective to be employed to the large-scale production of 3D stem cell-based tissue constructs. A 3D-culture platform that could realize the large-scale production of mesoderm lineage tissue constructs from embryonic stem cells (ESCs) is developed. ESCs are cultured using our previously established 3D-bioprocess platform which is amenable to mass-production of 3D ESC-based tissue constructs. Hepatocarcinoma cell line conditioned medium is introduced to the large-scale 3D culture to provide a specific biomolecular microenvironment to mimic in vivo mesoderm formation process. After 5 days of spontaneous differentiation period, the resulting 3D tissue constructs are composed of multipotent mesodermal progenitor cells verified by gene and molecular expression profiles. Subsequently the optimal time points to trigger terminal differentiation towards cardiomyogenesis or osteogenesis from the mesodermal tissue constructs is found. A simple and affordable 3D ESC-bioprocess that can reach the scalable production of mesoderm origin tissues with significantly improved correspondent tissue properties is demonstrated. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Optical sensor for rapid microbial detection

NASA Astrophysics Data System (ADS)

Al-Adhami, Mustafa; Tilahun, Dagmawi; Rao, Govind; Kostov, Yordan

2016-05-01

In biotechnology, the ability to instantly detect contaminants is key to running a reliable bioprocess. Bioprocesses are prone to be contaminated by cells that are abundant in our environment; detection and quantification of these cells would aid in the preservation of the bioprocess product. This paper discusses the design and development of a portable kinetics fluorometer which acts as a single-excitation, single-emission photometer that continuously measures fluorescence intensity of an indicator dye, and plots it. Resazurin is used as an indicator dye since the viable contaminant cells reduce Resazurin toResorufin, the latter being strongly fluorescent. A photodiode detects fluorescence change by generating current proportional to the intensity of the light that reached it, and a trans-impedance differential op-amp ensures amplification of the photodiodes' signal. A microfluidic chip was designed specifically for the device. It acts as a fully enclosed cuvette, which enhances the Resazurin reduction rate. E. coli in LB media, along with Resazurin were injected into the microfluidic chip. The optical sensor detected the presence of E. coli in the media based on the fluorescence change that occurred in the indicator dye in concentrations as low as 10 CFU/ml. A method was devised to detect and determine an approximate amount of contamination with this device. This paper discusses application of this method to detect and estimate sample contamination. This device provides fast, accurate, and inexpensive means to optically detect the presence of viable cells.

Production of polyol oils from soybean oil by Pseudomonas aeruginosa E03-12.

USDA-ARS?s Scientific Manuscript database

Soy-polyols are important starting materials for the manufacture of polymers such as polyurethane. We have been trying to develop a bioprocess for the production of polyol oils directly from soybean oil. We reported earlier the polyol products produced from soybean oil by Acinetobacter haemolyticus ...

Prospects for bioprocess development based on recent genome advances in lignocellulose degrading basidiomycetes

Treesearch

Chiaki Hori; Daniel Cullen

2016-01-01

Efficient and complete degradation of woody plant cell walls requires the concerted action of hydrolytic and oxidative systems possessed by a relatively small group of filamentous basidiomycetous fungi. Among these wood decay species, Phanerochaete chrysosporium was the first to be sequenced (Martinez et al. 2004). In...

By-products from the biodiesel chain as a substrate to citric acid production by solid-state fermentation.

PubMed

Schneider, Manuella; Zimmer, Gabriela F; Cremonese, Ezequiel B; de C de S Schneider, Rosana; Corbellini, Valeriano A

2014-07-01

In this study, we propose the use of tung cake for the production of organic acids, with an emphasis on citric acid by solid-state fermentation. We evaluated the conditions of production and the by-products from the biodiesel chain as raw materials involved in this bioprocess. First, we standardized the conditions of solid-state fermentation in tung cake with and without residual fat and with different concentrations of glycerine using the fungus Aspergillus niger The solid-state fermentation process was monitored for 7 days considering the biomass growth and pH level. Citric acid production was determined by high-performance liquid chromatography. Fungal development was better in the crude tung cake, consisting of 20% glycerine. The highest citric acid yield was 350 g kg(-1) of biomass. Therefore, the solid-state fermentation of the tung cake with glycerine led to citric acid production using the Aspergillus niger fungus. © The Author(s) 2014.

Dual-Responsive Metabolic Precursor and Light-Up AIEgen for Cancer Cell Bio-orthogonal Labeling and Precise Ablation.

PubMed

Hu, Fang; Yuan, Youyong; Wu, Wenbo; Mao, Duo; Liu, Bin

2018-06-05

Metabolic glycoengineering of unnatural glycans with bio-orthogonal chemical groups and a subsequent click reaction with fluorescent probes have been widely used in monitoring various bioprocesses. Herein, we developed a dual-responsive metabolic precursor that could specifically generate unnatural glycans with azide groups on the membrane of targeted cancer cells with high selectivity. Moreover, a water-soluble fluorescent light-up probe with aggregation-induced emission (AIE) was synthesized, which turned its fluorescence on upon a click reaction with azide groups on the cancer cell surface, enabling special cancer cell imaging with low background signal. Furthermore, the probe can generate 1 O 2 upon light irradiation, fulfilling its dual role as an imaging and therapeutic agent for cancer cells. Therefore, the concepts of the cancer-cell-specific metabolic precursor cRGD-S-Ac 3 ManNAz and the AIE light-up probe are promising in bio-orthogonal labeling and cancer-specific imaging and therapy.

Sonocrystallization and Its Application in Food and Bioprocessing

NASA Astrophysics Data System (ADS)

Gogate, Parag R.; Pandit, Aniruddha B.

The chapter aims at understanding in detail, the application of ultrasound for intensification of crystallization operation and covers different aspects such as basic mechanism of expected intensification, reactor designs and overview of existing literature related to food and bioprocess industry applications with an objective of presenting optimum guidelines for maximizing the efficacy of using ultrasound. A case study of lactose recovery from whey has also been discussed in details so as to give quantitative information about the effects of ultrasound in different stages of the crystallization operation and guidelines for optimization of different geometric and operating parameters. Overall it appears that use of ultrasound can significantly improve the crystallization operation by significant reduction in the processing time with generation of better quality crystals and also the recent developments in the design of large scale sonochemical reactors have enhanced the possibility of the application in actual commercial practice.

Current state of coenzyme Q(10) production and its applications.

PubMed

Jeya, Marimuthu; Moon, Hee-Jung; Lee, Jeong-Lim; Kim, In-Won; Lee, Jung-Kul

2010-02-01

Coenzyme Q(10) (CoQ(10)), an obligatory cofactor in the aerobic respiratory electron transfer for energy generation, is formed from the conjugation of a benzoquinone ring with a hydrophobic isoprenoid chain. CoQ(10) is now used as a nutritional supplement because of its antioxidant properties and is beneficial in the treatment of several human diseases when administered orally. Bioprocesses have been developed for the commercial production of CoQ(10) because of its increased demand, and these bioprocesses depend on microbes that produce high levels of CoQ(10) naturally. However, as knowledge of the biosynthetic enzymes and the regulatory mechanisms modulating CoQ(10) production increases, approaches arise for the genetic engineering of CoQ(10) production in Escherichia coli and Agrobacterium tumefaciens. This review focused on approaches for CoQ(10) production, strategies used to engineer CoQ(10) production in microbes, and potential applications of CoQ(10).

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

Beck, Ashley; Hunt, Kristopher; Bernstein, Hans C.

Interest in microbial communities for bioprocessing has surged in recent years based on the potential to optimize multiple tasks simultaneously and to enhance process productivity and stability. The presence and magnitude of these desirable system properties often result from interactions between functionally distinct community members. The importance of interactions, while appreciated by some disciplines for decades, has gained interest recently due to the development of ‘omics techniques, polymicrobial culturing approaches, and computational methods which has made the systems-level analysis of interacting components more tractable. This review defines and categorizes natural and engineered system components, interactions, and emergent properties, as wellmore » as presents three ecological theories relevant to microbial communities. Case studies are interpreted to illustrate components, interactions, emergent properties and agreement with theoretical concepts. A general foundation is laid to facilitate interpretation of current systems and to aid in future design of microbial systems for the next generation of bioprocesses.« less

Software sensors for bioprocesses.

PubMed

Bogaerts, Ph; Vande Wouwer, A

2003-10-01

State estimation is a significant problem in biotechnological processes, due to the general lack of hardware sensor measurements of the variables describing the process dynamics. The objective of this paper is to review a number of software sensor design methods, including extended Kalman filters, receding-horizon observers, asymptotic observers, and hybrid observers, which can be efficiently applied to bioprocesses. These several methods are illustrated with simulation and real-life case studies.

Elm tree (Ulmus parvifolia) bark bioprocessed with Mycelia of Shiitake (Lentinus edodes) mushrooms in liquid Culture: Composition and mechanism of protection against allergic asthma in mice

USDA-ARS?s Scientific Manuscript database

The present study investigated the antiasthma effect of a bioprocessed Ulmus parvifolia bark extract (BPUBE) from Lentinus edodes liquid mycelia culture against allergic asthma biomarkers in U266B1 leukemia cells and OVA-sensitized/challenged mice. BPUBE suppressed total IgE release from U266B1 cel...

Integrated continuous bioprocessing: Economic, operational, and environmental feasibility for clinical and commercial antibody manufacture.

PubMed

Pollock, James; Coffman, Jon; Ho, Sa V; Farid, Suzanne S

2017-07-01

This paper presents a systems approach to evaluating the potential of integrated continuous bioprocessing for monoclonal antibody (mAb) manufacture across a product's lifecycle from preclinical to commercial manufacture. The economic, operational, and environmental feasibility of alternative continuous manufacturing strategies were evaluated holistically using a prototype UCL decisional tool that integrated process economics, discrete-event simulation, environmental impact analysis, operational risk analysis, and multiattribute decision-making. The case study focused on comparing whole bioprocesses that used either batch, continuous or a hybrid combination of batch and continuous technologies for cell culture, capture chromatography, and polishing chromatography steps. The cost of goods per gram (COG/g), E-factor, and operational risk scores of each strategy were established across a matrix of scenarios with differing combinations of clinical development phase and company portfolio size. The tool outputs predict that the optimal strategy for early phase production and small/medium-sized companies is the integrated continuous strategy (alternating tangential flow filtration (ATF) perfusion, continuous capture, continuous polishing). However, the top ranking strategy changes for commercial production and companies with large portfolios to the hybrid strategy with fed-batch culture, continuous capture and batch polishing from a COG/g perspective. The multiattribute decision-making analysis highlighted that if the operational feasibility was considered more important than the economic benefits, the hybrid strategy would be preferred for all company scales. Further considerations outside the scope of this work include the process development costs required to adopt continuous processing. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 33:854-866, 2017. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers.

Verification of a new biocompatible single-use film formulation with optimized additive content for multiple bioprocess applications.

PubMed

Jurkiewicz, Elke; Husemann, Ute; Greller, Gerhard; Barbaroux, Magali; Fenge, Christel

2014-01-01

Single-use bioprocessing bags and bioreactors gained significant importance in the industry as they offer a number of advantages over traditional stainless steel solutions. However, there is continued concern that the plastic materials might release potentially toxic substances negatively impacting cell growth and product titers, or even compromise drug safety when using single-use bags for intermediate or drug substance storage. In this study, we have focused on the in vitro detection of potentially cytotoxic leachables originating from the recently developed new polyethylene (PE) multilayer film called S80. This new film was developed to guarantee biocompatibility for multiple bioprocess applications, for example, storage of process fluids, mixing, and cell culture bioreactors. For this purpose, we examined a protein-free cell culture medium that had been used to extract leachables from freshly gamma-irradiated sample bags in a standardized cell culture assay. We investigated sample bags from films generated to establish the operating ranges of the film extrusion process. Further, we studied sample bags of different age after gamma-irradiation and finally, we performed extended media extraction trials at cold room conditions using sample bags. In contrast to a nonoptimized film formulation, our data demonstrate no cytotoxic effect of the S80 polymer film formulation under any of the investigated conditions. The S80 film formulation is based on an optimized PE polymer composition and additive package. Full traceability alongside specifications and controls of all critical raw materials, and process controls of the manufacturing process, that is, film extrusion and gamma-irradiation, have been established to ensure lot-to-lot consistency. © 2014 American Institute of Chemical Engineers.

Integrated continuous bioprocessing: Economic, operational, and environmental feasibility for clinical and commercial antibody manufacture

PubMed Central

Pollock, James; Coffman, Jon; Ho, Sa V.

2017-01-01

This paper presents a systems approach to evaluating the potential of integrated continuous bioprocessing for monoclonal antibody (mAb) manufacture across a product's lifecycle from preclinical to commercial manufacture. The economic, operational, and environmental feasibility of alternative continuous manufacturing strategies were evaluated holistically using a prototype UCL decisional tool that integrated process economics, discrete‐event simulation, environmental impact analysis, operational risk analysis, and multiattribute decision‐making. The case study focused on comparing whole bioprocesses that used either batch, continuous or a hybrid combination of batch and continuous technologies for cell culture, capture chromatography, and polishing chromatography steps. The cost of goods per gram (COG/g), E‐factor, and operational risk scores of each strategy were established across a matrix of scenarios with differing combinations of clinical development phase and company portfolio size. The tool outputs predict that the optimal strategy for early phase production and small/medium‐sized companies is the integrated continuous strategy (alternating tangential flow filtration (ATF) perfusion, continuous capture, continuous polishing). However, the top ranking strategy changes for commercial production and companies with large portfolios to the hybrid strategy with fed‐batch culture, continuous capture and batch polishing from a COG/g perspective. The multiattribute decision‐making analysis highlighted that if the operational feasibility was considered more important than the economic benefits, the hybrid strategy would be preferred for all company scales. Further considerations outside the scope of this work include the process development costs required to adopt continuous processing. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 33:854–866, 2017 PMID:28480535

Efficient and reproducible mammalian cell bioprocesses without probes and controllers?

PubMed

Tissot, Stéphanie; Oberbek, Agata; Reclari, Martino; Dreyer, Matthieu; Hacker, David L; Baldi, Lucia; Farhat, Mohamed; Wurm, Florian M

2011-07-01

Bioprocesses for recombinant protein production with mammalian cells are typically controlled for several physicochemical parameters including the pH and dissolved oxygen concentration (DO) of the culture medium. Here we studied whether these controls are necessary for efficient and reproducible bioprocesses in an orbitally shaken bioreactor (OSR). Mixing, gas transfer, and volumetric power consumption (P(V)) were determined in both a 5-L OSR and a 3-L stirred-tank bioreactor (STR). The two cultivation systems had a similar mixing intensity, but the STR had a lower volumetric mass transfer coefficient of oxygen (k(L)a) and a higher P(V) than the OSR. Recombinant CHO cell lines expressing either tumor necrosis factor receptor as an Fc fusion protein (TNFR:Fc) or an anti-RhesusD monoclonal antibody were cultivated in the two systems. The 5-L OSR was operated in an incubator shaker with 5% CO(2) in the gas environment but without pH and DO control whereas the STR was operated with or without pH and DO control. Higher cell densities and recombinant protein titers were obtained in the OSR as compared to both the controlled and the non-controlled STRs. To test the reproducibility of a bioprocess in a non-controlled OSR, the two CHO cell lines were each cultivated in parallel in six 5-L OSRs. Similar cell densities, cell viabilities, and recombinant protein titers along with similar pH and DO profiles were achieved in each group of replicates. Our study demonstrated that bioprocesses can be performed in OSRs without pH or DO control in a highly reproducible manner, at least at the scale of operation studied here. Copyright © 2011 Elsevier B.V. All rights reserved.

Application of Hydrodynamic Cavitation for Food and Bioprocessing

NASA Astrophysics Data System (ADS)

Gogate, Parag R.

Hydrodynamic cavitation can be simply generated by the alterations in the flow field in high speed/high pressure devices and also by passage of the liquid through a constriction such as orifice plate, venturi, or throttling valve. Hydrodynamic cavitation results in the formation of local hot spots, release of highly reactive free radicals, and enhanced mass transfer rates due to turbulence generated as a result of liquid circulation currents. These conditions can be suitably applied for intensification of different bioprocessing applications in an energy-efficient manner as compared to conventionally used ultrasound-based reactors. The current chapter aims at highlighting different aspects related to hydrodynamic cavitation, including the theoretical aspects for optimization of operating parameters, reactor designs, and overview of applications relevant to food and bioprocessing. Some case studies highlighting the comparison of hydrodynamic cavitation and acoustic cavitation reactors will also be discussed.

New Laboratory Course for Senior-Level Chemical Engineering Students

ERIC Educational Resources Information Center

Aronson, Mark T.; Deitcher, Robert W.; Xi, Yuanzhou; Davis, Robert J.

2009-01-01

A new laboratory course has been developed at the University of Virginia for senior- level chemical engineering students. The new course is based on three 4-week long experiments in bioprocess engineering, energy conversion and catalysis, and polymer synthesis and characterization. The emphasis is on the integration of process steps and the…

Genome Sequence of the Alkaline-Tolerant Cellulomonas sp. Strain FA1

DOE PAGES

Cohen, Michael F.; Hu, Ping; Nguyen, My Vu; ...

2015-06-18

We present the genome of the cellulose-degrading Cellulomonas sp. strain FA1 isolated from an actively serpentinizing highly alkaline spring. Knowledge of this genome will enable studies into the molecular basis of plant material degradation in alkaline environments and inform the development of lignocellulose bioprocessing procedures for biofuel production.

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

Baker, Scott E.; Bruno, Kenneth S.; Butcher, Mark G.

In 2009, we continued to address barriers to fungal fermentation in the primary areas of morphology control, genomics, proteomics, fungal hyperproductivity, biomass-to-products via fungal based consolidated bioprocesses, and filamentous fungal ethanol. “Alternative renewable fuels from fungi” was added as a new subtask. Plans were also made to launch a new advanced strain development subtask in FY2010.

An Interactive Teaching System for Bond Graph Modeling and Simulation in Bioengineering

ERIC Educational Resources Information Center

Roman, Monica; Popescu, Dorin; Selisteanu, Dan

2013-01-01

The objective of the present work was to implement a teaching system useful in modeling and simulation of biotechnological processes. The interactive system is based on applications developed using 20-sim modeling and simulation software environment. A procedure for the simulation of bioprocesses modeled by bond graphs is proposed and simulators…

Bioremediation and degradation of CCA-treated wood waste.

Treesearch

Barbara L Illman; Vina W. Yang

2004-01-01

Bioprocessing CCA wood waste is an efficient and economical alternative to depositing the waste in landfills, especially if landfill restrictions on CCA waste are imposed nation wide. We have developed bioremediation and degradation technologies for microbial processing of CCA waste. The technologies are based on specially formulated inoculum of wood decay fungi,...

Process analytical technology (PAT) in insect and mammalian cell culture processes: dielectric spectroscopy and focused beam reflectance measurement (FBRM).

PubMed

Druzinec, Damir; Weiss, Katja; Elseberg, Christiane; Salzig, Denise; Kraume, Matthias; Pörtner, Ralf; Czermak, Peter

2014-01-01

Modern bioprocesses demand for a careful definition of the critical process parameters (CPPs) already during the early stages of process development in order to ensure high-quality products and satisfactory yields. In this context, online monitoring tools can be applied to recognize unfavorable changes of CPPs during the production processes and to allow for early interventions in order to prevent losses of production batches due to quality issues. Process analytical technologies such as the dielectric spectroscopy or focused beam reflectance measurement (FBRM) are possible online monitoring tools, which can be applied to monitor cell growth as well as morphological changes. Since the dielectric spectroscopy only captures cells with intact cell membranes, even information about dead cells with ruptured or leaking cell membranes can be derived. The following chapter describes the application of dielectric spectroscopy on various virus-infected and non-infected cell lines with respect to adherent as well as suspension cultures in common stirred tank reactors. The adherent mammalian cell lines Vero (African green monkey kidney cells) and hMSC-TERT (telomerase-immortalized human mesenchymal stem cells) are thereby cultured on microcarrier, which provide the required growth surface and allow the cultivation of these cells even in dynamic culture systems. In turn, the insect-derived cell lines S2 and Sf21 are used as examples for cells typically cultured in suspension. Moreover, the FBRM technology as a further monitoring tool for cell culture applications has been included in this chapter using the example of Drosophila S2 insect cells.

Bioprocessing of Cryopreservation for Large-Scale Banking of Human Pluripotent Stem Cells

PubMed Central

Ma, Teng

2012-01-01

Abstract Human pluripotent stem cell (hPSC)-derived cell therapy requires production of therapeutic cells in large quantity, which starts from thawing the cryopreserved cells from a working cell bank or a master cell bank. An optimal cryopreservation and thaw process determines the efficiency of hPSC expansion and plays a significant role in the subsequent lineage-specific differentiation. However, cryopreservation in hPSC bioprocessing has been a challenge due to the unique growth requirements of hPSC, the sensitivity to cryoinjury, and the unscalable cryopreservation procedures commonly used in the laboratory. Tremendous progress has been made to identify the regulatory pathways regulating hPSC responses during cryopreservation and the development of small molecule interventions that effectively improves the efficiency of cryopreservation. The adaption of these methods in current good manufacturing practices (cGMP)-compliant cryopreservation processes not only improves cell survival, but also their therapeutic potency. This review summarizes the advances in these areas and discusses the technical requirements in the development of cGMP-compliant hPSC cryopreservation process. PMID:23515461

Universal Capacitance Model for Real-Time Biomass in Cell Culture.

PubMed

Konakovsky, Viktor; Yagtu, Ali Civan; Clemens, Christoph; Müller, Markus Michael; Berger, Martina; Schlatter, Stefan; Herwig, Christoph

2015-09-02

: Capacitance probes have the potential to revolutionize bioprocess control due to their safe and robust use and ability to detect even the smallest capacitors in the form of biological cells. Several techniques have evolved to model biomass statistically, however, there are problems with model transfer between cell lines and process conditions. Errors of transferred models in the declining phase of the culture range for linear models around +100% or worse, causing unnecessary delays with test runs during bioprocess development. The goal of this work was to develop one single universal model which can be adapted by considering a potentially mechanistic factor to estimate biomass in yet untested clones and scales. The novelty of this work is a methodology to select sensitive frequencies to build a statistical model which can be shared among fermentations with an error between 9% and 38% (mean error around 20%) for the whole process, including the declining phase. A simple linear factor was found to be responsible for the transferability of biomass models between cell lines, indicating a link to their phenotype or physiology.

Consolidated bioprocessing method using thermophilic microorganisms

DOEpatents

Mielenz, Jonathan Richard

2016-02-02

The present invention is directed to a method of converting biomass to biofuel, and particularly to a consolidated bioprocessing method using a co-culture of thermophilic and extremely thermophilic microorganisms which collectively can ferment the hexose and pentose sugars produced by degradation of cellulose and hemicelluloses at high substrate conversion rates. A culture medium therefor is also provided as well as use of the methods to produce and recover cellulosic ethanol.

Genetic programming assisted stochastic optimization strategies for optimization of glucose to gluconic acid fermentation.

PubMed

Cheema, Jitender Jit Singh; Sankpal, Narendra V; Tambe, Sanjeev S; Kulkarni, Bhaskar D

2002-01-01

This article presents two hybrid strategies for the modeling and optimization of the glucose to gluconic acid batch bioprocess. In the hybrid approaches, first a novel artificial intelligence formalism, namely, genetic programming (GP), is used to develop a process model solely from the historic process input-output data. In the next step, the input space of the GP-based model, representing process operating conditions, is optimized using two stochastic optimization (SO) formalisms, viz., genetic algorithms (GAs) and simultaneous perturbation stochastic approximation (SPSA). These SO formalisms possess certain unique advantages over the commonly used gradient-based optimization techniques. The principal advantage of the GP-GA and GP-SPSA hybrid techniques is that process modeling and optimization can be performed exclusively from the process input-output data without invoking the detailed knowledge of the process phenomenology. The GP-GA and GP-SPSA techniques have been employed for modeling and optimization of the glucose to gluconic acid bioprocess, and the optimized process operating conditions obtained thereby have been compared with those obtained using two other hybrid modeling-optimization paradigms integrating artificial neural networks (ANNs) and GA/SPSA formalisms. Finally, the overall optimized operating conditions given by the GP-GA method, when verified experimentally resulted in a significant improvement in the gluconic acid yield. The hybrid strategies presented here are generic in nature and can be employed for modeling and optimization of a wide variety of batch and continuous bioprocesses.

Industrial biosystems engineering and biorefinery systems.

PubMed

Chen, Shulin

2008-06-01

The concept of Industrial Biosystems Engineering (IBsE) was suggested as a new engineering branch to be developed for meeting the needs for science, technology and professionals by the upcoming bioeconomy. With emphasis on systems, IBsE builds upon the interfaces between systems biology, bioprocessing, and systems engineering. This paper discussed the background, the suggested definition, the theoretical framework and methodologies of this new discipline as well as its challenges and future development.

Development and application of biotechnologies in the metal mining industry.

PubMed

Johnson, D Barrie

2013-11-01

Metal mining faces a number of significant economic and environmental challenges in the twenty-first century for which established and emerging biotechnologies may, at least in part, provide the answers. Bioprocessing of mineral ores and concentrates is already used in variously engineered formats to extract base (e.g., copper, cobalt, and nickel) and precious (gold and silver) metals in mines throughout the world, though it remains a niche technology. However, current projections of an increasing future need to use low-grade primary metal ores, to reprocess mine wastes, and to develop in situ leaching technologies to extract metals from deep-buried ore bodies, all of which are economically more amenable to bioprocessing than conventional approaches (e.g., pyrometallurgy), would suggest that biomining will become more extensively utilized in the future. Recent research has also shown that bioleaching could be used to process a far wider range of metal ores (e.g., oxidized ores) than has previously been the case. Biotechnologies are also being developed to control mine-related pollution, including securing mine wastes (rocks and tailings) by using "ecological engineering" approaches, and also to remediate and recover metals from waste waters, such as acid mine drainage. This article reviews the current status of biotechnologies within the mining sector and considers how these may be developed and applied in future years.

Characterisation and identification of bacteria using SERS.

PubMed

Jarvis, Roger M; Goodacre, Royston

2008-05-01

Within microbiology Raman spectroscopy is considered as a very important whole-organism fingerprinting technique, which is used to characterise, discriminate and identify microorganisms and assess how they respond to abiotic or biotic stress. Enhancing the sensitivity of Raman spectroscopy is very beneficial for the rapid analysis of bacteria (and indeed biological systems in general), where the ultimate goal is to achieve this without the need for lengthy cell culture. Bypassing this step would provide significant benefits in many areas such as medical, environmental and industrial microbiology, microbial systems biology, biological warfare countermeasures and bioprocess monitoring. In this tutorial review we will report on the advances made in bacterial studies, a relatively new and exciting application area for SERS.

Progress in bacterial cellulose matrices for biotechnological applications.

PubMed

Cacicedo, Maximiliano L; Castro, M Cristina; Servetas, Ioannis; Bosnea, Loulouda; Boura, Konstantina; Tsafrakidou, Panagiota; Dima, Agapi; Terpou, Antonia; Koutinas, Athanasios; Castro, Guillermo R

2016-08-01

Bacterial cellulose (BC) is an extracellular polymer produced by many microorganisms. The Komagataeibacter genus is the best producer using semi-synthetic media and agricultural wastes. The main advantages of BC are the nanoporous structure, high water content and free hydroxyl groups. Modification of BC can be made by two strategies: in-situ, during the BC production, and ex-situ after BC purification. In bioprocesses, multilayer BC nanocomposites can contain biocatalysts designed to be suitable for outside to inside cell activities. These nanocomposites biocatalysts can (i) increase productivity in bioreactors and bioprocessing, (ii) provide cell activities does not possess without DNA cloning and (iii) provide novel nano-carriers for cell inside activity and bioprocessing. In nanomedicine, BC matrices containing therapeutic molecules can be used for pathologies like skin burns, and implantable therapeutic devices. In nanoelectronics, semiconductors BC-based using salts and synthetic polymers brings novel films showing excellent optical and photochemical properties. Copyright © 2016 Elsevier Ltd. All rights reserved.

Additive Biotech-Chances, challenges, and recent applications of additive manufacturing technologies in biotechnology.

PubMed

Krujatz, Felix; Lode, Anja; Seidel, Julia; Bley, Thomas; Gelinsky, Michael; Steingroewer, Juliane

2017-10-25

The diversity and complexity of biotechnological applications are constantly increasing, with ever expanding ranges of production hosts, cultivation conditions and measurement tasks. Consequently, many analytical and cultivation systems for biotechnology and bioprocess engineering, such as microfluidic devices or bioreactors, are tailor-made to precisely satisfy the requirements of specific measurements or cultivation tasks. Additive manufacturing (AM) technologies offer the possibility of fabricating tailor-made 3D laboratory equipment directly from CAD designs with previously inaccessible levels of freedom in terms of structural complexity. This review discusses the historical background of these technologies, their most promising current implementations and the associated workflows, fabrication processes and material specifications, together with some of the major challenges associated with using AM in biotechnology/bioprocess engineering. To illustrate the great potential of AM, selected examples in microfluidic devices, 3D-bioprinting/biofabrication and bioprocess engineering are highlighted. Copyright © 2017 Elsevier B.V. All rights reserved.

Direct ethanol production from starch using a natural isolate, Scheffersomyces shehatae: Toward consolidated bioprocessing.

PubMed

Tanimura, Ayumi; Kikukawa, Minako; Yamaguchi, Shino; Kishino, Shigenobu; Ogawa, Jun; Shima, Jun

2015-04-22

Consolidated bioprocessing (CBP), which integrates enzyme production, saccharification and fermentation into a one-step process, is a promising strategy for cost-effective ethanol production from starchy biomass. To gain insights into starch-based ethanol production using CBP, an extensive screening was undertaken to identify naturally occurring yeasts that produce ethanol without the addition of any amylases. Three yeast strains were capable of producing a significant amount of ethanol. Quantitative assays revealed that Scheffersomyces shehatae JCM 18690 was the strain showing the highest ethanol production ability. This strain was able to utilize starch directly, and the ethanol concentration reached 9.21 g/L. We attribute the ethanol-producing ability of this strain to the high levels of glucoamylase activity, fermentation potential and ethanol stress tolerance. This study strongly suggests the possibility of starch-based ethanol production by consolidated bioprocessing using natural yeasts such as S. shehatae JCM 18690.

Comparative analysis of solid-state bioprocessing and enzymatic treatment of finger millet for mobilization of bound phenolics.

PubMed

Yadav, Geetanjali; Singh, Anshu; Bhattacharya, Patrali; Yuvraj, Jude; Banerjee, Rintu

2013-11-01

The present work investigates the probable bioprocessing technique to mobilize the bound phenolics naturally found in finger millet cell wall for enriching it with dietary antioxidants. Comparative study was performed between the exogenous enzymatic treatment and solid-state fermentation of grain (SSF) with a food grade organism Rhizopus oryzae. SSF results indicated that at the 6th day of incubation, total phenolic content (18.64 mg gallic acid equivalent/gds) and antioxidant property (DPPH radical scavenging activity of 39.03 %, metal chelating ability of 54 % and better reducing power) of finger millet were drastically enhanced when fermented with GRAS filamentous fungi. During the enzymatic bioprocessing, most of the phenolics released during the hydrolysis, leached out into the liquid portion rather than retaining them within the millet grain, resulting in overall loss of dietary antioxidant. The present study establishes the most effective strategy to enrich the finger millet with phenolic antioxidants.

Direct ethanol production from starch using a natural isolate, Scheffersomyces shehatae: Toward consolidated bioprocessing

PubMed Central

Tanimura, Ayumi; Kikukawa, Minako; Yamaguchi, Shino; Kishino, Shigenobu; Ogawa, Jun; Shima, Jun

2015-01-01

Consolidated bioprocessing (CBP), which integrates enzyme production, saccharification and fermentation into a one-step process, is a promising strategy for cost-effective ethanol production from starchy biomass. To gain insights into starch-based ethanol production using CBP, an extensive screening was undertaken to identify naturally occurring yeasts that produce ethanol without the addition of any amylases. Three yeast strains were capable of producing a significant amount of ethanol. Quantitative assays revealed that Scheffersomyces shehatae JCM 18690 was the strain showing the highest ethanol production ability. This strain was able to utilize starch directly, and the ethanol concentration reached 9.21 g/L. We attribute the ethanol-producing ability of this strain to the high levels of glucoamylase activity, fermentation potential and ethanol stress tolerance. This study strongly suggests the possibility of starch-based ethanol production by consolidated bioprocessing using natural yeasts such as S. shehatae JCM 18690. PMID:25901788

Techno-economic evaluation of a combined bioprocess for fermentative hydrogen production from food waste.

PubMed

Han, Wei; Fang, Jun; Liu, Zhixiang; Tang, Junhong

2016-02-01

In this study, the techno-economic evaluation of a combined bioprocess based on solid state fermentation for fermentative hydrogen production from food waste was carried out. The hydrogen production plant was assumed to be built in Hangzhou and designed for converting 3 ton food waste per day into hydrogen. The total capital cost (TCC) and the annual production cost (APC) were US$583092 and US$88298.1/year, respectively. The overall revenue after the tax was US$146473.6/year. The return on investment (ROI), payback period (PBP) and internal rate of return (IRR) of the plant were 26.75%, 5 years and 24.07%, respectively. The results exhibited that the combined bioprocess for hydrogen production from food waste was feasible. This is an important study for attracting investment and industrialization interest for hydrogen production from food waste in the industrial scale. Copyright © 2015 Elsevier Ltd. All rights reserved.

Membrane Bioprocesses for Pharmaceutical Micropollutant Removal from Waters

PubMed Central

de Cazes, Matthias; Abejón, Ricardo; Belleville, Marie-Pierre; Sanchez-Marcano, José

2014-01-01

The purpose of this review work is to give an overview of the research reported on bioprocesses for the treatment of domestic or industrial wastewaters (WW) containing pharmaceuticals. Conventional WW treatment technologies are not efficient enough to completely remove all pharmaceuticals from water. Indeed, these compounds are becoming an actual public health problem, because they are more and more present in underground and even in potable waters. Different types of bioprocesses are described in this work: from classical activated sludge systems, which allow the depletion of pharmaceuticals by bio-degradation and adsorption, to enzymatic reactions, which are more focused on the treatment of WW containing a relatively high content of pharmaceuticals and less organic carbon pollution than classical WW. Different aspects concerning the advantages of membrane bioreactors for pharmaceuticals removal are discussed, as well as the more recent studies on enzymatic membrane reactors to the depletion of these recalcitrant compounds. PMID:25295629

Mammalian cell culture monitoring using in situ spectroscopy: Is your method really optimised?

PubMed

André, Silvère; Lagresle, Sylvain; Hannas, Zahia; Calvosa, Éric; Duponchel, Ludovic

2017-03-01

In recent years, as a result of the process analytical technology initiative of the US Food and Drug Administration, many different works have been carried out on direct and in situ monitoring of critical parameters for mammalian cell cultures by Raman spectroscopy and multivariate regression techniques. However, despite interesting results, it cannot be said that the proposed monitoring strategies, which will reduce errors of the regression models and thus confidence limits of the predictions, are really optimized. Hence, the aim of this article is to optimize some critical steps of spectroscopic acquisition and data treatment in order to reach a higher level of accuracy and robustness of bioprocess monitoring. In this way, we propose first an original strategy to assess the most suited Raman acquisition time for the processes involved. In a second part, we demonstrate the importance of the interbatch variability on the accuracy of the predictive models with a particular focus on the optical probes adjustment. Finally, we propose a methodology for the optimization of the spectral variables selection in order to decrease prediction errors of multivariate regressions. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:308-316, 2017. © 2017 American Institute of Chemical Engineers.

Optical fiber extrinsic Fabry-Perot interferometric (EFPI)-based biosensors

NASA Astrophysics Data System (ADS)

Elster, Jennifer L.; Jones, Mark E.; Evans, Mishell K.; Lenahan, Shannon M.; Boyce, Christopher A.; Velander, William H.; VanTassell, Roger

2000-05-01

A novel system incorporating optical fiber extrinsic Fabry- Perot interferometric (EFPI)-based sensors for rapid detection of biological targets is presented. With the appropriate configuration, the EFPI senor is able to measure key environmental parameters by monitoring the interferometric fringes resulting from an optical path differences of reflected signals. The optical fiber EFPI sensor has been demonstrated for strain, pressure, and temperature measurements and can be readily modified for refractive index measurements by allowing solutions to flow into an open cavity. The sensor allows for highly sensitive, real-time, refractive index measurements and by applying affinity coatings containing ligands within this cavity, specific binding of target molecules can be accomplished. As target molecules bind to the coating, there is an increased density within the film, causing a measurable refractive index change that correlates to the concentration of detected target molecules. This sensor platform offers enhanced sensing capabilities for clinical diagnostics, pharmaceutical screening, environmental monitoring, food pathogen detection, biological warfare agent detection, and industrial bioprocessing. Promising applications also exist for process monitoring within the food/beverage, petroleum, and chemical industry.

Process Analytical Technology for Advanced Process Control in Biologics Manufacturing with the Aid of Macroscopic Kinetic Modeling.

PubMed

Kornecki, Martin; Strube, Jochen

2018-03-16

Productivity improvements of mammalian cell culture in the production of recombinant proteins have been made by optimizing cell lines, media, and process operation. This led to enhanced titers and process robustness without increasing the cost of the upstream processing (USP); however, a downstream bottleneck remains. In terms of process control improvement, the process analytical technology (PAT) initiative, initiated by the American Food and Drug Administration (FDA), aims to measure, analyze, monitor, and ultimately control all important attributes of a bioprocess. Especially, spectroscopic methods such as Raman or near-infrared spectroscopy enable one to meet these analytical requirements, preferably in-situ. In combination with chemometric techniques like partial least square (PLS) or principal component analysis (PCA), it is possible to generate soft sensors, which estimate process variables based on process and measurement models for the enhanced control of bioprocesses. Macroscopic kinetic models can be used to simulate cell metabolism. These models are able to enhance the process understanding by predicting the dynamic of cells during cultivation. In this article, in-situ turbidity (transmission, 880 nm) and ex-situ Raman spectroscopy (785 nm) measurements are combined with an offline macroscopic Monod kinetic model in order to predict substrate concentrations. Experimental data of Chinese hamster ovary cultivations in bioreactors show a sufficiently linear correlation (R² ≥ 0.97) between turbidity and total cell concentration. PLS regression of Raman spectra generates a prediction model, which was validated via offline viable cell concentration measurement (RMSE ≤ 13.82, R² ≥ 0.92). Based on these measurements, the macroscopic Monod model can be used to determine different process attributes, e.g., glucose concentration. In consequence, it is possible to approximately calculate (R² ≥ 0.96) glucose concentration based on online cell concentration measurements using turbidity or Raman spectroscopy. Future approaches will use these online substrate concentration measurements with turbidity and Raman measurements, in combination with the kinetic model, in order to control the bioprocess in terms of feeding strategies, by employing an open platform communication (OPC) network-either in fed-batch or perfusion mode, integrated into a continuous operation of upstream and downstream.

Process Analytical Technology for Advanced Process Control in Biologics Manufacturing with the Aid of Macroscopic Kinetic Modeling

PubMed Central

Kornecki, Martin; Strube, Jochen

2018-01-01

Productivity improvements of mammalian cell culture in the production of recombinant proteins have been made by optimizing cell lines, media, and process operation. This led to enhanced titers and process robustness without increasing the cost of the upstream processing (USP); however, a downstream bottleneck remains. In terms of process control improvement, the process analytical technology (PAT) initiative, initiated by the American Food and Drug Administration (FDA), aims to measure, analyze, monitor, and ultimately control all important attributes of a bioprocess. Especially, spectroscopic methods such as Raman or near-infrared spectroscopy enable one to meet these analytical requirements, preferably in-situ. In combination with chemometric techniques like partial least square (PLS) or principal component analysis (PCA), it is possible to generate soft sensors, which estimate process variables based on process and measurement models for the enhanced control of bioprocesses. Macroscopic kinetic models can be used to simulate cell metabolism. These models are able to enhance the process understanding by predicting the dynamic of cells during cultivation. In this article, in-situ turbidity (transmission, 880 nm) and ex-situ Raman spectroscopy (785 nm) measurements are combined with an offline macroscopic Monod kinetic model in order to predict substrate concentrations. Experimental data of Chinese hamster ovary cultivations in bioreactors show a sufficiently linear correlation (R2 ≥ 0.97) between turbidity and total cell concentration. PLS regression of Raman spectra generates a prediction model, which was validated via offline viable cell concentration measurement (RMSE ≤ 13.82, R2 ≥ 0.92). Based on these measurements, the macroscopic Monod model can be used to determine different process attributes, e.g., glucose concentration. In consequence, it is possible to approximately calculate (R2 ≥ 0.96) glucose concentration based on online cell concentration measurements using turbidity or Raman spectroscopy. Future approaches will use these online substrate concentration measurements with turbidity and Raman measurements, in combination with the kinetic model, in order to control the bioprocess in terms of feeding strategies, by employing an open platform communication (OPC) network—either in fed-batch or perfusion mode, integrated into a continuous operation of upstream and downstream. PMID:29547557

Strain and bioprocess improvement of a thermophilic anaerobe for the production of ethanol from wood

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

Herring, Christopher D.; Kenealy, William R.; Shaw, A. Joe

Here, the thermophilic, anaerobic bacterium Thermoanaerobacterium saccharolyticum digests hemicellulose and utilizes the major sugars present in biomass. It was previously engineered to produce ethanol at yields equivalent to yeast. While saccharolytic anaerobes have been long studied as potential biomass-fermenting organisms, development efforts for commercial ethanol production have not been reported.

Strain and bioprocess improvement of a thermophilic anaerobe for the production of ethanol from wood

DOE PAGES

Herring, Christopher D.; Kenealy, William R.; Shaw, A. Joe; ...

2016-06-16

Here, the thermophilic, anaerobic bacterium Thermoanaerobacterium saccharolyticum digests hemicellulose and utilizes the major sugars present in biomass. It was previously engineered to produce ethanol at yields equivalent to yeast. While saccharolytic anaerobes have been long studied as potential biomass-fermenting organisms, development efforts for commercial ethanol production have not been reported.

Bioprocessing in Space

NASA Technical Reports Server (NTRS)

Morrison, D. R. (Compiler)

1977-01-01

Proceedings are presented of the 1976 NASA Colloquium on bioprocessing in space. The program included general sessions and formal presentations on the following topics: NASA's Space Shuttle, Spacelab, and space-processing programs; the known unusual behavior of materials in space; space-processing experiment results; cell biology, gravity sensors in cells, space electrophoresis of living cells, new approaches to biosynthesis of biologicals from cell culture in space, and zero-g fermentation concepts; and upcoming flight opportunities and industrial application planning studies already underway.

Modeling the Downstream Processing of Monoclonal Antibodies Reveals Cost Advantages for Continuous Methods for a Broad Range of Manufacturing Scales.

PubMed

Hummel, Jonathan; Pagkaliwangan, Mark; Gjoka, Xhorxhi; Davidovits, Terence; Stock, Rick; Ransohoff, Thomas; Gantier, Rene; Schofield, Mark

2018-01-17

The biopharmaceutical industry is evolving in response to changing market conditions, including increasing competition and growing pressures to reduce costs. Single-use (SU) technologies and continuous bioprocessing have attracted attention as potential facilitators of cost-optimized manufacturing for monoclonal antibodies. While disposable bioprocessing has been adopted at many scales of manufacturing, continuous bioprocessing has yet to reach the same level of implementation. In this study, the cost of goods of Pall Life Science's integrated, continuous bioprocessing (ICB) platform is modeled, along with that of purification processes in stainless-steel and SU batch formats. All three models include costs associated with downstream processing only. Evaluation of the models across a broad range of clinical and commercial scenarios reveal that the cost savings gained by switching from stainless-steel to SU batch processing are often amplified by continuous operation. The continuous platform exhibits the lowest cost of goods across 78% of all scenarios modeled here, with the SU batch process having the lowest costs in the rest of the cases. The relative savings demonstrated by the continuous process are greatest at the highest feed titers and volumes. These findings indicate that existing and imminent continuous technologies and equipment can become key enablers for more cost effective manufacturing of biopharmaceuticals. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Turmeric Bioprocessed with Mycelia from the Shiitake Culinary-Medicinal Mushroom Lentinus edodes (Agaricomycetes) Protects Mice Against Salmonellosis.

PubMed

Kim, Sung Phil; Lee, Sang Jong; Nam, Seok Hyun; Friedman, Mendel

2017-01-01

This study investigated the suppressive mechanisms of an extract from bioprocessed Lentinus edodes mycelial liquid culture supplemented with turmeric (bioprocessed Curcuma longa extract [BPCLE]) against murine salmonellosis. The BPLCE extract from the bioprocessed mycelia of the Salmonella Typhimurium into murine RAW 264.7 macrophage cells, elimination of intracellular bacteria, and elevation of inducible nitric oxide synthase expression. Dietary administration of BPCLE activated leukocytes from the mice infected with Salmonella through the intraperitoneal route. The enzyme-linked immunosorbent assay of the cytokines produced by splenocytes from infected mice showed significant increases in the levels of Th1 cytokines, including interleukin (IL)-1β, IL-2, IL-6, and IL-12. Histology showed that dietary administration of BPCLE protected against necrosis of the liver resulting from a sublethal dose of Salmonella. In addition, the treatment (1) extended the lifespan of lethally infected mice, (2) suppressed the invasion of Salmonella into human Caco-2 colorectal adenocarcinoma cells, (3) increased excretion of the bacterium in the feces, (4) suppressed the translocation of the Salmonella to internal organs, and (5) increased total immunoglobulin A in both serum and intestinal fluids. BPCLE protected the mice against salmonellosis via cooperative effects that include the upregulation of the Th1 immune reaction, prevention of translocation of bacteria across the intestinal epithelial cells, and increased immunoglobulin A production in serum and intestinal fluids.

Development of a high-throughput microscale cell disruption platform for Pichia pastoris in rapid bioprocess design.

PubMed

Bláha, Benjamin A F; Morris, Stephen A; Ogonah, Olotu W; Maucourant, Sophie; Crescente, Vincenzo; Rosenberg, William; Mukhopadhyay, Tarit K

2018-01-01

The time and cost benefits of miniaturized fermentation platforms can only be gained by employing complementary techniques facilitating high-throughput at small sample volumes. Microbial cell disruption is a major bottleneck in experimental throughput and is often restricted to large processing volumes. Moreover, for rigid yeast species, such as Pichia pastoris, no effective high-throughput disruption methods exist. The development of an automated, miniaturized, high-throughput, noncontact, scalable platform based on adaptive focused acoustics (AFA) to disrupt P. pastoris and recover intracellular heterologous protein is described. Augmented modes of AFA were established by investigating vessel designs and a novel enzymatic pretreatment step. Three different modes of AFA were studied and compared to the performance high-pressure homogenization. For each of these modes of cell disruption, response models were developed to account for five different performance criteria. Using multiple responses not only demonstrated that different operating parameters are required for different response optima, with highest product purity requiring suboptimal values for other criteria, but also allowed for AFA-based methods to mimic large-scale homogenization processes. These results demonstrate that AFA-mediated cell disruption can be used for a wide range of applications including buffer development, strain selection, fermentation process development, and whole bioprocess integration. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:130-140, 2018. © 2017 American Institute of Chemical Engineers.

Batch dark fermentation from enzymatic hydrolyzed food waste for hydrogen production.

PubMed

Han, Wei; Ye, Min; Zhu, Ai Jun; Zhao, Hong Ting; Li, Yong Feng

2015-09-01

A combination bioprocess of solid-state fermentation (SSF) and dark fermentative hydrogen production from food waste was developed. Aspergillus awamori and Aspergillus oryzae were utilized in SSF from food waste to generate glucoamylase and protease which were used to hydrolyze the food waste suspension to get the nutrients-rich (glucose and free amino nitrogen (FAN)) hydrolysate. Both glucose and FAN increased with increasing of food waste mass ratio from 4% to 10% (w/v) and the highest glucose (36.9 g/L) and FAN (361.3mg/L) were observed at food waste mass ratio of 10%. The food waste hydrolysates were then used as the feedstock for dark fermentative hydrogen production by heat pretreated sludge. The best hydrogen yield of 39.14 ml H2/g food waste (219.91 ml H2/VSadded) was achieved at food waste mass ratio of 4%. The proposed combination bioprocess could effectively accelerate the hydrolysis rate, improve raw material utilization and enhance hydrogen yield. Copyright © 2015 Elsevier Ltd. All rights reserved.

KSC-99pp0290

NASA Image and Video Library

1999-03-09

In the KSC Life Sciences Building, Hangar L, Cape Canaveral Air Station, Mark Rupert, with BioServe Space Technologies, checks the canisters, or incubators, that will hold an experiment to fly on mission STS-93. The incubators will hold a mix of fruit fly embryos and larvae to examine the effects of microgravity and space flight on the development of neural connections between specific motor neurons and their targets in muscle fibers. The incubators are part of a Commercial Generic Bioprocessing Apparatus (CGBA), which can start bioprocessing reactions by mixing or heating a sample and can also initiate multiple-step, sequential reactions in a technique called phased processing. The primary payload of mission STS-93 is the Chandra X-ray Observatory, which will allow scientists from around the world to see previously invisible black holes and high-temperature gas clouds, giving the observatory the potential to rewrite the books on the structure and evolution of our universe. The target launch date for STS-93 is July 9, aboard Space Shuttle Columbia, from Launch Pad 39B

KSC-99pp0291

NASA Image and Video Library

1999-03-09

In the KSC Life Sciences Building, Hangar L, Cape Canaveral Air Station, Jake Freeman and Mark Rupert, with BioServe Space Technologies, check canisters, or incubators, that will hold fruit fly embryos and larvae for an experiment to fly on mission STS-93. The experiment will examine the effects of microgravity and space flight on the development of neural connections between specific motor neurons and their targets in muscle fibers. The incubators are part of the Commercial Generic Bioprocessing Apparatus (CGBA), which can start bioprocessing reactions by mixing or heating a sample and can also initiate multiple-step, sequential reactions in a technique called phased processing. The primary payload of mission STS-93 is the Chandra X-ray Observatory, which will allow scientists from around the world to see previously invisible black holes and high-temperature gas clouds, giving the observatory the potential to rewrite the books on the structure and evolution of our universe. The target launch date for STS-93 is July 9, aboard Space Shuttle Columbia, from Launch Pad 39B

Recent advances and strategies in process and strain engineering for the production of butyric acid by microbial fermentation.

PubMed

Luo, Hongzhen; Yang, Rongling; Zhao, Yuping; Wang, Zhaoyu; Liu, Zheng; Huang, Mengyu; Zeng, Qingwei

2018-04-01

Butyric acid is an important platform chemical, which is widely used in the fields of food, pharmaceutical, energy, etc. Microbial fermentation as an alternative approach for butyric acid production is attracting great attention as it is an environmentally friendly bioprocessing. However, traditional fermentative butyric acid production is still not economically competitive compared to chemical synthesis route, due to the low titer, low productivity, and high production cost. Therefore, reduction of butyric acid production cost by utilization of alternative inexpensive feedstock, and improvement of butyric acid production and productivity has become an important target. Recently, several advanced strategies have been developed for enhanced butyric acid production, including bioprocess techniques and metabolic engineering methods. This review provides an overview of advances and strategies in process and strain engineering for butyric acid production by microbial fermentation. Additionally, future perspectives on improvement of butyric acid production are also proposed. Copyright © 2018 Elsevier Ltd. All rights reserved.

Engineering cells for cell culture bioprocessing--physiological fundamentals.

PubMed

Seth, Gargi; Hossler, Patrick; Yee, Joon Chong; Hu, Wei-Shou

2006-01-01

In the past decade, we have witnessed a tremendous increase in the number of mammalian cell-derived therapeutic proteins with clinical applications. The success of making these life-saving biologics available to the public is partly due to engineering efforts to enhance process efficiency. To further improve productivity, much effort has been devoted to developing metabolically engineered producing cells, which possess characteristics favorable for large-scale bioprocessing. In this article we discuss the fundamental physiological basis for cell engineering. Different facets of cellular mechanisms, including metabolism, protein processing, and the balancing pathways of cell growth and apoptosis, contribute to the complex traits of favorable growth and production characteristics. We present our assessment of the current state of the art by surveying efforts that have already been undertaken in engineering cells for a more robust process. The concept of physiological homeostasis as a key determinant and its implications on cell engineering is emphasized. Integrating the physiological perspective with cell culture engineering will facilitate attainment of dream cells with superlative characteristics.

A continuous system for biocatalytic hydrogenation of CO2 to formate.

PubMed

Mourato, Cláudia; Martins, Mónica; da Silva, Sofia M; Pereira, Inês A C

2017-07-01

In this work a novel bioprocess for hydrogenation of CO 2 to formate was developed, using whole cell catalysis by a sulfate-reducing bacterium. Three Desulfovibrio species were tested (D. vulgaris Hildenborough, D. alaskensis G20, and D. desulfuricans ATCC 27774), of which D. desulfuricans showed the highest activity, producing 12mM of formate in batch, with a production rate of 0.09mMh -1 . Gene expression analysis indicated that among the three formate dehydrogenases and five hydrogenases, the cytoplasmic FdhAB and the periplasmic [FeFe] HydAB are the main enzymes expressed in D. desulfuricans in these conditions. The new bioprocess for continuous formate production by D. desulfuricans had a maximum specific formate production rate of 14mMg dcw -1 h -1 , and more than 45mM of formate were obtained with a production rate of 0.40mMh -1 . This is the first report of a continuous process for biocatalytic formate production. Copyright © 2017 Elsevier Ltd. All rights reserved.

Transporter engineering in biomass utilization by yeast.

PubMed

Hara, Kiyotaka Y; Kobayashi, Jyumpei; Yamada, Ryosuke; Sasaki, Daisuke; Kuriya, Yuki; Hirono-Hara, Yoko; Ishii, Jun; Araki, Michihiro; Kondo, Akihiko

2017-11-01

Biomass resources are attractive carbon sources for bioproduction because of their sustainability. Many studies have been performed using biomass resources to produce sugars as carbon sources for cell factories. Expression of biomass hydrolyzing enzymes in cell factories is an important approach for constructing biomass-utilizing bioprocesses because external addition of these enzymes is expensive. In particular, yeasts have been extensively engineered to be cell factories that directly utilize biomass because of their manageable responses to many genetic engineering tools, such as gene expression, deletion and editing. Biomass utilizing bioprocesses have also been developed using these genetic engineering tools to construct metabolic pathways. However, sugar input and product output from these cells are critical factors for improving bioproduction along with biomass utilization and metabolic pathways. Transporters are key components for efficient input and output activities. In this review, we focus on transporter engineering in yeast to enhance bioproduction from biomass resources. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Application of metabolic engineering for the biotechnological production of L-valine.

PubMed

Oldiges, Marco; Eikmanns, Bernhard J; Blombach, Bastian

2014-07-01

The branched chain amino acid L-valine is an essential nutrient for higher organisms, such as animals and humans. Besides the pharmaceutical application in parenteral nutrition and as synthon for the chemical synthesis of e.g. herbicides or anti-viral drugs, L-valine is now emerging into the feed market, and significant increase of sales and world production is expected. In accordance, well-known microbial production bacteria, such as Escherichia coli and Corynebacterium glutamicum strains, have recently been metabolically engineered for efficient L-valine production under aerobic or anaerobic conditions, and the respective cultivation and production conditions have been optimized. This review summarizes the state of the art in L-valine biosynthesis and its regulation in E. coli and C. glutamicum with respect to optimal metabolic network for microbial L-valine production, genetic strain engineering and bioprocess development for L-valine production, and finally, it will shed light on emerging technologies that have the potential to accelerate strain and bioprocess engineering in the near future.

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

Microgravity: New opportunities to facilitate biotechnology development

NASA Astrophysics Data System (ADS)

Johnson, Terry; Todd, Paul; Stodieck, Louis S.

1996-03-01

New opportunities exist to use the microgravity environment to facilitate biotechnology development. BioServe Space Technologies Center for the Commercial Development of Space offers access to microgravity environments for companies who wish to perform research or develop products in three specific life-science fields: Biomedical and Pharmaceutical Research, Biotechnology and Bioprocessing Research, and Agricultural and Environmental Research. Examples of each include physiological testing of new pharmaceutical countermeasures against symptoms that are exaggerated in space flight, crystallization and testing of novel, precompetitive biopharmaceutical substances in a convection-free environment, and closed life-support system product development.

Commercial Generic Bioprocessing Apparatus Science Insert - 03

NASA Technical Reports Server (NTRS)

Moreno, Nancy; Stodieck, Louis; Cushing, Paula; Stowe, Mark; Hamilton, Mary Ann; Werner, Ken

2008-01-01

Commercial Generic Bioprocessing Apparatus Science Insert - 03 (CSI-03) is the third set of investigations in the CSI program series. The CSI program provides the K-12 community opportunities to utilize the unique microgravity environment of the International Space Station as part of the regular classroom to encourage learning and interest in science, technology, engineering and math. CSI-03 will examine the complete life cycle of the painted lady butterfly and the ability of an orb weaving spider to spin a web, eat and remain healthy in space.

Crewmember in the middeck with Commercial Generic Bioprocessing experiment.

NASA Image and Video Library

1993-01-19

STS054-30-009 (13 Jan 1993) --- Astronaut Susan J. Helms communicates with ground controllers about the Commercial Generic Bioprocessing Apparatus (CGBA) on Endeavour's middeck. The mission specialist holds samples from the CGBA in her left hand. Sleep restraints can be seen in their temporary stow position in the left part of the frame, near the airlock hatch. Also onboard the spacecraft for the six-day mission were astronauts John H. Casper, Donald R. McMonagle, Gregory J. Harbaugh and Mario Runco Jr.

Microbial ecology of fermentative hydrogen producing bioprocesses: useful insights for driving the ecosystem function.

PubMed

Cabrol, Lea; Marone, Antonella; Tapia-Venegas, Estela; Steyer, Jean-Philippe; Ruiz-Filippi, Gonzalo; Trably, Eric

2017-03-01

One of the most important biotechnological challenges is to develop environment friendly technologies to produce new sources of energy. Microbial production of biohydrogen through dark fermentation, by conversion of residual biomass, is an attractive solution for short-term development of bioH2 producing processes. Efficient biohydrogen production relies on complex mixed communities working in tight interaction. Species composition and functional traits are of crucial importance to maintain the ecosystem service. The analysis of microbial community revealed a wide phylogenetic diversity that contributes in different-and still mostly unclear-ways to hydrogen production. Bridging this gap of knowledge between microbial ecology features and ecosystem functionality is essential to optimize the bioprocess and develop strategies toward a maximization of the efficiency and stability of substrate conversion. The aim of this review is to provide a comprehensive overview of the most up-to-date biodata available and discuss the main microbial community features of biohydrogen engineered ecosystems, with a special emphasis on the crucial role of interactions and the relationships between species composition and ecosystem service. The elucidation of intricate relationships between community structure and ecosystem function would make possible to drive ecosystems toward an improved functionality on the basis of microbial ecology principles. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Adsorptive separation in bioprocess engineering

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

Huang, E.W.Y.

1987-01-01

The invention and development of an energy-efficient separation technique for recovery of desired chemicals from biomass conversion would greatly enhance the economic viability of this bioprocess. Adsorptive separation of several chemicals from aqueous solution was studied in this thesis. The desired species were recovered from the dilute aqueous solution by using crosslinked polyvinylpyridine resin to effect selective sorption. The sorbed chemicals were then removed from the resin by either thermal regeneration or elution with some appropriate desorbents. The effects of temperature, pH value, and solute concentration on resin swelling were investigated. The adsorption equilibrium isotherms, resin capacities and resin selectivitiesmore » of methanol, ethanol, 1-propanol, isopropanol, glycerol, acetone, 1-butanol, tert-butanol, and 2,3-butanediol were determined to study the homologies. Furthermore, acetic acid, butyric acid, hydrochloric acid, lactic acid, and sulfuric acid were recovered from very dilute aqueous solutions. The concentration of the sorbed chemical in the stationary phase can be many times higher than in the mobile phase for some acids. Finally, different types of equilibrium isotherms were used to fit the experimental data. A mathematical model was developed by using the theory of interference to predict the breakthrough curves and the process efficiency to provide information for large-scale process design and development.« less

Cell culture experiments planned for the space bioreactor

NASA Technical Reports Server (NTRS)

Morrison, Dennis R.; Cross, John H.

1987-01-01

Culturing of cells in a pilot-scale bioreactor remains to be done in microgravity. An approach is presented based on several studies of cell culture systems. Previous and current cell culture research in microgravity which is specifically directed towards development of a space bioprocess is described. Cell culture experiments planned for a microgravity sciences mission are described in abstract form.

Identification of molecular species of polyol oils produced from soybean oil by Pseudomonas aeruginosa e03-12 nrrl b-59991

USDA-ARS?s Scientific Manuscript database

The objective of this study is to develop a bioprocess for the production of polyol oils directly from soybean oil. We reported earlier methods for microbial screening and production of polyol oils from soybean oil (Hou and Lin, 2013). The polyol oil produced by Acinetobacter haemolyticus A01-35 (NR...

Large-scale production of diesel-like biofuels - process design as an inherent part of microorganism development.

PubMed

Cuellar, Maria C; Heijnen, Joseph J; van der Wielen, Luuk A M

2013-06-01

Industrial biotechnology is playing an important role in the transition to a bio-based economy. Currently, however, industrial implementation is still modest, despite the advances made in microorganism development. Given that the fuels and commodity chemicals sectors are characterized by tight economic margins, we propose to address overall process design and efficiency at the start of bioprocess development. While current microorganism development is targeted at product formation and product yield, addressing process design at the start of bioprocess development means that microorganism selection can also be extended to other critical targets for process technology and process scale implementation, such as enhancing cell separation or increasing cell robustness at operating conditions that favor the overall process. In this paper we follow this approach for the microbial production of diesel-like biofuels. We review current microbial routes with both oleaginous and engineered microorganisms. For the routes leading to extracellular production, we identify the process conditions for large scale operation. The process conditions identified are finally translated to microorganism development targets. We show that microorganism development should be directed at anaerobic production, increasing robustness at extreme process conditions and tailoring cell surface properties. All the same time, novel process configurations integrating fermentation and product recovery, cell reuse and low-cost technologies for product separation are mandatory. This review provides a state-of-the-art summary of the latest challenges in large-scale production of diesel-like biofuels. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An Intelligent Automation Platform for Rapid Bioprocess Design.

PubMed

Wu, Tianyi; Zhou, Yuhong

2014-08-01

Bioprocess development is very labor intensive, requiring many experiments to characterize each unit operation in the process sequence to achieve product safety and process efficiency. Recent advances in microscale biochemical engineering have led to automated experimentation. A process design workflow is implemented sequentially in which (1) a liquid-handling system performs high-throughput wet lab experiments, (2) standalone analysis devices detect the data, and (3) specific software is used for data analysis and experiment design given the user's inputs. We report an intelligent automation platform that integrates these three activities to enhance the efficiency of such a workflow. A multiagent intelligent architecture has been developed incorporating agent communication to perform the tasks automatically. The key contribution of this work is the automation of data analysis and experiment design and also the ability to generate scripts to run the experiments automatically, allowing the elimination of human involvement. A first-generation prototype has been established and demonstrated through lysozyme precipitation process design. All procedures in the case study have been fully automated through an intelligent automation platform. The realization of automated data analysis and experiment design, and automated script programming for experimental procedures has the potential to increase lab productivity. © 2013 Society for Laboratory Automation and Screening.

An Intelligent Automation Platform for Rapid Bioprocess Design

PubMed Central

Wu, Tianyi

2014-01-01

Bioprocess development is very labor intensive, requiring many experiments to characterize each unit operation in the process sequence to achieve product safety and process efficiency. Recent advances in microscale biochemical engineering have led to automated experimentation. A process design workflow is implemented sequentially in which (1) a liquid-handling system performs high-throughput wet lab experiments, (2) standalone analysis devices detect the data, and (3) specific software is used for data analysis and experiment design given the user’s inputs. We report an intelligent automation platform that integrates these three activities to enhance the efficiency of such a workflow. A multiagent intelligent architecture has been developed incorporating agent communication to perform the tasks automatically. The key contribution of this work is the automation of data analysis and experiment design and also the ability to generate scripts to run the experiments automatically, allowing the elimination of human involvement. A first-generation prototype has been established and demonstrated through lysozyme precipitation process design. All procedures in the case study have been fully automated through an intelligent automation platform. The realization of automated data analysis and experiment design, and automated script programming for experimental procedures has the potential to increase lab productivity. PMID:24088579

Advances in Consolidated Bioprocessing Using Clostridium thermocellum and Thermoanaerobacter saccharolyticum

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

Lynd, Lee R.; Guss, Adam M.; Himmel, Mike

2016-11-01

Recent advances are addressed pertaining to consolidated bioprocessing (CBP) of plant cell walls to ethanol using two thermophilic, saccharolytic bacteria: the cellulose-fermenting Clostridium thermocellum and the hemicellulose- fermenting ermoanaerobacterium saccharolyticum. On the basis of the largest comparative dataset assembled to date, it appears that C. thermocellum is substantially more effective at solubilizing unpretreated plant cell walls than industry-standard fungal cellulase, and that this is particularly the case for more recalcitrant feedstocks. e distinctive central metabolism of C. thermocellum appears to involve more extensive energy coupling (e.g., on the order of 5 ATP per glucosyl moiety) than most fermentative anaerobes. Ethanolmore » yields and titers realized by engineered strains of T. saccharolyticum meet standards for industrial feasibility and provide an important proof of concept as well as a model that may be emulated in other organisms. Progress has also been made with C. thermocellum, although not yet to this extent. e current state of strain development is summarized and outstanding challenges for commercial application are discussed. We speculate that CBP organism development is more promising starting with naturally occurring cellulolytic microbes as compared to starting with noncellulolytic hosts.« less

Refolding of proteins from inclusion bodies: rational design and recipes.

PubMed

Basu, Anindya; Li, Xiang; Leong, Susanna Su Jan

2011-10-01

The need to develop protein biomanufacturing platforms that can deliver proteins quickly and cost-effectively is ever more pressing. The rapid rate at which genomes can now be sequenced demands efficient protein production platforms for gene function identification. There is a continued need for the biotech industry to deliver new and more effective protein-based drugs to address new diseases. Bacterial production platforms have the advantage of high expression yields, but insoluble expression of many proteins necessitates the development of diverse and optimised refolding-based processes. Strategies employed to eliminate insoluble expression are reviewed, where it is concluded that inclusion bodies are difficult to eliminate for various reasons. Rational design of refolding systems and recipes are therefore needed to expedite production of recombinant proteins. This review article discusses efforts towards rational design of refolding systems and recipes, which can be guided by the development of refolding screening platforms that yield both qualitative and quantitative information on the progression of a given refolding process. The new opportunities presented by light scattering technologies for developing rational protein refolding buffer systems which in turn can be used to develop new process designs armed with better monitoring and controlling functionalities are discussed. The coupling of dynamic and static light scattering methodologies for incorporation into future bioprocess designs to ensure delivery of high-quality refolded proteins at faster rates is also discussed.

Monitoring key organic indoor pollutants and their elimination in a biotrickling biofilter.

PubMed

Saucedo-Lucero, José Octavio; Revah, Sergio

2018-04-01

A biotrickling filter was evaluated to treat the air of the interior of a bioprocess research laboratory. Initially, various solid-phase microextraction (SPME) fibers were used to identify and quantify the volatile organic pollutants and hexane, methyl isobutyl ketone, benzene, toluene, and xylene were further selected as indicators due to their prevalence and relative abundance. The system treated organic loading rates between 0.16 mg carbon  m -3  h -1 and close to 30 mg carbon  m -3  h -1 achieving removal efficiencies (RE) over 85% during 136 operational days. Respirometry experiments demonstrated that moderate acidification (below 5.0), due to microbial activity, adversely affected biofilter performance and consequently pH control was necessary to maintain performance.

Cellulosome-based, Clostridium-derived multi-functional enzyme complexes for advanced biotechnology tool development: advances and applications.

PubMed

Hyeon, Jeong Eun; Jeon, Sang Duck; Han, Sung Ok

2013-11-01

The cellulosome is one of nature's most elegant and elaborate nanomachines and a key biological and biotechnological macromolecule that can be used as a multi-functional protein complex tool. Each protein module in the cellulosome system is potentially useful in an advanced biotechnology application. The high-affinity interactions between the cohesin and dockerin domains can be used in protein-based biosensors to improve both sensitivity and selectivity. The scaffolding protein includes a carbohydrate-binding module (CBM) that attaches strongly to cellulose substrates and facilitates the purification of proteins fused with the dockerin module through a one-step CBM purification method. Although the surface layer homology (SLH) domain of CbpA is not present in other strains, replacement of the cell surface anchoring domain allows a foreign protein to be displayed on the surface of other strains. The development of a hydrolysis enzyme complex is a useful strategy for consolidated bioprocessing (CBP), enabling microorganisms with biomass hydrolysis activity. Thus, the development of various configurations of multi-functional protein complexes for use as tools in whole-cell biocatalyst systems has drawn considerable attention as an attractive strategy for bioprocess applications. This review provides a detailed summary of the current achievements in Clostridium-derived multi-functional complex development and the impact of these complexes in various areas of biotechnology. Copyright © 2013 Elsevier Inc. All rights reserved.

Biosimilars advancements: Moving on to the future.

PubMed

Tsuruta, Lilian Rumi; Lopes dos Santos, Mariana; Moro, Ana Maria

2015-01-01

Many patents for the first biologicals derived from recombinant technology and, more recently, monoclonal antibodies (mAbs) are expiring. Naturally, biosimilars are becoming an increasingly important area of interest for the pharmaceutical industry worldwide, not only for emergent countries that need to import biologic products. This review shows the evolution of biosimilar development regarding regulatory, manufacturing bioprocess, comparability, and marketing. The regulatory landscape is evolving globally, whereas analytical structure and functional analyses provide the foundation of a biosimilar development program. The challenges to develop and demonstrate biosimilarity should overcome the inherent differences in the bioprocess manufacturing and physicochemical and biological characterization of a biosimilar compared to several lots of the reference product. The implementation of approaches, such as Quality by Design (QbD), will provide products with defined specifications in relation to quality, purity, safety, and efficacy that were not possible when the reference product was developed. Actually, the need to prove comparability to the reference product by the biosimilar industry has increased the knowledge about the product and the production-process associated by the use of powerful analytical tools. The technological challenges to make copies of biologic products while attending regulatory and market demands are expected to help innovation in the direction of attaining more productive manufacturing processes. © 2015 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers.

Stoichiometric and kinetic analysis of extreme halophilic Archaea on various substrates in a corrosion resistant bioreactor.

PubMed

Lorantfy, Bettina; Seyer, Bernhard; Herwig, Christoph

2014-01-25

Extreme halophilic Archaea are extremophile species which can thrive in hypersaline environments of up to 3-5 M sodium chloride concentration. Although their ecology and physiology are widely identified on the microbiological level, little emphasis has been laid on quantitative bioprocess development with extreme halophiles. The goal of this study was to establish, on the one hand, a methodological basis for quantitative bioprocess analysis of extreme halophilic Archaea with an extreme halophilic strain as an example. Firstly, as a novel usage, a corrosion resistant bioreactor setup for extreme halophiles has been implemented. Then, paying special attention to total bioprocess quantification approaches, an indirect method for biomass quantification using on-line process signals was introduced. Subsequently, robust quantitative data evaluation methods for halophiles could be developed, providing defined and controlled cultivation conditions in the bioreactor and therefore obtaining suitable quality of on-line as well as off-line datasets. On the other hand, new physiological results of extreme halophiles in bioreactor have also been obtained based on the quantitative methodological tools. For the first time, quantitative data on stoichiometry and kinetics were collected and evaluated on different carbon sources. The results on various substrates were interpreted, with proposed metabolic mechanisms, by linking to the reported primary carbon metabolism of extreme halophilic Archaea. Moreover, results of chemostat cultures demonstrated that extreme halophilic organisms show Monod-kinetics on different sole carbon sources. A diauxic growth pattern was described on a mixture of substrates in batch cultivations. In addition, the methodologies presented here enable one to characterize the utilized strain Haloferax mediterranei (HFX) as a potential new host organism. Thus, this study offers a strong methodological basis as well as a fundamental physiological assessment for bioreactor quantification of extreme halophiles that can serve as primary knowledge for applications of extreme halophiles in biotechnology. Copyright © 2013 Elsevier B.V. All rights reserved.

Consortia-mediated bioprocessing of cellulose to ethanol with a symbiotic Clostridium phytofermentans/yeast co-culture.

PubMed

Zuroff, Trevor R; Xiques, Salvador Barri; Curtis, Wayne R

2013-04-29

Lignocellulosic ethanol is a viable alternative to petroleum-based fuels with the added benefit of potentially lower greenhouse gas emissions. Consolidated bioprocessing (simultaneous enzyme production, hydrolysis and fermentation; CBP) is thought to be a low-cost processing scheme for lignocellulosic ethanol production. However, no single organism has been developed which is capable of high productivity, yield and titer ethanol production directly from lignocellulose. Consortia of cellulolytic and ethanologenic organisms could be an attractive alternate to the typical single organism approaches but implementation of consortia has a number of challenges (e.g., control, stability, productivity). Ethanol is produced from α-cellulose using a consortium of C. phytofermentans and yeast that is maintained by controlled oxygen transport. Both Saccharomyces cerevisiae cdt-1 and Candida molischiana "protect" C. phytofermentans from introduced oxygen in return for soluble sugars released by C. phytofermentans hydrolysis. Only co-cultures were able to degrade filter paper when mono- and co-cultures were incubated at 30°C under semi-aerobic conditions. Using controlled oxygen delivery by diffusion through neoprene tubing at a calculated rate of approximately 8 μmol/L hour, we demonstrate establishment of the symbiotic relationship between C. phytofermentans and S. cerevisiae cdt-1 and maintenance of populations of 105 to 106 CFU/mL for 50 days. Comparable symbiotic population dynamics were observed in scaled up 500 mL bioreactors as those in 50 mL shake cultures. The conversion of α-cellulose to ethanol was shown to improve with additional cellulase indicating a limitation in hydrolysis rate. A co-culture of C. phytofermentans and S. cerevisiae cdt-1 with added endoglucanase produced approximately 22 g/L ethanol from 100 g/L α-cellulose compared to C. phytofermentans and S. cerevisiae cdt-1 mono-cultures which produced approximately 6 and 9 g/L, respectively. This work represents a significant step toward developing consortia-based bioprocessing systems for lignocellulosic biofuels production which utilize scalable, environmentally-mediated symbiosis mechanisms to provide consortium stability.

Mixo-IBR BP1A Report

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

Ahrens, Toby; Van der Meulen, Leslie

The objective of the initial budget period was to determine whether BioProcess Algae (BPA) had all of the data, information, facilities, and agreements presented in its proposal response to DE-FOA-0000739. BPA was to demonstrate heterotrophic lipid production, demonstrate the production of fuels by metathesis of algal oils, and develop data to justify the scale-up effort proposed for the Pilot Mixotrophic Algae Integrated Biorefinery.

Bioprocess for treating coproduced oily sands

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

Munnecke, D.M.; Ireland, J.

1996-12-31

The production of oil from certain oil fields creates significant amounts of oily sand which in many regulatory jurisdictions is regulated as a hazardous material, thus disposal costs can be significant. Environmental BioTechnologies, Inc. (San Carlos, CA) has developed a physical/biological treatment process that is able to economically treat these coproduced sands and produce a product that contains less than 2,000 ppm total petroleum hydrocarbons.

The role of thermodynamics in biochemical engineering

NASA Astrophysics Data System (ADS)

von Stockar, Urs

2013-09-01

This article is an adapted version of the introductory chapter of a book whose publication is imminent. It bears the title "Biothermodynamics - The role of thermodynamics in biochemical engineering." The aim of the paper is to give a very short overview of the state of biothermodynamics in an engineering context as reflected in this book. Seen from this perspective, biothermodynamics may be subdivided according to the scale used to formalize the description of the biological system into three large areas: (i) biomolecular thermodynamics (most fundamental scale), (ii) thermodynamics of metabolism (intermediary scale), and (iii) whole-cell thermodynamics ("black-box" description of living entities). In each of these subareas, the main available theoretical approaches and the current and the potential applications are discussed. Biomolecular thermodynamics (i) is especially well developed and is obviously highly pertinent for the development of downstream processing. Its use ought to be encouraged as much as possible. The subarea of thermodynamics of live cells (iii), although scarcely applied in practice, is also expected to enhance bioprocess research and development, particularly in predicting culture performances, for understanding the driving forces for cellular growth, and in developing, monitoring, and controlling cellular cultures. Finally, there is no question that thermodynamic analysis of cellular metabolism (ii) is a promising tool for systems biology and for many other applications, but quite a large research effort is still needed before it may be put to practical use.

The temporal analysis of yeast exponential phase using shotgun proteomics as a fermentation monitoring technique

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

Huang, Eric L.; Orsat, Valerie; Shah, Manesh B

2012-01-01

System biology and bioprocess technology can be better understood using shotgun proteomics as a monitoring system during the fermentation. We demonstrated a shotgun proteomic method to monitor the temporal yeast proteome in early, middle and late exponential phases. Our study identified a total of 1389 proteins combining all 2D-LC-MS/MS runs. The temporal Saccharomyces cerevisiae proteome was enriched with proteolysis, radical detoxification, translation, one-carbon metabolism, glycolysis and TCA cycle. Heat shock proteins and proteins associated with oxidative stress response were found throughout the exponential phase. The most abundant proteins observed were translation elongation factors, ribosomal proteins, chaperones and glycolytic enzymes. Themore » high abundance of the H-protein of the glycine decarboxylase complex (Gcv3p) indicated the availability of glycine in the environment. We observed differentially expressed proteins and the induced proteins at mid-exponential phase were involved in ribosome biogenesis, mitochondria DNA binding/replication and transcriptional activator. Induction of tryptophan synthase (Trp5p) indicated the abundance of tryptophan during the fermentation. As fermentation progressed toward late exponential phase, a decrease in cell proliferation was implied from the repression of ribosomal proteins, transcription coactivators, methionine aminopeptidase and translation-associated proteins.« less

A Novel Approach for Using Dielectric Spectroscopy to Predict Viable Cell Volume (VCV) in Early Process Development

PubMed Central

Downey, Brandon J; Graham, Lisa J; Breit, Jeffrey F; Glutting, Nathaniel K

2014-01-01

Online monitoring of viable cell volume (VCV) is essential to the development, monitoring, and control of bioprocesses. The commercial availability of steam-sterilizable dielectric-spectroscopy probes has enabled successful adoption of this technology as a key noninvasive method to measure VCV for cell-culture processes. Technological challenges still exist, however. For some cell lines, the technique's accuracy in predicting the VCV from probe-permittivity measurements declines as the viability of the cell culture decreases. To investigate the cause of this decrease in accuracy, divergences in predicted vs. actual VCV measurements were directly related to the shape of dielectric frequency scans collected during a cell culture. The changes in the shape of the beta dispersion, which are associated with changes in cell state, are quantified by applying a novel “area ratio” (AR) metric to frequency-scanning data from the dielectric-spectroscopy probes. The AR metric is then used to relate the shape of the beta dispersion to single-frequency permittivity measurements to accurately predict the offline VCV throughout an entire fed-batch run, regardless of cell state. This work demonstrates the possible feasibility of quantifying the shape of the beta dispersion, determined from frequency-scanning data, for enhanced measurement of VCV in mammalian cell cultures by applying a novel shape-characterization technique. In addition, this work demonstrates the utility of using changes in the shape of the beta dispersion to quantify cell health. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:479–487, 2014 PMID:24851255

Bioprocess integration for human mesenchymal stem cells: From up to downstream processing scale-up to cell proteome characterization.

PubMed

Cunha, Bárbara; Aguiar, Tiago; Carvalho, Sofia B; Silva, Marta M; Gomes, Ricardo A; Carrondo, Manuel J T; Gomes-Alves, Patrícia; Peixoto, Cristina; Serra, Margarida; Alves, Paula M

2017-04-20

To deliver the required cell numbers and doses to therapy, scaling-up production and purification processes (at least to the liter-scale) while maintaining cells' characteristics is compulsory. Therefore, the aim of this work was to prove scalability of an integrated streamlined bioprocess compatible with current good manufacturing practices (cGMP) comprised by cell expansion, harvesting and volume reduction unit operations using human mesenchymal stem cells (hMSC) isolated from bone marrow (BM-MSC) and adipose tissue (AT-MSC). BM-MSC and AT-MSC expansion and harvesting steps were scaled-up from spinner flasks to 2L scale stirred tank single-use bioreactor using synthetic microcarriers and xeno-free medium, ensuring high cellular volumetric productivities (50×10 6 cellL -1 day -1 ), expansion factors (14-16 fold) and cell recovery yields (80%). For the concentration step, flat sheet cassettes (FSC) and hollow fiber cartridges (HF) were compared showing a fairly linear scale-up, with a need to slightly decrease the permeate flux (30-50 LMH, respectively) to maximize cell recovery yield. Nonetheless, FSC allowed to recover 18% more cells after a volume reduction factor of 50. Overall, at the end of the entire bioprocess more than 65% of viable (>95%) hMSC could be recovered without compromising cell's critical quality attributes (CQA) of viability, identity and differentiation potential. Alongside the standard quality assays, a proteomics workflow based on mass spectrometry tools was established to characterize the impact of processing on hMSC's CQA; These analytical tools constitute a powerful tool to be used in process design and development. Copyright © 2017 Elsevier B.V. All rights reserved.

Economic analysis of pilot-scale production of B-phycoerythrin.

PubMed

Torres-Acosta, Mario A; Ruiz-Ruiz, Federico; Aguilar-Yáñez, José M; Benavides, Jorge; Rito-Palomares, Marco

2016-11-01

β-Phycoerythrin is a color protein with several applications, from food coloring to molecular labeling. Depending on the application, different purity is required, affecting production cost and price. Different production and purification strategies for B-phycoerythrin have been developed, the most studied are based on the production using Porphyridium cruentum and purified using chromatographic techniques or aqueous two-phase systems. The use of the latter can result in a less expensive and intensive recovery of the protein, but there is lack of a proper economic analysis to study the effect of using aqueous two-phase systems in a scaled-up process. This study analyzed the production of B-Phycoerythrin using real data obtained during the scale-up of a bioprocess using specialized software (BioSolve, Biopharm Services, UK). First, a sensitivity analysis was performed to identify critical parameters for the production cost, then a Monte Carlo analysis to emulate real processes by adding uncertainty to the identified parameters. Next, the bioprocess was analyzed to determine its financial attractiveness and possible optimization strategies were tested and discussed. Results show that aqueous two-phase systems retain their advantages of low cost and intensive recovery (54.56%); the costs of production per gram calculated (before titer optimization: US$15,709 and after optimization: US$2,374) allowed to obtain profit (in the range of US$millions in a 10-year period) for a potential company taking this production method by comparing the production cost against commercial prices. The bioprocess analyzed is a promising and profitable method for the generation of a highly purified B-phycoerythrin. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1472-1479, 2016. © 2016 American Institute of Chemical Engineers.

Challenges and Advances for Genetic Engineering of Non-model Bacteria and Uses in Consolidated Bioprocessing

PubMed Central

Yan, Qiang; Fong, Stephen S.

2017-01-01

Metabolic diversity in microorganisms can provide the basis for creating novel biochemical products. However, most metabolic engineering projects utilize a handful of established model organisms and thus, a challenge for harnessing the potential of novel microbial functions is the ability to either heterologously express novel genes or directly utilize non-model organisms. Genetic manipulation of non-model microorganisms is still challenging due to organism-specific nuances that hinder universal molecular genetic tools and translatable knowledge of intracellular biochemical pathways and regulatory mechanisms. However, in the past several years, unprecedented progress has been made in synthetic biology, molecular genetics tools development, applications of omics data techniques, and computational tools that can aid in developing non-model hosts in a systematic manner. In this review, we focus on concerns and approaches related to working with non-model microorganisms including developing molecular genetics tools such as shuttle vectors, selectable markers, and expression systems. In addition, we will discuss: (1) current techniques in controlling gene expression (transcriptional/translational level), (2) advances in site-specific genome engineering tools [homologous recombination (HR) and clustered regularly interspaced short palindromic repeats (CRISPR)], and (3) advances in genome-scale metabolic models (GSMMs) in guiding design of non-model species. Application of these principles to metabolic engineering strategies for consolidated bioprocessing (CBP) will be discussed along with some brief comments on foreseeable future prospects. PMID:29123506

KSC-99pp0292

NASA Image and Video Library

1999-03-09

In the KSC Life Sciences Building, Hangar L, Cape Canaveral Air Station, Dr. Haig Keshishian checks fruit fly larvae in a petri dish. The larvae are part of an experiment that is a secondary payload on mission STS-93. The experiment will examine the effects of microgravity and space flight on the development of neural connections between specific motor neurons and their targets in muscle fibers. Dr. Keshishian, from Yale University, is the principle investigator for the experiment. The larvae will be contained in incubators that are part of a Commercial Generic Bioprocessing Apparatus (CGBA), which can start bioprocessing reactions by mixing or heating a sample and can also initiate multiple-step, sequential reactions in a technique called phased processing. The primary payload of mission STS-93 is the Chandra X-ray Observatory, which will allow scientists from around the world to see previously invisible black holes and high-temperature gas clouds, giving the observatory the potential to rewrite the books on the structure and evolution of our universe. The target launch date for STS-93 is July 9, aboard Space Shuttle Columbia, from Launch Pad 39B

KSC-99pp0293

NASA Image and Video Library

1999-03-09

In the KSC Life Sciences Building, Hangar L, Cape Canaveral Air Station, Shawn Bengtson, with Lockheed Martin, checks population cages containing fruit flies. The larvae of the flies are part of an experiment that is a secondary payload on mission STS-93. The experiment will examine the effects of microgravity and space flight on the development of neural connections between specific motor neurons and their targets in muscle fibers. That information could lead to understanding the effect of microgravity on human nervous system connectivity. The larvae will be contained in incubators that are part of a Commercial Generic Bioprocessing Apparatus (CGBA), which can start bioprocessing reactions by mixing or heating a sample and can also initiate multiple-step, sequential reactions in a technique called phased processing. The primary payload of mission STS-93 is the Chandra X-ray Observatory, which will allow scientists from around the world to see previously invisible black holes and high-temperature gas clouds, giving the observatory the potential to rewrite the books on the structure and evolution of our universe. The target launch date for STS-93 is July 9, aboard Space Shuttle Columbia, from Launch Pad 39B

Treatment of supermarket vegetable wastes to be used as alternative substrates in bioprocesses.

PubMed

Díaz, Ana Isabel; Laca, Amanda; Laca, Adriana; Díaz, Mario

2017-09-01

Fruits and vegetables have the highest wastage rates at retail and consumer levels. These wastes have promising potential for being used as substrates in bioprocesses. However, an effective hydrolysis of carbohydrates that form these residues has to be developed before the biotransformation. In this work, vegetable wastes from supermarket (tomatoes, green peppers and potatoes) have been separately treated by acid, thermal and enzymatic hydrolysis processes in order to maximise the concentration of fermentable sugars in the final broth. For all substrates, thermal and enzymatic processes have shown to be the most effective. A new combined hydrolysis procedure including these both treatments was also assayed and the enzymatic step was successfully modelled. With this combined hydrolysis, the percentage of reducing sugars extracted was increased, in comparison with the amount extracted from non-hydrolysed samples, approximately by 30% in the case of tomato and green peeper wastes. For potato wastes this percentage increased from values lower than 1% to 77%. In addition, very low values of fermentation inhibitors were found in the final broth. Copyright © 2017. Published by Elsevier Ltd.

An experiment to fly on mission STS-93 is prepared at Life Sciences Building, CCAS

NASA Technical Reports Server (NTRS)

1999-01-01

In the KSC Life Sciences Building, Hangar L, Cape Canaveral Air Station, Mark Rupert, with BioServe Space Technologies, checks the canisters, or incubators, that will hold an experiment to fly on mission STS-93. The incubators will hold a mix of fruit fly embryos and larvae to examine the effects of microgravity and space flight on the development of neural connections between specific motor neurons and their targets in muscle fibers. The incubators are part of a Commercial Generic Bioprocessing Apparatus (CGBA), which can start bioprocessing reactions by mixing or heating a sample and can also initiate multiple-step, sequential reactions in a technique called phased processing. The primary payload of mission STS-93 is the Chandra X-ray Observatory, which will allow scientists from around the world to see previously invisible black holes and high-temperature gas clouds, giving the observatory the potential to rewrite the books on the structure and evolution of our universe. The target launch date for STS-93 is July 9, aboard Space Shuttle Columbia, from Launch Pad 39B.

An experiment to fly on mission STS-93 is prepared at Life Sciences Building, CCAS

NASA Technical Reports Server (NTRS)

1999-01-01

In the KSC Life Sciences Building, Hangar L, Cape Canaveral Air Station, Jake Freeman and Mark Rupert, with BioServe Space Technologies, check canisters, or incubators, that will hold fruit fly embryos and larvae for an experiment to fly on mission STS-93. The experiment will examine the effects of microgravity and space flight on the development of neural connections between specific motor neurons and their targets in muscle fibers. The incubators are part of the Commercial Generic Bioprocessing Apparatus (CGBA), which can start bioprocessing reactions by mixing or heating a sample and can also initiate multiple-step, sequential reactions in a technique called phased processing. The primary payload of mission STS-93 is the Chandra X-ray Observatory, which will allow scientists from around the world to see previously invisible black holes and high-temperature gas clouds, giving the observatory the potential to rewrite the books on the structure and evolution of our universe. The target launch date for STS-93 is July 9, aboard Space Shuttle Columbia, from Launch Pad 39B.

Characterization of functionalized multiwalled carbon nanotubes for use in an enzymatic sensor.

PubMed

Guadarrama-Fernández, Leonor; Chanona-Pérez, Jorge; Manzo-Robledo, Arturo; Calderón-Domínguez, Georgina; Martínez-Rivas, Adrián; Ortiz-López, Jaime; Vargas-García, Jorge Roberto

2014-10-01

Carbon nanotubes (CNT) have proven to be materials with great potential for the construction of biosensors. Development of fast, simple, and low cost biosensors to follow reactions in bioprocesses, or to detect food contaminants such as toxins, chemical compounds, and microorganisms, is presently an important research topic. This report includes microscopy and spectroscopy to characterize raw and chemically modified multiwall carbon nanotubes (MWCNTs) synthesized by chemical vapor deposition with the intention of using them as the active transducer in bioprocessing sensors. MWCNT were simultaneously purified and functionalized by an acid mixture involving HNO3-H2SO4 and amyloglucosidase attached onto the chemically modified MWCNT surface. A 49.0% decrease in its enzymatic activity was observed. Raw, purified, and enzyme-modified MWCNTs were analyzed by scanning and transmission electron microscopy and Raman and X-ray photoelectron spectroscopy. These studies confirmed purification and functionalization of the CNTs. Finally, cyclic voltammetry electrochemistry was used for electrical characterization of CNTs, which showed promising results that can be useful for construction of electrochemical biosensors applied to biological areas.

Designing industrial yeasts for the consolidated bioprocessing of starchy biomass to ethanol

PubMed Central

Favaro, Lorenzo; Jooste, Tania; Basaglia, Marina; Rose, Shaunita H.; Saayman, Maryna; Görgens, Johann F.; Casella, Sergio; van Zyl, Willem H.

2013-01-01

Consolidated bioprocessing (CBP), which integrates enzyme production, saccharification and fermentation into a one step process, is a promising strategy for the effective ethanol production from cheap lignocellulosic and starchy materials. CBP requires a highly engineered microbial strain able to both hydrolyze biomass with enzymes produced on its own and convert the resulting simple sugars into high-titer ethanol. Recently, heterologous production of cellulose and starch-degrading enzymes has been achieved in yeast hosts, which has realized direct processing of biomass to ethanol. However, essentially all efforts aimed at the efficient heterologous expression of saccharolytic enzymes in yeast have involved laboratory strains and much of this work has to be transferred to industrial yeasts that provide the fermentation capacity and robustness desired for large scale bioethanol production. Specifically, the development of an industrial CBP amylolytic yeast would allow the one-step processing of low-cost starchy substrates into ethanol. This article gives insight in the current knowledge and achievements on bioethanol production from starchy materials with industrial engineered S. cerevisiae strains. PMID:22989992

Bioprocess design guided by in situ substrate supply and product removal: process intensification for synthesis of (S)-1-(2-chlorophenyl)ethanol.

PubMed

Schmölzer, Katharina; Mädje, Katharina; Nidetzky, Bernd; Kratzer, Regina

2012-03-01

We report herein on bioprocess development guided by the hydrophobicities of substrate and product. Bioreductions of o-chloroacetophenone are severely limited by instability of the catalyst in the presence of aromatic substrate and (S)-1-(2-chlorophenyl)ethanol. In situ substrate supply and product removal was used to protect the utilized Escherichia coli whole cell catalyst based on Candida tenuis xylose reductase during the reaction. Further engineering at the levels of the catalyst and the reaction media was matched to low substrate concentrations in the aqueous phase. Productivities obtained in aqueous batch reductions were 21-fold improved by addition of 20% (v/v) hexane, NAD(+), expression engineering, cell permeabilization and pH optimization. Reduction of 300 mM substrate was accomplished in 97% yield and use of the co-solvent hexane in subsequent extraction steps led to 88% recovery. Product loss due to high catalyst loading was minimized by using the same extractant in bioreduction and product isolation. Copyright © 2012 Elsevier Ltd. All rights reserved.

Hybrid semi-parametric mathematical systems: bridging the gap between systems biology and process engineering.

PubMed

Teixeira, Ana P; Carinhas, Nuno; Dias, João M L; Cruz, Pedro; Alves, Paula M; Carrondo, Manuel J T; Oliveira, Rui

2007-12-01

Systems biology is an integrative science that aims at the global characterization of biological systems. Huge amounts of data regarding gene expression, proteins activity and metabolite concentrations are collected by designing systematic genetic or environmental perturbations. Then the challenge is to integrate such data in a global model in order to provide a global picture of the cell. The analysis of these data is largely dominated by nonparametric modelling tools. In contrast, classical bioprocess engineering has been primarily founded on first principles models, but it has systematically overlooked the details of the embedded biological system. The full complexity of biological systems is currently assumed by systems biology and this knowledge can now be taken by engineers to decide how to optimally design and operate their processes. This paper discusses possible methodologies for the integration of systems biology and bioprocess engineering with emphasis on applications involving animal cell cultures. At the mathematical systems level, the discussion is focused on hybrid semi-parametric systems as a way to bridge systems biology and bioprocess engineering.

Techno-economic analysis of biocatalytic processes for production of alkene expoxides

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

Borole, Abhijeet P

2007-01-01

A techno-economic analysis of two different bioprocesses was conducted, one for the conversion of propylene to propylene oxide (PO) and other for conversion of styrene to styrene expoxide (SO). The first process was a lipase-mediated chemo-enzymatic reaction, whereas the second one was a one-step enzymatic process using chloroperoxidase. The PO produced through the chemo-enzymatic process is a racemic product, whereas the latter process (based on chloroperoxidase) produces an enantio-pure product. The former process thus falls under the category of high-volume commodity chemical (PO); whereas the latter is a low-volume, high-value product (SO).A simulation of the process was conducted using themore » bioprocess engineering software SuperPro Designer v6.0 (Intelligen, Inc., Scotch Plains, NJ) to determine the economic feasibility of the process. The purpose of the exercise was to compare biocatalytic processes with existing chemical processes for production of alkene expoxides. The results show that further improvements are needed in improving biocatalyst stability to make these bioprocesses competitive with chemical processes.« less

Alternative Feedstocks Program Technical and Economic Assessment: Thermal/Chemical and Bioprocessing Components

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

Bozell, J. J.; Landucci, R.

This resource document on biomass to chemicals opportunities describes the development of a technical and market rationale for incorporating renewable feedstocks into the chemical industry in both a qualitative and quantitative sense. The term "renewable feedstock?s" can be defined to include a huge number of materials such as agricultural crops rich in starch, lignocellulosic materials (biomass), or biomass material recovered from a variety of processing wastes.

Production of Laccase by Recombinant Yarrowia lipolytica from Molasses: Bioprocess Development Using Statistical Modeling and Increase Productivity in Shake-Flask and Bioreactor Cultures.

PubMed

Darvishi, Farshad; Moradi, Marzieh; Madzak, Catherine; Jolivalt, Claude

2017-03-01

Laccases are used in numerous applications, from green degradation of various xenobiotic compounds, waste detoxification, textile dye bleaching, and delignification of lignocellulose materials to biofuel production. In this study, the recombinant Yarrowia lipolytica YL4 strain carrying the white-rot fungus Trametes versicolor laccase IIIb gene was used for laccase production from beet molasses as an agro-industrial residue. Response surface methodology was used to statistical optimization of the production of laccase by Y. lipolytica using an industrial medium containing molasses which allows a six times increase in laccase activity compared to primary medium contains glucose after 144 h. In bioreactor cultivation after 48 h, laccase production reached to 3.7- and 22.5-fold more than optimized and primary media in shake-flask cultures, respectively. Laccase productivity in bioreactor (0.0937 U/h) was higher than shake-flask culture (0.0084 U/h). The present study provides valuable information about statistical optimization of bioprocess development for cost-effective production of laccase and other heterologous proteins in Y. lipolytica from beet molasses as sole carbon source, thus allowing the valorization and decreasing environmental pollution of this agro-industrial waste.

Validation of a novel sequential cultivation method for the production of enzymatic cocktails from Trichoderma strains.

PubMed

Florencio, C; Cunha, F M; Badino, A C; Farinas, C S

2015-02-01

The development of new cost-effective bioprocesses for the production of cellulolytic enzymes is needed in order to ensure that the conversion of biomass becomes economically viable. The aim of this study was to determine whether a novel sequential solid-state and submerged fermentation method (SF) could be validated for different strains of the Trichoderma genus. Cultivation of the Trichoderma reesei Rut-C30 reference strain under SF using sugarcane bagasse as substrate was shown to be favorable for endoglucanase (EGase) production, resulting in up to 4.2-fold improvement compared with conventional submerged fermentation. Characterization of the enzymes in terms of the optimum pH and temperature for EGase activity and comparison of the hydrolysis profiles obtained using a synthetic substrate did not reveal any qualitative differences among the different cultivation conditions investigated. However, the thermostability of the EGase was influenced by the type of carbon source and cultivation system. All three strains of Trichoderma tested (T. reesei Rut-C30, Trichoderma harzianum, and Trichoderma sp INPA 666) achieved higher enzymatic productivity when cultivated under SF, hence validating the proposed SF method for use with different Trichoderma strains. The results suggest that this bioprocess configuration is a very promising development for the cellulosic biofuels industry.

GlycoExtractor: a web-based interface for high throughput processing of HPLC-glycan data.

PubMed

Artemenko, Natalia V; Campbell, Matthew P; Rudd, Pauline M

2010-04-05

Recently, an automated high-throughput HPLC platform has been developed that can be used to fully sequence and quantify low concentrations of N-linked sugars released from glycoproteins, supported by an experimental database (GlycoBase) and analytical tools (autoGU). However, commercial packages that support the operation of HPLC instruments and data storage lack platforms for the extraction of large volumes of data. The lack of resources and agreed formats in glycomics is now a major limiting factor that restricts the development of bioinformatic tools and automated workflows for high-throughput HPLC data analysis. GlycoExtractor is a web-based tool that interfaces with a commercial HPLC database/software solution to facilitate the extraction of large volumes of processed glycan profile data (peak number, peak areas, and glucose unit values). The tool allows the user to export a series of sample sets to a set of file formats (XML, JSON, and CSV) rather than a collection of disconnected files. This approach not only reduces the amount of manual refinement required to export data into a suitable format for data analysis but also opens the field to new approaches for high-throughput data interpretation and storage, including biomarker discovery and validation and monitoring of online bioprocessing conditions for next generation biotherapeutics.

The Draft Genome Sequence of Clostridium sp. Strain NJ4, a Bacterium Capable of Producing Butanol from Inulin Through Consolidated Bioprocessing.

PubMed

Jiang, Yujia; Lu, Jiasheng; Chen, Tianpeng; Yan, Wei; Dong, Weiliang; Zhou, Jie; Zhang, Wenming; Ma, Jiangfeng; Jiang, Min; Xin, Fengxue

2018-05-23

A novel butanogenic Clostridium sp. NJ4 was successfully isolated and characterized, which could directly produce relatively high titer of butanol from inulin through consolidated bioprocessing (CBP). The assembled draft genome of strain NJ4 is 4.09 Mp, containing 3891 encoded protein sequences with G+C content of 30.73%. Among these annotated genes, a levanase, a hypothetical inulinase, and two bifunctional alcohol/aldehyde dehydrogenases (AdhE) were found to play key roles in the achievement of ABE production from inulin through CBP.

Cell separation and electrofusion in space

NASA Technical Reports Server (NTRS)

Morrison, D. R.; Hofmann, G. A.

1990-01-01

In microgravity, free-fluid electrophoretic methods for separating living cells and proteins are improved significantly by the absence of gravity-driven phenomena. Cell fusion, culture, and other bioprocessing steps are being investigated to understand the limits of earth-based processing. A multistep space bioprocess is described that includes electrophoretic separation of human target cells, single-cell manipulations using receptor-specific antibodies, electrofusion to produce immortal hybridomas, gentle suspension culture, and monoclonal antibody recovery using continuous-flow electrophoresis or recirculating isoelectric focusing. Improvements in several key steps already have been demonstrated by space experiments, and others will be studied on Space Station Freedom.

Bioprocessing of ores: Application to space resources

NASA Technical Reports Server (NTRS)

Johansson, Karl R.

1992-01-01

The role of microorganisms in the oxidation and leaching of various ores (especially those of copper, iron, and uranium) is well known. This role is increasingly being applied by the mining, metallurgy, and sewage industries in the bioconcentration of metal ions from natural receiving waters and from waste waters. It is concluded that bioprocessing using bacteria in closed reactors may be a variable option for the recovery of metals from the lunar regolith. Obviously, considerable research must be done to define the process, specify the appropriate bacteria, determine the necessary conditions and limitations, and evaluate the overall feasibility.

Staying alive! Sensors used for monitoring cell health in bioreactors.

PubMed

O'Mara, P; Farrell, A; Bones, J; Twomey, K

2018-01-01

Current and next generation sensors such as pH, dissolved oxygen (dO) and temperature sensors that will help drive the use of single-use bioreactors in industry are reviewed. The current trend in bioreactor use is shifting from the traditional fixed bioreactors to the use of single-use bioreactors (SUBs). However as the shift in paradigm occurs there is now a greater need for sensor technology to play 'catch up' with the innovation of bioreactor technology. Many of the sensors still in use today rely on technology created in the 1960's such as the Clark-type dissolved oxygen sensor or glass pH electrodes. This is due to the strict requirements of sensors to monitor bioprocesses resulting in the use of traditional well understood methods, making it difficult to incorporate new sensor technology into industry. A number of advances in sensor technology have been achieved in recent years, a few of these advances and future research will also be discussed in this review. Copyright © 2017 Elsevier B.V. All rights reserved.

Model-based cell number quantification using online single-oxygen sensor data for tissue engineering perfusion bioreactors.

PubMed

Lambrechts, T; Papantoniou, I; Sonnaert, M; Schrooten, J; Aerts, J-M

2014-10-01

Online and non-invasive quantification of critical tissue engineering (TE) construct quality attributes in TE bioreactors is indispensable for the cost-effective up-scaling and automation of cellular construct manufacturing. However, appropriate monitoring techniques for cellular constructs in bioreactors are still lacking. This study presents a generic and robust approach to determine cell number and metabolic activity of cell-based TE constructs in perfusion bioreactors based on single oxygen sensor data in dynamic perfusion conditions. A data-based mechanistic modeling technique was used that is able to correlate the number of cells within the scaffold (R(2)  = 0.80) and the metabolic activity of the cells (R(2)  = 0.82) to the dynamics of the oxygen response to step changes in the perfusion rate. This generic non-destructive measurement technique is effective for a large range of cells, from as low as 1.0 × 10(5) cells to potentially multiple millions of cells, and can open-up new possibilities for effective bioprocess monitoring. © 2014 Wiley Periodicals, Inc.

Recent developments in biodesulfurization of fossil fuels.

PubMed

Xu, Ping; Feng, Jinhui; Yu, Bo; Li, Fuli; Ma, Cuiqing

2009-01-01

The emission of sulfur oxides can have adverse effects on the environment. Biodesulfurization of fossil fuels is attracting more and more attention because such a bioprocess is environmentally friendly. Some techniques of desulfurization have been used or studied to meet the stricter limitation on sulfur content in China. Recent advances have demonstrated the mechanism and developments for biodesulfurization of gasoline, diesel and crude oils by free cells or immobilized cells. Genetic technology was also used to improve sulfur removal efficiencies. In this review, we summarize recent progress mainly in China on petroleum biodesulfurization.

An integrated biotechnology platform for developing sustainable chemical processes.

PubMed

Barton, Nelson R; Burgard, Anthony P; Burk, Mark J; Crater, Jason S; Osterhout, Robin E; Pharkya, Priti; Steer, Brian A; Sun, Jun; Trawick, John D; Van Dien, Stephen J; Yang, Tae Hoon; Yim, Harry

2015-03-01

Genomatica has established an integrated computational/experimental metabolic engineering platform to design, create, and optimize novel high performance organisms and bioprocesses. Here we present our platform and its use to develop E. coli strains for production of the industrial chemical 1,4-butanediol (BDO) from sugars. A series of examples are given to demonstrate how a rational approach to strain engineering, including carefully designed diagnostic experiments, provided critical insights about pathway bottlenecks, byproducts, expression balancing, and commercial robustness, leading to a superior BDO production strain and process.

Consortia-mediated bioprocessing of cellulose to ethanol with a symbiotic Clostridium phytofermentans/yeast co-culture

PubMed Central

2013-01-01

Background Lignocellulosic ethanol is a viable alternative to petroleum-based fuels with the added benefit of potentially lower greenhouse gas emissions. Consolidated bioprocessing (simultaneous enzyme production, hydrolysis and fermentation; CBP) is thought to be a low-cost processing scheme for lignocellulosic ethanol production. However, no single organism has been developed which is capable of high productivity, yield and titer ethanol production directly from lignocellulose. Consortia of cellulolytic and ethanologenic organisms could be an attractive alternate to the typical single organism approaches but implementation of consortia has a number of challenges (e.g., control, stability, productivity). Results Ethanol is produced from α-cellulose using a consortium of C. phytofermentans and yeast that is maintained by controlled oxygen transport. Both Saccharomyces cerevisiae cdt-1 and Candida molischiana “protect” C. phytofermentans from introduced oxygen in return for soluble sugars released by C. phytofermentans hydrolysis. Only co-cultures were able to degrade filter paper when mono- and co-cultures were incubated at 30°C under semi-aerobic conditions. Using controlled oxygen delivery by diffusion through neoprene tubing at a calculated rate of approximately 8 μmol/L hour, we demonstrate establishment of the symbiotic relationship between C. phytofermentans and S. cerevisiae cdt-1 and maintenance of populations of 105 to 106 CFU/mL for 50 days. Comparable symbiotic population dynamics were observed in scaled up 500 mL bioreactors as those in 50 mL shake cultures. The conversion of α-cellulose to ethanol was shown to improve with additional cellulase indicating a limitation in hydrolysis rate. A co-culture of C. phytofermentans and S. cerevisiae cdt-1 with added endoglucanase produced approximately 22 g/L ethanol from 100 g/L α-cellulose compared to C. phytofermentans and S. cerevisiae cdt-1 mono-cultures which produced approximately 6 and 9 g/L, respectively. Conclusion This work represents a significant step toward developing consortia-based bioprocessing systems for lignocellulosic biofuels production which utilize scalable, environmentally-mediated symbiosis mechanisms to provide consortium stability. PMID:23628342

Genome-editing applications of CRISPR–Cas9 to promote in vitro studies of Alzheimer’s disease

PubMed Central

Shim, Kyu Hwan; Bagyinszky, Eva

2018-01-01

Genetic variations play an important role in the clinical presentation and progression of Alzheimer’s disease (AD), especially early-onset Alzheimer’s disease. Hundreds of mutations have been reported with the majority resulting from alterations in β-amyloid precursor protein (APP), presenilin 1 (PSEN1), or presenilin 2 (PSEN2) genes. The roles of these mutations in the pathogenesis of AD have been classically confirmed or refuted through functional studies, where the mutations are cloned, inserted into cell lines, and monitored for changes in various properties including cell survival, amyloid production, or Aβ42/40 ratio. However, these verification studies tend to be expensive, time consuming, and inconsistent. Recently, the clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR–Cas9) system was developed, which improves sequence-specific gene editing in cell lines, organs, and animals. CRISPR–Cas9 is a promising tool for the generation of models of human genetic diseases and could facilitate the establishment of new animal AD models and the observation of dynamic bioprocesses in AD. Here, we recapitulated the history of CRISPR technology, recent progress, and, especially, its potential applications in AD-related genetic, animal modeling, and functional studies. PMID:29445268

Enzymatic Processes in Marine Biotechnology.

PubMed

Trincone, Antonio

2017-03-25

In previous review articles the attention of the biocatalytically oriented scientific community towards the marine environment as a source of biocatalysts focused on the habitat-related properties of marine enzymes. Updates have already appeared in the literature, including marine examples of oxidoreductases, hydrolases, transferases, isomerases, ligases, and lyases ready for food and pharmaceutical applications. Here a new approach for searching the literature and presenting a more refined analysis is adopted with respect to previous surveys, centering the attention on the enzymatic process rather than on a single novel activity. Fields of applications are easily individuated: (i) the biorefinery value-chain, where the provision of biomass is one of the most important aspects, with aquaculture as the prominent sector; (ii) the food industry, where the interest in the marine domain is similarly developed to deal with the enzymatic procedures adopted in food manipulation; (iii) the selective and easy extraction/modification of structurally complex marine molecules, where enzymatic treatments are a recognized tool to improve efficiency and selectivity; and (iv) marine biomarkers and derived applications (bioremediation) in pollution monitoring are also included in that these studies could be of high significance for the appreciation of marine bioprocesses.

Enzymatic Processes in Marine Biotechnology

PubMed Central

Trincone, Antonio

2017-01-01

In previous review articles the attention of the biocatalytically oriented scientific community towards the marine environment as a source of biocatalysts focused on the habitat-related properties of marine enzymes. Updates have already appeared in the literature, including marine examples of oxidoreductases, hydrolases, transferases, isomerases, ligases, and lyases ready for food and pharmaceutical applications. Here a new approach for searching the literature and presenting a more refined analysis is adopted with respect to previous surveys, centering the attention on the enzymatic process rather than on a single novel activity. Fields of applications are easily individuated: (i) the biorefinery value-chain, where the provision of biomass is one of the most important aspects, with aquaculture as the prominent sector; (ii) the food industry, where the interest in the marine domain is similarly developed to deal with the enzymatic procedures adopted in food manipulation; (iii) the selective and easy extraction/modification of structurally complex marine molecules, where enzymatic treatments are a recognized tool to improve efficiency and selectivity; and (iv) marine biomarkers and derived applications (bioremediation) in pollution monitoring are also included in that these studies could be of high significance for the appreciation of marine bioprocesses. PMID:28346336

Whole cell entrapment techniques.

PubMed

Trelles, Jorge A; Rivero, Cintia W

2013-01-01

Microbial whole cells are efficient, ecological, and low-cost catalysts that have been successfully applied in the pharmaceutical, environmental, and alimentary industries, among others. Microorganism immobilization is a good way to carry out the bioprocess under preparative conditions. The main advantages of this methodology lie in their high operational stability, easy upstream separation and bioprocess scale-up feasibility. Cell entrapment is the most widely used technique for whole cell immobilization. This technique-in which the cells are included within a rigid network-is porous enough to allow the diffusion of substrates and products, protects the selected microorganism from the reaction medium, and has high immobilization efficiency (100 % in most cases).

On-Line Ion Exchange Liquid Chromatography as a Process Analytical Technology for Monoclonal Antibody Characterization in Continuous Bioprocessing.

PubMed

Patel, Bhumit A; Pinto, Nuno D S; Gospodarek, Adrian; Kilgore, Bruce; Goswami, Kudrat; Napoli, William N; Desai, Jayesh; Heo, Jun H; Panzera, Dominick; Pollard, David; Richardson, Daisy; Brower, Mark; Richardson, Douglas D

2017-11-07

Combining process analytical technology (PAT) with continuous production provides a powerful tool to observe and control monoclonal antibody (mAb) fermentation and purification processes. This work demonstrates on-line liquid chromatography (on-line LC) as a PAT tool for monitoring a continuous biologics process and forced degradation studies. Specifically, this work focused on ion exchange chromatography (IEX), which is a critical separation technique to detect charge variants. Product-related impurities, including charge variants, that impact function are classified as critical quality attributes (CQAs). First, we confirmed no significant differences were observed in the charge heterogeneity profile of a mAb through both at-line and on-line sampling and that the on-line method has the ability to rapidly detect changes in protein quality over time. The robustness and versatility of the PAT methods were tested by sampling from two purification locations in a continuous mAb process. The PAT IEX methods used with on-line LC were a weak cation exchange (WCX) separation and a newly developed shorter strong cation exchange (SCX) assay. Both methods provided similar results with the distribution of percent acidic, main, and basic species remaining unchanged over a 2 week period. Second, a forced degradation study showed an increase in acidic species and a decrease in basic species when sampled on-line over 7 days. These applications further strengthen the use of on-line LC to monitor CQAs of a mAb continuously with various PAT IEX analytical methods. Implementation of on-line IEX will enable faster decision making during process development and could potentially be applied to control in biomanufacturing.

A Thermophilic Ionic Liquid-Tolerant Cellulase Cocktail for the Production of Cellulosic Biofuels

PubMed Central

Park, Joshua I.; Steen, Eric J.; Burd, Helcio; Evans, Sophia S.; Redding-Johnson, Alyssa M.; Batth, Tanveer; Benke, Peter I.; D'haeseleer, Patrik; Sun, Ning; Sale, Kenneth L.; Keasling, Jay D.; Lee, Taek Soon; Petzold, Christopher J.; Mukhopadhyay, Aindrila; Singer, Steven W.; Simmons, Blake A.; Gladden, John M.

2012-01-01

Generation of biofuels from sugars in lignocellulosic biomass is a promising alternative to liquid fossil fuels, but efficient and inexpensive bioprocessing configurations must be developed to make this technology commercially viable. One of the major barriers to commercialization is the recalcitrance of plant cell wall polysaccharides to enzymatic hydrolysis. Biomass pretreatment with ionic liquids (ILs) enables efficient saccharification of biomass, but residual ILs inhibit both saccharification and microbial fuel production, requiring extensive washing after IL pretreatment. Pretreatment itself can also produce biomass-derived inhibitory compounds that reduce microbial fuel production. Therefore, there are multiple points in the process from biomass to biofuel production that must be interrogated and optimized to maximize fuel production. Here, we report the development of an IL-tolerant cellulase cocktail by combining thermophilic bacterial glycoside hydrolases produced by a mixed consortia with recombinant glycoside hydrolases. This enzymatic cocktail saccharifies IL-pretreated biomass at higher temperatures and in the presence of much higher IL concentrations than commercial fungal cocktails. Sugars obtained from saccharification of IL-pretreated switchgrass using this cocktail can be converted into biodiesel (fatty acid ethyl-esters or FAEEs) by a metabolically engineered strain of E. coli. During these studies, we found that this biodiesel-producing E. coli strain was sensitive to ILs and inhibitors released by saccharification. This cocktail will enable the development of novel biomass to biofuel bioprocessing configurations that may overcome some of the barriers to production of inexpensive cellulosic biofuels. PMID:22649505

Recombinant and epitope-based vaccines on the road to the market and implications for vaccine design and production.

PubMed

Oyarzún, Patricio; Kobe, Bostjan

2016-03-03

Novel vaccination approaches based on rational design of B- and T-cell epitopes - epitope-based vaccines - are making progress in the clinical trial pipeline. The epitope-focused recombinant protein-based malaria vaccine (termed RTS,S) is a next-generation approach that successfully reached phase-III trials, and will potentially become the first commercial vaccine against a human parasitic disease. Progress made on methods such as recombinant DNA technology, advanced cell-culture techniques, immunoinformatics and rational design of immunogens are driving the development of these novel concepts. Synthetic recombinant proteins comprising both B- and T-cell epitopes can be efficiently produced through modern biotechnology and bioprocessing methods, and can enable the induction of large repertoires of immune specificities. In particular, the inclusion of appropriate CD4+ T-cell epitopes is increasingly considered a key vaccine component to elicit robust immune responses, as suggested by results coming from HIV-1 clinical trials. In silico strategies for vaccine design are under active development to address genetic variation in pathogens and several broadly protective "universal" influenza and HIV-1 vaccines are currently at different stages of clinical trials. Other methods focus on improving population coverage in target populations by rationally considering specificity and prevalence of the HLA proteins, though a proof-of-concept in humans has not been demonstrated yet. Overall, we expect immunoinformatics and bioprocessing methods to become a central part of the next-generation epitope-based vaccine development and production process.

Extractables analysis of single-use flexible plastic biocontainers.

PubMed

Marghitoiu, Liliana; Liu, Jian; Lee, Hans; Perez, Lourdes; Fujimori, Kiyoshi; Ronk, Michael; Hammond, Matthew R; Nunn, Heather; Lower, Asher; Rogers, Gary; Nashed-Samuel, Yasser

2015-01-01

Studies of the extractable profiles of bioprocessing components have become an integral part of drug development efforts to minimize possible compromise in process performance, decrease in drug product quality, and potential safety risk to patients due to the possibility of small molecules leaching out from the components. In this study, an effective extraction solvent system was developed to evaluate the organic extractable profiles of single-use bioprocess equipment, which has been gaining increasing popularity in the biopharmaceutical industry because of the many advantages over the traditional stainless steel-based bioreactors and other fluid mixing and storage vessels. The chosen extraction conditions were intended to represent aggressive conditions relative to the application of single-use bags in biopharmaceutical manufacture, in which aqueous based systems are largely utilized. Those extraction conditions, along with a non-targeted analytical strategy, allowed for the generation and identification of an array of extractable compounds; a total of 53 organic compounds were identified from four types of commercially available single-use bags, the majority of which are degradation products of polymer additives. The success of this overall extractables analysis strategy was reflected partially by the effectiveness in the extraction and identification of a compound that was later found to be highly detrimental to mammalian cell growth. The usage of single-use bioreactors has been increasing in biopharmaceutical industry because of the appealing advantages that it promises regarding to the cleaning, sterilization, operational flexibility, and so on, during manufacturing of biologics. However, compared to its conventional counterparts based mainly on stainless steel, single-use bioreactors are more susceptible to potential problems associated with compound leaching into the bioprocessing fluid. As a result, extractable profiling of the single-use system has become essential in the qualification of such systems for its use in drug manufacturing. The aim of this study is to evaluate the effectiveness of an extraction solvent system developed to study the extraction profile of single-use bioreactors in which aqueous-based systems are largely used. The results showed that with a non-targeted analytical approach, the extraction solvent allowed the generation and identification of an array of extractable compounds from four commercially available single-use bioreactors. Most of extractables are degradation products of polymer additives, among which was a compound that was later found to be highly detrimental to mammalian cell growth. © PDA, Inc. 2015.

Developing a mesophilic co-culture for direct conversion of cellulose to butanol in consolidated bioprocess.

PubMed

Wang, Zhenyu; Cao, Guangli; Zheng, Ju; Fu, Defeng; Song, Jinzhu; Zhang, Junzheng; Zhao, Lei; Yang, Qian

2015-01-01

Consolidated bioprocessing (CBP) of butanol production from cellulosic biomass is a promising strategy for cost saving compared to other processes featuring dedicated cellulase production. CBP requires microbial strains capable of hydrolyzing biomass with enzymes produced on its own with high rate and high conversion and simultaneously produce a desired product at high yield. However, current reported butanol-producing candidates are unable to utilize cellulose as a sole carbon source and energy source. Consequently, developing a co-culture system using different microorganisms by taking advantage of their specific metabolic capacities to produce butanol directly from cellulose in consolidated bioprocess is of great interest. This study was mainly undertaken to find complementary organisms to the butanol producer that allow simultaneous saccharification and fermentation of cellulose to butanol in their co-culture under mesophilic condition. Accordingly, a highly efficient and stable consortium N3 on cellulose degradation was first developed by multiple subcultures. Subsequently, the functional microorganisms with 16S rRNA sequences identical to the denaturing gradient gel electrophoresis (DGGE) profile were isolated from consortium N3. The isolate Clostridium celevecrescens N3-2 exhibited higher cellulose-degrading capability was thus chosen as the partner strain for butanol production with Clostridium acetobutylicum ATCC824. Meanwhile, the established stable consortium N3 was also investigated to produce butanol by co-culturing with C. acetobutylicum ATCC824. Butanol was produced from cellulose when C. acetobutylicum ATCC824 was co-cultured with either consortium N3 or C. celevecrescens N3-2. Co-culturing C. acetobutylicum ATCC824 with the stable consortium N3 resulted in a relatively higher butanol concentration, 3.73 g/L, and higher production yield, 0.145 g/g of glucose equivalent. The newly isolated microbial consortium N3 and strain C. celevecrescens N3-2 displayed effective degradation of cellulose and produced considerable amounts of butanol when they were co-cultured with C. acetobutylicum ATCC824. This is the first report of application of co-culture to produce butanol directly from cellulose under mesophilic condition. Our results indicated that co-culture of mesophilic cellulolytic microbe and butanol-producing clostridia provides a technically feasible and more simplified way for producing butanol directly from cellulose.

Comparison of time-gated surface-enhanced Raman spectroscopy (TG-SERS) and classical SERS based monitoring of Escherichia coli cultivation samples.

PubMed

Kögler, Martin; Paul, Andrea; Anane, Emmanuel; Birkholz, Mario; Bunker, Alex; Viitala, Tapani; Maiwald, Michael; Junne, Stefan; Neubauer, Peter

2018-06-08

The application of Raman spectroscopy as a monitoring technique for bioprocesses is severely limited by a large background signal originating from fluorescing compounds in the culture media. Here we compare time-gated Raman (TG-Raman)-, continuous wave NIR-process Raman (NIR-Raman) and continuous wave micro-Raman (micro-Raman) approaches in combination with surface enhanced Raman spectroscopy (SERS) for their potential to overcome this limit. For that purpose, we monitored metabolite concentrations of Escherichia coli bioreactor cultivations in cell-free supernatant samples. We investigated concentration transients of glucose, acetate, AMP and cAMP at alternating substrate availability, from deficiency to excess. Raman and SERS signals were compared to off-line metabolite analysis of carbohydrates, carboxylic acids and nucleotides. Results demonstrate that SERS, in almost all cases, led to a higher number of identifiable signals and better resolved spectra. Spectra derived from the TG-Raman were comparable to those of micro-Raman resulting in well-discernable Raman peaks, which allowed for the identification of a higher number of compounds. In contrast, NIR-Raman provided a superior performance for the quantitative evaluation of analytes, both with and without SERS nanoparticles when using multivariate data analysis. This article is protected by copyright. All rights reserved. © 2018 American Institute of Chemical Engineers.

Bioproduction of mushroom mycelium of Agaricus bisporus by commercial submerged fermentation for the production of meat analogue.

PubMed

Kim, Kyoungju; Choi, Byungsun; Lee, Inhee; Lee, Hyeyoung; Kwon, Soonhyang; Oh, Kyoungyoung; Kim, Augustine Yonghwi

2011-07-01

As worldwide interest in healthy and delicious meat analogues increases, the texture of these products has become an important indicator of quality. Mycoprotein as fungal mycelium could provide a distinctive chewing sensation; however, the unfavorable consumer perception of fungal mycelium demands the production of meat analogues with true mushroom mycelium. The industrial and economical bioprocess was developed using an inexpensive medium (30 g L(-1) sugar cane extract (SCE), 10 g L(-1) NaNO(3) and 5 g L(-1) yeast extract) and A. bisporus Suksung. The SCE was maintained at around 10 g L(-1) to minimize osmotic shock. The maximum mycelium production of 15.0 g L(-1) (dry weight) was reached within 4 days. Scanning electron microscopic analysis showed fibrous and directional structure rather than a more typical pellet structure. Meat analogues with mushroom mycelium had better textural properties, being higher in hardness, springiness, and chewiness and with preferable umami characteristics compared to meat analogues utilizing soy protein. The overall acceptance of meat analogues prepared with mycelium and soy protein, and a ground beef patty, were 5, 2 and 10, respectively. The development of an industrial bioprocess for A. bisporus mycelium allowed the production of a highly acceptable meat analogue having not only superior textural properties but also umami characteristics when compared to that of soy protein. Copyright © 2011 Society of Chemical Industry.

Microencapsulation Technology: A Powerful Tool for Integrating Expansion and Cryopreservation of Human Embryonic Stem Cells

PubMed Central

Malpique, Rita; Brito, Catarina; Jensen, Janne; Bjorquist, Petter; Carrondo, Manuel J. T.; Alves, Paula M.

2011-01-01

The successful implementation of human embryonic stem cells (hESCs)-based technologies requires the production of relevant numbers of well-characterized cells and their efficient long-term storage. In this study, cells were microencapsulated in alginate to develop an integrated bioprocess for expansion and cryopreservation of pluripotent hESCs. Different three-dimensional (3D) culture strategies were evaluated and compared, specifically, microencapsulation of hESCs as: i) single cells, ii) aggregates and iii) immobilized on microcarriers. In order to establish a scalable bioprocess, hESC-microcapsules were cultured in stirred tank bioreactors. The combination of microencapsulation and microcarrier technology resulted in a highly efficient protocol for the production and storage of pluripotent hESCs. This strategy ensured high expansion ratios (an approximately twenty-fold increase in cell concentration) and high cell recovery yields (>70%) after cryopreservation. When compared with non-encapsulated cells, cell survival post-thawing demonstrated a three-fold improvement without compromising hESC characteristics. Microencapsulation also improved the culture of hESC aggregates by protecting cells from hydrodynamic shear stress, controlling aggregate size and maintaining cell pluripotency for two weeks. This work establishes that microencapsulation technology may prove a powerful tool for integrating the expansion and cryopreservation of pluripotent hESCs. The 3D culture strategy developed herein represents a significant breakthrough towards the implementation of hESCs in clinical and industrial applications. PMID:21850261

Nutrition and bioprocess development for efficient biosynthesis of an antitumor compound from marine-derived fungus.

PubMed

Zhou, Weiqiang; Cai, Menghao; Zhou, Jiushun; Jiang, Tao; Zhou, Jiao; Wang, Meixia; Zhou, Xiangshan; Zhang, Yuanxing

2013-10-01

An integrated nutrition and bioprocess strategy was developed for improving the biosynthesis of an antitumor compound, 1403C, by a marine-derived fungus, Halorosellinia sp. (no. 1403). First, statistical design strategies were synthetically applied to optimize the nutritional composition. The resulting 1403C production reached 2.07 g/l, which was 143.5 % higher than the original production. However, it only produced 0.44 g/l of 1403C in 5-l bioreactor fermentation. Thus, the operating parameters including culture pH, dissolved oxygen, agitation speed, impeller type and inoculum level were considered to improve the fermentation process, and an effective control strategy for 1403C production by Halorosellinia sp. submerged in a 5-l bioreactor was established. When inoculating 0.22 g/l dry biomass, controlling dissolved oxygen not lower than 30 % during the growth phase but ranging between 30 and 40 % during the stationary phase, using a double-layer six-flat-blade Rushton disc turbine agitated at 400 rpm, keeping short-term low pH and rapid-rising pH with glucose starvation, the highest 1403C production was finally obtained at 1.32 g/l, which was promoted by 200 % compared to before optimization. Fermentation scale-up was finally performed in a 500-l bioreactor, and 1403C production of 1.09 g/l was obtained.

MOSAIC: a chemical-genetic interaction data repository and web resource for exploring chemical modes of action.

PubMed

Nelson, Justin; Simpkins, Scott W; Safizadeh, Hamid; Li, Sheena C; Piotrowski, Jeff S; Hirano, Hiroyuki; Yashiroda, Yoko; Osada, Hiroyuki; Yoshida, Minoru; Boone, Charles; Myers, Chad L

2018-04-01

Chemical-genomic approaches that map interactions between small molecules and genetic perturbations offer a promising strategy for functional annotation of uncharacterized bioactive compounds. We recently developed a new high-throughput platform for mapping chemical-genetic (CG) interactions in yeast that can be scaled to screen large compound collections, and we applied this system to generate CG interaction profiles for more than 13 000 compounds. When integrated with the existing global yeast genetic interaction network, CG interaction profiles can enable mode-of-action prediction for previously uncharacterized compounds as well as discover unexpected secondary effects for known drugs. To facilitate future analysis of these valuable data, we developed a public database and web interface named MOSAIC. The website provides a convenient interface for querying compounds, bioprocesses (Gene Ontology terms) and genes for CG information including direct CG interactions, bioprocesses and gene-level target predictions. MOSAIC also provides access to chemical structure information of screened molecules, chemical-genomic profiles and the ability to search for compounds sharing structural and functional similarity. This resource will be of interest to chemical biologists for discovering new small molecule probes with specific modes-of-action as well as computational biologists interested in analysing CG interaction networks. MOSAIC is available at http://mosaic.cs.umn.edu. hisyo@riken.jp, yoshidam@riken.jp, charlie.boone@utoronto.ca or chadm@umn.edu. Supplementary data are available at Bioinformatics online.

An experiment to fly on mission STS-93 is prepared at Life Sciences Building, CCAS

NASA Technical Reports Server (NTRS)

1999-01-01

In the KSC Life Sciences Building, Hangar L, Cape Canaveral Air Station, Shawn Bengtson, with Lockheed Martin, checks population cages containing fruit flies. The larvae of the flies are part of an experiment that is a secondary payload on mission STS-93. The experiment will examine the effects of microgravity and space flight on the development of neural connections between specific motor neurons and their targets in muscle fibers. That information could lead to understanding the effect of microgravity on human nervous system connectivity. The larvae will be contained in incubators that are part of a Commercial Generic Bioprocessing Apparatus (CGBA), which can start bioprocessing reactions by mixing or heating a sample and can also initiate multiple-step, sequential reactions in a technique called phased processing. The primary payload of mission STS-93 is the Chandra X-ray Observatory, which will allow scientists from around the world to see previously invisible black holes and high-temperature gas clouds, giving the observatory the potential to rewrite the books on the structure and evolution of our universe. The target launch date for STS-93 is July 9, aboard Space Shuttle Columbia, from Launch Pad 39B.

Advanced Chemical Design for Efficient Lignin Bioconversion

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

Xie, Shangxian; Sun, Qining; Pu, Yunqiao

Here, lignin depolymerization mainly involves redox reactions relying on the effective electron transfer. Even though electron mediators were previously used for delignification of paper pulp, no study has established a bioprocess to fragment and solubilize the lignin with an effective laccase–mediator system, in particular, for subsequent microbial bioconversion. Efficient lignin depolymerization was achieved by screening proper electron mediators with laccase to attain a nearly 6-fold increase of kraft lignin solubility compared to the control kraft lignin without laccase treatment. Chemical analysis suggested the release of a low molecular weight fraction of kraft lignin into the solution phase. Moreover, NMR analysismore » revealed that an efficient enzyme–mediator system can promote the lignin degradation. More importantly, the fundamental mechanisms guided the development of an efficient lignin bioconversion process, where solubilized lignin from laccase–HBT treatment served as a superior substrate for bioconversion by Rhodococcus opacus PD630. The cell growth was increased by 10 6 fold, and the lipid titer reached 1.02 g/L. Overall, the study has manifested that an efficient enzyme–mediator–microbial system can be exploited to establish a bioprocess to solubilize lignin, cleave lignin linkages, modify the structure, and produce substrates amenable to bioconversion.« less

P-MASS and P-GBA: Two new hardware developments for growing plants in space

NASA Technical Reports Server (NTRS)

Hoehn, Alexander; Luttges, Marvin W.; Robinson, Michael C.; Stodieck, Louis S.; Kliss, Mark H.

1994-01-01

Plant growth, and especially plant performance experiments in microgravity are limited by the currently available plant growth facilities (low light levels, inadequate nutrient delivery and atmosphere conditioning systems, insufficient science instrumentation, infrequent flight opportunities). In addition, mission durations of 10 to 14 days aboard the NSTS Space Shuttle allow for only brief periods of microgravity exposure with respect to the life cycle of a plant. Based on seed germination experiments, using the Generic BioProcessing Apparatus hardware (GBA), two new payloads have been designed specifically for plant growth. These payloads provide new opportunities for plant gravitational and space biology research and emphasize the investigation of plant performance (photosynthesis, biomass accumulations) in microgravity. The Plant-Module for Autonomous Space Support (P-MASS) was designed to utilize microgravity exposure times in excess of 30 days on the first flight of the recoverable COMET satellite (Commercial Experiment Transporter). The Plant-Generic Bioprocessing Apparatus (P-GBA), is designed for the National Space Transportation System (NSTS) Space Shuttle middeck and the SPACEHAB environment. The P-GBA is an evolution from the GBA hardware and P-MASS (plant chamber and instrumentation). The available light levels of both payloads more than double currently available capabilities.

Advanced Chemical Design for Efficient Lignin Bioconversion

DOE PAGES

Xie, Shangxian; Sun, Qining; Pu, Yunqiao; ...

2017-01-30

Here, lignin depolymerization mainly involves redox reactions relying on the effective electron transfer. Even though electron mediators were previously used for delignification of paper pulp, no study has established a bioprocess to fragment and solubilize the lignin with an effective laccase–mediator system, in particular, for subsequent microbial bioconversion. Efficient lignin depolymerization was achieved by screening proper electron mediators with laccase to attain a nearly 6-fold increase of kraft lignin solubility compared to the control kraft lignin without laccase treatment. Chemical analysis suggested the release of a low molecular weight fraction of kraft lignin into the solution phase. Moreover, NMR analysismore » revealed that an efficient enzyme–mediator system can promote the lignin degradation. More importantly, the fundamental mechanisms guided the development of an efficient lignin bioconversion process, where solubilized lignin from laccase–HBT treatment served as a superior substrate for bioconversion by Rhodococcus opacus PD630. The cell growth was increased by 10 6 fold, and the lipid titer reached 1.02 g/L. Overall, the study has manifested that an efficient enzyme–mediator–microbial system can be exploited to establish a bioprocess to solubilize lignin, cleave lignin linkages, modify the structure, and produce substrates amenable to bioconversion.« less

An experiment to fly on mission STS-93 is prepared at Life Sciences Building, CCAS

NASA Technical Reports Server (NTRS)

1999-01-01

In the KSC Life Sciences Building, Hangar L, Cape Canaveral Air Station, Dr. Haig Keshishian checks fruit fly larvae in a petri dish. The larvae are part of an experiment that is a secondary payload on mission STS-93. The experiment will examine the effects of microgravity and space flight on the development of neural connections between specific motor neurons and their targets in muscle fibers. Dr. Keshishian, from Yale University, is the principle investigator for the experiment. The larvae will be contained in incubators that are part of a Commercial Generic Bioprocessing Apparatus (CGBA), which can start bioprocessing reactions by mixing or heating a sample and can also initiate multiple-step, sequential reactions in a technique called phased processing. The primary payload of mission STS-93 is the Chandra X-ray Observatory, which will allow scientists from around the world to see previously invisible black holes and high- temperature gas clouds, giving the observatory the potential to rewrite the books on the structure and evolution of our universe. The target launch date for STS-93 is July 9, aboard Space Shuttle Columbia, from Launch Pad 39B.

Peptidomic Identification of Cysteine-Rich Peptides from Plants.

PubMed

Hemu, Xinya; Serra, Aida; Darwis, Dina A; Cornvik, Tobias; Sze, Siu Kwan; Tam, James P

2018-01-01

Plant cysteine-rich peptides (CRPs) constitute a majority of plant-derived peptides with high molecular diversity. This protocol describes a rapid and efficient peptidomic approach to identify a whole spectrum of CRPs in a plant extract and decipher their molecular diversity and bioprocessing mechanism. Cyclotides from C. ternatea are used as the model CRPs to demonstrate our methodology. Cyclotides exist naturally in both cyclic and linear forms, although the linear forms (acyclotide) are generally present at much lower concentrations. Both cyclotides and acyclotides require linearization of their backbone prior to fragmentation and sequencing. A novel and practical three-step chemoenzymatic treatment was developed to linearize and distinguish both forms: (1) N-terminal acetylation that pre-labels the acyclotides; (2) conversion of Cys into pseudo-Lys through aziridine-mediated S-alkylation to reduce disulfide bonds and to increase the net charge of peptides; and (3) opening of cyclic backbones by the novel asparaginyl endopeptidase butelase 2 that cleaves at the native bioprocessing site. The treated peptides are subsequently analyzed by liquid chromatography coupled to mass spectrometry using electron transfer dissociation fragmentation and sequences are identified by matching the MS/MS spectra directly with the transcriptomic database.

The art of CHO cell engineering: A comprehensive retrospect and future perspectives.

PubMed

Fischer, Simon; Handrick, René; Otte, Kerstin

2015-12-01

Chinese hamster ovary (CHO) cells represent the most frequently applied host cell system for industrial manufacturing of recombinant protein therapeutics. CHO cells are capable of producing high quality biologics exhibiting human-like post-translational modifications in gram quantities. However, production processes for biopharmaceuticals using mammalian cells still suffer from cellular limitations such as limited growth, low productivity and stress resistance as well as higher expenses compared to bacterial or yeast based expression systems. Besides bioprocess, media and vector optimizations, advances in host cell engineering technologies comprising introduction, knock-out or post-transcriptional silencing of engineering genes have paved the way for remarkable achievements in CHO cell line development. Furthermore, thorough analysis of cellular pathways and mechanisms important for bioprocessing steadily unravels novel target molecules which might be addressed by functional genomic tools in order to establish superior production cell factories. This review provides a comprehensive summary of the most fundamental achievements in CHO cell engineering over the past three decades. Finally, the authors discuss the potential of novel and innovative methodologies that might contribute to further enhancement of existing CHO based production platforms for biopharmaceutical manufacturing in the future. Copyright © 2015 Elsevier Inc. All rights reserved.

Rapid Analysis of Carbohydrates in Bioprocess Samples: An Evaluation of the CarboPac SA10 for HPAE-PAD Analysis by Interlaboratory Comparison

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

Sevcik, R. S.; Hyman, D. A.; Basumallich, L.

2013-01-01

A technique for carbohydrate analysis for bioprocess samples has been developed, providing reduced analysis time compared to current practice in the biofuels R&D community. The Thermofisher CarboPac SA10 anion-exchange column enables isocratic separation of monosaccharides, sucrose and cellobiose in approximately 7 minutes. Additionally, use of a low-volume (0.2 mL) injection valve in combination with a high-volume detection cell minimizes the extent of sample dilution required to bring sugar concentrations into the linear range of the pulsed amperometric detector (PAD). Three laboratories, representing academia, industry, and government, participated in an interlaboratory study which analyzed twenty-one opportunistic samples representing biomass pretreatment, enzymaticmore » saccharification, and fermentation samples. The technique's robustness, linearity, and interlaboratory reproducibility were evaluated and showed excellent-to-acceptable characteristics. Additionally, quantitation by the CarboPac SA10/PAD was compared with the current practice method utilizing a HPX-87P/RID. While these two methods showed good agreement a statistical comparison found significant quantitation difference between them, highlighting the difference between selective and universal detection modes.« less

Economic comparison between conventional and disposables-based technology for the production of biopharmaceuticals.

PubMed

Novais, J L; Titchener-Hooker, N J; Hoare, M

2001-10-20

Time to market, cost effectiveness, and flexibility are key issues in today's biopharmaceutical market. Bioprocessing plants based on fully disposable, presterilized, and prevalidated components appear as an attractive alternative to conventional stainless steel plants, potentially allowing for shorter implementation times, smaller initial investments, and increased flexibility. To evaluate the economic case of such an alternative it was necessary to develop an appropriate costing model which allows an economic comparison between conventional and disposables-based engineering to be made. The production of an antibody fragment from an E. coli fermentation was used to provide a case study for both routes. The conventional bioprocessing option was costed through available models, which were then modified to account for the intrinsic differences observed in a disposables-based option. The outcome of the analysis indicates that the capital investment required for a disposables-based option is substantially reduced at less than 60% of that for a conventional option. The disposables-based running costs were evaluated as being 70% higher than those of the conventional equivalent. Despite this higher value, the net present value (NPV) of the disposables-based plant is positive and within 25% of that for the conventional plant. Sensitivity analysis performed on key variables indicated the robustness of the economic analysis presented. In particular a 9-month reduction in time to market arising from the adoption of a disposables-based approach, results in a NPV which is identical to that of the conventional option. Finally, the effect of any possible loss in yield resulting from the use of disposables was also examined. This had only a limited impact on the NPV: for example, a 50% lower yield in the disposable chromatography step results in a 10% reduction of the disposable NPV. The results provide the necessary framework for the economic comparison of disposables and conventional bioprocessing technologies. Copyright 2001 John Wiley & Sons, Inc.

ASTM and ASME-BPE Standards--Complying with the Needs of the Pharmaceutical Industry.

PubMed

Huitt, William M

2011-01-01

Designing and building a pharmaceutical facility requires the owner, engineer of record, and constructor to be knowledgeable with regard to the industry codes and standards that apply to this effort. Up until 1997 there were no industry standards directed at the needs and requirements of the pharmaceutical industry. Prior to that time it was a patchwork effort at resourcing and adopting nonpharmaceutical-related codes and standards and then modifying them in order to meet the more stringent requirements of the Food and Drug Administration (FDA). In 1997 the American Society of Mechanical Engineers (ASME) published the first Bioprocessing Equipment (BPE) Standard. Through harmonization efforts this relatively new standard has brought together, scrutinized, and refined industry accepted methodologies together with FDA compliance requirements, and has established an American National Standard that provides a comprehensive set of standards that are integral to the pharmaceutical industry. This article describes various American National Standards, including those developed and published by the American Society for Testing and Materials (ASTM), and how they apply to the pharmaceutical industry. It goes on to discuss the harmonization effort that takes place between the various standards developers in an attempt to prevent conflicts and omissions between the many standards. Also included are examples of tables and figures taken from the ASME-BPE Standard. These examples provide the reader with insight to the relevant content of the ASME-BPE Standard. Designing and building a pharmaceutical facility requires the owner, engineer of record, and constructor to be knowledgeable with regard to the industry codes and standards that apply to this effort. Up until 1997 there were no industry standards directed at the needs and requirements of the pharmaceutical industry. Prior to that time it was a patchwork effort at resourcing and adopting nonpharmaceutical-related codes and standards and then modifying them in order to meet the more stringent requirements of the Food and Drug Administration (FDA). In 1997 the American Society of Mechanical Engineers (ASME) published the first Bioprocessing Equipment (BPE) Standard. In its initial development and ongoing maintenance it works with other American National Standards developers to harmonize the many standards associated with the design, engineering, and construction of bioprocessing facilities. This harmonization effort has established a comprehensive set of standards for the betterment of the pharmaceutical industry at large. This effort is, and will remain, very important as technology, along with new and improved product and processes, evolve into the future.

Bioprocessing of lignite coals using reductive microorganisms

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

Crawford, D.L.

In order to convert lignite coals into liquid fuels, gases or chemical feedstock, the macromolecular structure of the coal must be broken down into low molecular weight fractions prior to further modification. Our research focused on this aspect of coal bioprocessing. We isolated, characterized and studied the lignite coal-depolymerizing organisms Streptomyces viridosporus T7A, Pseudomonas sp. DLC-62, unidentified bacterial strain DLC-BB2 and Gram-positive Bacillus megaterium strain DLC-21. In this research we showed that these bacteria are able to solubilize and depolymerize lignite coals using a combination of biological mechanisms including the excretion of coal solublizing basic chemical metabolites and extracellular coalmore » depolymerizing enzymes.« less

Trends in Process Analytical Technology: Present State in Bioprocessing.

PubMed

Jenzsch, Marco; Bell, Christian; Buziol, Stefan; Kepert, Felix; Wegele, Harald; Hakemeyer, Christian

2017-08-04

Process analytical technology (PAT), the regulatory initiative for incorporating quality in pharmaceutical manufacturing, is an area of intense research and interest. If PAT is effectively applied to bioprocesses, this can increase process understanding and control, and mitigate the risk from substandard drug products to both manufacturer and patient. To optimize the benefits of PAT, the entire PAT framework must be considered and each elements of PAT must be carefully selected, including sensor and analytical technology, data analysis techniques, control strategies and algorithms, and process optimization routines. This chapter discusses the current state of PAT in the biopharmaceutical industry, including several case studies demonstrating the degree of maturity of various PAT tools. Graphical Abstract Hierarchy of QbD components.

A systematic petri net approach for multiple-scale modeling and simulation of biochemical processes.

PubMed

Chen, Ming; Hu, Minjie; Hofestädt, Ralf

2011-06-01

A method to exploit hybrid Petri nets for modeling and simulating biochemical processes in a systematic way was introduced. Both molecular biology and biochemical engineering aspects are manipulated. With discrete and continuous elements, the hybrid Petri nets can easily handle biochemical factors such as metabolites concentration and kinetic behaviors. It is possible to translate both molecular biological behavior and biochemical processes workflow into hybrid Petri nets in a natural manner. As an example, penicillin production bioprocess is modeled to illustrate the concepts of the methodology. Results of the dynamic of production parameters in the bioprocess were simulated and observed diagrammatically. Current problems and post-genomic perspectives were also discussed.

Online analysis and process control in recombinant protein production (review).

PubMed

Palmer, Shane M; Kunji, Edmund R S

2012-01-01

Online analysis and control is essential for efficient and reproducible bioprocesses. A key factor in real-time control is the ability to measure critical variables rapidly. Online in situ measurements are the preferred option and minimize the potential loss of sterility. The challenge is to provide sensors with a good lifespan that withstand harsh bioprocess conditions, remain stable for the duration of a process without the need for recalibration, and offer a suitable working range. In recent decades, many new techniques that promise to extend the possibilities of analysis and control, not only by providing new parameters for analysis, but also through the improvement of accepted, well practiced, measurements have arisen.

Design of Cyclic Peptide Based Glucose Receptors and Their Application in Glucose Sensing.

PubMed

Li, Chao; Chen, Xin; Zhang, Fuyuan; He, Xingxing; Fang, Guozhen; Liu, Jifeng; Wang, Shuo

2017-10-03

Glucose assay is of great scientific significance in clinical diagnostics and bioprocess monitoring, and to design a new glucose receptor is necessary for the development of more sensitive, selective, and robust glucose detection techniques. Herein, a series of cyclic peptide (CP) glucose receptors were designed to mimic the binding sites of glucose binding protein (GBP), and CPs' sequence contained amino acid sites Asp, Asn, His, Asp, and Arg, which constituted the first layer interactions of GBP. The properties of these CPs used as a glucose receptor or substitute for the GBP were studied by using a quartz crystal microbalance (QCM) technique. It was found that CPs can form a self-assembled monolayer at the Au quartz electrode surface, and the monolayer's properties were characterized by using cyclic voltammetry, electrochemical impedance spectroscopy, and atomic force microscopy. The CPs' binding affinity to saccharide (i.e., galactose, fructose, lactose, sucrose, and maltose) was investigated, and the CPs' sensitivity and selectivity toward glucose were found to be dependent upon the configuration,i.e., the amino acids sequence of the CPs. The cyclic unit with a cyclo[-CNDNHCRDNDC-] sequence gave the highest selectivity and sensitivity for glucose sensing. This work suggests that a synthetic peptide bearing a particular functional sequence could be applied for developing a new generation of glucose receptors and would find huge application in biological, life science, and clinical diagnostics fields.

Bioprocessing feasibility analysis. [thymic hormone bioassay and electrophoresis

NASA Technical Reports Server (NTRS)

1978-01-01

The biology and pathophysiology of the thymus gland is discussed and a clinical procedure for thymic hormone assay is described. The separation of null lymphocytes from mice spleens and the functional characteristics of the cells after storage and transportation were investigated to develop a clinical procedure for thymic hormone assay, and to determine whether a ground-based approach will provide the desired end-product in sufficient quantities, or whether the microgravity of space should be exploited for more economical preparation of the hormone.

Anaerobic bioprocessing of low-rank coals. [Veillonella alcalescens and Propionibacterium acidipropionici

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

Jain, M.K.; Narayan, R.; Han, O.

1992-01-30

The overall goal of this project is to find biological methods to remove carboxylic functionalities from low-rank coals under ambient conditions and to assess the properties of these modified coals towards coal liquefaction. The main objectives of this quarter were: (1) continuation of microbial consortia development, (2) evaluation of the isolated organisms for decarboxylation, (3) selection of best performing culture (known cultures vs. new isolates), and (4) coal decarboxylation using activated carbon as blanks. The project began on September 12, 1990.

Increasing the antioxidant activity, total phenolic and flavonoid contents by optimizing the germination conditions of amaranth seeds.

PubMed

Perales-Sánchez, Janitzio X K; Reyes-Moreno, Cuauhtémoc; Gómez-Favela, Mario A; Milán-Carrillo, Jorge; Cuevas-Rodríguez, Edith O; Valdez-Ortiz, Angel; Gutiérrez-Dorado, Roberto

2014-09-01

The aim of this study was to optimize the germination conditions of amaranth seeds that would maximize the antioxidant activity (AoxA), total phenolic (TPC), and flavonoid (TFC) contents. To optimize the germination bioprocess, response surface methodology was applied over three response variables (AoxA, TPC, TFC). A central composite rotable experimental design with two factors [germination temperature (GT), 20-45 ºC; germination time (Gt), 14-120 h] in five levels was used; 13 treatments were generated. The amaranth seeds were soaked in distilled water (25 °C/6 h) before germination. The sprouts from each treatment were dried (50 °C/8 h), cooled, and ground to obtain germinated amaranth flours (GAF). The best combination of germination bioprocess variables for producing optimized GAF with the highest AoxA [21.56 mmol trolox equivalent (TE)/100 g sample, dw], TPC [247.63 mg gallic acid equivalent (GAE)/100 g sample, dw], and TFC [81.39 mg catechin equivalent (CAE)/100 g sample, dw] was GT = 30 ºC/Gt = 78 h. The germination bioprocess increased AoxA, TPC, and TFC in 300-470, 829, and 213%, respectively. The germination is an effective strategy to increase the TPC and TFC of amaranth seeds for enhancing functionality with improved antioxidant activity.

A unique noninvasive approach to monitoring dissolved O2 and CO2 in cell culture.

PubMed

Chatterjee, Madhubanti; Ge, Xudong; Uplekar, Shaunak; Kostov, Yordan; Croucher, Leah; Pilli, Manohar; Rao, Govind

2015-01-01

Although online monitoring of dissolved oxygen (DO) and carbon dioxide (DCO2 ) is highly desirable in bioprocesses, small-scale bioreactors are usually not monitored due to the lack of suitable sensors. Traditional electrochemical sensors are usually not used because they are bulky and invasive. Disposable optical sensors are small and only partially invasive, but there are concerns regarding the toxicity of the patch and the phototoxicity of the illuminating light. Here we present a novel, noninvasive, rate-based technique for monitoring DO and DCO2 in cell cultures. A silicone sampling loop which allowed the diffusion of O2 and CO2 through its wall was inserted inside a bioreactor, and then flushed with N2 until the CO2 and O2 inside the loop were completely removed. The gas inside the loop was then allowed to recirculate through gas impermeable tubing to the O2 and CO2 sensors. We have shown that by measuring the initial diffusion rate we were able to determine the partial pressures of the two gases in the culture. The technique could be readily automated and measurements could be made in minutes. It was tested in demonstration experiments by growing murine hybridoma cells in a T-flask and a spinner-flask at 37°C. The results were comparable to those measured with commercially available fluorescence-based patch sensors. These results show that the rate-based method is an effective way to monitor small-scale cell cultures. This measurement mechanism can be easily built into disposable cell culture vessels for facile use. © 2014 Wiley Periodicals, Inc.

Elm Tree (Ulmus parvifolia) Bark Bioprocessed with Mycelia of Shiitake (Lentinus edodes) Mushrooms in Liquid Culture: Composition and Mechanism of Protection against Allergic Asthma in Mice.

PubMed

Kim, Sung Phil; Lee, Sang Jong; Nam, Seok Hyun; Friedman, Mendel

2016-02-03

Mushrooms can break down complex plant materials into smaller, more digestible and bioactive compounds. The present study investigated the antiasthma effect of an Ulmus parvifolia bark extract bioprocessed in Lentinus edodes liquid mycelium culture (BPUBE) against allergic asthma in chicken egg ovalbumin (OVA)-sensitized/challenged mice. BPUBE suppressed total IgE release from U266B1 cells in a dose-dependent manner without cytotoxicity. Inhibitory activity of BPUBE against OVA-specific IgE secretion in bronchoalveolar lavage fluid (BALF) was observed in OVA-sensitized/challenged asthmatic mice. BPUBE also inhibited OVA-specific IgG and IgG1 secretion into serum from the allergic mice, suggesting the restoration of a Th2-biased immune reaction to a Th1/Th2-balanced status, as indicated by the Th1/Th2 as well as regulatory T cell (Treg) cytokine profile changes caused by BPUBE in serum or BALF. Inflammatory cell counts in BALF and lung histology showed that leukocytosis and eosinophilia induced by OVA-sensitization/challenge were inhibited by the oral administration of BPUBE. Amelioration of eosinophil infiltration near the trachea was associated with reduced eotaxin and vascular cell adhesion molecule-1 (VCAM-1) levels. Changes in proinflammatory mediator levels in BALF suggest that BPUBE decreased OVA-sensitization-induced elevation of leukotriene C4 (LTC4) and prostaglandin D2 (PGD2). The finding that asthma-associated biomarker levels of OVA-sensitized/challenged mice were much more inhibited with BPUBE treatment than NPUBE (not-bioprocessed Ulmus parvifolia extract) treatment suggested the production of new bioactive compounds by the mushroom mycelia that may be involved in enhancing the observed antiasthmatic properties. The possible relation of the composition determined by proximate analysis and GC/MS to observed bioactivity is discussed. The results suggest that the elm tree (Ulmus parvifolia) bark bioprocessed with mycelia of shiitake (Lentinus edodes) mushrooms has the potential to prevent and/or treat allergic asthma.

Reproducible culture and differentiation of mouse embryonic stem cells using an automated microwell platform☆

PubMed Central

Hussain, Waqar; Moens, Nathalie; Veraitch, Farlan S.; Hernandez, Diana; Mason, Chris; Lye, Gary J.

2013-01-01

The use of embryonic stem cells (ESCs) and their progeny in high throughput drug discovery and regenerative medicine will require production at scale of well characterized cells at an appropriate level of purity. The adoption of automated bioprocessing techniques offers the possibility to overcome the lack of consistency and high failure rates seen with current manual protocols. To build the case for increased use of automation this work addresses the key question: “can an automated system match the quality of a highly skilled and experienced person working manually?” To answer this we first describe an integrated automation platform designed for the ‘hands-free’ culture and differentiation of ESCs in microwell formats. Next we outline a framework for the systematic investigation and optimization of key bioprocess variables for the rapid establishment of validatable Standard Operating Procedures (SOPs). Finally the experimental comparison between manual and automated bioprocessing is exemplified by expansion of the murine Oct-4-GiP ESC line over eight sequential passages with their subsequent directed differentiation into neural precursors. Our results show that ESCs can be effectively maintained and differentiated in a highly reproducible manner by the automated system described. Statistical analysis of the results for cell growth over single and multiple passages shows up to a 3-fold improvement in the consistency of cell growth kinetics with automated passaging. The quality of the cells produced was evaluated using a panel of biological markers including cell growth rate and viability, nutrient and metabolite profiles, changes in gene expression and immunocytochemistry. Automated processing of the ESCs had no measurable negative effect on either their pluripotency or their ability to differentiate into the three embryonic germ layers. Equally important is that over a 6-month period of culture without antibiotics in the medium, we have not had any cases of culture contamination. This study thus confirms the benefits of adopting automated bioprocess routes to produce cells for therapy and for use in basic discovery research. PMID:23956681

STS-69 flight day 6 highlights

NASA Astrophysics Data System (ADS)

1995-09-01

After being awakened by the Beatles song, 'A Hard Days Night', the flightcrew of the STS-69 mission, Cmdr. Dave Walker, Pilot Ken Cockrell, and Mission Specialists Jim Voss, Jim Newman, and Mike Gernhardt, began their sixth day in orbit by monitoring the free orbiting Wake Shield Facility (WSF). Later Cmdr. Walker conducted an interview with television reporters from Atlanta and Boston, answering questions about the mission and general questions about NASA's space program. The crew filmed a video fo themselves performing daily routines (eating, shaving, exercising), as well as some of the physiological experiments, and shuttle equipment maintenance and checkout. One of the secondary experiments included the Commercial Generic Bioprocessing Apparatus-7 (CGBA-7), which served as an incubator and experiment station for a variety of tests (agricultural, pharmaceutical, biomedical, and environmental). Earth views included some cloud cover, the Gulf of Mexico, Texas, and the Atlantic Ocean.

STS-69 Flight Day 6 Highlights

NASA Technical Reports Server (NTRS)

1995-01-01

After being awakened by the Beatles song, 'A Hard Days Night', the flightcrew of the STS-69 mission, Cmdr. Dave Walker, Pilot Ken Cockrell, and Mission Specialists Jim Voss, Jim Newman, and Mike Gernhardt, began their sixth day in orbit by monitoring the free orbiting Wake Shield Facility (WSF). Later Cmdr. Walker conducted an interview with television reporters from Atlanta and Boston, answering questions about the mission and general questions about NASA's space program. The crew filmed a video for themselves performing daily routines (eating, shaving, exercising), as well as some of the physiological experiments, and shuttle equipment maintenance and checkout. One of the secondary experiments included the Commercial Generic Bioprocessing Apparatus-7 (CGBA-7), which served as an incubator and experiment station for a variety of tests (agricultural, pharmaceutical, biomedical, and environmental). Earth views included some cloud cover, the Gulf of Mexico, Texas, and the Atlantic Ocean.

Microtiter miniature shaken bioreactor system as a scale-down model for process development of production of therapeutic alpha-interferon2b by recombinant Escherichia coli.

PubMed

Tan, Joo Shun; Abbasiliasi, Sahar; Kadkhodaei, Saeid; Tam, Yew Joon; Tang, Teck-Kim; Lee, Yee-Ying; Ariff, Arbakariya B

2018-01-04

Demand for high-throughput bioprocessing has dramatically increased especially in the biopharmaceutical industry because the technologies are of vital importance to process optimization and media development. This can be efficiently boosted by using microtiter plate (MTP) cultivation setup embedded into an automated liquid-handling system. The objective of this study was to establish an automated microscale method for upstream and downstream bioprocessing of α-IFN2b production by recombinant Escherichia coli. The extraction performance of α-IFN2b by osmotic shock using two different systems, automated microscale platform and manual extraction in MTP was compared. The amount of α-IFN2b extracted using automated microscale platform (49.2 μg/L) was comparable to manual osmotic shock method (48.8 μg/L), but the standard deviation was 2 times lower as compared to manual osmotic shock method. Fermentation parameters in MTP involving inoculum size, agitation speed, working volume and induction profiling revealed that the fermentation conditions for the highest production of α-IFN2b (85.5 μg/L) was attained at inoculum size of 8%, working volume of 40% and agitation speed of 1000 rpm with induction at 4 h after the inoculation. Although the findings at MTP scale did not show perfect scalable results as compared to shake flask culture, but microscale technique development would serve as a convenient and low-cost solution in process optimization for recombinant protein.

KENNEDY SPACE CENTER, FLA. - The second International Microgravity Laboratory-2 (IML-2) is off to an ontime start as the Space Shuttle Columbia lifts off from Launch Pad 39A at 12:43:00 p.m. EDT. On board are a crew of seven and more than 80 investigations developed by more than 200 scientists from 13 countries. The IML-2 complement includes materials science, bioprocessing, space and radiation biology, and human physiology experiments that will be carried out over the course of the 14-day flight. The commander of Space Shuttle Mission STS-65 is Robert D. Cabana. James D. Halsell Jr. is the pilot; the payload commander is Richard J. Hieb; the three mission specialists are Carl E. Walz, Leroy Chiao and Donald A. Thomas. Dr. Chiaki Mukai, representing NASDA, the National Space Development Agency of Japan, is the payload specialist. Mukai becomes the first Japanese woman to fly into space.

NASA Image and Video Library

1994-07-08

KENNEDY SPACE CENTER, FLA. - The second International Microgravity Laboratory-2 (IML-2) is off to an ontime start as the Space Shuttle Columbia lifts off from Launch Pad 39A at 12:43:00 p.m. EDT. On board are a crew of seven and more than 80 investigations developed by more than 200 scientists from 13 countries. The IML-2 complement includes materials science, bioprocessing, space and radiation biology, and human physiology experiments that will be carried out over the course of the 14-day flight. The commander of Space Shuttle Mission STS-65 is Robert D. Cabana. James D. Halsell Jr. is the pilot; the payload commander is Richard J. Hieb; the three mission specialists are Carl E. Walz, Leroy Chiao and Donald A. Thomas. Dr. Chiaki Mukai, representing NASDA, the National Space Development Agency of Japan, is the payload specialist. Mukai becomes the first Japanese woman to fly into space.

Strategies for the profiling, characterisation and detailed structural analysis of N-linked oligosaccharides.

PubMed

Tharmalingam, Tharmala; Adamczyk, Barbara; Doherty, Margaret A; Royle, Louise; Rudd, Pauline M

2013-02-01

Many post-translational modifications, including glycosylation, are pivotal for the structural integrity, location and functional activity of glycoproteins. Sub-populations of proteins that are relocated or functionally changed by such modifications can change resting proteins into active ones, mediating specific effector functions, as in the case of monoclonal antibodies. To ensure safe and efficacious drugs it is essential to employ appropriate robust, quantitative analytical strategies that can (i) perform detailed glycan structural analysis, (ii) characterise specific subsets of glycans to assess known critical features of therapeutic activities (iii) rapidly profile glycan pools for at-line monitoring or high level batch to batch screening. Here we focus on these aspects of glycan analysis, showing how state-of-the-art technologies are required at all stages during the production of recombinant glycotherapeutics. These data can provide insights into processing pathways and suggest markers for intervention at critical control points in bioprocessing and also critical decision points in disease and drug monitoring in patients. Importantly, these tools are now enabling the first glycome/genome studies in large populations, allowing the integration of glycomics into other 'omics platforms in a systems biology context.

A Graphene-enhanced imaging of microRNA with enzyme-free signal amplification of catalyzed hairpin assembly in living cells.

PubMed

Liu, Haiyun; Tian, Tian; Ji, Dandan; Ren, Na; Ge, Shenguang; Yan, Mei; Yu, Jinghua

2016-11-15

In situ imaging of miRNA in living cells could help us to monitor the miRNA expression in real time and obtain accurate information for studying miRNA related bioprocesses and disease. Given the low-level expression of miRNA, amplification strategies for intracellular miRNA are imperative. Here, we propose an amplification strategy with a non-destructive enzyme-free manner in living cells using catalyzed hairpin assembly (CHA) based on graphene oxide (GO) for cellular miRNA imaging. The enzyme-free CHA exhibits stringent recognition and excellent signal amplification of miRNA in the living cells. GO is a good candidate as a fluorescence quencher and cellular carrier. Taking the advantages of the CHA and GO, we can monitor the miRNA at low level in living cells with a simple, sensitive and real-time manner. Finally, imaging of miRNAs in the different expression cells is realized. The novel method could supply an effective tool to visualize intracellular low-level miRNAs and help us to further understand the role of miRNAs in cellular processes. Copyright © 2016 Elsevier B.V. All rights reserved.

Materials processing in space, 1980 science planning document. [crystal growth, containerless processing, solidification, bioprocessing, and ultrahigh vacuum processes

NASA Technical Reports Server (NTRS)

Naumann, R. J.

1980-01-01

The scientific aspects of the Materials Processing in Space program are described with emphasis on the major categories of interest: (1) crystal growth; (2) solidification of metals, alloys, and composites; (3) fluids and chemical processes; (4) containerless processing, glasses, and refractories; (5) ultrahigh vacuum processes; and (6) bioprocessing. An index is provided for each of these areas. The possible contributions that materials science experiments in space can make to the various disciplines are summarized, and the necessity for performing experiments in space is justified. What has been learned from previous experiments relating to space processing, current investigations, and remaining issues that require resolution are discussed. Recommendations for the future direction of the program are included.

Bioprocessing of lignite coals using reductive microorganisms. Final technical report, September 30, 1988--March 29, 1992

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

Crawford, D.L.

In order to convert lignite coals into liquid fuels, gases or chemical feedstock, the macromolecular structure of the coal must be broken down into low molecular weight fractions prior to further modification. Our research focused on this aspect of coal bioprocessing. We isolated, characterized and studied the lignite coal-depolymerizing organisms Streptomyces viridosporus T7A, Pseudomonas sp. DLC-62, unidentified bacterial strain DLC-BB2 and Gram-positive Bacillus megaterium strain DLC-21. In this research we showed that these bacteria are able to solubilize and depolymerize lignite coals using a combination of biological mechanisms including the excretion of coal solublizing basic chemical metabolites and extracellular coalmore » depolymerizing enzymes.« less

Intelligent modelling of bioprocesses: a comparison of structured and unstructured approaches.

PubMed

Hodgson, Benjamin J; Taylor, Christopher N; Ushio, Misti; Leigh, J R; Kalganova, Tatiana; Baganz, Frank

2004-12-01

This contribution moves in the direction of answering some general questions about the most effective and useful ways of modelling bioprocesses. We investigate the characteristics of models that are good at extrapolating. We trained three fully predictive models with different representational structures (differential equations, differential equations with inheritance of rates and a network of reactions) on Saccharopolyspora erythraea shake flask fermentation data using genetic programming. The models were then tested on unseen data outside the range of the training data and the resulting performances were compared. It was found that constrained models with mathematical forms analogous to internal mass balancing and stoichiometric relations were superior to flexible unconstrained models, even though no a priori knowledge of this fermentation was used.

Learning-based controller for biotechnology processing, and method of using

DOEpatents

Johnson, John A.; Stoner, Daphne L.; Larsen, Eric D.; Miller, Karen S.; Tolle, Charles R.

2004-09-14

The present invention relates to process control where some of the controllable parameters are difficult or impossible to characterize. The present invention relates to process control in biotechnology of such systems, but not limited to. Additionally, the present invention relates to process control in biotechnology minerals processing. In the inventive method, an application of the present invention manipulates a minerals bioprocess to find local exterma (maxima or minima) for selected output variables/process goals by using a learning-based controller for bioprocess oxidation of minerals during hydrometallurgical processing. The learning-based controller operates with or without human supervision and works to find processor optima without previously defined optima due to the non-characterized nature of the process being manipulated.

Direct Ethanol Production from Lignocellulosic Sugars and Sugarcane Bagasse by a Recombinant Trichoderma reesei Strain HJ48

PubMed Central

Huang, Jun; Chen, Dong; Wei, Yutuo; Wang, Qingyan; Li, Zhenchong; Chen, Ying; Huang, Ribo

2014-01-01

Trichoderma reesei can be considered as a candidate for consolidated bioprocessing (CBP) microorganism. However, its ethanol yield needs to be improved significantly. Here the ethanol production of T. reesei CICC 40360 was improved by genome shuffling while simultaneously enhancing the ethanol resistance. The initial mutant population was generated by nitrosoguanidine treatment of the spores, and an improved population producing more than fivefold ethanol than wild type was obtained by genome shuffling. The results show that the shuffled strain HJ48 can efficiently convert lignocellulosic sugars to ethanol under aerobic conditions. Furthermore, it was able to produce ethanol directly from sugarcane bagasse, demonstrating that the shuffled strain HJ48 is a suitable microorganism for consolidated bioprocessing. PMID:24995362

Direct ethanol production from lignocellulosic sugars and sugarcane bagasse by a recombinant Trichoderma reesei strain HJ48.

PubMed

Huang, Jun; Chen, Dong; Wei, Yutuo; Wang, Qingyan; Li, Zhenchong; Chen, Ying; Huang, Ribo

2014-01-01

Trichoderma reesei can be considered as a candidate for consolidated bioprocessing (CBP) microorganism. However, its ethanol yield needs to be improved significantly. Here the ethanol production of T. reesei CICC 40360 was improved by genome shuffling while simultaneously enhancing the ethanol resistance. The initial mutant population was generated by nitrosoguanidine treatment of the spores, and an improved population producing more than fivefold ethanol than wild type was obtained by genome shuffling. The results show that the shuffled strain HJ48 can efficiently convert lignocellulosic sugars to ethanol under aerobic conditions. Furthermore, it was able to produce ethanol directly from sugarcane bagasse, demonstrating that the shuffled strain HJ48 is a suitable microorganism for consolidated bioprocessing.

Application of an Online-Biomass Sensor in an Optical Multisensory Platform Prototype for Growth Monitoring of Biotechnical Relevant Microorganism and Cell Lines in Single-Use Shake Flasks

PubMed Central

Ude, Christian; Schmidt-Hager, Jörg; Findeis, Michael; John, Gernot Thomas; Scheper, Thomas; Beutel, Sascha

2014-01-01

In the context of this work we evaluated a multisensory, noninvasive prototype platform for shake flask cultivations by monitoring three basic parameters (pH, pO2 and biomass). The focus lies on the evaluation of the biomass sensor based on backward light scattering. The application spectrum was expanded to four new organisms in addition to E. coli K12 and S. cerevisiae [1]. It could be shown that the sensor is appropriate for a wide range of standard microorganisms, e.g., L. zeae, K. pastoris, A. niger and CHO-K1. The biomass sensor signal could successfully be correlated and calibrated with well-known measurement methods like OD600, cell dry weight (CDW) and cell concentration. Logarithmic and Bleasdale-Nelder derived functions were adequate for data fitting. Measurements at low cell concentrations proved to be critical in terms of a high signal to noise ratio, but the integration of a custom made light shade in the shake flask improved these measurements significantly. This sensor based measurement method has a high potential to initiate a new generation of online bioprocess monitoring. Metabolic studies will particularly benefit from the multisensory data acquisition. The sensor is already used in labscale experiments for shake flask cultivations. PMID:25232914

Coupling curvature-dependent and shear stress-stimulated neotissue growth in dynamic bioreactor cultures: a 3D computational model of a complete scaffold.

PubMed

Guyot, Y; Papantoniou, I; Luyten, F P; Geris, L

2016-02-01

The main challenge in tissue engineering consists in understanding and controlling the growth process of in vitro cultured neotissues toward obtaining functional tissues. Computational models can provide crucial information on appropriate bioreactor and scaffold design but also on the bioprocess environment and culture conditions. In this study, the development of a 3D model using the level set method to capture the growth of a microporous neotissue domain in a dynamic culture environment (perfusion bioreactor) was pursued. In our model, neotissue growth velocity was influenced by scaffold geometry as well as by flow- induced shear stresses. The neotissue was modeled as a homogenous porous medium with a given permeability, and the Brinkman equation was used to calculate the flow profile in both neotissue and void space. Neotissue growth was modeled until the scaffold void volume was filled, thus capturing already established experimental observations, in particular the differences between scaffold filling under different flow regimes. This tool is envisaged as a scaffold shape and bioprocess optimization tool with predictive capacities. It will allow controlling fluid flow during long-term culture, whereby neotissue growth alters flow patterns, in order to provide shear stress profiles and magnitudes across the whole scaffold volume influencing, in turn, the neotissue growth.

Endowing non-cellulolytic microorganisms with cellulolytic activity aiming for consolidated bioprocessing.

PubMed

Yamada, Ryosuke; Hasunuma, Tomohisa; Kondo, Akihiko

2013-11-01

With the exhaustion of fossil fuels and with the environmental issues they pose, utilization of abundant lignocellulosic biomass as a feedstock for biofuels and bio-based chemicals has recently become an attractive option. Lignocellulosic biomass is primarily composed of cellulose, hemicellulose, and lignin and has a very rigid and complex structure. It is accordingly much more expensive to process than starchy grains because of the need for extensive pretreatment and relatively large amounts of cellulases for efficient hydrolysis. Efficient and cost-effective methods for the production of biofuels and chemicals from lignocellulose are required. A consolidated bioprocess (CBP), which integrates all biological steps consisting of enzyme production, saccharification, and fermentation, is considered a promising strategy for reducing production costs. Establishing an efficient CBP using lignocellulosic biomass requires both lignocellulose degradation into glucose and efficient production of biofuels or chemicals from glucose. With this aim, many researchers are attempting to endow selected microorganisms with lignocellulose-assimilating ability. In this review, we focus on studies aimed at conferring lignocellulose-assimilating ability not only to yeast strains but also to bacterial strains by recombinant technology. Recent developments in improvement of enzyme productivity by microorganisms and in improvement of the specific activity of cellulase are emphasized. Copyright © 2013 Elsevier Inc. All rights reserved.

A method to estimate the biomass of Spirulina platensis cultivated on a solid medium.

PubMed

Pelizer, Lúcia Helena; Moraes, Iracema de Oliveira

2014-01-01

This paper presents a method to estimate the biomass of Spirulina cultivated on solid medium with sugarcane bagasse as a support, in view of the difficulty in determining biomass concentrations in bioprocesses, particularly those conducted in semi-solid or solid media. The genus Spirulina of the family Oscillatoriaceae comprises the group of multicellular filamentous cyanobacteria (blue-green microalgae). Spirulina is used as fish feed in aquaculture, as a food supplement, a source of vitamins, pigments, antioxidants and fatty acids. Therefore, its growth parameters are extremely important in studies of the development and optimization of bioprocesses. For studies of biomass growth, Spirulina platensis was cultured on solid medium using sugarcane bagasse as a support. The biomass thus produced was estimated by determining the protein content of the material grown during the process, based on the ratio of dry weight to protein content obtained in the surface growth experiments. The protein content of the biomass grown in Erlenmeyer flasks on surface medium was examined daily to check the influence of culture time on the protein content of the biomass. The biomass showed an average protein content of 42.2%. This methodology enabled the concentration of biomass adhering to the sugarcane bagasse to be estimated from the indirect measurement of the protein content associated with cell growth.

Predictive control of hollow-fiber bioreactors for the production of monoclonal antibodies.

PubMed

Dowd, J E; Weber, I; Rodriguez, B; Piret, J M; Kwok, K E

1999-05-20

The selection of medium feed rates for perfusion bioreactors represents a challenge for process optimization, particularly in bioreactors that are sampled infrequently. When the present and immediate future of a bioprocess can be adequately described, predictive control can minimize deviations from set points in a manner that can maximize process consistency. Predictive control of perfusion hollow-fiber bioreactors was investigated in a series of hybridoma cell cultures that compared operator control to computer estimation of feed rates. Adaptive software routines were developed to estimate the current and predict the future glucose uptake and lactate production of the bioprocess at each sampling interval. The current and future glucose uptake rates were used to select the perfusion feed rate in a designed response to deviations from the set point values. The routines presented a graphical user interface through which the operator was able to view the up-to-date culture performance and assess the model description of the immediate future culture performance. In addition, fewer samples were taken in the computer-estimated cultures, reducing labor and analytical expense. The use of these predictive controller routines and the graphical user interface decreased the glucose and lactate concentration variances up to sevenfold, and antibody yields increased by 10% to 43%. Copyright 1999 John Wiley & Sons, Inc.

At-line process analytical technology (PAT) for more efficient scale up of biopharmaceutical microfiltration unit operations.

PubMed

Watson, Douglas S; Kerchner, Kristi R; Gant, Sean S; Pedersen, Joseph W; Hamburger, James B; Ortigosa, Allison D; Potgieter, Thomas I

2016-01-01

Tangential flow microfiltration (MF) is a cost-effective and robust bioprocess separation technique, but successful full scale implementation is hindered by the empirical, trial-and-error nature of scale-up. We present an integrated approach leveraging at-line process analytical technology (PAT) and mass balance based modeling to de-risk MF scale-up. Chromatography-based PAT was employed to improve the consistency of an MF step that had been a bottleneck in the process used to manufacture a therapeutic protein. A 10-min reverse phase ultra high performance liquid chromatography (RP-UPLC) assay was developed to provide at-line monitoring of protein concentration. The method was successfully validated and method performance was comparable to previously validated methods. The PAT tool revealed areas of divergence from a mass balance-based model, highlighting specific opportunities for process improvement. Adjustment of appropriate process controls led to improved operability and significantly increased yield, providing a successful example of PAT deployment in the downstream purification of a therapeutic protein. The general approach presented here should be broadly applicable to reduce risk during scale-up of filtration processes and should be suitable for feed-forward and feed-back process control. © 2015 American Institute of Chemical Engineers.

New challenges and opportunities for industrial biotechnology.

PubMed

Chen, Guo-Qiang

2012-08-20

Industrial biotechnology has not developed as fast as expected due to some challenges including the emergences of alternative energy sources, especially shale gas, natural gas hydrate (or gas hydrate) and sand oil et al. The weaknesses of microbial or enzymatic processes compared with the chemical processing also make industrial biotech products less competitive with the chemical ones. However, many opportunities are still there if industrial biotech processes can be as similar as the chemical ones. Taking advantages of the molecular biology and synthetic biology methods as well as changing process patterns, we can develop bioprocesses as competitive as chemical ones, these including the minimized cells, open and continuous fermentation processes et al.

Development of a commercial scale process for production of 1,4-butanediol from sugar.

PubMed

Burgard, Anthony; Burk, Mark J; Osterhout, Robin; Van Dien, Stephen; Yim, Harry

2016-12-01

A sustainable bioprocess for the production of 1,4-butanediol (BDO) from carbohydrate feedstocks was developed. BDO is a chemical intermediate that goes into a variety of products including automotive parts, electronics, and apparel, and is currently manufactured commercially through energy-intensive petrochemical processes using fossil raw materials. This review highlights the development of an Escherichia coli strain and an overall process that successfully performed at commercial scale for direct production of bio-BDO from dextrose. Achieving such high level performance required an integrated technology platform enabling detailed engineering of enzyme, pathway, metabolic network, and organism, as well as development of effective fermentation and downstream recovery processes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Developments in biotechnological research in Austria

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

Kubicek, C.P.

1996-12-01

Austria is a small European country with a small number of universities and biotechnological industries, but with great efforts in the implementation of environmental consciousness and corresponding legal standards. This review attempts to describe the biotechnological landscape of Austria, thereby focusing on the highlights in research by industry, universities, and research laboratories, as published during 1990 to early 1995. These will include microbial metabolite (organic acids, antibiotics) and biopolymer (polyhydroxibutyrate, S-layers) production; enzyme (cellulases, hemicellulases, ligninases) technology and biocatalysis; environmental biotechnology; plant breeding and plant protection; mammalian cell products; fermenter design; and bioprocess engineering. 234 refs.

Preliminary benefit analysis of biological space processing

NASA Technical Reports Server (NTRS)

Perrine, J.

1976-01-01

The value of weightlessness in bioprocessing is assessed. The ecomonic benefits are assessed for space processing urokinase and human lymphocytes for treatment of end stage renal disease and thromboembolisms.

Evaluation of conditions necessary for successful bioprocessing of gray water in a microgravity environment

NASA Astrophysics Data System (ADS)

Urban, James E.; Supra, Laura; MacKnight, Allen

2000-01-01

A unique combination of researchers are investigating biological and engineering aspects of a biological wastewater treatment system which could effectively function to treat gray water in a microgravity environment such as that on the International Space Station and human-occupied interplanetary spacecraft. As part of the effort, 23 bacterial strains have been isolated from a bioprocessor operating at unit gravity and various strain combinations have been tested in microgravity for survivability and reduction of total organic carbon in ersatz gray water. All tested strains survive equally well in microgravity and unit gravity and each is capable of reducing TOC in microgravity. While the results reported are encouraging, they also reveal that current testing procedures and equipment are inadequate for fully evaluating bioprocessing in microgravity. .

Elucidating the Structural Changes to Populus Lignin during Consolidated Bioprocessing with Clostridium thermocellum

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

Akinosho, Hannah O.; Yoo, Chang Geun; Dumitrache, Alexandru

During consolidated bioprocessing (CBP), Clostridium thermocellum hydrolyzes several plant cell wall components. Cellulose hydrolysis, specifically, liberates sugars for fermentation, which generates ethanol, acetate, hydrogen, and other products. While several studies indicate that C. thermocellum hydrolyzes carbohydrates in biomass, the structural changes to lignin during CBP remain unclear. In this paper, the whole plant cell walls of untreated and C. thermocellum-treated Populus trichocarpa were characterized using NMR and FTIR. The results suggest that C. thermocellum reduces the β-O-4 linkage content and increases the lignin S/G ratio. Finally, this investigation indicates that C. thermocellum not only modifies lignin in order to accessmore » cellulose but also leaves behind a suitable lignin substrate for value-added applications in the cellulosic ethanol production scheme.« less

Characterization of Inulin Hydrolyzing Enzyme(s) in Oleaginous Yeast Trichosporon cutaneum in Consolidated Bioprocessing of Microbial Lipid Fermentation.

PubMed

Wang, Juan; Zhang, Huizhan; Bao, Jie

2015-11-01

Oleaginous yeast Trichosporon cutaneum CGMCC 2.1374 was found to utilize inulin directly for microbial lipid fermentation without a hydrolysis step. The potential inulinase-like enzyme(s) in T. cutaneum CGMCC 2.1374 were characterized and compared with other inulinase enzymes produced by varied yeast strains. The consolidated bioprocessing (CBP) for lipid accumulated using inulin was optimized with 4.79 g/L of lipid produced from 50 g/L inulin with the lipid content of 33.6% in dry cells. The molecular weight of the enzyme was measured which was close to invertase in Saccharomyces cerevisiae. The study provided information for inulin hydrolyzing enzyme(s) in oleaginous yeasts, as well as a preliminary CBP process for lipid production from inulin feedstock.

Cellulosic ethanol: status and innovation

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

Lynd, Lee R.; Liang, Xiaoyu; Biddy, Mary J.

Although the purchase price of cellulosic feedstocks is competitive with petroleum on an energy basis, the cost of lignocellulose conversion to ethanol using today's technology is high. Cost reductions can be pursued via either in-paradigm or new-paradigm innovation. Here, as an example of new-paradigm innovation, consolidated bioprocessing using thermophilic bacteria combined with milling during fermentation (cotreatment) is analyzed. Acknowledging the nascent state of this approach, our analysis indicates potential for radically improved cost competitiveness and feasibility at smaller scale compared to current technology, arising from (a) R&D-driven advances (consolidated bioprocessing with cotreatment in lieu of thermochemical pretreatment and added fungalmore » cellulase), and (b) configurational changes (fuel pellet coproduction instead of electricity, gas boiler(s) in lieu of a solid fuel boiler).« less

Cellulosic ethanol: status and innovation

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

Lynd, Lee R.; Liang, Xiaoyu; Biddy, Mary J.

Although the purchase price of cellulosic feedstocks is competitive with petroleum on an energy basis, the cost of lignocellulose conversion to ethanol using today’s technology is high. Cost reductions can be pursued via either in-paradigm or new-paradigm innovation. As an example of new-paradigm innovation, consolidated bioprocessing using thermophilic bacteria combined with milling during fermentation (cotreatment) is analyzed. Acknowledging the nascent state of this approach, our analysis indicates potential for radically improved cost competitiveness and feasibility at smaller scale compared to current technology, arising from (a) R&D-driven advances (consolidated bioprocessing with cotreatment in lieu of thermochemical pretreatment and added fungal cellulase),more » and (b) configurational changes (fuel pellet coproduction instead of electricity, gas boiler(s) in lieu of a solid fuel boiler).« less

Cellulosic ethanol: status and innovation

DOE PAGES

Lynd, Lee R.; Liang, Xiaoyu; Biddy, Mary J.; ...

2017-05-18

Although the purchase price of cellulosic feedstocks is competitive with petroleum on an energy basis, the cost of lignocellulose conversion to ethanol using today's technology is high. Cost reductions can be pursued via either in-paradigm or new-paradigm innovation. Here, as an example of new-paradigm innovation, consolidated bioprocessing using thermophilic bacteria combined with milling during fermentation (cotreatment) is analyzed. Acknowledging the nascent state of this approach, our analysis indicates potential for radically improved cost competitiveness and feasibility at smaller scale compared to current technology, arising from (a) R&D-driven advances (consolidated bioprocessing with cotreatment in lieu of thermochemical pretreatment and added fungalmore » cellulase), and (b) configurational changes (fuel pellet coproduction instead of electricity, gas boiler(s) in lieu of a solid fuel boiler).« less

Survey of selected topics relevant to bioprocess engineering. Technical note (Final)

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

Hubbard, J.B.; Clark, E.J.; Levelt Sengers, J.M.H.

1990-05-01

The following is a collection of reports on topics considered important and generic in biotechnology and bioprocess engineering: (1) Isoelectric points of proteins; (2) Solubility and mass transfer of oxygen in bioreactors; (3) Solubility and mass transfer of carbon dioxide in bioreactors. The reports arose from a survey of the past and current biotechnology literature with special effort given to a critique of data measurement quality. The format is as follows. The technological importance of a topic is briefly discussed, followed by a critical review of relevant physical properties, data presentation, and measurement techniques. A conclusions and recommendations section summarizesmore » the findings and contains specific recommendations for future research projects. The last section consists of an annotated bibliography and references pertaining to the survey.« less

Elucidating the Structural Changes to Populus Lignin during Consolidated Bioprocessing with Clostridium thermocellum

DOE PAGES

Akinosho, Hannah O.; Yoo, Chang Geun; Dumitrache, Alexandru; ...

2017-07-20

During consolidated bioprocessing (CBP), Clostridium thermocellum hydrolyzes several plant cell wall components. Cellulose hydrolysis, specifically, liberates sugars for fermentation, which generates ethanol, acetate, hydrogen, and other products. While several studies indicate that C. thermocellum hydrolyzes carbohydrates in biomass, the structural changes to lignin during CBP remain unclear. In this paper, the whole plant cell walls of untreated and C. thermocellum-treated Populus trichocarpa were characterized using NMR and FTIR. The results suggest that C. thermocellum reduces the β-O-4 linkage content and increases the lignin S/G ratio. Finally, this investigation indicates that C. thermocellum not only modifies lignin in order to accessmore » cellulose but also leaves behind a suitable lignin substrate for value-added applications in the cellulosic ethanol production scheme.« less

Production of erythrocytes from directly isolated or Delta1 Notch ligand expanded CD34+ hematopoietic progenitor cells: process characterization, monitoring and implications for manufacture.

PubMed

Glen, Katie E; Workman, Victoria L; Ahmed, Forhad; Ratcliffe, Elizabeth; Stacey, Adrian J; Thomas, Robert J

2013-09-01

Economic ex vivo manufacture of erythrocytes at 10(12) cell doses requires an efficiently controlled bio-process capable of extensive proliferation and high terminal density. High-resolution characterization of the process would identify production strategies for increased efficiency, monitoring and control. CD34(+) cord blood cells or equivalent cells that had been pre-expanded for 7 days with Delta1 Notch ligand were placed in erythroid expansion and differentiation conditions in a micro-scale ambr suspension bioreactor. Multiple culture parameters were varied, and phenotype markers and metabolites measured to identify conserved trends and robust monitoring markers. The cells exhibited a bi-modal erythroid differentiation pattern with an erythroid marker peak after 2 weeks and 3 weeks of culture; differentiation was comparatively weighted toward the second peak in Delta1 pre-expanded cells. Both differentiation events were strengthened by omission of stem cell factor and dexamethasone. The cumulative cell proliferation and death, or directly measured CD45 expression, enabled monitoring of proliferative rate of the cells. The metabolic activities of the cultures (glucose, glutamine and ammonia consumption or production) were highly variable but exhibited systematic change synchronized with the change in differentiation state. Erythroid differentiation chronology is partly determined by the heterogeneous CD34(+) progenitor compartment with implications for input control; Delta1 ligand-mediated progenitor culture can alter differentiation profile with control benefits for engineering production strategy. Differentiation correlated changes in cytokine response, markers and metabolic state will enable scientifically designed monitoring and timing of manufacturing process steps. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

miRNA engineering of CHO cells facilitates production of difficult-to-express proteins and increases success in cell line development.

PubMed

Fischer, Simon; Marquart, Kim F; Pieper, Lisa A; Fieder, Juergen; Gamer, Martin; Gorr, Ingo; Schulz, Patrick; Bradl, Harald

2017-07-01

In recent years, coherent with growing biologics portfolios also the number of complex and thus difficult-to-express (DTE) therapeutic proteins has increased considerably. DTE proteins challenge bioprocess development and can include various therapeutic protein formats such as monoclonal antibodies (mAbs), multi-specific affinity scaffolds (e.g., bispecific antibodies), cytokines, or fusion proteins. Hence, the availability of robust and versatile Chinese hamster ovary (CHO) host cell factories is fundamental for high-yielding bioprocesses. MicroRNAs (miRNAs) have emerged as potent cell engineering tools to improve process performance of CHO manufacturing cell lines. However, there has not been any report demonstrating the impact of beneficial miRNAs on industrial cell line development (CLD) yet. To address this question, we established novel CHO host cells constitutively expressing a pro-productive miRNA: miR-557. Novel host cells were tested in two independent CLD campaigns using two different mAb candidates including a normal as well as a DTE antibody. Presence of miR-557 significantly enhanced each process step during CLD in a product independent manner. Stable expression of miR-557 increased the probability to identify high-producing cell clones. Furthermore, production cell lines derived from miR-557 expressing host cells exhibited significantly increased final product yields in fed-batch cultivation processes without compromising product quality. Strikingly, cells co-expressing miR-557 and a DTE antibody achieved a twofold increase in product titer compared to clones co-expressing a negative control miRNA. Thus, host cell engineering using miRNAs represents a promising tool to overcome limitations in industrial CLD especially with regard to DTE proteins. Biotechnol. Bioeng. 2017;114: 1495-1510. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

A real-time multi-channel monitoring system for stem cell culture process.

PubMed

Xicai Yue; Drakakis, E M; Lim, M; Radomska, A; Hua Ye; Mantalaris, A; Panoskaltsis, N; Cass, A

2008-06-01

A novel, up to 128 channels, multi-parametric physiological measurement system suitable for monitoring hematopoietic stem cell culture processes and cell cultures in general is presented in this paper. The system aims to measure in real-time the most important physical and chemical culture parameters of hematopoietic stem cells, including physicochemical parameters, nutrients, and metabolites, in a long-term culture process. The overarching scope of this research effort is to control and optimize the whole bioprocess by means of the acquisition of real-time quantitative physiological information from the culture. The system is designed in a modular manner. Each hardware module can operate as an independent gain programmable, level shift adjustable, 16 channel data acquisition system specific to a sensor type. Up to eight such data acquisition modules can be combined and connected to the host PC to realize the whole system hardware. The control of data acquisition and the subsequent management of data is performed by the system's software which is coded in LabVIEW. Preliminary experimental results presented here show that the system not only has the ability to interface to various types of sensors allowing the monitoring of different types of culture parameters. Moreover, it can capture dynamic variations of culture parameters by means of real-time multi-channel measurements thus providing additional information on both temporal and spatial profiles of these parameters within a bioreactor. The system is by no means constrained in the hematopoietic stem cell culture field only. It is suitable for cell growth monitoring applications in general.

Bioprocessing in space

NASA Technical Reports Server (NTRS)

Westgate, P.; Kohlmann, K.; Hendrickson, R.; Ladisch, M. R.; Mitchell, C. A. (Principal Investigator)

1992-01-01

Two approaches for biomass processing in Controlled Ecological Life Support Systems are compared in a literature survey. The approaches are based on (1) total oxidation of plant matter and (2) the potential of bioregenerative recovery.

Anaerobic bioprocessing of organic wastes.

PubMed

Verstraete, W; de Beer, D; Pena, M; Lettinga, G; Lens, P

1996-05-01

Anaerobic digestion of dissolved, suspended and solid organics has rapidly evolved in the last decades but nevertheless still faces several scientific unknowns. In this review, some fundamentals of bacterial conversions and adhesion are addressed initially. It is argued in the light of ΔG-values of reactions, and in view of the minimum energy quantum per mol, that anaerobic syntrophs must have special survival strategies in order to support their existence: redistributing the available energy between the partners, reduced end-product fermentation reactions and special cell-to-cell physiological interactions. In terms of kinetics, it appears that both reaction rates and residual substrate thresholds are strongly related to minimum ΔG-values. These new fundamental insights open perspectives for efficient design and operation of anaerobic bioprocesses. Subsequently, an overview is given of the current anaerobic biotechnology. For treating wastewaters, a novel and high performance new system has been introduced during the last decade; the upflow anaerobic sludge blanket system (UASB). This reactor concept requires anaerobic consortia to grow in a dense and eco-physiologically well-organized way. The microbial principles of such granular sludge growth are presented. Using a thermodynamic approach, the formation of different types of aggregates is explained. The application of this bioprocess in worldwide wastewater treatment is indicated. Due to the long retention times of the active biomass, the UASB is also suitable for the development of bacterial consortia capable of degrading xenobiotics. Operating granular sludge reactors at high upflow velocities (5-6 m/h) in expanded granular sludge bed (EGSB) systems enlarges the application field to very low strength wastewaters (chemical oxygen demand < 1 g/l) and psychrophilic temperatures (10°C). For the treatment of organic suspensions, there is currently a tendency to evolve from the conventional mesophilic continuously stirred tank system to the thermophilic configuration, as the latter permits higher conversion rates and easier sanitation. Integration of ultrafiltration in anaerobic slurry digestion facilitates operation at higher volumetric loading rates and at shorter residence times. With respect to organic solids, the recent trend in society towards source separated collection of biowaste has opened a broad range of new application areas for solid state anaerobic fermentation.

Monitoring the bio-stimulation of hydrocarbon-contaminated soils by measurements of soil electrical properties, and CO2 content and its 13C/12C isotopic signature

NASA Astrophysics Data System (ADS)

Noel, C.; Gourry, J.; Ignatiadis, I.; Colombano, S.; Dictor, M.; Guimbaud, C.; Chartier, M.; Dumestre, A.; Dehez, S.; Naudet, V.

2013-12-01

Hydrocarbon contaminated soils represent an environmental issue as it impacts on ecosystems and aquifers. Where significant subsurface heterogeneity exists, conventional intrusive investigations and groundwater sampling can be insufficient to obtain a robust monitoring of hydrocarbon contaminants, as the information they provide is restricted to vertical profiles at discrete locations, with no information between sampling points. In order to obtain wider information in space volume on subsurface modifications, complementary methods can be used like geophysics. Among geophysical methods, geoelectrical techniques such as electrical resistivity (ER) and induced polarization (IP) seem the more promising, especially to study the effects of biodegradation processes. Laboratory and field geoelectrical experiments to characterize soils contaminated by oil products have shown that mature hydrocarbon-contaminated soils are characterized by enhanced electrical conductivity although hydrocarbons are electrically resistive. This high bulk conductivity is due to bacterial impacts on geological media, resulting in changes in the chemical and physical properties and thus, to the geophysical properties of the ground. Moreover, microbial activity induced CO2 production and isotopic deviation of carbon. Indeed, produced CO2 will reflect the pollutant isotopic signature. Thus, the ratio δ13C(CO2) will come closer to δ13C(hydrocarbon). BIOPHY, project supported by the French National Research Agency (ANR), proposes to use electrical methods and gas analyses to develop an operational and non-destructive method for monitoring in situ biodegradation of hydrocarbons in order to optimize soil treatment. Demonstration field is located in the South of Paris (France), where liquid fuels (gasoline and diesel) leaked from some tanks in 1997. In order to stimulate biodegradation, a trench has been dug to supply oxygen to the water table and thus stimulate aerobic metabolic bioprocesses. ER and IP surveys are performed regularly to monitor the stimulated biodegradation and progress of remediation until soil cleanup. Microbial activity is characterized by CO2 production increase and δ13C isotopic deviation, in the produced CO2 measured by infrared laser spectroscopy, and by an evolution of electrical conductivity and IP responses in correlation with microbiological and chemical analyses.

D-stat culture for studying the metabolic shifts from oxidative metabolism to lipid accumulation and citric acid production in Yarrowia lipolytica.

PubMed

Ochoa-Estopier, Abril; Guillouet, Stéphane E

2014-01-20

Lipid accumulation in oleaginous yeasts is triggered by nutrient imbalance in the culture medium between the carbon source in excess and the nitrogen source in limiting concentration. However Yarrowia lipolytica when cultivated on glucose as the sole carbon source, mainly produces citric acid upon nitrogen limitation over lipid accumulation (only 5-10% triacylglycerol). Therefore for developing bioprocess for the production of triacylglycerol from renewable carbon source as glucose it is of first importance to control this imbalance in order to avoid citric acid production during TAG accumulation. Using D-stat cultivation system, where the N/C was linearly decreased using a constant change rate we were able to identify the N/C ratio inducing TAG accumulation (0.085NmolCmol(-1)) and citric acid (0.021NmolCmol(-1)). We therefore demonstrated that it was possible to accumulate lipids without excretion citric acid as long as the N/C was within this indicated range. Moreover enzyme specific activities measurement during the D-stat indicated that ATP-citrate lyase, malic enzyme and acetyl-coA carboxylase were strongly induced at the onset of lipid accumulation and showed different patterns when citric acid was excreted. Our results give relevant information for future industrial bioprocess development concerning the production of lipids using renewable carbohydrate substrates as an alternative way to produce synthons for fuel or chemical industry. By controlling the N/C over the fermentation process on glucose Y. lipolytica can accumulate lipids without excreting citric acid. Copyright © 2013 Elsevier B.V. All rights reserved.

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.

Induction of fungal laccase production under solid state bioprocessing of new agroindustrial waste and its application on dye decolorization.

PubMed

Akpinar, Merve; Ozturk Urek, Raziye

2017-06-01

Lignocellulosic wastes are generally produced in huge amounts worldwide. Peach waste of these obtained from fruit juice industry was utilized as the substrate for laccase production by Pleurotus eryngii under solid state bioprocessing (SSB). Its chemical composition was determined and this bioprocess was carried out under stationary conditions at 28 °C. The effects of different compounds; copper, iron, Tween 80, ammonium nitrate and manganese, and their variable concentrations on laccase production were investigated in detail. The optimum production of laccase (43,761.33 ± 3845 U L -1 ) was achieved on the day of 20 by employing peach waste of 5.0 g and 70 µM Cu 2+ , 18 µM Fe 2+ , 0.025% (v/v) Tween 80, 4.0 g L -1 ammonium nitrate, 750 µM Mn 2+ as the inducers. The dye decolorization also researched to determine the degrading capability of laccase produced from peach culture under the above-mentioned conditions. Within this scope of the study, methyl orange, tartrazine, reactive red 2 and reactive black dyes were treated with this enzyme. The highest decolorization was performed with methyl orange as 43 ± 2.8% after 5 min of treatment when compared to other dyes. Up to now, this is the first report on the induction of laccase production by P. eryngii under SSB using peach waste as the substrate.

Bio-processing of solid wastes and secondary resources for metal extraction - A review

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

Lee, Jae-chun; Pandey, Banshi Dhar, E-mail: bd_pandey@yahoo.co.uk; CSIR - National Metallurgical Laboratory, Jamshedpur 831007

2012-01-15

Highlights: Black-Right-Pointing-Pointer Review focuses on bio-extraction of metals from solid wastes of industries and consumer goods. Black-Right-Pointing-Pointer Bio-processing of certain effluents/wastewaters with metals is also included in brief. Black-Right-Pointing-Pointer Quantity/composition of wastes are assessed, and microbes used and leaching conditions included. Black-Right-Pointing-Pointer Bio-recovery using bacteria, fungi and archaea is highlighted for resource recycling. Black-Right-Pointing-Pointer Process methodology/mechanism, R and D direction and scope of large scale use are briefly included. - Abstract: Metal containing wastes/byproducts of various industries, used consumer goods, and municipal waste are potential pollutants, if not treated properly. They may also be important secondary resources if processed inmore » eco-friendly manner for secured supply of contained metals/materials. Bio-extraction of metals from such resources with microbes such as bacteria, fungi and archaea is being increasingly explored to meet the twin objectives of resource recycling and pollution mitigation. This review focuses on the bio-processing of solid wastes/byproducts of metallurgical and manufacturing industries, chemical/petrochemical plants, electroplating and tanning units, besides sewage sludge and fly ash of municipal incinerators, electronic wastes (e-wastes/PCBs), used batteries, etc. An assessment has been made to quantify the wastes generated and its compositions, microbes used, metal leaching efficiency etc. Processing of certain effluents and wastewaters comprising of metals is also included in brief. Future directions of research are highlighted.« less

Microbial Enzymes: Tools for Biotechnological Processes

PubMed Central

Adrio, Jose L.; Demain, Arnold L.

2014-01-01

Microbial enzymes are of great importance in the development of industrial bioprocesses. Current applications are focused on many different markets including pulp and paper, leather, detergents and textiles, pharmaceuticals, chemical, food and beverages, biofuels, animal feed and personal care, among others. Today there is a need for new, improved or/and more versatile enzymes in order to develop more novel, sustainable and economically competitive production processes. Microbial diversity and modern molecular techniques, such as metagenomics and genomics, are being used to discover new microbial enzymes whose catalytic properties can be improved/modified by different strategies based on rational, semi-rational and random directed evolution. Most industrial enzymes are recombinant forms produced in bacteria and fungi. PMID:24970208

New challenges and opportunities for industrial biotechnology

PubMed Central

2012-01-01

Industrial biotechnology has not developed as fast as expected due to some challenges including the emergences of alternative energy sources, especially shale gas, natural gas hydrate (or gas hydrate) and sand oil et al. The weaknesses of microbial or enzymatic processes compared with the chemical processing also make industrial biotech products less competitive with the chemical ones. However, many opportunities are still there if industrial biotech processes can be as similar as the chemical ones. Taking advantages of the molecular biology and synthetic biology methods as well as changing process patterns, we can develop bioprocesses as competitive as chemical ones, these including the minimized cells, open and continuous fermentation processes et al. PMID:22905695

Perspectives on biotechnological applications of archaea

PubMed Central

Schiraldi, Chiara; Giuliano, Mariateresa; De Rosa, Mario

2002-01-01

Many archaea colonize extreme environments. They include hyperthermophiles, sulfur-metabolizing thermophiles, extreme halophiles and methanogens. Because extremophilic microorganisms have unusual properties, they are a potentially valuable resource in the development of novel biotechnological processes. Despite extensive research, however, there are few existing industrial applications of either archaeal biomass or archaeal enzymes. This review summarizes current knowledge about the biotechnological uses of archaea and archaeal enzymes with special attention to potential applications that are the subject of current experimental evaluation. Topics covered include cultivation methods, recent achievements in genomics, which are of key importance for the development of new biotechnological tools, and the application of wild-type biomasses, engineered microorganisms, enzymes and specific metabolites in particular bioprocesses of industrial interest. PMID:15803645

Perspectives on biotechnological applications of archaea.

PubMed

Schiraldi, Chiara; Giuliano, Mariateresa; De Rosa, Mario

2002-09-01

Many archaea colonize extreme environments. They include hyperthermophiles, sulfur-metabolizing thermophiles, extreme halophiles and methanogens. Because extremophilic microorganisms have unusual properties, they are a potentially valuable resource in the development of novel biotechnological processes. Despite extensive research, however, there are few existing industrial applications of either archaeal biomass or archaeal enzymes. This review summarizes current knowledge about the biotechnological uses of archaea and archaeal enzymes with special attention to potential applications that are the subject of current experimental evaluation. Topics covered include cultivation methods, recent achievements in genomics, which are of key importance for the development of new biotechnological tools, and the application of wild-type biomasses, engineered microorganisms, enzymes and specific metabolites in particular bioprocesses of industrial interest.

Comparison of Fiber Optic and Conduit Attenuated Total Reflection (ATR) Fourier Transform Infrared (FT-IR) Setup for In-Line Fermentation Monitoring.

PubMed

Koch, Cosima; Posch, Andreas E; Herwig, Christoph; Lendl, Bernhard

2016-12-01

The performance of a fiber optic and an optical conduit in-line attenuated total reflection mid-infrared (IR) probe during in situ monitoring of Penicillium chrysogenum fermentation were compared. The fiber optic probe was connected to a sealed, portable, Fourier transform infrared (FT-IR) process spectrometer via a plug-and-play interface. The optical conduit, on the other hand, was connected to a FT-IR process spectrometer via a knuckled probe with mirrors that had to be adjusted prior to each fermentation, which were purged with dry air. Penicillin V (PenV) and its precursor phenoxyacetic acid (POX) concentrations were determined by online high-performance liquid chromatography and the obtained concentrations were used as reference to build partial least squares regression models. Cross-validated root-mean-square errors of prediction were found to be 0.2 g L -1 (POX) and 0.19 g L -1 (PenV) for the fiber optic setup and 0.17 g L -1 (both POX and PenV) for the conduit setup. Higher noise-levels and spectrum-to-spectrum variations of the fiber optic setup lead to higher noise of estimated (i.e., unknown) POX and PenV concentrations than was found for the conduit setup. It seems that trade-off has to be made between ease of handling (fiber optic setup) and measurement accuracy (optical conduit setup) when choosing one of these systems for bioprocess monitoring. © The Author(s) 2016.

A Small-scale Model to Assess the Risk of Leachables from Single-use Bioprocess Containers through Protein Quality Characterization.

PubMed

Xiao, Nina J; Medley, Colin D; Shieh, Ian C; Downing, Gregory; Pizarro, Shelly; Liu, Jun; Patel, Ankit R

Leachables from single-use bioprocess containers (BPCs) are a source of process-related impurities that have the potential to alter product quality of biotherapeutics and affect patient health. Leachables often exist at very low concentrations, making it difficult to detect their presence and challenging to assess their impact on protein quality. A small-scale stress model based on assessing protein stability was developed to evaluate the potential risks associated with storing biotherapeutics in disposable bags caused by the presence of leachables. Small-scale BPCs were filled with protein solution at high surface area-to-volume ratios (≥3× the surface area-to-volume ratio of manufacturing-scale BPCs) and incubated at stress temperatures (e.g., 25 °C or 30 °C for up to 12 weeks) along with an appropriate storage vessel (e.g., glass vial or stainless steel) as a control for side-by-side comparison. Changes in protein size variants measured by size exclusion chromatography, capillary electrophoresis, and particle formation for two monoclonal antibodies using both the small-scale stress model and a control revealed a detrimental effect of gamma-irradiated BPCs on protein aggregation and significant BPC difference between earlier and later batches. It was found that preincubation of the empty BPCs prior to protein storage improved protein stability, suggesting the presence of volatile or heat-sensitive leachables (heat-labile or thermally degraded). In addition, increasing the polysorbate 20 concentration lowered, but did not completely mitigate, the leachable-protein interactions, indicating the presence of a hydrophobic leachable. Overall, this model can inform the risk of BPC leachables on biotherapeutics during routine manufacturing and assist in making decisions on the selection of a suitable BPC for the manufacturing process by assessing changes in product quality. Leachables from single-use systems often exist in small quantities and are difficult to detect with existing analytical methods. The presence of relevant detrimental leachables from single-use bioprocess containers (BPCs) can be indirectly detected by studying the stability of monoclonal antibodies via changes by size exclusion chromatography, capillary electrophoresis sodium dodecyl sulfate, and visible/sub-visible particles using a small-scale stress model containing high surface area-to-volume ratio at elevated temperature alongside with an appropriate control (e.g., glass vials or stainless steel containers). These changes in protein quality attributes allowed the evaluation of potential risks associated with adopting single-use bioprocess containers for storage as well as bag quality and bag differences between earlier and later batches. These leachables appear to be generated during the bag sterilization process by gamma irradiation. Improvements in protein stability after storage in "preheated" bags indicated that these leachables may be thermally unstable or volatile. The effect of surfactant levels, storage temperatures, surface area-to-volume ratios, filtration, and buffer exchange on leachables and protein stability were also assessed. © PDA, Inc. 2016.

Brine shrimp development in space: ground-based data to shuttle flight results

NASA Technical Reports Server (NTRS)

Spooner, B. S.; DeBell, L.; Hawkins, L.; Metcalf, J.; Guikema, J. A.; Rosowski, J.

1992-01-01

The brine shrimp, Artemia salina, has been used as a model system to assess microgravity effects on developing organisms. Following fertilization and early development, the egg can arrest in early gastrula as a dehydrated cyst stage that is stable to harsh environments over long time periods. When salt water is added, the cysts can reactivate, with embryonic development and egg hatching occurring in about 24 h. A series of larval molts or instars, over about a 2 week period, results in the adult crustacean. We have assessed these developmental events in a closed syringe system, a bioprocessing module, in ground-based studies, and have conducted preliminary in-orbit experiments aboard the Space Shuttle Atlantis during the flights of STS-37 and STS-43. Although the in-flight data are limited, spectacular degrees of development have been achieved.

Ethanol production from renewable resources.

PubMed

Gong, C S; Cao, N J; Du, J; Tsao, G T

1999-01-01

Vast amounts of renewable biomass are available for conversion to liquid fuel, ethanol. In order to convert biomass to ethanol, the efficient utilization of both cellulose-derived and hemicellulose-derived carbohydrates is essential. Six-carbon sugars are readily utilized for this purpose. Pentoses, on the other hand, are more difficult to convert. Several metabolic factors limit the efficient utilization of pentoses (xylose and arabinose). Recent developments in the improvement of microbial cultures provide the versatility of conversion of both hexoses and pentoses to ethanol more efficiently. In addition, novel bioprocess technologies offer a promising prospective for the efficient conversion of biomass and recovery of ethanol.

Quarterly progress report for the Chemical and Energy Research Section of the Chemical Technology Division, April--June 1997

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

Jubin, R.T.

The Chemical and Energy Research Section conducts basic and applied research and development in chemical engineering, applied chemistry, and bioprocessing, with an emphasis on energy-driven technologies and advanced chemical separations for nuclear and waste applications. The report describes the various tasks performed within six major areas of research: Hot Cell Operations, Process Chemistry and thermodynamics, Separations and Materials Synthesis, Solution Thermodynamics, biotechnology Research, and Molecular Studies. The name of a technical contact is included with each task described, and readers are encouraged to contact these individuals if they need additional information.

Analysis of Bioprocesses. Dynamic Modeling is a Must.

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

Ramkrishna, Doraiswami; Song, Hyun-Seob

2016-01-01

The goal of this paper is to report on the performance of a promising dynamic framework based on the cybernetic concepts which have evolved over three decades. We present case studies of successful dynamic simulations of wild-type strains as well as specific KO mutants on bacteria and yeast. An extensive metabolic engineering effort, including genome scale networks, is called for to secure the methodology and realize its full potential. Towards this end, the software AUMIC is under active further development to enable speedy applications. Its wide use will be enabled by a publication that is shortly due.

Biosynthesis of Gold Nanoparticles Using Pseudomonas Aeruginosa

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

Abd El-Aziz, M.; Badr, Y.; Mahmoud, M. A.

2007-02-14

Pseudomonas aeruginosa were used for extracellular biosynthesis of gold nanoparticles (Au NPs). Consequently, Au NPs were formed due to reduction of gold ion by bacterial cell supernatant of P. aeruginos ATCC 90271, P. aeruginos (2) and P. aeruginos (1). The UV-Vis. and fluorescence spectra of the bacterial as well as chemical prepared Au NPs were recorded. Transmission electron microscopy (TEM) micrograph showed the formation of well-dispersed gold nanoparticles in the range of 15-30 nm. The process of reduction being extracellular and may lead to the development of an easy bioprocess for synthesis of Au NPs.

Research and Application of Marine Microbial Enzymes: Status and Prospects

PubMed Central

Zhang, Chen; Kim, Se-Kwon

2010-01-01

Over billions of years, the ocean has been regarded as the origin of life on Earth. The ocean includes the largest range of habitats, hosting the most life-forms. Competition amongst microorganisms for space and nutrients in the marine environment is a powerful selective force, which has led to evolution. The evolution prompted the marine microorganisms to generate multifarious enzyme systems to adapt to the complicated marine environments. Therefore, marine microbial enzymes can offer novel biocatalysts with extraordinary properties. This review deals with the research and development work investigating the occurrence and bioprocessing of marine microbial enzymes. PMID:20631875

Metabolic Engineering of Clostridium cellulolyticum for Production of Isobutanol from Cellulose▿

PubMed Central

Higashide, Wendy; Li, Yongchao; Yang, Yunfeng; Liao, James C.

2011-01-01

Producing biofuels directly from cellulose, known as consolidated bioprocessing, is believed to reduce costs substantially compared to a process in which cellulose degradation and fermentation to fuel are accomplished in separate steps. Here we present a metabolic engineering example for the development of a Clostridium cellulolyticum strain for isobutanol synthesis directly from cellulose. This strategy exploits the host's natural cellulolytic activity and the amino acid biosynthesis pathway and diverts its 2-keto acid intermediates toward alcohol synthesis. Specifically, we have demonstrated the first production of isobutanol to approximately 660 mg/liter from crystalline cellulose by using this microorganism. PMID:21378054

Bioprocessing of Biodiesel Industry Effluent by Immobilized Bacteria to Produce Value-Added Products.

PubMed

Prakash, Jyotsana; Gupta, Rahul Kumar; Xx, Priyanka; Kalia, Vipin Chandra

2018-05-01

Biodiesel industrial effluent rich in crude glycerol (CG) was processed to produce value-added product. Under continuous culture system, Bacillus amyloliquefaciens strain CD16 immobilized within its biofilm, produced 3.2 L H 2 /day/L feed, over a period of 60 days at a hydraulic retention time of 2 days. The effective H 2 yield by B. amyloliquefaciens strain CD16 was 165 L/L CG. This H 2 yield was 1.18-fold higher than that observed with non-biofilm forming Bacillus thuringiensis strain EGU45. Bioprocessing of the effluent released after this stage, by recycling it up to 25% did not have any adverse effect on H 2 production by strain EGU45; however, a 25% reduction in yield was recorded with strain CD16. Biofilm forming H 2 producers thus proved effective as self-immobilizing system leading to enhanced process efficiency.

A novel approach of integrated bioprocessing of cane molasses for production of prebiotic and functional bioproducts.

PubMed

Sharma, Manisha; Patel, Satya Narayan; Lata, Kusum; Singh, Umesh; Krishania, Meena; Sangwan, Rajender S; Singh, Sudhir P

2016-11-01

In this work, the sugar industry by-product cane molasses was investigated as feedstock for acceptor reactions by dextransucrase from Leuconostoc mesenteroides MTCC 10508, leading to the biosynthesis of oligosaccharides. The starch industry corn fiber residue was used as a source for acceptor molecules, maltose, in the reaction. Production of approximately 124g oligosaccharides (DP3-DP6) per kg of fresh molasses was achieved. Further, cane molasses based medium was demonstrated as a sole carbon source for L. mesenteroides growth and dextransucrase production. d-Fructose released by dextransucrase activity as processing by-product was transformed into the functional monosaccharide with zero caloric value, d-psicose, by inducing its epimerization. Quantitative analysis approximated 37g d-psicose per kg of fresh molasses. Thus, the study established a novel approach of integrated bioprocessing of cane molasses into prebiotic and functional food additives. Copyright © 2016 Elsevier Ltd. All rights reserved.

Consolidated bioprocessing for production of polyhydroxyalkanotes from red algae Gelidium amansii.

PubMed

Sawant, Shailesh S; Salunke, Bipinchandra K; Kim, Beom Soo

2018-04-01

Noncompetitive carbon sources such as algae are unconventional and promising raw material for sustainable biofuel production. The capability of one marine bacterium, Saccharophagus degradans 2-40 to degrade red seaweed Gelidium amansii for production of polyhydroxyalkanoates (PHA) was evaluated in this study. S. degradans can readily attach to algae, degrade algal carbohydrates, and utilize that material as main carbon source. Minimal media containing 8g/L G. amansii were used for the growth of S. degradans. The PHA content obtained was 17-27% of dry cell weight by pure culture of S. degradans and co-culture of S. degradans and Bacillus cereus, a contaminant found with S. degradans cultures. The PHA type was found to be poly(3-hydroxybutyrate) by gas chromatography and Fourier transform-infrared spectroscopy. This work demonstrates PHA production through consolidated bioprocessing of insoluble, untreated red algae by bacterial pure culture and co-culture. Copyright © 2017 Elsevier B.V. All rights reserved.

Production of a generic microbial feedstock for lignocellulose biorefineries through sequential bioprocessing.

PubMed

Chang, Chen-Wei; Webb, Colin

2017-03-01

Lignocellulosic materials, mostly from agricultural and forestry residues, provide a potential renewable resource for sustainable biorefineries. Reducing sugars can be produced only after a pre-treatment stage, which normally involves chemicals but can be biological. In this case, two steps are usually necessary: solid-state cultivation of fungi for deconstruction, followed by enzymatic hydrolysis using cellulolytic enzymes. In this research, the utilisation of solid-state bioprocessing using the fungus Trichoderma longibrachiatum was implemented as a simultaneous microbial pretreatment and in-situ enzyme production method for fungal autolysis and further enzyme hydrolysis of fermented solids. Suspending the fermented solids in water at 50°C led to the highest hydrolysis yields of 226mg/g reducing sugar and 7.7mg/g free amino nitrogen (FAN). The resultant feedstock was shown to be suitable for the production of various products including ethanol. Copyright © 2016 Elsevier Ltd. All rights reserved.

Consolidated bioprocessing of microalgal biomass to carboxylates by a mixed culture of cow rumen bacteria using anaerobic sequencing batch reactor (ASBR).

PubMed

Zhao, Baisuo; Liu, Jie; Frear, Craig; Holtzapple, Mark; Chen, Shulin

2016-12-01

This study employed mixed-culture consolidated bioprocessing (CBP) to digest microalgal biomass in an anaerobic sequencing batch reactor (ASBR). The primary objectives are to evaluate the impact of hydraulic residence time (HRT) on the productivity of carboxylic acids and to characterize the bacterial community. HRT affects the production rate and patterns of carboxylic acids. For the 5-L laboratory-scale fermentation, a 12-day HRT was selected because it offered the highest productivity of carboxylic acids and it synthesized longer chains. The variability of the bacterial community increased with longer HRT (R 2 =0.85). In the 5-L laboratory-scale fermentor, the most common phyla were Firmicutes (58.3%), Bacteroidetes (27.4%), and Proteobacteria (11.9%). The dominant bacterial classes were Clostridia (29.8%), Bacteroidia (27.4%), Tissierella (26.2%), and Betaproteobacteria (8.9%). Copyright © 2016 Elsevier Ltd. All rights reserved.

Improved ethanol production at high temperature by consolidated bioprocessing using Saccharomyces cerevisiae strain engineered with artificial zinc finger protein.

PubMed

Khatun, M Mahfuza; Yu, Xinshui; Kondo, Akihiko; Bai, Fengwu; Zhao, Xinqing

2017-12-01

In this work, the consolidated bioprocessing (CBP) yeast Saccharomyces cerevisiae MNII/cocδBEC3 was transformed by an artificial zinc finger protein (AZFP) library to improve its thermal tolerance, and the strain MNII-AZFP with superior growth at 42°C was selected. Improved degradation of acid swollen cellulose by 45.9% led to an increase in ethanol production, when compared to the control strain. Moreover, the fermentation of Jerusalem artichoke stalk (JAS) by MNII-AZFP was shortened by 12h at 42°C with a concomitant improvement in ethanol production. Comparative transcriptomics analysis suggested that the AZFP in the mutant exerted beneficial effect by modulating the expression of multiple functional genes. These results provide a feasible strategy for efficient ethanol production from JAS and other cellulosic biomass through CBP based-fermentation at elevated temperatures. Copyright © 2017 Elsevier Ltd. All rights reserved.

An overview: Recycling of solid barley waste generated as a by-product in distillery and brewery.

PubMed

Nigam, Poonam Singh

2017-04-01

This overview has focused on the options available for the utilisation of residual-biomass generated in distillery and brewery for the production of added-value products. Bio-processing approaches have been reviewed and discussed for the economical bioconversion and utilisation of this waste for the production of bioproducts, such as lactic acid, enzymes, xylitol and animal feed. Though this overview provides several options for the bioprocessing of this residual material, a more suitable one could be chosen according to the processing-facilities available and the amount of residue available in local area. The feasibility of any chosen process should be evaluated on the basis of cost of material available, its local utilisation for animal feed, and the overall economical advantages that could be gained by changing its current traditional landfill use to produce higher added value products. Copyright © 2017 Elsevier Ltd. All rights reserved.

Dynamic metabolic modeling for a MAB bioprocess.

PubMed

Gao, Jianying; Gorenflo, Volker M; Scharer, Jeno M; Budman, Hector M

2007-01-01

Production of monoclonal antibodies (MAb) for diagnostic or therapeutic applications has become an important task in the pharmaceutical industry. The efficiency of high-density reactor systems can be potentially increased by model-based design and control strategies. Therefore, a reliable kinetic model for cell metabolism is required. A systematic procedure based on metabolic modeling is used to model nutrient uptake and key product formation in a MAb bioprocess during both the growth and post-growth phases. The approach combines the key advantages of stoichiometric and kinetic models into a complete metabolic network while integrating the regulation and control of cellular activity. This modeling procedure can be easily applied to any cell line during both the cell growth and post-growth phases. Quadratic programming (QP) has been identified as a suitable method to solve the underdetermined constrained problem related to model parameter identification. The approach is illustrated for the case of murine hybridoma cells cultivated in stirred spinners.

Predicting yields for autotrophic and cometabolic processes

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

Andrews, G.

1995-12-31

The goal of bioprocess engineering is to state how the optimum design and control strategy for a bioprocess follow from the metabolism of the particular microorganism. A necessary step toward this goal is to show how the parameters used in quantitative descriptions of a process (e.g., yield and maintenance coefficients) are related to those describing the metabolism [e.g., Y{sub ATP}, (P/O)]. The {open_quotes}yield equation{close_quotes} approach to this problem involves dividing metabolism into the separate pathways for catabolism, anabolism, respiration, and product formation and balancing the production and consumption of reducing equivalents and ATP. The general approach, demonstrated previously for heterotrophicmore » cell growth and products of fermentation, is illustrated by three new examples: the cell yield for chemoautotrophic iron-oxidizing bacteria, the cometabolic degradation of chloroform by methanotrophic bacteria, and the theoretical yield of succinic acid from glucose.« less

A novel ionic liquid-tolerant Fusarium oxysporum BN secreting ionic liquid-stable cellulase: consolidated bioprocessing of pretreated lignocellulose containing residual ionic liquid.

PubMed

Xu, Jiaxing; Wang, Xinfeng; Hu, Lei; Xia, Jun; Wu, Zhen; Xu, Ning; Dai, Benlin; Wu, Bin

2015-04-01

In this study, microbial communities from chemicals polluted microhabitats were cultured with the addition of imidazolium-based ionic liquid (IL) to enrich for IL-tolerant microbes. A strain of Fusarium oxysporum BN producing cellulase from these enrichments was capable of growing in 10% (w/v) 1-ethyl-3-methylimidazolium phosphinate, much higher than the normal IL concentrations in the lignocellulose regenerated from ILs. Cellulase secreted by the strain showed high resistance to ILs based on phosphate and sulfate radicals, evidencing of a high conformational stability in relevant media. Gratifyingly, F. oxysporum BN can directly convert IL-pretreated rice straw to bioethanol via consolidated bioprocessing (I-CBP). At optimum fermentation condition, a maximum ethanol yield of 0.125 g ethanol g(-1) of rice straw was finally obtained, corresponding to 64.2% of the theoretical yield. Copyright © 2015 Elsevier Ltd. All rights reserved.

Ex situ bioremediation of oil-contaminated soil.

PubMed

Lin, Ta-Chen; Pan, Po-Tsen; Cheng, Sheng-Shung

2010-04-15

An innovative bioprocess method, Systematic Environmental Molecular Bioremediation Technology (SEMBT) that combines bioaugmentation and biostimulation with a molecular monitoring microarray biochip, was developed as an integrated bioremediation technology to treat S- and T-series biopiles by using the landfarming operation and reseeding process to enhance the bioremediation efficiency. After 28 days of the bioremediation process, diesel oil (TPH(C10-C28)) and fuel oil (TPH(C10-C40)) were degraded up to approximately 70% and 63% respectively in the S-series biopiles. When the bioaugmentation and biostimulation were applied in the beginning of bioremediation, the microbial concentration increased from approximately 10(5) to 10(6) CFU/g dry soil along with the TPH biodegradation. Analysis of microbial diversity in the contaminated soils by microarray biochips revealed that Acinetobacter sp. and Pseudomonas aeruginosa were the predominant groups in indigenous consortia, while the augmented consortia were Gordonia alkanivorans and Rhodococcus erythropolis in both series of biopiles during bioremediation. Microbial respiration as influenced by the microbial activity reflected directly the active microbial population and indirectly the biodegradation of TPH. Field experimental results showed that the residual TPH concentration in the complex biopile was reduced to less than 500 mg TPH/kg dry soil. The above results demonstrated that the SEMBT technology is a feasible alternative to bioremediate the oil-contaminated soil. Crown Copyright 2009. Published by Elsevier B.V. All rights reserved.

An automated perfusion bioreactor for the streamlined production of engineered osteogenic grafts.

PubMed

Ding, Ming; Henriksen, Susan S; Wendt, David; Overgaard, Søren

2016-04-01

A computer-controlled perfusion bioreactor was developed for the streamlined production of engineered osteogenic grafts. This system automated the required bioprocesses, from the initial filling of the system through the phases of cell seeding and prolonged cell/tissue culture. Flow through chemo-optic micro-sensors allowed to non-invasively monitor the levels of oxygen and pH in the perfused culture medium throughout the culture period. To validate its performance, freshly isolated ovine bone marrow stromal cells were directly seeded on porous scaffold granules (hydroxyapatite/β-tricalcium-phosphate/poly-lactic acid), bypassing the phase of monolayer cell expansion in flasks. Either 10 or 20 days after culture, engineered cell-granule grafts were implanted in an ectopic mouse model to quantify new bone formation. After four weeks of implantation, histomorphometry showed more bone in bioreactor-generated grafts than cell-free granule controls, while bone formation did not show significant differences between 10 days and 20 days of incubation. The implanted granules without cells had no bone formation. This novel perfusion bioreactor has revealed the capability of activation larger viable bone graft material, even after shorter incubation time of graft material. This study has demonstrated the feasibility of engineering osteogenic grafts in an automated bioreactor system, laying the foundation for a safe, regulatory-compliant, and cost-effective manufacturing process. © 2015 Wiley Periodicals, Inc.

Exometabolomics and MSI: deconstructing how cells interact to transform their small molecule environment.

PubMed

Silva, Leslie P; Northen, Trent R

2015-08-01

Metabolism is at the heart of many biotechnologies from biofuels to medical diagnostics. Metabolomic methods that provide glimpses into cellular metabolism have rapidly developed into a critical component of the biotechnological development process. Most metabolomics methods have focused on what is happening inside the cell. Equally important are the biochemical transformations of the cell, and their effect on other cells and their environment; the exometabolome. Exometabolomics is therefore gaining popularity as a robust approach for obtaining rich phenotypic data, and being used in bioprocessing and biofuel development. Mass spectrometry imaging approaches, including several nanotechnologies, provide complimentary information by localizing metabolic processes within complex biological matrices. Together, the two technologies can provide new insights into the metabolism and interactions of cells. Published by Elsevier Ltd.

Economic analysis of uricase production under uncertainty: Contrast of chromatographic purification and aqueous two-phase extraction (with and without PEG recycle).

PubMed

Torres-Acosta, Mario A; Aguilar-Yáñez, José M; Rito-Palomares, Marco; Titchener-Hooker, Nigel J

2016-01-01

Uricase is the enzyme responsible for the breakdown of uric acid, the key molecule leading to gout in humans, into allantoin, but it is absent in humans. It has been produced as a PEGylated pharmaceutical where the purification is performed through three sequential chromatographic columns. More recently an aqueous two-phase system (ATPS) was reported that could recover Uricase with high yield and purity. Although the use of ATPS can decrease cost and time, it also generates a large amount of waste. The ability, therefore, to recycle key components of ATPS is of interest. Economic modelling is a powerful tool that allows the bioprocess engineer to compare possible outcomes and find areas where further research or optimization might be required without recourse to extensive experiments and time. This research provides an economic analysis using the commercial software BioSolve of the strategies for Uricase production: chromatographic and ATPS, and includes a third bioprocess that uses material recycling. The key parameters that affect the process the most were located via a sensitivity analysis and evaluated with a Monte Carlo analysis. Results show that ATPS is far less expensive than chromatography, but that there is an area where the cost of production of both bioprocesses overlap. Furthermore, recycling does not impact the cost of production. This study serves to provide a framework for the economic analysis of Uricase production using alternative techniques. © 2015 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers.

Economic analysis of uricase production under uncertainty: Contrast of chromatographic purification and aqueous two‐phase extraction (with and without PEG recycle)

PubMed Central

Torres‐Acosta, Mario A.; Aguilar‐Yáñez, José M.; Rito‐Palomares, Marco

2015-01-01

Uricase is the enzyme responsible for the breakdown of uric acid, the key molecule leading to gout in humans, into allantoin, but it is absent in humans. It has been produced as a PEGylated pharmaceutical where the purification is performed through three sequential chromatographic columns. More recently an aqueous two‐phase system (ATPS) was reported that could recover Uricase with high yield and purity. Although the use of ATPS can decrease cost and time, it also generates a large amount of waste. The ability, therefore, to recycle key components of ATPS is of interest. Economic modelling is a powerful tool that allows the bioprocess engineer to compare possible outcomes and find areas where further research or optimization might be required without recourse to extensive experiments and time. This research provides an economic analysis using the commercial software BioSolve of the strategies for Uricase production: chromatographic and ATPS, and includes a third bioprocess that uses material recycling. The key parameters that affect the process the most were located via a sensitivity analysis and evaluated with a Monte Carlo analysis. Results show that ATPS is far less expensive than chromatography, but that there is an area where the cost of production of both bioprocesses overlap. Furthermore, recycling does not impact the cost of production. This study serves to provide a framework for the economic analysis of Uricase production using alternative techniques. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:126–133, 2016 PMID:26561271

Allogeneic Cell Therapy Bioprocess Economics and Optimization: Single-Use Cell Expansion Technologies

PubMed Central

Simaria, Ana S; Hassan, Sally; Varadaraju, Hemanthram; Rowley, Jon; Warren, Kim; Vanek, Philip; Farid, Suzanne S

2014-01-01

For allogeneic cell therapies to reach their therapeutic potential, challenges related to achieving scalable and robust manufacturing processes will need to be addressed. A particular challenge is producing lot-sizes capable of meeting commercial demands of up to 109 cells/dose for large patient numbers due to the current limitations of expansion technologies. This article describes the application of a decisional tool to identify the most cost-effective expansion technologies for different scales of production as well as current gaps in the technology capabilities for allogeneic cell therapy manufacture. The tool integrates bioprocess economics with optimization to assess the economic competitiveness of planar and microcarrier-based cell expansion technologies. Visualization methods were used to identify the production scales where planar technologies will cease to be cost-effective and where microcarrier-based bioreactors become the only option. The tool outputs also predict that for the industry to be sustainable for high demand scenarios, significant increases will likely be needed in the performance capabilities of microcarrier-based systems. These data are presented using a technology S-curve as well as windows of operation to identify the combination of cell productivities and scale of single-use bioreactors required to meet future lot sizes. The modeling insights can be used to identify where future R&D investment should be focused to improve the performance of the most promising technologies so that they become a robust and scalable option that enables the cell therapy industry reach commercially relevant lot sizes. The tool outputs can facilitate decision-making very early on in development and be used to predict, and better manage, the risk of process changes needed as products proceed through the development pathway. Biotechnol. Bioeng. 2014;111: 69–83. © 2013 Wiley Periodicals, Inc. PMID:23893544

Linking genes to microbial growth kinetics: an integrated biochemical systems engineering approach.

PubMed

Koutinas, Michalis; Kiparissides, Alexandros; Silva-Rocha, Rafael; Lam, Ming-Chi; Martins Dos Santos, Vitor A P; de Lorenzo, Victor; Pistikopoulos, Efstratios N; Mantalaris, Athanasios

2011-07-01

The majority of models describing the kinetic properties of a microorganism for a given substrate are unstructured and empirical. They are formulated in this manner so that the complex mechanism of cell growth is simplified. Herein, a novel approach for modelling microbial growth kinetics is proposed, linking biomass growth and substrate consumption rates to the gene regulatory programmes that control these processes. A dynamic model of the TOL (pWW0) plasmid of Pseudomonas putida mt-2 has been developed, describing the molecular interactions that lead to the transcription of the upper and meta operons, known to produce the enzymes for the oxidative catabolism of m-xylene. The genetic circuit model was combined with a growth kinetic model decoupling biomass growth and substrate consumption rates, which are expressed as independent functions of the rate-limiting enzymes produced by the operons. Estimation of model parameters and validation of the model's predictive capability were successfully performed in batch cultures of mt-2 fed with different concentrations of m-xylene, as confirmed by relative mRNA concentration measurements of the promoters encoded in TOL. The growth formation and substrate utilisation patterns could not be accurately described by traditional Monod-type models for a wide range of conditions, demonstrating the critical importance of gene regulation for the development of advanced models closely predicting complex bioprocesses. In contrast, the proposed strategy, which utilises quantitative information pertaining to upstream molecular events that control the production of rate-limiting enzymes, predicts the catabolism of a substrate and biomass formation and could be of central importance for the design of optimal bioprocesses. Copyright © 2011 Elsevier Inc. All rights reserved.

The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli

PubMed Central

2011-01-01

Background Escherichia coli is a model prokaryote, an important pathogen, and a key organism for industrial biotechnology. E. coli W (ATCC 9637), one of four strains designated as safe for laboratory purposes, has not been sequenced. E. coli W is a fast-growing strain and is the only safe strain that can utilize sucrose as a carbon source. Lifecycle analysis has demonstrated that sucrose from sugarcane is a preferred carbon source for industrial bioprocesses. Results We have sequenced and annotated the genome of E. coli W. The chromosome is 4,900,968 bp and encodes 4,764 ORFs. Two plasmids, pRK1 (102,536 bp) and pRK2 (5,360 bp), are also present. W has unique features relative to other sequenced laboratory strains (K-12, B and Crooks): it has a larger genome and belongs to phylogroup B1 rather than A. W also grows on a much broader range of carbon sources than does K-12. A genome-scale reconstruction was developed and validated in order to interrogate metabolic properties. Conclusions The genome of W is more similar to commensal and pathogenic B1 strains than phylogroup A strains, and therefore has greater utility for comparative analyses with these strains. W should therefore be the strain of choice, or 'type strain' for group B1 comparative analyses. The genome annotation and tools created here are expected to allow further utilization and development of E. coli W as an industrial organism for sucrose-based bioprocesses. Refinements in our E. coli metabolic reconstruction allow it to more accurately define E. coli metabolism relative to previous models. PMID:21208457

Allogeneic cell therapy bioprocess economics and optimization: single-use cell expansion technologies.

PubMed

Simaria, Ana S; Hassan, Sally; Varadaraju, Hemanthram; Rowley, Jon; Warren, Kim; Vanek, Philip; Farid, Suzanne S

2014-01-01

For allogeneic cell therapies to reach their therapeutic potential, challenges related to achieving scalable and robust manufacturing processes will need to be addressed. A particular challenge is producing lot-sizes capable of meeting commercial demands of up to 10(9) cells/dose for large patient numbers due to the current limitations of expansion technologies. This article describes the application of a decisional tool to identify the most cost-effective expansion technologies for different scales of production as well as current gaps in the technology capabilities for allogeneic cell therapy manufacture. The tool integrates bioprocess economics with optimization to assess the economic competitiveness of planar and microcarrier-based cell expansion technologies. Visualization methods were used to identify the production scales where planar technologies will cease to be cost-effective and where microcarrier-based bioreactors become the only option. The tool outputs also predict that for the industry to be sustainable for high demand scenarios, significant increases will likely be needed in the performance capabilities of microcarrier-based systems. These data are presented using a technology S-curve as well as windows of operation to identify the combination of cell productivities and scale of single-use bioreactors required to meet future lot sizes. The modeling insights can be used to identify where future R&D investment should be focused to improve the performance of the most promising technologies so that they become a robust and scalable option that enables the cell therapy industry reach commercially relevant lot sizes. The tool outputs can facilitate decision-making very early on in development and be used to predict, and better manage, the risk of process changes needed as products proceed through the development pathway. © 2013 Wiley Periodicals, Inc.

NABIR Assessment Element, Expanded Rapid, Comprehensive, Lipid Biomarker Analysis for Subsurface, Community Composition and Nutritional/Physiological Status as Monitors of Remediation and Detoxification Effectiveness

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

David C. White

2005-09-14

NABIR funding at the University of Tennessee Center for Biomarker Analysis (CBA) has led to several key contributions to the investigation of bioremediation of metals and radionuclides. This lab has played an integral part in assessing microbial communities at the field scale at the ORNL FRC (Istok et al., 2004) and two UMTRA sites (Anderson et al., 2003, Chang et al., 2001). Our work over the period of the grant has resulted in 42-peer reviewed publications, 62 presentations (14 of which were international), and one patent pending. Currently CBA has 2 papers in press. The main objectives relating to themore » field portion of this program were to provide comprehensive biomarker analysis for NABIR collaborators to enhance the understanding of microbial geo-bioprocesses involved in the effective immobilization of metals (We have worked with and published or currently are publishing with 10 groups of NAIBR investigators). The laboratory portion of our research centered on methods development and has led to three major innovations that could result in a systematic way of evaluating sites for potential bioremediation. The first of these is the development of an in situ sampling device (Peacock et al., 2004, Anderson et al., 2003, Istok et al., 2004) for the collection and concentration of microbial biomass. The second is the development of expanded lipid analysis based on the significantly greater sensitivity and selectivity of the LC/MS/MS that allows the analysis of respiratory quinones, diglycerides, sterols, intact phospholipids, poly-hydroxyalkonates, and potentially archaeol, and caldarchaeols from archea. These new analyses are accomplished more rapidly and with increased sensitivities and resolution than in the past (Lytle et al., 2000a, 2000b, 2001a, Geyer et al., 2004). The third advance is the coupling of lipid analysis with 13C enrichment experiments (Lytle et al., 2001b, Geyer et al. 2005). With this technique it is now possible to follow the active portion of the in situ microbial community with a resolution heretofore not possible. These three advances in technology have been initially demonstrated at the NABIR Field Research Center (FRC) in Oak Ridge, TN and at the UMTRA Old Rifle site in Colorado. Microbial communities are of primary importance in the use of bioimmobilization strategies for metals and radionuclides from contaminated groundwater and sediments. These communities represent a potentially transformable agent that is able to affect virtually all biogeochemical pathways. Microorganisms can alter metal chemistry and mobility through reduction, accumulation, and immobilization and have been shown to be responsible for mineral formation and dissolution. Research is directed to provide collaborating NABIR investigators a rapid, comprehensive, and cost-effective suite of biomarker measurements to quantify microbial community structure, activity, and effectiveness, thereby providing defensible evidence that a desired bioprocess is occurring or may occur at a given site.« less

Carotenoids from microalgae: A review of recent developments.

PubMed

Gong, Mengyue; Bassi, Amarjeet

2016-12-01

Carotenoids have been receiving increasing attention due to their potential health benefits. Microalgae are recognized as a natural source of carotenoids and other beneficial byproducts. However, the production of micro-algal carotenoids is not yet sufficiently cost-effective to compete with traditional chemical synthetic methods and other technologies such as extraction from plant based sources. This review presents the recent biotechnological developments in microalgal carotenoid production. The current technologies involved in their bioprocessing including cultivation, harvesting, extraction, and purification are discussed with a specific focus on downstream processing. The recent advances in chemical and biochemical synthesis of carotenoids are also reviewed for a better understanding of suitable and economically feasible biotechnological strategies. Some possible future directions are also proposed. Copyright © 2016 Elsevier Inc. All rights reserved.

Bioreactor and process design for biohydrogen production.

PubMed

Show, Kuan-Yeow; Lee, Duu-Jong; Chang, Jo-Shu

2011-09-01

Biohydrogen is regarded as an attractive future clean energy carrier due to its high energy content and environmental-friendly conversion. It has the potential for renewable biofuel to replace current hydrogen production which rely heavily on fossil fuels. While biohydrogen production is still in the early stage of development, there have been a variety of laboratory- and pilot-scale systems developed with promising potential. This work presents a review of advances in bioreactor and bioprocess design for biohydrogen production. The state-of-the art of biohydrogen production is discussed emphasizing on production pathways, factors affecting biohydrogen production, as well as bioreactor configuration and operation. Challenges and prospects of biohydrogen production are also outlined. Copyright © 2011 Elsevier Ltd. All rights reserved.

Development of at-line assay to monitor charge variants of MAbs during production.

PubMed

St Amand, M M; Ogunnaike, B A; Robinson, A S

2014-01-01

One major challenge currently facing the biopharmaceutical industry is to understand how MAb microheterogeneity affects therapeutic efficacy, potency, immunogenicity, and clearance. MAb micro-heterogeneity can result from post-translational modifications such as sialylation, galactosylation, C-terminal lysine cleavage, glycine amidation, and tryptophan oxidation, each of which can generate MAb charge variants; such heterogeneity can affect pharmacokinetics (PK) considerably. Implementation of appropriate on-line quality control strategies may help to regulate bioprocesses, thus enabling more homogenous material with desired post-translational modifications and PK behavior. However, one major restriction to implementation of quality control strategies is the availability of techniques for obtaining on-line or at-line measurements of these attributes. In this work, we describe the development of an at-line assay to separate MAb charge variants in near real-time, which could ultimately be used to implement on-line quality control strategies for MAb production. The assay consists of a 2D-HPLC method with sequential in-line Protein A and WCX-10 HPLC column steps. To perform the 2D-HPLC assay at-line, the two columns steps were integrated into a single method using a novel system configuration that allowed parallel flow over column 1 or column 2 or sequential flow from column 1 to column 2. A bioreactor system was also developed such that media samples could be removed automatically from bioreactor vessels during production and delivered to the 2D-HPLC for analysis. With this at-line HPLC assay, we have demonstrated that MAb microheterogeneity occurs throughout the cell cycle whether the host cell line is grown under different or the same nominal culture conditions. © 2013 American Institute of Chemical Engineers.

Characterization of alanine to valine sequence variants in the Fc region of nivolumab biosimilar produced in Chinese hamster ovary cells.

PubMed

Li, Yantao; Fu, Tuo; Liu, Tao; Guo, Huaizu; Guo, Qingcheng; Xu, Jin; Zhang, Dapeng; Qian, Weizhu; Dai, Jianxin; Li, Bohua; Guo, Yajun; Hou, Sheng; Wang, Hao

2016-07-01

Nivolumab is a therapeutic fully human IgG4 antibody to programmed death 1 (PD-1). In this study, a nivolumab biosimilar, which was produced in our laboratory, was analyzed and characterized. Sequence variants that contain undesired amino acid sequences may cause concern during biosimilar bioprocess development. We found that low levels of sequence variants were detected in the heavy chain of the nivolumab biosimilar by ultra performance liquid chromatography (UPLC) and tandem mass spectrometry. It was further identified with UPLC-MS/MS by IdeS or trypsin digestion. The sequence variant was confirmed through addition of synthetic mutant peptide. Subsequently, the mixing base signal of normal and mutant sequence was detected through DNA sequencing. The relative levels of mutant A424V in the Fc region of the heavy chain have been detected and demonstrated to be 12.25% and 13.54%, via base peak intensity (BPI) and UV chromatography of the tryptic peptide mapping, respectively. A424V variant was also quantified by real-time PCR (RT-PCR) at the DNA and RNA level, which was 19.2% and 16.8%, respectively. The relative content of the mutant was consistent at the DNA, RNA and protein level, indicating that the A424V mutation may have little influence at transcriptional or translational levels. These results demonstrate that orthogonal state-of-the-art techniques such as LC- UV- MS and RT-PCR should be implemented to characterize recombinant proteins and cell lines for development of biosimilars. Our study suggests that it is important to establish an integrated and effective analytical method to monitor and characterize sequence variants during antibody drug development, especially for antibody biosimilar products.

Stirred tank bioreactor culture combined with serum-/xenogeneic-free culture medium enables an efficient expansion of umbilical cord-derived mesenchymal stem/stromal cells.

PubMed

Mizukami, Amanda; Fernandes-Platzgummer, Ana; Carmelo, Joana G; Swiech, Kamilla; Covas, Dimas T; Cabral, Joaquim M S; da Silva, Cláudia L

2016-08-01

Mesenchymal stem/stromal cells (MSC) are being widely explored as promising candidates for cell-based therapies. Among the different human MSC origins exploited, umbilical cord represents an attractive and readily available source of MSC that involves a non-invasive collection procedure. In order to achieve relevant cell numbers of human MSC for clinical applications, it is crucial to develop scalable culture systems that allow bioprocess control and monitoring, combined with the use of serum/xenogeneic (xeno)-free culture media. In the present study, we firstly established a spinner flask culture system combining gelatin-based Cultispher(®) S microcarriers and xeno-free culture medium for the expansion of umbilical cord matrix (UCM)-derived MSC. This system enabled the production of 2.4 (±1.1) x10(5) cells/mL (n = 4) after 5 days of culture, corresponding to a 5.3 (±1.6)-fold increase in cell number. The established protocol was then implemented in a stirred-tank bioreactor (800 mL working volume) (n = 3) yielding 115 million cells after 4 days. Upon expansion under stirred conditions, cells retained their differentiation ability and immunomodulatory potential. The development of a scalable microcarrier-based stirred culture system, using xeno-free culture medium that suits the intrinsic features of UCM-derived MSC represents an important step towards a GMP compliant large-scale production platform for these promising cell therapy candidates. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bioprocesses. [in the marine environment

NASA Technical Reports Server (NTRS)

Ditoro, D. M.; Iverson, R. L.; Mccarthy, J. J.

1980-01-01

The application of remote sensing techniques to the study of eutrophication in natural waters and the location and characterization of fronts is considered. The specific problem to be studied is examined along with the feasibility and capabability of remote sensing techniques for each application.

Methanol-Independent Protein Expression by AOX1 Promoter with trans-Acting Elements Engineering and Glucose-Glycerol-Shift Induction in Pichia pastoris

PubMed Central

Wang, Jinjia; Wang, Xiaolong; Shi, Lei; Qi, Fei; Zhang, Ping; Zhang, Yuanxing; Zhou, Xiangshan; Song, Zhiwei; Cai, Menghao

2017-01-01

The alcohol oxidase 1 promoter (PAOX1) of Pichia pastoris is commonly used for high level expression of recombinant proteins. While the safety risk of methanol and tough process control for methanol induction usually cause problems especially in large-scale fermentation. By testing the functions of trans-acting elements of PAOX1 and combinatorially engineering of them, we successfully constructed a methanol-free PAOX1 start-up strain, in which, three transcription repressors were identified and deleted and, one transcription activator were overexpressed. The strain expressed 77% GFP levels in glycerol compared to the wide-type in methanol. Then, insulin precursor (IP) was expressed, taking which as a model, we developed a novel glucose-glycerol-shift induced PAOX1 start-up for this methanol-free strain. A batch phase with glucose of 40 g/L followed by controlling residual glucose not lower than 20 g/L was compatible for supporting cell growth and suppressing PAOX1. Then, glycerol induction was started after glucose used up. Accordingly, an optimal bioprocess was further determined, generating a high IP production of 2.46 g/L in a 5-L bioreactor with dramatical decrease of oxygen consumption and heat evolution comparing with the wild-type in methanol. This mutant and bioprocess represent a safe and efficient alternative to the traditional glycerol-repressed/methanol-induced PAOX1 system. PMID:28150747

Bioprocessing of Stichococcus bacillaris strain siva2011.

PubMed

Sivakumar, Ganapathy; Jeong, Kwangkook; Lay, Jackson O

2014-01-01

Globally, the development of a cost-effective long-term renewable energy infrastructure is one of the most challenging problems faced by society today. Microalgae are rich in potential biofuel substrates such as lipids, including triacylglycerols (TAGs). Some of these algae also biosynthesize small molecule hydrocarbons. These hydrocarbons can often be used as liquid fuels, often with more versatility and by a more direct approach than some TAGs. However, the appropriate TAGs, accumulated from microalgae biomass, can be used as substrates for different kinds of renewable liquid fuels such as biodiesel and jet fuel. This article describes the isolation and identification of a lipid-rich, hydrocarbon-producing alga, Stichococcus bacillaris strain siva2011, together with its bioprocessing, hydrocarbon and fatty acid methyl ester (FAME) profiles. The S. bacillaris strain siva2011 was scaled-up in an 8 L bioreactor with 0.2% CO2. The C16:0, C16:3, C18:1, C18:2 and C18:3 were 112.2, 9.4, 51.3, 74.1 and 69.2 mg/g dry weight (DW), respectively. This new strain produced a significant amount of biomass of 3.79 g/L DW on day 6 in the 8 L bioreactor and also produced three hydrocarbons. A new oil-rich microalga S. bacillaris strain siva2011 was discovered and its biomass has been scaled-up in a newly designed balloon-type bioreactor. The TAGs and hydrocarbons produced by this organism could be used as substrates for jet fuel or biodiesel.

Characterization of simultaneous heat and mass transfer phenomena for water vapour condensation on a solid surface in an abiotic environment--application to bioprocesses.

PubMed

Tiwari, Akhilesh; Kondjoyan, Alain; Fontaine, Jean-Pierre

2012-07-01

The phenomenon of heat and mass transfer by condensation of water vapour from humid air involves several key concepts in aerobic bioreactors. The high performance of bioreactors results from optimised interactions between biological processes and multiphase heat and mass transfer. Indeed in various processes such as submerged fermenters and solid-state fermenters, gas/liquid transfer need to be well controlled, as it is involved at the microorganism interface and for the control of the global process. For the theoretical prediction of such phenomena, mathematical models require heat and mass transfer coefficients. To date, very few data have been validated concerning mass transfer coefficients from humid air inflows relevant to those bioprocesses. Our study focussed on the condensation process of water vapour and developed an experimental set-up and protocol to study the velocity profiles and the mass flux on a small size horizontal flat plate in controlled environmental conditions. A closed circuit wind tunnel facility was used to control the temperature, hygrometry and hydrodynamics of the flow. The temperature of the active surface was controlled and kept isothermal below the dew point to induce condensation, by the use of thermoelectricity. The experiments were performed at ambient temperature for a relative humidity between 35-65% and for a velocity of 1.0 ms⁻¹. The obtained data are analysed and compared to available theoretical calculations on condensation mass flux.

Optimization of human granulocyte macrophage-colony stimulating factor (hGM-CSF) expression using asparaginase and xylanase gene's signal sequences in Escherichia coli.

PubMed

Khasa, Yogender Pal; Khushoo, Amardeep; Tapryal, Suman; Mukherjee, K J

2011-09-01

The toxicity of the recombinant protein towards the expression host remains a significant deterrent for bioprocess development. In this study, the expression of human granulocyte macrophage-colony stimulating factor (hGM-CSF), which is known to be toxic to its host, was enhanced many folds using a combination of genetic and bioprocess strategies in Escherichia coli. The N terminus attachment of endoxylanase and asparaginase signal sequences from Bacillus subtilis and E. coli, respectively, in combination with and without His-tag, considerably improved expression levels. Induction and media optimization studies in shake flask cultures resulted in a maximal hGM-CSF concentration of 365 mg/L in the form of inclusion bodies (IBs) with a specific product yield (Y (P/X)) of 120 mg/g dry cell weight in case of the asparaginase signal. Culturing the cells in nutrient rich Terrific broth maintained the specific product yields (Y (P/X)) while a 6.6-fold higher volumetric concentration of both product and biomass was obtained. The purification and refolding steps were optimized resulting in a 95% pure protein with a fairly high refolding yield of 45%. The biological activity of the refolded protein was confirmed by a cell proliferation assay on hGM-CSF dependent human erythroleukemia TF-1 cells. This study demonstrated that this indeed is a viable route for the efficient production of hGM-CSF.

Impedance microflow cytometry for viability studies of microorganisms

NASA Astrophysics Data System (ADS)

Di Berardino, Marco; Hebeisen, Monika; Hessler, Thomas; Ziswiler, Adrian; Largiadèr, Stephanie; Schade, Grit

2011-02-01

Impedance-based Coulter counters and its derivatives are widely used cell analysis tools in many laboratories and use normally DC or low frequency AC to perform these electrical analyses. The emergence of micro-fabrication technologies in the last decade, however, provides a new means of measuring electrical properties of cells. Microfluidic approaches combined with impedance spectroscopy measurements in the radio frequency (RF) range increase sensitivity and information content and thus push single cell analyses beyond simple cell counting and sizing applications towards multiparametric cell characterization. Promising results have been shown already in the fields of cell differentiation and blood analysis. Here we emphasize the potential of this technology by presenting new data obtained from viability studies on microorganisms. Impedance measurements of several yeast and bacteria strains performed at frequencies around 10 MHz enable an easy discrimination between dead and viable cells. Moreover, cytotoxic effects of antibiotics and other reagents, as well as cell starvation can also be monitored easily. Control analyses performed with conventional flow cytometers using various fluorescent dyes (propidium iodide, oxonol) indicate a good correlation and further highlight the capability of this device. The label-free approach makes on the one hand the use of usually expensive fluorochromes obsolete, on the other hand practically eliminates laborious sample preparation procedures. Until now, online cell monitoring was limited to the determination of viable biomass, which provides rather poor information of a cell culture. Impedance microflow cytometry, besides other aspects, proposes a simple solution to these limitations and might become an important tool for bioprocess monitoring applications in the biotech industry.

Catalysis and biocatalysis program

NASA Technical Reports Server (NTRS)

1991-01-01

The annual report presents the fiscal year (FY) 1990 research activities and accomplishments for the Catalysis and Biocatalysis Program of the Advanced Industrial Concepts Division (AICD), Office of Industrial Technologies of the Department of Energy (DOE). The mission of the AICD is to create a balanced program of high risk, long term, directed interdisciplinary research and development that will improve energy efficiency and enhance fuel flexibility in the industrial sector. The Catalysis and Biocatalysis Program's technical activities were organized into five work elements: the Molecular Modeling and Catalysis by Design element; the Applied Microbiology and Genetics element; the Bioprocess Engineering element; the Separations and Novel Chemical Processes element; and the Process Design and Analysis element.

Immunotoxicology and the new biotechnology.

PubMed

Cavagnaro, J

1987-01-01

The use of recombinant DNA techniques, cell fusion and novel bioprocessing in the pharmaceutical industry has assisted the development of many kinds of diagnostic and therapeutic products. Some directly affect the immune system (e.g. interleukins, interferons, tumour necrosis factor, and colony stimulating factors). Others (e.g. peptides, cytokines, growth factors, H2-receptor antagonists, non-steroidal anti-inflammatory drugs and neurohormonal agents) have immunological reactivity even though they are not designed to be immune modulators. The need to define the immunotoxicological potential of these products during preclinical safety evaluation was among the topics discussed at a recent meeting. Copyright © 1987. Published by Elsevier B.V.

Bioremediation of Industrial Waste Through Enzyme Producing Marine Microorganisms.

PubMed

Sivaperumal, P; Kamala, K; Rajaram, R

Bioremediation process using microorganisms is a kind of nature-friendly and cost-effective clean green technology. Recently, biodegradation of industrial wastes using enzymes from marine microorganisms has been reported worldwide. The prospectus research activity in remediation area would contribute toward the development of advanced bioprocess technology. To minimize industrial wastes, marine enzymes could constitute a novel alternative in terms of waste treatment. Nowadays, the evidence on the mechanisms of bioremediation-related enzymes from marine microorganisms has been extensively studied. This review also will provide information about enzymes from various marine microorganisms and their complexity in the biodegradation of comprehensive range of industrial wastes. © 2017 Elsevier Inc. All rights reserved.

Comparative genomics of biotechnologically important yeasts

USDA-ARS?s Scientific Manuscript database

Saccharomyces cerevisiae, is used in the vast majority of the world’s bioprocesses, and its economic significance is unchallenged. It, however, represents only a small slice of yeast physiological diversity. Many other yeasts, are used in lesser known, but commercially important processes that take ...

INTEGRATED BIOPROCESS SYSTEMS FOR LOW-COST ENVIRONMENTAL REMEDIATION AND SUSTAINABLE BIOFERTILIZER PRODUCTION

EPA Science Inventory

Preliminary techno-economic analysis of these processes will be undertaken, utilizing the literature and including key supporting data and proof-of-principle experiments. The emphasis on low-cost bioreactors and operation greatly enhances the economic feasibility and practica...

Sequential capillary electrophoresis analysis using optically gated sample injection and UV/vis detection.

PubMed

Liu, Xiaoxia; Tian, Miaomiao; Camara, Mohamed Amara; Guo, Liping; Yang, Li

2015-10-01

We present sequential CE analysis of amino acids and L-asparaginase-catalyzed enzyme reaction, by combing the on-line derivatization, optically gated (OG) injection and commercial-available UV-Vis detection. Various experimental conditions for sequential OG-UV/vis CE analysis were investigated and optimized by analyzing a standard mixture of amino acids. High reproducibility of the sequential CE analysis was demonstrated with RSD values (n = 20) of 2.23, 2.57, and 0.70% for peak heights, peak areas, and migration times, respectively, and the LOD of 5.0 μM (for asparagine) and 2.0 μM (for aspartic acid) were obtained. With the application of the OG-UV/vis CE analysis, sequential online CE enzyme assay of L-asparaginase-catalyzed enzyme reaction was carried out by automatically and continuously monitoring the substrate consumption and the product formation every 12 s from the beginning to the end of the reaction. The Michaelis constants for the reaction were obtained and were found to be in good agreement with the results of traditional off-line enzyme assays. The study demonstrated the feasibility and reliability of integrating the OG injection with UV/vis detection for sequential online CE analysis, which could be of potential value for online monitoring various chemical reaction and bioprocesses. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Grouping annotations on the subcellular layered interactome demonstrates enhanced autophagy activity in a recurrent experimental autoimmune uveitis T cell line.

PubMed

Jia, Xiuzhi; Li, Jingbo; Shi, Dejing; Zhao, Yu; Dong, Yucui; Ju, Huanyu; Yang, Jinfeng; Sun, Jianhua; Li, Xia; Ren, Huan

2014-01-01

Human uveitis is a type of T cell-mediated autoimmune disease that often shows relapse-remitting courses affecting multiple biological processes. As a cytoplasmic process, autophagy has been seen as an adaptive response to cell death and survival, yet the link between autophagy and T cell-mediated autoimmunity is not certain. In this study, based on the differentially expressed genes (GSE19652) between the recurrent versus monophasic T cell lines, whose adoptive transfer to susceptible animals may result in respective recurrent or monophasic uveitis, we proposed grouping annotations on a subcellular layered interactome framework to analyze the specific bioprocesses that are linked to the recurrence of T cell autoimmunity. That is, the subcellular layered interactome was established by the Cytoscape and Cerebral plugin based on differential expression, global interactome, and subcellular localization information. Then, the layered interactomes were grouping annotated by the ClueGO plugin based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases. The analysis showed that significant bioprocesses with autophagy were orchestrated in the cytoplasmic layered interactome and that mTOR may have a regulatory role in it. Furthermore, by setting up recurrent and monophasic uveitis in Lewis rats, we confirmed by transmission electron microscopy that, in comparison to the monophasic disease, recurrent uveitis in vivo showed significantly increased autophagy activity and extended lymphocyte infiltration to the affected retina. In summary, our framework methodology is a useful tool to disclose specific bioprocesses and molecular targets that can be attributed to a certain disease. Our results indicated that targeted inhibition of autophagy pathways may perturb the recurrence of uveitis.

Object-oriented programming for the biosciences.

PubMed

Wiechert, W; Joksch, B; Wittig, R; Hartbrich, A; Höner, T; Möllney, M

1995-10-01

The development of software systems for the biosciences is always closely connected to experimental practice. Programs must be able to handle the inherent complexity and heterogeneous structure of biological systems in combination with the measuring equipment. Moreover, a high degree of flexibility is required to treat rapidly changing experimental conditions. Object-oriented methodology seems to be well suited for this purpose. It enables an evolutionary approach to software development that still maintains a high degree of modularity. This paper presents experience with object-oriented technology gathered during several years of programming in the fields of bioprocess development and metabolic engineering. It concentrates on the aspects of experimental support, data analysis, interaction and visualization. Several examples are presented and discussed in the general context of the experimental cycle of knowledge acquisition, thus pointing out the benefits and problems of object-oriented technology in the specific application field of the biosciences. Finally, some strategies for future development are described.

Technical Support to SBIR Phase II Project: Improved Conversion of Cellulose Waste to Ethanol Using a Dual Bioreactor System: Cooperative Research and Development Final Report, CRADA Number CRD-08-310

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

Zhang, M.

2013-04-01

Over-dependence on fossil fuel has spurred research on alternative energy. Inedible plant materials such as grass and corn stover represent abundant renewable natural resources that can be transformed into biofuel. Problems in enzymatic conversion of biomass to sugars include the use of incomplete synergistic enzymes, end-product inhibition, and adsorption and loss of enzymes necessitating their use in large quantities. Technova Corporation will develop a defined consortium of natural microorganisms that will efficiently break down biomass to energy-rich soluble sugars, and convert them to cleaner-burning ethanol fuel. The project will also develop a novel biocatalytic hybrid reactor system dedicated to thismore » bioprocess, which embodies recent advances in nanotechnology. NREL will participate to develop a continuous fermentation process.« less

Space biosynthesis systems

NASA Technical Reports Server (NTRS)

Nyiri, L. K.; Toth, G. M.

1976-01-01

Model reactions based on chemical, enzymatic or cellular conversion of D glucose into d gluconic acid are designed to unequivocally define the advantages of microgravity on reaction mechanisms, mass-transfers and separation of organic chemicals and to serve as procedures to test the performance characteristics of space bioprocessing equipment.

Candidate substances for space bioprocessing methodology and data specification for benefit evaluation

NASA Technical Reports Server (NTRS)

1978-01-01

Analytical and quantitative economic techniques are applied to the evaluation of the economic benefits of a wide range of substances for space bioprocessing. On the basis of expected clinical applications, as well as the size of the patient that could be affected by the clinical applications, eight substances are recommended for further benefit evaluation. Results show that a transitional probability methodology can be used to model at least one clinical application for each of these substances. In each recommended case, the disease and its therapy are sufficiently well understood and documented, and the statistical data is available to operate the model and produce estimates of the impact of new therapy systems on the cost of treatment, morbidity, and mortality. Utilizing the morbidity and mortality information produced by the model, a standard economic technique called the Value of Human Capital is used to estimate the social welfare benefits that could be attributable to the new therapy systems.

Citrus waste as feedstock for bio-based products recovery: Review on limonene case study and energy valorization.

PubMed

Negro, Viviana; Mancini, Giuseppe; Ruggeri, Bernardo; Fino, Debora

2016-08-01

The citrus peels and residue of fruit juices production are rich in d-limonene, a cyclic terpene characterized by antimicrobial activity, which could hamper energy valorization bioprocess. Considering that limonene is used in nutritional, pharmaceutical and cosmetic fields, citrus by-products processing appear to be a suitable feedstock either for high value product recovery or energy bio-processes. This waste stream, more than 10MTon at 2013 in European Union (AIJN, 2014), can be considered appealing, from the view point of conducting a key study on limonene recovery, as its content of about 1%w/w of high value-added molecule. Different processes are currently being studied to recover or remove limonene from citrus peel to both prevent pollution and energy resources recovery. The present review is aimed to highlight pros and contras of different approaches suggesting an energy sustainability criterion to select the most effective one for materials and energy valorization. Copyright © 2016 Elsevier Ltd. All rights reserved.

Enhanced simultaneous saccharification and fermentation of pretreated beech wood by in situ treatment with the white rot fungus Irpex lacteus in a membrane aerated biofilm reactor.

PubMed

Brethauer, Simone; Robert Lawrence, Shahab; Michael Hans-Peter, Studer

2017-08-01

The aim of the present study was to investigate the combination of steam pretreatment and biological treatment with lignin degrading fungal strains in order to enable efficient bioprocessing of beech wood to ethanol. In a sequential process of steam and fungal pretreatment followed by enzymatic hydrolysis, Irpex lacteus almost doubled the glucose yield for mildly pretreated beech wood, but could not improve yields for more severely pretreated substrates. However, when simultaneous saccharification and fermentation is combined with in situ I. lacteus treatment, which is enabled by the application of a membrane aerated biofilm reactor, ethanol yields of optimally steam pretreated beech could be improved from 65 to 80%. Generally, in situ fungal treatment during bioprocessing of lignocellulose is an interesting method to harness the versatile abilities of white rot fungi. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Invitro Study on the Fluid From Banana Stem Bioprocess as Direct Fed Microbial

NASA Astrophysics Data System (ADS)

Mutaqin, B. K.; Tanuwiria, U. H.; Hernawan, E.

2018-02-01

The purpose of this research was to study the liquid produced by the bioprocess of banana stem as a Direct Fed Microbial (DFM) in order to enhance local sheep productivity invitro. Studying was the use of DFM in two invitro feeds. The object observed in this research was fermentability and digestibility value. The method was experimental with the experimental design, i.e. factorial experimental design with two factors. The first factor was DFM, the levels of which were 0, 0,2, 0,4 and 0,6%, while the second factor was two feed types (complete feed and Pennisetum purpureum only) with the treatment of threefold repetition. This research showed that fermentability and digestibility value were influenced by the DFM in the invitro complete feed. The research result analyzed using MANOVA with further testing using Duncan Test. The conclusion of the research result were shows the interaction DFM in the complete feed improve fermentability and digestibility values and DFM 0,6% shows the highest value.

Thermostability in endoglucanases is fold-specific

PubMed Central

2011-01-01

Background Endoglucanases are usually considered to be synergistically involved in the initial stages of cellulose breakdown-an essential step in the bioprocessing of lignocellulosic plant materials into bioethanol. Despite their economic importance, we currently lack a basic understanding of how some endoglucanases can sustain their ability to function at elevated temperatures required for bioprocessing, while others cannot. In this study, we present a detailed comparative analysis of both thermophilic and mesophilic endoglucanases in order to gain insights into origins of thermostability. We analyzed the sequences and structures for sets of endoglucanase proteins drawn from the Carbohydrate-Active enZymes (CAZy) database. Results Our results demonstrate that thermophilic endoglucanases and their mesophilic counterparts differ significantly in their amino acid compositions. Strikingly, these compositional differences are specific to protein folds and enzyme families, and lead to differences in intramolecular interactions in a fold-dependent fashion. Conclusions Here, we provide fold-specific guidelines to control thermostability in endoglucanases that will aid in making production of biofuels from plant biomass more efficient. PMID:21291533

Bio-processing of solid wastes and secondary resources for metal extraction - A review.

PubMed

Lee, Jae-Chun; Pandey, Banshi Dhar

2012-01-01

Metal containing wastes/byproducts of various industries, used consumer goods, and municipal waste are potential pollutants, if not treated properly. They may also be important secondary resources if processed in eco-friendly manner for secured supply of contained metals/materials. Bio-extraction of metals from such resources with microbes such as bacteria, fungi and archaea is being increasingly explored to meet the twin objectives of resource recycling and pollution mitigation. This review focuses on the bio-processing of solid wastes/byproducts of metallurgical and manufacturing industries, chemical/petrochemical plants, electroplating and tanning units, besides sewage sludge and fly ash of municipal incinerators, electronic wastes (e-wastes/PCBs), used batteries, etc. An assessment has been made to quantify the wastes generated and its compositions, microbes used, metal leaching efficiency etc. Processing of certain effluents and wastewaters comprising of metals is also included in brief. Future directions of research are highlighted. Copyright © 2011 Elsevier Ltd. All rights reserved.

Fueling the Future with Fungal Genomics

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

Grigoriev, Igor V.; Cullen, Daniel; Hibbett, David

Fungi play important roles across the range of current and future biofuel production processes. From crop/feedstock health to plant biomass saccharification, enzyme production to bioprocesses for producing ethanol, higher alcohols or future hydrocarbon biofuels, fungi are involved. Research and development are underway to understand the underlying biological processes and improve them to make bioenergy production efficient on an industrial scale. Genomics is the foundation of the systems biology approach that is being used to accelerate the research and development efforts across the spectrum of topic areas that impact biofuels production. In this review, we discuss past, current and future advancesmore » made possible by genomic analyses of the fungi that impact plant/feedstock health, degradation of lignocellulosic biomass and fermentation of sugars to ethanol, hydrocarbon biofuels and renewable chemicals.« less

Suspension cell culture in microgravity and development of a space bioreactor

NASA Technical Reports Server (NTRS)

Morrison, Dennis R.

1987-01-01

NASA has methodically developed unique suspension type cell and recovery apparatus culture systems for bioprocess technology experiments and production of biological products in microgravity. The first space bioreactor has been designed for microprocessor control, no gaseous headspace, circulation and resupply of culture medium, and slow mixing in very low shear regimes. Various ground based bioreactors are being used to test reactor vessel design, on-line sensors, effects of shear, nutrient supply, and waste removal from continuous culture of human cells attached to microcarriers. The small (500 ml) bioreactor is being constructed for flight experiments in the Shuttle middeck to verify systems operation under microgravity conditions and to measure the efficiencies of mass transport, gas transfer, oxygen consumption, and control of low shear stress on cells.

Impact of synthetic biology and metabolic engineering on industrial production of fine chemicals.

PubMed

Jullesson, David; David, Florian; Pfleger, Brian; Nielsen, Jens

2015-11-15

Industrial bio-processes for fine chemical production are increasingly relying on cell factories developed through metabolic engineering and synthetic biology. The use of high throughput techniques and automation for the design of cell factories, and especially platform strains, has played an important role in the transition from laboratory research to industrial production. Model organisms such as Saccharomyces cerevisiae and Escherichia coli remain widely used host strains for industrial production due to their robust and desirable traits. This review describes some of the bio-based fine chemicals that have reached the market, key metabolic engineering tools that have allowed this to happen and some of the companies that are currently utilizing these technologies for developing industrial production processes. Copyright © 2015 Elsevier Inc. All rights reserved.

Process relevant screening of cellulolytic organisms for consolidated bioprocessing.

PubMed

Antonov, Elena; Schlembach, Ivan; Regestein, Lars; Rosenbaum, Miriam A; Büchs, Jochen

2017-01-01

Although the biocatalytic conversion of cellulosic biomass could replace fossil oil for the production of various compounds, it is often not economically viable due to the high costs of cellulolytic enzymes. One possibility to reduce costs is consolidated bioprocessing (CBP), integrating cellulase production, hydrolysis of cellulose, and the fermentation of the released sugars to the desired product into one process step. To establish such a process, the most suitable cellulase-producing organism has to be identified. Thereby, it is crucial to evaluate the candidates under target process conditions. In this work, the chosen model process was the conversion of cellulose to the platform chemical itaconic acid by a mixed culture of a cellulolytic fungus with Aspergillus terreus as itaconic acid producer. Various cellulase producers were analyzed by the introduced freeze assay that measures the initial carbon release rate, quantifying initial cellulase activity under target process conditions. Promising candidates were then characterized online by monitoring their respiration activity metabolizing cellulose to assess the growth and enzyme production dynamics. The screening of five different cellulase producers with the freeze assay identified Trichoderma   reesei and Penicillium   verruculosum as most promising. The measurement of the respiration activity revealed a retarded induction of cellulase production for P.   verruculosum but a similar cellulase production rate afterwards, compared to T.   reesei . The freeze assay measurement depicted that P.   verruculosum reaches the highest initial carbon release rate among all investigated cellulase producers. After a modification of the cultivation procedure, these results were confirmed by the respiration activity measurement. To compare both methods, a correlation between the measured respiration activity and the initial carbon release rate of the freeze assay was introduced. The analysis revealed that the different initial enzyme/cellulose ratios as well as a discrepancy in cellulose digestibility are the main differences between the two approaches. With two complementary methods to quantify cellulase activity and the dynamics of cellulase production for CBP applications, T.   reesei and P.   verruculosum were identified as compatible candidates for the chosen model process. The presented methods can easily be adapted to screen for suitable cellulose degrading organisms for various other applications.

Application of holographic sub-wavelength diffraction gratings for monitoring of kinetics of bioprocesses

NASA Astrophysics Data System (ADS)

Tamulevičius, Tomas; Šeperys, Rimas; Andrulevičius, Mindaugas; Kopustinskas, Vitoldas; Meškinis, Šarūnas; Tamulevičius, Sigitas; Mikalayeva, Valeryia; Daugelavičius, Rimantas

2012-09-01

In this work we present a refractive index (RI) sensor based on a sub-wavelength holographic diffraction grating. The sensor chip was fabricated by dry etching of the finely spaced (d = 428 nm) diffraction grating in SiOx doped diamond like carbon (DLC) film. It is shown that employing a fabricated sensor chip, and using the proposed method of analysis of data, one can inspect kinetics of processes in liquids occurring in the vicinity of the grating surface. The method is based on the spectral composition analysis of polarized polychromatic light reflected from the sub-wavelength diffraction grating. The RI measurement system was tested with different model liquid analytes including 25 wt.%, 50 wt.% sugar water solutions, 10 °C, 50 °C distilled water, also Gram-positive bacteria Bacillus subtilis interaction with ion-permeable channels forming antibiotic gramicidin D and a murolytic enzyme lysozyme. Analysis of the data set of specular reflection spectra enabled us to follow the kinetics of the RI changes in the analyte with millisecond resolution. Detectable changes in the effective RI were not worse than Δn = 10-4.

Cascading and Parallelising Curvilinear Inertial Focusing Systems for High Volume, Wide Size Distribution, Separation and Concentration of Particles

PubMed Central

Miller, B.; Jimenez, M.; Bridle, H.

2016-01-01

Inertial focusing is a microfluidic based separation and concentration technology that has expanded rapidly in the last few years. Throughput is high compared to other microfluidic approaches although sample volumes have typically remained in the millilitre range. Here we present a strategy for achieving rapid high volume processing with stacked and cascaded inertial focusing systems, allowing for separation and concentration of particles with a large size range, demonstrated here from 30 μm–300 μm. The system is based on curved channels, in a novel toroidal configuration and a stack of 20 devices has been shown to operate at 1 L/min. Recirculation allows for efficient removal of large particles whereas a cascading strategy enables sequential removal of particles down to a final stage where the target particle size can be concentrated. The demonstration of curved stacked channels operating in a cascaded manner allows for high throughput applications, potentially replacing filtration in applications such as environmental monitoring, industrial cleaning processes, biomedical and bioprocessing and many more. PMID:27808244

Production of hydroxyl fatty acids, polyol oils, and diacylglycerol by bioprocess

USDA-ARS?s Scientific Manuscript database

Hydroxy fatty acids (HFA), originally found in plant systems, are good examples of the structurally modified lipids, rendering special properties such as higher viscosity and reactivity compared to normal fatty acids. Based on these properties, HFAs possess high industrial potentials in a wide range...

Production of polyol oils from soybean oil through bioprocess

USDA-ARS?s Scientific Manuscript database

Soy-polyol oils (oxygenated acylglycerols) are important starting materials for the manufacture of polymers such as polyurethane. Currently, they are produced by a two-step chemical process involving epoxidation and then the subsequent opening of the oxirane ring. The objective of this study is to d...

NASA Tech Briefs, July 2010

NASA Technical Reports Server (NTRS)

2010-01-01

Topics covered include: Wirelessly Interrogated Wear or Temperature Sensors; Processing Nanostructured Sensors Using Microfabrication Techniques; Optical Pointing Sensor; Radio-Frequency Tank Eigenmode Sensor for Propellant Quantity Gauging; High-Temperature Optical Sensor; Integral Battery Power Limiting Circuit for Intrinsically Safe Applications; Configurable Multi-Purpose Processor; Squeezing Alters Frequency Tuning of WGM Optical Resonator; Automated Computer Access Request System; Range Safety for an Autonomous Flight Safety System; Fast and Easy Searching of Files in Unisys 2200 Computers; Parachute Drag Model; Evolutionary Scheduler for the Deep Space Network; Modular Habitats Comprising Rigid and Inflatable Modules; More About N2O-Based Propulsion and Breathable-Gas Systems; Ultrasonic/Sonic Rotary-Hammer Drills; Miniature Piezoelectric Shaker for Distribution of Unconsolidated Samples to Instrument Cells; Lunar Soil Particle Separator; Advanced Aerobots for Scientific Exploration; Miniature Bioreactor System for Long-Term Cell Culture; Electrochemical Detection of Multiple Bioprocess Analytes; Fabrication and Modification of Nanoporous Silicon Particles; High-Altitude Hydration System; Photon Counting Using Edge-Detection Algorithm; Holographic Vortex Coronagraph; Optical Structural Health Monitoring Device; Fuel-Cell Power Source Based on Onboard Rocket Propellants; Polar Lunar Regions: Exploiting Natural and Augmented Thermal Environments; Simultaneous Spectral Temporal Adaptive Raman Spectrometer - SSTARS; Improved Speed and Functionality of a 580-GHz Imaging Radar; Bolometric Device Based on Fluxoid Quantization; Algorithms for Learning Preferences for Sets of Objects; Model for Simulating a Spiral Software-Development Process; Algorithm That Synthesizes Other Algorithms for Hashing; Algorithms for High-Speed Noninvasive Eye-Tracking System; and Adapting ASPEN for Orbital Express.

A novel cell factory for efficient production of ethanol from dairy waste.

PubMed

Liu, Jianming; Dantoft, Shruti Harnal; Würtz, Anders; Jensen, Peter Ruhdal; Solem, Christian

2016-01-01

Sustainable and economically feasible ways to produce ethanol or other liquid fuels are becoming increasingly relevant due to the limited supply of fossil fuels and the environmental consequences associated with their consumption. Microbial production of fuel compounds has gained a lot of attention and focus has mostly been on developing bio-processes involving non-food plant biomass feedstocks. The high cost of the enzymes needed to degrade such feedstocks into its constituent sugars as well as problems due to various inhibitors generated in pretreatment are two challenges that have to be addressed if cost-effective processes are to be established. Various industries, especially within the food sector, often have waste streams rich in carbohydrates and/or other nutrients, and these could serve as alternative feedstocks for such bio-processes. The dairy industry is a good example, where large amounts of cheese whey or various processed forms thereof are generated. Because of their nutrient-rich nature, these substrates are particularly well suited as feedstocks for microbial production. We have generated a Lactococcus lactis strain which produces ethanol as its sole fermentation product from the lactose contained in residual whey permeate (RWP), by introducing lactose catabolism into a L. lactis strain CS4435 (MG1363 Δ(3) ldh, Δpta, ΔadhE, pCS4268), where the carbon flow has been directed toward ethanol instead of lactate. To achieve growth and ethanol production on RWP, we added corn steep liquor hydrolysate (CSLH) as the nitrogen source. The outcome was efficient ethanol production with a titer of 41 g/L and a yield of 70 % of the theoretical maximum using a fed-batch strategy. The combination of a low-cost medium from industrial waste streams and an efficient cell factory should make the developed process industrially interesting. A process for the production of ethanol using L. lactis and a cheap renewable feedstock was developed. The results demonstrate that it is possible to achieve sustainable bioconversion of waste products from the dairy industry (RWP) and corn milling industry (CSLH) to ethanol and the process developed shows great potential for commercial realization.

Process development of human multipotent stromal cell microcarrier culture using an automated high-throughput microbioreactor.

PubMed

Rafiq, Qasim A; Hanga, Mariana P; Heathman, Thomas R J; Coopman, Karen; Nienow, Alvin W; Williams, David J; Hewitt, Christopher J

2017-10-01

Microbioreactors play a critical role in process development as they reduce reagent requirements and can facilitate high-throughput screening of process parameters and culture conditions. Here, we have demonstrated and explained in detail, for the first time, the amenability of the automated ambr15 cell culture microbioreactor system for the development of scalable adherent human mesenchymal multipotent stromal/stem cell (hMSC) microcarrier culture processes. This was achieved by first improving suspension and mixing of the microcarriers and then improving cell attachment thereby reducing the initial growth lag phase. The latter was achieved by using only 50% of the final working volume of medium for the first 24 h and using an intermittent agitation strategy. These changes resulted in >150% increase in viable cell density after 24 h compared to the original process (no agitation for 24 h and 100% working volume). Using the same methodology as in the ambr15, similar improvements were obtained with larger scale spinner flask studies. Finally, this improved bioprocess methodology based on a serum-based medium was applied to a serum-free process in the ambr15, resulting in >250% increase in yield compared to the serum-based process. At both scales, the agitation used during culture was the minimum required for microcarrier suspension, N JS . The use of the ambr15, with its improved control compared to the spinner flask, reduced the coefficient of variation on viable cell density in the serum containing medium from 7.65% to 4.08%, and the switch to serum free further reduced these to 1.06-0.54%, respectively. The combination of both serum-free and automated processing improved the reproducibility more than 10-fold compared to the serum-based, manual spinner flask process. The findings of this study demonstrate that the ambr15 microbioreactor is an effective tool for bioprocess development of hMSC microcarrier cultures and that a combination of serum-free medium, control, and automation improves both process yield and consistency. Biotechnol. Bioeng. 2017;114: 2253-2266. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Process development of human multipotent stromal cell microcarrier culture using an automated high‐throughput microbioreactor

PubMed Central

Hanga, Mariana P.; Heathman, Thomas R. J.; Coopman, Karen; Nienow, Alvin W.; Williams, David J.; Hewitt, Christopher J.

2017-01-01

ABSTRACT Microbioreactors play a critical role in process development as they reduce reagent requirements and can facilitate high‐throughput screening of process parameters and culture conditions. Here, we have demonstrated and explained in detail, for the first time, the amenability of the automated ambr15 cell culture microbioreactor system for the development of scalable adherent human mesenchymal multipotent stromal/stem cell (hMSC) microcarrier culture processes. This was achieved by first improving suspension and mixing of the microcarriers and then improving cell attachment thereby reducing the initial growth lag phase. The latter was achieved by using only 50% of the final working volume of medium for the first 24 h and using an intermittent agitation strategy. These changes resulted in >150% increase in viable cell density after 24 h compared to the original process (no agitation for 24 h and 100% working volume). Using the same methodology as in the ambr15, similar improvements were obtained with larger scale spinner flask studies. Finally, this improved bioprocess methodology based on a serum‐based medium was applied to a serum‐free process in the ambr15, resulting in >250% increase in yield compared to the serum‐based process. At both scales, the agitation used during culture was the minimum required for microcarrier suspension, NJS. The use of the ambr15, with its improved control compared to the spinner flask, reduced the coefficient of variation on viable cell density in the serum containing medium from 7.65% to 4.08%, and the switch to serum free further reduced these to 1.06–0.54%, respectively. The combination of both serum‐free and automated processing improved the reproducibility more than 10‐fold compared to the serum‐based, manual spinner flask process. The findings of this study demonstrate that the ambr15 microbioreactor is an effective tool for bioprocess development of hMSC microcarrier cultures and that a combination of serum‐free medium, control, and automation improves both process yield and consistency. Biotechnol. Bioeng. 2017;114: 2253–2266. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:28627713

Bioprocessing of Stichococcus bacillaris strain siva2011

PubMed Central

2014-01-01

Background Globally, the development of a cost-effective long-term renewable energy infrastructure is one of the most challenging problems faced by society today. Microalgae are rich in potential biofuel substrates such as lipids, including triacylglycerols (TAGs). Some of these algae also biosynthesize small molecule hydrocarbons. These hydrocarbons can often be used as liquid fuels, often with more versatility and by a more direct approach than some TAGs. However, the appropriate TAGs, accumulated from microalgae biomass, can be used as substrates for different kinds of renewable liquid fuels such as biodiesel and jet fuel. Results This article describes the isolation and identification of a lipid-rich, hydrocarbon-producing alga, Stichococcus bacillaris strain siva2011, together with its bioprocessing, hydrocarbon and fatty acid methyl ester (FAME) profiles. The S. bacillaris strain siva2011 was scaled-up in an 8 L bioreactor with 0.2% CO2. The C16:0, C16:3, C18:1, C18:2 and C18:3 were 112.2, 9.4, 51.3, 74.1 and 69.2 mg/g dry weight (DW), respectively. This new strain produced a significant amount of biomass of 3.79 g/L DW on day 6 in the 8 L bioreactor and also produced three hydrocarbons. Conclusions A new oil-rich microalga S. bacillaris strain siva2011 was discovered and its biomass has been scaled-up in a newly designed balloon-type bioreactor. The TAGs and hydrocarbons produced by this organism could be used as substrates for jet fuel or biodiesel. PMID:24731690

Use of focused acoustics for cell disruption to provide ultra scale-down insights of microbial homogenization and its bioprocess impact--recovery of antibody fragments from rec E. coli.

PubMed

Li, Qiang; Aucamp, Jean P; Tang, Alison; Chatel, Alex; Hoare, Mike

2012-08-01

An ultra scale-down (USD) device that provides insight of how industrial homogenization impacts bioprocess performance is desirable in the biopharmaceutical industry, especially at the early stage of process development where only a small quantity of material is available. In this work, we assess the effectiveness of focused acoustics as the basis of an USD cell disruption method to mimic and study high-pressure, step-wise homogenization of rec Escherichia coli cells for the recovery of an intracellular protein, antibody fragment (Fab'). The release of both Fab' and of overall protein follows first-order reaction kinetics with respect to time of exposure to focused acoustics. The rate constant is directly proportional to applied electrical power input per unit volume. For nearly total protein or Fab' release (>99%), the key physical properties of the disruptate produced by focused acoustics, such as cell debris particle size distribution and apparent viscosity show good agreement with those for homogenates produced by high-pressure homogenization operated to give the same fractional release. The only key difference is observed for partial disruption of cells where focused acoustics yields a disruptate of lower viscosity than homogenization, evidently due to a greater extent of polynucleic acids degradation. Verification of this USD approach to cell disruption by high-pressure homogenization is achieved using USD centrifugation to demonstrate the same sedimentation characteristics of disruptates prepared using both the scaled-down focused acoustic and the pilot-scale homogenization methods for the same fraction of protein release. Copyright © 2012 Wiley Periodicals, Inc.

Process economics of renewable biorefineries: butanol and ethanol production in integrated bioprocesses from lignocellulosics and other industrial by-products

USDA-ARS?s Scientific Manuscript database

This chapter provides process economic details on production of butanol from lignocellulosic biomass and glycerol in integrated bioreactors where numerous unit operations are combined. In order to compare various processes, economic evaluations were performed using SuperPro Designer Software (versio...

Establishing a Taxonometric Structure for the Study of Biotechnology in Secondary School Technology Education.

ERIC Educational Resources Information Center

Wells, John G.

1994-01-01

A Delphi panel of 19 experts identified 8 main knowledge areas of biotechnology: bioprocessing, foundations, genetic engineering, agriculture, biochemistry, medicine, environment, and bioethics. Round 2 elicited 84 subdivisions and round 3 adjusted the ratings. The resulting classification suggests a different context and focus for technology…

Positive Effects of Converting a Food and Bioprocessing Analysis Course to an Inquiry-Guided Approach

USDA-ARS?s Scientific Manuscript database

Food science laboratory courses are traditionally taught as a series of preplanned laboratories with known endpoints. In contrast, inquiry-guided (IG) laboratories allow students to ask questions, think through problems, design experiments, then adapt and learn in response to unexpected results. T...

Improved lignocellulose conversion to biofuels with thermophilic bacteria and thermostable enzymes.

PubMed

Bhalla, Aditya; Bansal, Namita; Kumar, Sudhir; Bischoff, Kenneth M; Sani, Rajesh K

2013-01-01

Second-generation feedstock, especially nonfood lignocellulosic biomass is a potential source for biofuel production. Cost-intensive physical, chemical, biological pretreatment operations and slow enzymatic hydrolysis make the overall process of lignocellulosic conversion into biofuels less economical than available fossil fuels. Lignocellulose conversions carried out at ≤ 50 °C have several limitations. Therefore, this review focuses on the importance of thermophilic bacteria and thermostable enzymes to overcome the limitations of existing lignocellulosic biomass conversion processes. The influence of high temperatures on various existing lignocellulose conversion processes and those that are under development, including separate hydrolysis and fermentation, simultaneous saccharification and fermentation, and extremophilic consolidated bioprocess are also discussed. Copyright © 2012 Elsevier Ltd. All rights reserved.

Gold-modified indium tin oxide as a transparent window in optoelectronic diagnostics of electrochemically active biofilms.

PubMed

Schmidt, Igor; Gad, Alaaeldin; Scholz, Gregor; Boht, Heidi; Martens, Michael; Schilling, Meinhard; Suryo Wasisto, Hutomo; Waag, Andreas; Schröder, Uwe

2017-08-15

Microbial electrochemical technologies (METs) are one of the emerging green bioenergy domains that are utilizing microorganisms for wastewater treatment or electrosynthesis. Real-time monitoring of bioprocess during operation is a prerequisite for understanding and further improving bioenergy harvesting. Optical methods are powerful tools for this, but require transparent, highly conductive and biocompatible electrodes. Whereas indium tin oxide (ITO) is a well-known transparent conductive oxide, it is a non-ideal platform for biofilm growth. Here, a straightforward approach of surface modification of ITO anodes with gold (Au) is demonstrated, to enhance direct microbial biofilm cultivation on their surface and to improve the produced current densities. The trade-off between the electrode transmittance (critical for the underlying integrated sensors) and the enhanced growth of biofilms (crucial for direct monitoring) is studied. Au-modified ITO electrodes show a faster and reproducible biofilm growth with three times higher maximum current densities and about 6.9 times thicker biofilms compared to their unmodified ITO counterparts. The electrochemical analysis confirms the enhanced performance and the reversibility of the ITO/Au electrodes. The catalytic effect of Au on the ITO surface seems to be the key factor of the observed performance improvement since the changes in the electrode conductivity and their surface wettability are relatively small and in the range of ITO. An integrated platform for the ITO/Au transparent electrode with light-emitting diodes was fabricated and its feasibility for optical biofilm thickness monitoring is demonstrated. Such transparent electrodes with embedded catalytic metals can serve as multifunctional windows for biofilm diagnostic microchips. Copyright © 2017 Elsevier B.V. All rights reserved.

Microfabricated Genomic Analysis System

NASA Technical Reports Server (NTRS)

Gonda, Steve; Elms, Rene

2005-01-01

Genetic sequencing and many genetic tests and assays require electrophoretic separation of DNA. In this technique, DNA fragments are separated by size as they migrate through a sieving gel under the influence of an applied electric field. In order to conduct these analyses on-orbit, it is essential to acquire the capability to efficiently perform electrophoresis in a microgravity environment. Conventional bench top electrophoresis equipment is large and cumbersome and does not lead itself to on-orbit utilization. Much of the previous research regarding on-orbit electrophoresis involved altering conventional electrophoresis equipment for bioprocessing, purification, and/or separation technology applications. A new and more efficient approach to on-orbit electrophoresis is the use of a microfabricated electrophoresis platform. These platforms are much smaller, less expensive to produce and operate, use less power, require smaller sample sizes (nanoliters), and achieve separation in a much shorter distance (a few centimeters instead of 10 s or 100 s of centimeters.) In contrast to previous applications, this platform would be utilized as an analytical tool for life science/medical research, environmental monitoring, and medical diagnoses. Identification of infectious agents as well as radiation related damage are significant to NASA s efforts to maintain, study, and monitor crew health during and in support of near-Earth and interplanetary missions. The capability to perform genetic assays on-orbit is imperative to conduct relevant and insightful biological and medical research, as well as continuing NASA s search for life elsewhere. This technology would provide an essential analytical tool for research conducted in a microgravity environment (Shuttle, ISS, long duration/interplanetary missions.) In addition, this technology could serve as a critical and invaluable component of a biosentinel system to monitor space environment genotoxic insults to include radiation.

Rhamnolipids know-how: Looking for strategies for its industrial dissemination.

PubMed

Lovaglio, R B; Silva, V L; Ferreira, H; Hausmann, R; Contiero, J

2015-12-01

Despite the numerous advantages of biosurfactants, such as low toxicity, biodegradability and high stability, these compounds are not widely used because of the high cost of production. Details about genetics, regulation and biosynthesis of rhamnolipids by Pseudomonas aeruginosa, are extremely important to the development of bioprocesses involving the synthesis of these compounds. The holding of such knowledge associated with the use of metabolic engineering tools allow modification of producing strains and the development of synthetic routes, with the purpose of increasing the production of rhamnolipids. Considering the need to obtain this know-how, this review provides information on the rhamnolipids, covering genetics, biosynthesis of hydrophobic and hydrophilic portions, and regulation, plus some future strategies that would contribute to the expansion of the production of this green surfactant. Copyright © 2015 Elsevier Inc. All rights reserved.

Consolidated bioprocessing strategy for ethanol production from Jerusalem artichoke tubers by Kluyveromyces marxianus under high gravity conditions.

PubMed

Yuan, W J; Chang, B L; Ren, J G; Liu, J P; Bai, F W; Li, Y Y

2012-01-01

Developing an innovative process for ethanol fermentation from Jerusalem artichoke tubers under very high gravity (VHG) conditions. A consolidated bioprocessing (CBP) strategy that integrated inulinase production, saccharification of inulin contained in Jerusalem artichoke tubers and ethanol production from sugars released from inulin by the enzyme was developed with the inulinase-producing yeast Kluyveromyces marxianus Y179 and fed-batch operation. The impact of inoculum age, aeration, the supplementation of pectinase and nutrients on the ethanol fermentation performance of the CBP system was studied. Although inulinase activities increased with the extension of the seed incubation time, its contribution to ethanol production was negligible because vigorously growing yeast cells harvested earlier carried out ethanol fermentation more efficiently. Thus, the overnight incubation that has been practised in ethanol production from starch-based feedstocks is recommended. Aeration facilitated the fermentation process, but compromised ethanol yield because of the negative Crabtree effect of the species, and increases the risk of contamination under industrial conditions. Therefore, nonaeration conditions are preferred for the CBP system. Pectinase supplementation reduced viscosity of the fermentation broth and improved ethanol production performance, particularly under high gravity conditions, but the enzyme cost should be carefully balanced. Medium optimization was performed, and ethanol concentration as high as 94·2 g l(-1) was achieved when 0·15 g l(-1) K(2) HPO(4) was supplemented, which presents a significant progress in ethanol production from Jerusalem artichoke tubers. A CBP system using K. marxianus is suitable for efficient ethanol production from Jerusalem artichoke tubers under VHG conditions. Jerusalem artichoke tubers are an alternative to grain-based feedstocks for ethanol production. The high ethanol concentration achieved using K. marxianus with the CBP system not only saves energy consumption for ethanol distillation, but also significantly reduces the amount of waste distillage discharged from the distillation system. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

Total biosynthesis of deoxynucleoside triphosphates using deoxynucleoside monophosphate kinases for PCR application.

PubMed

Bao, Jie; Ryu, Dewey D Y

2007-09-01

Polymerase chain reaction (PCR) and other PCR applications for DNA synthesis require deoxynucleoside triphosphates (dNTP) as the essential precursors and substrates. Currently, the dNTP is commercially produced by a chemical method which is environmentally hazardous and costly due to its low yields in both the synthetic reaction and purification processes. In this study, a enzyme technology for the total integrated biosynthesis of all dNTP components is presented. The bioprocess technology developed and reported here involves two sequential enzymatic phosphorylation reactions coupled with the cofactor regeneration starting from deoxynucleoside monophosphates (dNMP) to deoxynucleoside diphosphates (dNDP) in the first reaction step and to dNTP in the second reaction step in the same bioreactor. The four genes encoding these deoxynucleoside monophosphate kinases were cloned into the recombinant E. coli and expressed using the recombinant E. coli strains. The reaction mechanisms and kinetics of the four kinase enzymes are studied and reported. The total enzymatic syntheses of the four dNTP products were carried out in four separate operations under the high substrate concentrations which emulate the practical application. The optimal process conditions were carefully investigated and complete conversion of dNMP to dNTP at high substrate concentration have been achieved. The purity and quality of dNTP products obtained from this work were analyzed and found to be at least equivalent or better than the commercially available dNTP products. The PCR application of dNTP products obtained from this work were also evaluated for isolating and amplifying genes of different sizes from different organisms. The PCR performance test also showed an equivalent quality as compared to the commercially available dNTP. The bioprocess technology developed and reported here for production of dNTP will provide economically competitive and environmentally friendly viable technology for the industry and research community as compared to the chemical technology currently in use.

Genomic and transcriptomic analysis of carbohydrate utilization by Paenibacillus sp. JDR-2: systems for bioprocessing plant polysaccharides

Treesearch

Neha Sawhney; Casey Crooks; Virginia Chow; James F. Preston; Franz St. John

2016-01-01

Background: Polysaccharides comprising plant biomass are potential resources for conversion to fuels and chemicals. These polysaccharides include xylans derived from the hemicellulose of hardwoods and grasses, soluble beta-glucans from cereals and starch as the primary form of energy storage in plants. Paenibacillus sp...

Reduction of total glucosinolates in canola meal via thermal treatment and fungal bioprocessing

USDA-ARS?s Scientific Manuscript database

On a worldwide basis, canola (Brassica napus) meal is second only to soybean meal as a protein source for livestock. A general limitation of Brassica spp. meals is the presence of glucosinolates (GLS). GLS and the enzyme myrosinase are compartmentally stored separately in the plant. Upon disruption ...

Using Aspen to Teach Chromatographic Bioprocessing: A Case Study in Weak Partitioning Chromatography for Biotechnology Applications

ERIC Educational Resources Information Center

Evans, Steven T.; Huang, Xinqun; Cramer, Steven M.

2010-01-01

The commercial simulator Aspen Chromatography was employed to study and optimize an important new industrial separation process, weak partitioning chromatography. This case study on antibody purification was implemented in a chromatographic separations course. Parametric simulations were performed to investigate the effect of operating parameters…

Lignin Exhibits Recalcitrance-Associated Features Following the Consolidated Bioprocessing of Populus trichocarpa Natural Variants

DOE PAGES

Akinosho, Hannah; Yee, Kelsey; Rodriguez, Miguel; ...

2017-11-21

Because cellulosic ethanol production remains cost-prohibitive„ advances in consolidated bioprocessing (CBP) have been directed towards lifting this restriction. CBP reduces the need for added enzymes and can potentially slash ethanol production costs through process integration. Clostridium thermocellum, a CBP microorganism, organizes its enzymes in a multi-enzyme complex - a stark contrast to fungal enzymes. Nonetheless, recalcitrance may limit the extent of biomass deconstruction. Here in this study, six Populus were treated with C. thermocellum (ATCC 27405) and characterized to determine structural changes that resulted from CBP. The 2D HSQC NMR spectra of lignin-enriched residues revealed that higher S/G ratio (2.6)more » and fewer carbon-carbon interunit linkages (generally 2–5%) were present in the top performing poplar. Furthermore, cellulose degree of polymerization data suggests that C. thermocellum likely circumvents long chain cellulose, while cellulose crystallinity and hemicellulose molecular weight data do not provide a direct indication of features connected to recalcitrance. Hence, C. thermocellum is similarly impacted by the proposed lignin properties that negatively impact biomass deconstruction using fungal enzymes.« less

Bioprocessing of wheat and paddy straw for their nutritional up-gradation.

PubMed

Sharma, Rakesh Kumar; Arora, Daljit Singh

2014-07-01

Solid-state bioprocessing of agricultural residues seems to be an emerging and effective method for the production of high quality animal feed. Seven strains of white-rot fungi were selected to degrade wheat and paddy straw (PS) under solid-state conditions. Degradation of different components, i.e., hemicellulose, cellulose and lignin was evaluated along with nutritional parameters including; in vitro digestibility, crude protein, amino acids, total phenolic contents (TPC) etc. Effect of nitrogen-rich supplements on degradation of lignocellulosics was evaluated using two best selected fungal strains (Phlebia brevispora and Phlebia floridensis). The best selected conditions were used to upscale the process up to 200 g batches of wheat and PS. Lignin was selectively degraded up to 30 % with a limited loss of 11-12 % in total organic matter. Finally, the degraded agro-residues demonstrated 50-62 % enhancement in their digestibility. Two-threefold enhancement in other nutritional quality (amino acids, TPCs and antioxidant activity) fortifies the process. Thus the method is quite helpful to design an effective solid-state fermentation system to improve the nutritive quality of agricultural residues by simultaneous production of lignocellulolytic enzyme production and antioxidants.

The effect of packing hydrophilization on bacterial attachment and the relationship with the performance of biotrickling filters.

PubMed

Prado, Oscar J; Popat, Sudeep C; Chen, Gexin; Walker, Sharon L; Lafuente, Javier; Gabriel, David; Deshusses, Marc A

2009-08-15

Many bioprocesses depend on the effective formation of a biofilm on a solid support. In the present study, three different surface treatments (sandblasting, pure-O(2) plasma, and He-O(2) plasma treatments) were conducted on polypropylene (PP) Pall rings used as a support in biotrickling filters for air pollution control. The intent was to modify the ring surface and/or electrochemical properties in order to possibly improve cell adhesion, wetting properties, and possibly reduce the start-up time and increase the performance of the biotrickling filters. The surface treatments were found to generally increase the hydrophilicity and the zeta potential of the surfaces. However, the startup and performance of lab-scale biotrickling filters packed with treated Pall rings were not significantly different than the control with untreated rings. Cell and colloid deposition experiments conducted in flow cells showed that the treated surfaces and the hydrodynamic conditions were not favorable for cell deposition indicating that there could be significant opportunities for improving packings used in environmental bioprocess applications. Copyright 2009 Wiley Periodicals, Inc.

One-Step Bioprocess of Inulin to Product Inulo-Oligosaccharides Using Bacillus subtilis Secreting an Extracellular Endo-Inulinase.

PubMed

Jiang, Ruifan; Qiu, Yibin; Huang, Weiwei; Zhang, Li; Xue, Feng; Ni, Hao; Mei, Difen; Gao, Jian; Xu, Hong

2018-06-18

Inulo-oligosaccharides (IOSs), a novel food additive and health product, represent a promising alternative to antibiotics. As prebiotics, IOSs can be obtained from inulin by endo-inulinase-mediated hydrolysis. Nonetheless, enzymatic catalysis is not feasible industrially because of the required catalytic conditions and cost. In this study, a 2331-bp optimized gene inuQ (from Pseudomonas mucidolens) encoding endo-inulinase was cloned into shuttle vector PHY300PLK and transfected into Bacillus subtilis WB800-R, with the simultaneous deletion of gene sacC encoding levanase. The maximal IOS yield after hydrolysis of the crude extract of inulin was 67.84 ± 0.72 g/L for a recombinant strain with the signal peptide nprB from alkaline protease and promoter P 43 . The conversion rate reached 75.38%. For the major IOSs, the degree of polymerization was between 3 and 5. This study offers a simple and efficient one-step bioprocess for IOS production from inulin through secretion of an extracellular heterologous endo-inulinase by B. subtilis.

Analysis of the efficiency of recombinant Escherichia coli strain cultivation in a gas-vortex bioreactor.

PubMed

Savelyeva, Anna V; Nemudraya, Anna A; Podgornyi, Vladimir F; Laburkina, Nadezhda V; Ramazanov, Yuriy A; Repkov, Andrey P; Kuligina, Elena V; Richter, Vladimir A

2017-09-01

The levels of aeration and mass transfer are critical parameters required for an efficient aerobic bioprocess, and directly depend on the design features of exploited bioreactors. A novel apparatus, using gas vortex for aeration and mass transfer processes, was constructed in the Center of Vortex Technologies (Novosibirsk, Russia). In this paper, we compared the efficiency of recombinant Escherichia coli strain cultivation using novel gas-vortex technology with conventional bioprocess technologies such as shake flasks and bioreactors with mechanical stirrers. We demonstrated that the system of aeration and agitation used in gas-vortex bioreactors provides 3.6 times higher volumetric oxygen transfer coefficient in comparison with mechanical bioreactor. The use of gas-vortex bioreactor for recombinant E. coli strain cultivation allows to increase the efficiency of target protein expression at 2.2 times for BL21(DE3)/pFK2 strain and at 3.5 times for auxotrophic C600/pRT strain (in comparison with stirred bioreactor). © 2016 International Union of Biochemistry and Molecular Biology, Inc.

A two-stage bioprocess for hydrogen and methane production from rice straw bioethanol residues.

PubMed

Cheng, Hai-Hsuan; Whang, Liang-Ming; Wu, Chao-Wei; Chung, Man-Chien

2012-06-01

This study evaluates a two-stage bioprocess for recovering hydrogen and methane while treating organic residues of fermentative bioethanol from rice straw. The obtained results indicate that controlling a proper volumetric loading rate, substrate-to-biomass ratio, or F/M ratio is important to maximizing biohydrogen production from rice straw bioethanol residues. Clostridium tyrobutyricum, the identified major hydrogen-producing bacteria enriched in the hydrogen bioreactor, is likely utilizing lactate and acetate for biohydrogen production. The occurrence of acetogenesis during biohydrogen fermentation may reduce the B/A ratio and lead to a lower hydrogen production. Organic residues remained in the effluent of hydrogen bioreactor can be effectively converted to methane with a rate of 2.8 mmol CH(4)/gVSS/h at VLR of 4.6 kg COD/m(3)/d. Finally, approximately 75% of COD in rice straw bioethanol residues can be removed and among that 1.3% and 66.1% of COD can be recovered in the forms of hydrogen and methane, respectively. Copyright © 2012 Elsevier Ltd. All rights reserved.

Reduction of Cr(6+) to Cr(3+) in a packed-bed bioreactor

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

Turick, C.E.; Apel, W.A.; Camp, C.E.

1997-12-31

Hexavalent chromium, Cr(6{sup +}), is a common and toxic pollutant in soils and waters. Reduction of the mobile Cr(6{sup +}) to the less mobile and less toxic trivalent chromium, Cr(3{sup +}), can be achieved with conventional chemical reduction technologies. Alternatively, Cr(6{sup +}) can be biochemically reduced to Cr(3{sup +}) by anaerobic microbial consortia which appear to use Cr(6{sup +}) as a terminal electron acceptor. A bioprocess for Cr(6{sup +}) reduction has been demonstrated using a packed-bed bioreactor containing ceramic packing, and then compared to a similar bioreactor containing DuPont Bio-Sep beads. An increase in volumetric productivity from 4 mg Cr(6{supmore » +})/L/h to 260 mg Cr(6{sup +})/L/h, probably due to an increase in biomass density, was obtained using Bio-Sep beads. The beads contain internal macropores which were shown by scanning electron microscopy to house dense concentrations of bacteria. Comparisons to conventional Cr(6{sup +}) treatment technologies indicate that a bioprocess has several economic and operational advantages. 7 refs., 4 figs.« less

Production and characterization of poly-3-hydroxybutyrate from Bacillus cereus PS 10.

PubMed

Sharma, Priyanka; Bajaj, Bijender Kumar

2015-11-01

Usage of renewable raw materials for production of fully degradable bioplastics (bacterial poly-3-hydroxybutyrate, PHB) has gained immense research impetus considering recalcitrant nature of petroleum based plastics, dwindling fossil fuel feed stocks, and associated green house gas emissions. However, high production cost of PHB is the major bottleneck for its wide range industrial applications. In current study, Bacillus cereus PS 10, a recent isolate, efficiently utilized molasses, an abundantly available by-product from sugar industries as sole carbon source for growth and PHB production. Most influential bioprocess variables i.e. molasses, pH and NH4Cl were identified based on Plackett-Burman-designed experiments. Design of experiment approach (response surface methodology) was further employed for optimization of these bioprocess variables, and an enhanced PHB yield (57.5%) was obtained. PHB produced by Bacillus cereus PS 10 was investigated using various physico-chemical approaches viz. thermogravimetric analysis, proton and carbon NMR ((1)H and (13)C) spectroscopy, melting point, elemental analysis and polarimetry for its detail characterization, and assessment for industrial application potential. Copyright © 2015 Elsevier B.V. All rights reserved.

Soybean P34 Probable Thiol Protease Probably Has Proteolytic Activity on Oleosins.

PubMed

Zhao, Luping; Kong, Xiangzhen; Zhang, Caimeng; Hua, Yufei; Chen, Yeming

2017-07-19

P34 probable thiol protease (P34) and Gly m Bd 30K (30K) show high relationship with the protease of 24 kDa oleosin of soybean oil bodies. In this study, 9 day germinated soybean was used to separate bioprocessed P34 (P32) from bioprocessed 30K (28K). Interestingly, P32 existed as dimer, whereas 28K existed as monomer; a P32-rich sample had proteolytic activity and high cleavage site specificity (Lys-Thr of 24 kDa oleosin), whereas a 28K-rich sample showed low proteolytic activity; the P32-rich sample contained one thiol protease. After mixing with purified oil bodies, all P32 dimers were dissociated and bound to 24 kDa oleosins to form P32-24 kDa oleosin complexes. By incubation, 24 kDa oleosin was preferentially hydrolyzed, and two hydrolyzed products (HPs; 17 and 7 kDa) were confirmed. After most of 24 kDa oleosin was hydrolyzed, some P32 existed as dimer, and the other as P32-17 kDa HP. It was suggested that P32 was the protease.

Extracellular Electron Uptake: Among Autotrophs and Mediated by Surfaces.

PubMed

Tremblay, Pier-Luc; Angenent, Largus T; Zhang, Tian

2017-04-01

Autotrophic microbes can acquire electrons from solid donors such as steel, other microbial cells, or electrodes. Based on this feature, bioprocesses are being developed for the microbial electrosynthesis (MES) of useful products from the greenhouse gas CO 2 . Extracellular electron-transfer mechanisms involved in the acquisition of electrons from metals by electrical microbially influenced corrosion (EMIC), from other living cells by interspecies electron transfer (IET), or from an electrode during MES rely on: (i) mediators such as H 2 ; (ii) physical contact through electron-transfer proteins; or (iii) mediator-generating enzymes detached from cells. This review explores the interactions of autotrophs with solid electron donors and their importance in nature and for biosustainable technologies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Microorganisms in inorganic chemical analysis.

PubMed

Godlewska-Zyłkiewicz, Beata

2006-01-01

There are innumerable strains of microbes (bacteria, yeast and fungi) that degrade or transform chemicals and compounds into simpler, safer or less toxic substances. These bioprocesses have been used for centuries in the treatment of municipal wastes, in wine, cheese and bread making, and in bioleaching and metal recovery processes. Recent literature shows that microorganisms can be also used as effective sorbents for solid phase extraction procedures. This review reveals that fundamental nonanalytical studies on the parameters and conditions of biosorption processes and on metal-biomass interactions often result in efficient analytical procedures and biotechnological applications. Some selected examples illustrate the latest developments in the biosorption of metals by microbial biomass, which have opened the door to the application of microorganisms to analyte preconcentration, matrix separation and speciation analysis.

Expression of a heat-stable NADPH-dependent alcohol dehydrogenase in Caldicellulosiruptor bescii results in furan aldehyde detoxification

DOE PAGES

Chung, Daehwan; Verbeke, Tobin J.; Cross, Karissa L.; ...

2015-07-22

Compounds such as furfural and 5-hydroxymethylfurfural (5-HMF) are generated through the dehydration of xylose and glucose, respectively, during dilute-acid pretreatment of lignocellulosic biomass and are also potent microbial growth and fermentation inhibitors. The enzymatic reduction of these furan aldehydes to their corresponding, and less toxic, alcohols is an engineering approach that has been successfully implemented in both Saccharomyces cerevisiae and ethanologenicEscherichia coli, but has not yet been investigated in thermophiles relevant to biofuel production through consolidated bioprocessing (CBP). Developing CBP-relevant biocatalysts that are either naturally resistant to such inhibitors, or are amenable to engineered resistance, is therefore, an important componentmore » in making biofuels production from lignocellulosic biomass feasible.« less

Applications of systems biology towards microbial fuel production.

PubMed

Gowen, Christopher M; Fong, Stephen S

2011-10-01

Harnessing the immense natural diversity of biological functions for economical production of fuel has enormous potential benefits. Inevitably, however, the native capabilities for any given organism must be modified to increase the productivity or efficiency of a biofuel bioprocess. From a broad perspective, the challenge is to sufficiently understand the details of cellular functionality to be able to prospectively predict and modify the cellular function of a microorganism. Recent advances in experimental and computational systems biology approaches can be used to better understand cellular level function and guide future experiments. With pressure to quickly develop viable, renewable biofuel processes a balance must be maintained between obtaining depth of biological knowledge and applying that knowledge. Copyright © 2011 Elsevier Ltd. All rights reserved.

The dynamics of hydroponic crops for simulation studies of the CELSS initial reference configurations

NASA Technical Reports Server (NTRS)

Volk, Tyler

1992-01-01

The goal of this research is to develop a progressive series of mathematical models for the CELSS hydroponic crops. These models will systematize the experimental findings from the crop researchers in the CELSS Program into a form useful to investigate system-level considerations, for example, dynamic studies of the CELSS Initial Reference Configurations. The crop models will organize data from different crops into a common modeling framework. This is the fifth semiannual report for this project. The following topics are discussed: (1) use of field crop models to explore phasic control of CELSS crops for optimizing yield; (2) seminar presented at Purdue CELSS NSCORT; and (3) paper submitted on analysis of bioprocessing of inedible plant materials.

[Improving industrial microbial stress resistance by metabolic engineering: a review].

PubMed

Fu, Ruiyan; Li, Yin

2010-09-01

Metabolic engineering is a technologic platform for industrial strain improvement and aims not only at modifying microbial metabolic fluxes, but also improving the physiological performance of industrial microbes. Microbes will meet multiple stresses in industrial processes. Consequently, elicited gene responses might result in a decrease in overall cell fitness and the efficiency of biotransformation. Thus, it is crucial to develop robust and productive microbial strains that can be integrated into industrial-scale bioprocesses. In this review, we focus on the progress of these novel methods and strategies for engineering stress-tolerance phenotypes referring to rational metabolic engineering and inverse metabolic engineering in recent years. In addition, we also address problems existing in this area and future research needs of microbial physiological functionality engineering.

Bio-functionalization of conductive textile materials with redox enzymes

NASA Astrophysics Data System (ADS)

Kahoush, M.; Behary, N.; Cayla, A.; Nierstrasz, V.

2017-10-01

In recent years, immobilization of oxidoreductase enzymes on electrically conductive materials has played an important role in the development of sustainable bio-technologies. Immobilization process allows the re-use of these bio-catalysts in their final applications. In this study, different methods of immobilizing redox enzymes on conductive textile materials were used to produce bio-functionalized electrodes. These electrodes can be used for bio-processes and bio-sensing in eco-designed applications in domains such as medicine and pollution control. However, the main challenge facing the stability and durability of these electrodes is the maintenance of the enzymatic activity after the immobilization. Hence, preventing the enzyme’s denaturation and leaching is a critical factor for the success of the immobilization processes.

Commercial biotechnology processing thermal control for transfer payloads to/from the International Space Station

NASA Astrophysics Data System (ADS)

Jennings, William M.; Vellinger, John C.; Deuser, Mark S.

2000-01-01

Biotechnology is undergoing a period of rapid and sustained growth, a trend which is expected to continue as the general population ages and as new medical treatments and products are conceived. As pharmaceutical and biomedical companies continue to search for improved methods of production and, for answers to basic research questions, they will seek out new avenues of research. Space processing on the International Space Station (ISS) offers such an opportunity! Space is rapidly becoming an industrial laboratory for biotechnology research and processing. Space bioprocessing offers exciting possibilities for developing new pharmaceuticals and medical treatments which can be used to benefit mankind on Earth. It also represents a new economic frontier for the private sector. .

Microfluidic tools toward industrial biotechnology.

PubMed

Oliveira, Aline F; Pessoa, Amanda C S N; Bastos, Reinaldo G; de la Torre, Lucimara G

2016-11-01

Microfluidics is a technology that operates with small amounts of fluids and makes possible the investigation of cells, enzymes, and biomolecules and encapsulation of biocatalysts in a greater variety of conditions than permitted using conventional methods. This review discusses technological possibilities that can be applied in the field of industrial biotechnology, presenting the principal definitions and fundamental aspects of microfluidic parameters to better understand advanced approaches. Specifically, concentration gradient generators, droplet-based microfluidics, and microbioreactors are explored as useful tools that can contribute to industrial biotechnology. These tools present potential applications, inclusive as commercial platforms to optimizing in bioprocesses development as screening cells, encapsulating biocatalysts, and determining critical kinetic parameters. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1372-1389, 2016. © 2016 American Institute of Chemical Engineers.

Sophisticated Cloning, Fermentation, and Purification Technologies for an Enhanced Therapeutic Protein Production: A Review

PubMed Central

Gupta, Sanjeev K.; Shukla, Pratyoosh

2017-01-01

The protein productions strategies are crucial towards the development of application based research and elucidating the novel purification strategies for industrial production. Currently, there are few innovative avenues are studies for cloning, upstream, and purification through efficient bioprocess development. Such strategies are beneficial for industries as well as proven to be vital for effectual therapeutic protein development. Though, these techniques are well documented, but, there is scope of addition to current knowledge with novel and new approaches and it will pave new avenues in production of recombinant microbial and non-microbial proteins including secondary metabolites. In this review, we have focussed on the recent development in clone selection, various modern fermentation and purification technologies and future directions in these emerging areas. Moreover, we have also highlighted notable perspectives and challenges involved in the bioengineering of such proteins, including quality by design, gene editing and pioneering ideas. The biopharmaceutical industries continue to shift towards more flexible, automated platforms and economical product development, which in turn can help in developing the cost effective processes and affordable drug development for a large community. PMID:28725194

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

The Effects of Antifoam Agent on Dead End Filtration Process

NASA Astrophysics Data System (ADS)

Mohamad Pauzi, S.; Ahmad, N.; Yahya, M. F.; Arifin, M. A.

2018-05-01

The formation of foam as a result from introducing gases during cell culture process in the bioprocess industry has indirectly affected the throughput of the product of interest. Due to that, antifoams were developed and established as one of the means to minimize the formation of foam in the cell culture. There are many types of antifoams but the silicone-type of antifoams are widely used in the bioprocess industry. Although the establishment of antifoam has aided the cell culture process, the impacts of its presence in the cell culture to the downstream process especially the dead end filtration is not widely discussed. The findings in the study emphasized on the dead end filtration performance that includes flux rate profile and the resulted filtration capacity. In this study, the concentrations of antifoam injected into the solution were varied from 0.2% v/v – 1.0% v/v and the solutions were filtered using constant flow method. The resulted maximum pressure readings and final flux rates indicated that the resistance exerted to the feed flow rate increased as the concentration of antifoam loaded in the solution increased. This later has led to the decline in the flux rates with percentage reduction between 32 – 68%. The calculated filter capacity for flux rate of 1000LMH ranged from 53 – 63L/m2 while it is in the range of 40 – 43L/m2 for flux rate of 2000LMH. The presence of antifoam agents in the feed load was determined to have negative effects on the dead end filtration performance and it may reduce the efficiency of the dead end filtration process.

Manufacturing validation of biologically functional T cells targeted to CD19 antigen for autologous adoptive cell therapy

PubMed Central

Hollyman, Daniel; Stefanski, Jolanta; Przybylowski, Mark; Bartido, Shirley; Borquez-Ojeda, Oriana; Taylor, Clare; Yeh, Raymond; Capacio, Vanessa; Olszewska, Malgorzata; Hosey, James; Sadelain, Michel; Brentjens, Renier J.; Rivière, Isabelle

2009-01-01

Summary Based on promising pre-clinical data demonstrating the eradication of systemic B cell malignancies by CD19-targeted T lymphocytes in vivo in SCID beige mouse models, we are launching Phase 1 clinical trials in patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). We present here the validation of the bioprocess we developed for the production and expansion of clinical grade autologous T cells derived from patients with CLL. We demonstrate that T cells genetically modified with a replication-defective gammaretroviral vector derived from the Moloney murine leukemia virus encoding a chimeric antigen receptor (CAR) targeted to CD19 (1928z) can be expanded with Dynabeads® CD3/CD28. This bioprocess allows us to generate clinical doses of 1928z+ T cells in approximately 2 to 3 weeks in a large-scale semi-closed culture system using the Wave bioreactor. These 1928z+ T cells remain biologically functional not only in vitro but also in SCID beige mice bearing disseminated tumors. The validation requirements in terms of T cell expansion, T cell transduction with the 1928z CAR, biological activity, quality control testing and release criteria were met for all four validation runs using apheresis products from patients with CLL. Additionally, following expansion of the T cells, the diversity of the skewed Vβ T cell receptor repertoire was significantly restored. This validated process will be used in phase I clinical trials in patients with chemo-refractory CLL and in patients with relapsed ALL. It can also be adapted for other clinical trials involving the expansion and transduction of patient or donor T cells using any chimeric antigen receptor or T cell receptor. PMID:19238016

%22Trojan Horse%22 strategy for deconstruction of biomass for biofuels production.

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

Simmons, Blake Alexander; Sinclair, Michael B.; Yu, Eizadora

2011-02-01

Production of renewable biofuels to displace fossil fuels currently consumed in the transportation sector is a pressing multiagency national priority (DOE/USDA/EERE). Currently, nearly all fuel ethanol is produced from corn-derived starch. Dedicated 'energy crops' and agricultural waste are preferred long-term solutions for renewable, cheap, and globally available biofuels as they avoid some of the market pressures and secondary greenhouse gas emission challenges currently facing corn ethanol. These sources of lignocellulosic biomass are converted to fermentable sugars using a variety of chemical and thermochemical pretreatments, which disrupt cellulose and lignin cross-links, allowing exogenously added recombinant microbial enzymes to more efficiently hydrolyzemore » the cellulose for 'deconstruction' into glucose. This process is plagued with inefficiencies, primarily due to the recalcitrance of cellulosic biomass, mass transfer issues during deconstruction, and low activity of recombinant deconstruction enzymes. Costs are also high due to the requirement for enzymes and reagents, and energy-intensive cumbersome pretreatment steps. One potential solution to these problems is found in synthetic biology-engineered plants that self-produce a suite of cellulase enzymes. Deconstruction can then be integrated into a one-step process, thereby increasing efficiency (cellulose-cellulase mass-transfer rates) and reducing costs. The unique aspects of our approach are the rationally engineered enzymes which become Trojan horses during pretreatment conditions. During this study we rationally engineered Cazy enzymes and then integrated them into plant cells by multiple transformation techniques. The regenerated plants were assayed for first expression of these messages and then for the resulting proteins. The plants were then subjected to consolidated bioprocessing and characterized in detail. Our results and possible implications of this work on developing dedicated energy crops and their advantage in a consolidated bioprocessing system.« less

Manufacturing validation of biologically functional T cells targeted to CD19 antigen for autologous adoptive cell therapy.

PubMed

Hollyman, Daniel; Stefanski, Jolanta; Przybylowski, Mark; Bartido, Shirley; Borquez-Ojeda, Oriana; Taylor, Clare; Yeh, Raymond; Capacio, Vanessa; Olszewska, Malgorzata; Hosey, James; Sadelain, Michel; Brentjens, Renier J; Rivière, Isabelle

2009-01-01

On the basis of promising preclinical data demonstrating the eradication of systemic B-cell malignancies by CD19-targeted T lymphocytes in vivo in severe combined immunodeficient-beige mouse models, we are launching phase I clinical trials in patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia. We present here the validation of the bioprocess which we developed for the production and expansion of clinical grade autologous T cells derived from patients with CLL. We demonstrate that T cells genetically modified with a replication-defective gammaretroviral vector derived from the Moloney murine leukemia virus encoding a chimeric antigen receptor (CAR) targeted to CD19 (1928z) can be expanded with Dynabeads CD3/CD28. This bioprocess allows us to generate clinical doses of 1928z+ T cells in approximately 2 to 3 weeks in a large-scale semiclosed culture system using the Wave Bioreactor. These 1928z+ T cells remain biologically functional not only in vitro but also in severe combined immunodeficient-beige mice bearing disseminated tumors. The validation requirements in terms of T-cell expansion, T-cell transduction with the 1928z CAR, biologic activity, quality control testing, and release criteria were met for all 4 validation runs using apheresis products from patients with CLL. Additionally, after expansion of the T cells, the diversity of the skewed Vbeta T-cell receptor repertoire was significantly restored. This validated process will be used in phase I clinical trials in patients with chemorefractory CLL and in patients with relapsed acute lymphoblastic leukemia. It can also be adapted for other clinical trials involving the expansion and transduction of patient or donor T cells using any CAR or T-cell receptor.

Bioprocess development workflow: Transferable physiological knowledge instead of technological correlations.

PubMed

Reichelt, Wieland N; Haas, Florian; Sagmeister, Patrick; Herwig, Christoph

2017-01-01

Microbial bioprocesses need to be designed to be transferable from lab scale to production scale as well as between setups. Although substantial effort is invested to control technological parameters, usually the only true constant parameter is the actual producer of the product: the cell. Hence, instead of solely controlling technological process parameters, the focus should be increasingly laid on physiological parameters. This contribution aims at illustrating a workflow of data life cycle management with special focus on physiology. Information processing condenses the data into physiological variables, while information mining condenses the variables further into physiological descriptors. This basis facilitates data analysis for a physiological explanation for observed phenomena in productivity. Targeting transferability, we demonstrate this workflow using an industrially relevant Escherichia coli process for recombinant protein production and substantiate the following three points: (1) The postinduction phase is independent in terms of productivity and physiology from the preinduction variables specific growth rate and biomass at induction. (2) The specific substrate uptake rate during induction phase was found to significantly impact the maximum specific product titer. (3) The time point of maximum specific titer can be predicted by an easy accessible physiological variable: while the maximum specific titers were reached at different time points (19.8 ± 7.6 h), those maxima were reached all within a very narrow window of cumulatively consumed substrate dSn (3.1 ± 0.3 g/g). Concluding, this contribution provides a workflow on how to gain a physiological view on the process and illustrates potential benefits. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:261-270, 2017. © 2016 American Institute of Chemical Engineers.

Positive Effects of Converting a Food and Bioprocessing Analysis Course to an Inquiry-Guided Approach

ERIC Educational Resources Information Center

Harris, Gabriel K.; Cvitkusic, Sanja; Draut, Amanda S.; Hathorn, Chelani S.; Stephens, Amanda M.; Constanza, Karen E.; Leonardelli, Michael J.; Watkins, Ruth H.; Dean, Lisa O.; Hentz, Nathaniel G.

2012-01-01

Food science laboratory courses are traditionally taught as a series of preplanned laboratories with known endpoints. In contrast, inquiry-guided (IG) laboratories allow students to ask questions, think through problems, design experiments, then adapt and learn in response to unexpected results. This study examined the effects of converting the…

Biomass Structure and Its Contributions to Recalcitrance During Consolidated Bioprocessing with Clostridium Thermocellum

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

Akinosho, Hannah O.

Each study provided compelling reasoning to strongly consider lignin’s part in limiting CBP efficiencies at the laboratory and eventually the industrial scale. To make cellulosic ethanol production feasible with CBP, lignin structure and content must be manipulated with genetic modifications or carefully selected in natural variants to combat these difficulties.

Space bioreactor: Design/process flow

NASA Technical Reports Server (NTRS)

Cross, John H.

1987-01-01

The design of the space bioreactor stems from three considerations. First, and foremost, it must sustain cells in microgravity. Closely related is the ability to take advantage of the weightlessness and microgravity. Lastly, it should fit into a bioprocess. The design of the space bioreactor is described in view of these considerations. A flow chart of the bioreactor is presented and discussed.

A polysaccharide isolated from the liquid culture of Lentinus edodes (shiitake) mushroom mycelia containing black rice bran protects mice against a Salmonella lipopolysaccharide-induced endotoxemia

USDA-ARS?s Scientific Manuscript database

Endotoxemia (sepsis, septic shock) is an inflammatory, virulent disease that results mainly from bacterial infection. The present study investigates the inhibitory effect of the bio-processed polysaccharide (BPP) isolated from the edible Lentinus edodes liquid mycelial mushroom culture supplemented...

A novel perfused rotary bioreactor for cardiomyogenesis of embryonic stem cells.

PubMed

Teo, Ailing; Mantalaris, Athanasios; Song, Kedong; Lim, Mayasari

2014-05-01

Developments in bioprocessing technology play an important role for overcoming challenges in cardiac tissue engineering. To this end, our laboratory has developed a novel rotary perfused bioreactor for supporting three-dimensional cardiac tissue engineering. The dynamic culture environments provided by our novel perfused rotary bioreactor and/or the high-aspect rotating vessel produced constructs with higher viability and significantly higher cell numbers (up to 4 × 10(5) cells/bead) than static tissue culture flasks. Furthermore, cells in the perfused rotary bioreactor showed earlier gene expressions of cardiac troponin-T, α- and β-myosin heavy chains with higher percentages of cardiac troponin-I-positive cells and better uniformity of sacromeric α-actinin expression. A dynamic and perfused environment, as provided by this bioreactor, provides a superior culture performance in cardiac differentiation for embryonic stem cells particularly for larger 3D constructs.

[Preface for special issue on bio-based materials (2016)].

PubMed

Weng, Yunxuan

2016-06-25

Bio-based materials are new materials or chemicals with renewable biomass as raw materials such as grain, legume, straw, bamboo and wood powder. This class of materials includes bio-based polymer, biobased fiber, glycotechnology products, biobased rubber and plastics produced by biomass thermoplastic processing and basic biobased chemicals, for instance, bio-alcohols, organic acids, alkanes, and alkenes, obtained by bio-synthesis, bio-processing and bio-refinery. Owing to its environmental friendly and resource conservation, bio-based materials are becoming a new dominant industry taking the lead in the world scientific and technological innovation and economic development. An overview of bio-based materials development is reported in this special issue, and the industrial status and research progress of the following aspects, including biobased fiber, polyhydroxyalkanoates, biodegradable mulching film, bio-based polyamide, protein based biomedical materials, bio-based polyurethane, and modification and processing of poly(lactic acid), are introduced.

Large Scale Production of Stem Cells and Their Derivatives

NASA Astrophysics Data System (ADS)

Zweigerdt, Robert

Stem cells have been envisioned to become an unlimited cell source for regenerative medicine. Notably, the interest in stem cells lies beyond direct therapeutic applications. They might also provide a previously unavailable source of valuable human cell types for screening platforms, which might facilitate the development of more efficient and safer drugs. The heterogeneity of stem cell types as well as the numerous areas of application suggests that differential processes are mandatory for their in vitro culture. Many of the envisioned applications would require the production of a high number of stem cells and their derivatives in scalable, well-defined and potentially clinical compliant manner under current good manufacturing practice (cGMP). In this review we provide an overview on recent strategies to develop bioprocesses for the expansion, differentiation and enrichment of stem cells and their progenies, presenting examples for adult and embryonic stem cells alike.

High-throughput strategies for the discovery and engineering of enzymes for biocatalysis.

PubMed

Jacques, Philippe; Béchet, Max; Bigan, Muriel; Caly, Delphine; Chataigné, Gabrielle; Coutte, François; Flahaut, Christophe; Heuson, Egon; Leclère, Valérie; Lecouturier, Didier; Phalip, Vincent; Ravallec, Rozenn; Dhulster, Pascal; Froidevaux, Rénato

2017-02-01

Innovations in novel enzyme discoveries impact upon a wide range of industries for which biocatalysis and biotransformations represent a great challenge, i.e., food industry, polymers and chemical industry. Key tools and technologies, such as bioinformatics tools to guide mutant library design, molecular biology tools to create mutants library, microfluidics/microplates, parallel miniscale bioreactors and mass spectrometry technologies to create high-throughput screening methods and experimental design tools for screening and optimization, allow to evolve the discovery, development and implementation of enzymes and whole cells in (bio)processes. These technological innovations are also accompanied by the development and implementation of clean and sustainable integrated processes to meet the growing needs of chemical, pharmaceutical, environmental and biorefinery industries. This review gives an overview of the benefits of high-throughput screening approach from the discovery and engineering of biocatalysts to cell culture for optimizing their production in integrated processes and their extraction/purification.

Two-stage dilute-acid and organic-solvent lignocellulosic pretreatment for enhanced bioprocessing.

PubMed

Brodeur, G; Telotte, J; Stickel, J J; Ramakrishnan, S

2016-11-01

A two stage pretreatment approach for biomass is developed in the current work in which dilute acid (DA) pretreatment is followed by a solvent based pretreatment (N-methyl morpholine N oxide - NMMO). When the combined pretreatment (DAWNT) is applied to sugarcane bagasse and corn stover, the rates of hydrolysis and overall yields (>90%) are seen to dramatically improve and under certain conditions 48h can be taken off the time of hydrolysis with the additional NMMO step to reach similar conversions. DAWNT shows a 2-fold increase in characteristic rates and also fractionates different components of biomass - DA treatment removes the hemicellulose while the remaining cellulose is broken down by enzymatic hydrolysis after NMMO treatment to simple sugars. The remaining residual solid is high purity lignin. Future work will focus on developing a full scale economic analysis of DAWNT for use in biomass fractionation. Copyright © 2016 Elsevier Ltd. All rights reserved.

NREL Advancements in Methane Conversion Lead to Cleaner Air, Useful Products

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

2016-06-01

Researchers at NREL leveraged the recent on-site development of gas fermentation capabilities and novel genetic tools to directly convert methane to lactic acid using an engineered methanotrophic bacterium. The results provide proof-of-concept data for a gas-to-liquids bioprocess that concurrently produces fuels and chemicals from methane. NREL researchers developed genetic tools to express heterologous genes in methanotrophic organisms, which have historically been difficult to genetically engineer. Using these tools, researchers demonstrated microbial conversion of methane to lactate, a high-volume biochemical precursor predominantly utilized for the production of bioplastics. Methane biocatalysis offers a means to concurrently liquefy and upgrade natural gas andmore » renewable biogas, enabling their utilization in conventional transportation and industrial manufacturing infrastructure. Producing chemicals and fuels from methane expands the suite of products currently generated from biorefineries, municipalities, and agricultural operations, with the potential to increase revenue and significantly reduce greenhouse gas emissions.« less

Two-stage dilute-acid and organic-solvent lignocellulosic pretreatment for enhanced bioprocessing

DOE PAGES

Brodeur, G.; Telotte, J.; Stickel, J. J.; ...

2016-08-26

A two stage pretreatment approach for biomass is developed in the current work in which dilute acid (DA) pretreatment is followed by a solvent based pretreatment (N-methyl morpholine N oxide -- NMMO). When the combined pretreatment (DAWNT) is applied to sugarcane bagasse and corn stover, the rates of hydrolysis and overall yields (>90%) are seen to dramatically improve and under certain conditions 48 h can be taken off the time of hydrolysis with the additional NMMO step to reach similar conversions. DAWNT shows a 2-fold increase in characteristic rates and also fractionates different components of biomass -- DA treatment removesmore » the hemicellulose while the remaining cellulose is broken down by enzymatic hydrolysis after NMMO treatment to simple sugars. The remaining residual solid is high purity lignin. Lastly, future work will focus on developing a full scale economic analysis of DAWNT for use in biomass fractionation.« less

Genetic tool development underpins recent advances in thermophilic whole‐cell biocatalysts

PubMed Central

Taylor, M. P.; van Zyl, L.; Tuffin, I. M.; Leak, D. J.; Cowan, D. A.

2011-01-01

Summary The environmental value of sustainably producing bioproducts from biomass is now widely appreciated, with a primary target being the economic production of fuels such as bioethanol from lignocellulose. The application of thermophilic prokaryotes is a rapidly developing niche in this field, driven by their known catabolic versatility with lignocellulose‐derived carbohydrates. Fundamental to the success of this work has been the development of reliable genetic and molecular systems. These technical tools are now available to assist in the development of other (hyper)thermophilic strains with diverse phenotypes such as hemicellulolytic and cellulolytic properties, branched chain alcohol production and other ‘valuable bioproduct’ synthetic capabilities. Here we present an insight into the historical limitations, recent developments and current status of a number of genetic systems for thermophiles. We also highlight the value of reliable genetic methods for increasing our knowledge of thermophile physiology. We argue that the development of robust genetic systems is paramount in the evolution of future thermophilic based bioprocesses and make suggestions for future approaches and genetic targets that will facilitate this process. PMID:21310009

Evaluation of software sensors for on-line estimation of culture conditions in an Escherichia coli cultivation expressing a recombinant protein.

PubMed

Warth, Benedikt; Rajkai, György; Mandenius, Carl-Fredrik

2010-05-03

Software sensors for monitoring and on-line estimation of critical bioprocess variables have mainly been used with standard bioreactor sensors, such as electrodes and gas analyzers, where algorithms in the software model have generated the desired state variables. In this article we propose that other on-line instruments, such as NIR probes and on-line HPLC, should be used to make more reliable and flexible software sensors. Five software sensor architectures were compared and evaluated: (1) biomass concentration from an on-line NIR probe, (2) biomass concentration from titrant addition, (3) specific growth rate from titrant addition, (4) specific growth rate from the NIR probe, and (5) specific substrate uptake rate and by-product rate from on-line HPLC and NIR probe signals. The software sensors were demonstrated on an Escherichia coli cultivation expressing a recombinant protein, green fluorescent protein (GFP), but the results could be extrapolated to other production organisms and product proteins. We conclude that well-maintained on-line instrumentation (hardware sensors) can increase the potential of software sensors. This would also strongly support the intentions with process analytical technology and quality-by-design concepts. 2010 Elsevier B.V. All rights reserved.

Origins of cell-to-cell bioprocessing diversity and implications of the extracellular environment revealed at the single-cell level

DOE PAGES

Vasdekis, A. E.; Silverman, A. M.; Stephanopoulos, G.

2015-12-14

We probed the lipid expression dynamics of the oleaginous yeast Yarrowia Lipolytica. We observed that neutral lipid expression is sporadic. By performing single-cell analysis, we found that such noise emanates from the metabolic reaction level. Our results provide an alternative insight into the regulation and phenotypic variability of lipogenesis.

Bioprocessing of common beans in diets for tilapia: in vivo digestibility and antinutritional factors.

PubMed

Valdez-González, Francisco; Gutiérrez-Dorado, Roberto; Hernández-Llamas, Alfredo; García-Ulloa, Manuel; Sánchez-Magaña, Luís; Cuevas-Rodríguez, Breidy; Rodríguez-González, Hervey

2017-09-01

Bioprocessing of ingredients by solid-state fermentation is a low-cost technique for preparing diets. It is performed by adding microorganisms such as Rhizopus oligosporus to bean grains, achieving minimal degradation of nutrients and a significant improvement in digestibility. In particular, fermentation induces favorable changes in beans by reducing enzyme inhibitors, such as phytates and tannins. Fermentation significantly (P < 0.05) increased the protein content and digestibility of dry matter and protein compared with whole bean grains, and decreased the content of lipids, ash and phytic acid. Hardening did not have a significant (P > 0.05) effect on the chemical content of beans and digestibility of diets. The dehulled bean meal significantly (P < 0.05) increased protein and lipid content and digestibility of dry matter and protein of beans, and decreased fiber, ash and tannin content. The chemical content of beans and digestibility of ingredients compare favorably with those reported by other authors, indicating the benefits of fermentation and dehulling. We concluded that bean meal obtained from fermentation or dehulling represents a low-cost alternative for diets for tilapia. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Manufacturing recombinant proteins in kg-ton quantities using animal cells in bioreactors.

PubMed

De Jesus, Maria; Wurm, Florian M

2011-06-01

Mammalian cells in bioreactors as production host are the focus of this review. We wish to briefly describe today's technical status and to highlight emerging trends in the manufacture of recombinant therapeutic proteins, focusing on Chinese hamster ovary (CHO) cells. CHO cells are the manufacturing host system of choice for more than 70% of protein pharmaceuticals on the market [21]. The current global capacity to grow mammalian cells in bioreactors stands at about 0.5 million liters, whereby the largest vessels can have a working volume of about 20,000l. We are focusing in this article on the upstream part of protein manufacturing. Over the past 25 years, volumetric yields for recombinant cell lines have increased about 20-fold mainly as the result of improvements in media and bioprocess design. Future yield increases are expected to come from improved gene delivery methods, from improved, possibly genetically modified host systems, and from further improved bioprocesses in bioreactors. Other emerging trends in protein manufacturing that are discussed include the use of disposal bioreactors and transient gene expression. We specifically highlight here current research in our own laboratories. Copyright © 2011 Elsevier B.V. All rights reserved.

Observation of non-linear biomass-capacitance correlations: reasons and implications for bioprocess control.

PubMed

Maskow, Thomas; Röllich, Anita; Fetzer, Ingo; Yao, Jun; Harms, Hauke

2008-09-15

Electrical capacitance has been discussed as a real time measure for living biomass concentration in technical bioreactors such as brewery (fermentation) tanks. Commonly, a linear correlation between biomass concentration and capacitance is assumed. While following the growth and subsequent lipid formation of the yeast Arxula adeninivorans we observed non-linearity between biomass concentration and capacitance. Capacitance deviation from linearity coincided with incipient lipid formation and depended on the intracellular lipid content. As the extent of deviation between capacitance and biomass concentration was proportional to the lipid concentration, it was considered as a quantitative measure of intracellular product formation. The correlation between shifts in dielectric relaxation (summarized as characteristic frequency of the Cole-Cole equation) and lipid content could not be explained by interfacial polarization on the lipid droplets alone. However, the parameters of the Cole-Cole equation were found to be a clear indicator for different phases of growth and lipid production. Integrating all results in a redundancy analysis (RDA), we were able to accurately describe the formation of cellular lipid inclusions. Our measurements are thus potentially valuable as components of future bioprocess control strategies targeting intracellular products such as proteins or biopolyesters.

Partitioning in aqueous two-phase systems: Analysis of strengths, weaknesses, opportunities and threats.

PubMed

Soares, Ruben R G; Azevedo, Ana M; Van Alstine, James M; Aires-Barros, M Raquel

2015-08-01

For half a century aqueous two-phase systems (ATPSs) have been applied for the extraction and purification of biomolecules. In spite of their simplicity, selectivity, and relatively low cost they have not been significantly employed for industrial scale bioprocessing. Recently their ability to be readily scaled and interface easily in single-use, flexible biomanufacturing has led to industrial re-evaluation of ATPSs. The purpose of this review is to perform a SWOT analysis that includes a discussion of: (i) strengths of ATPS partitioning as an effective and simple platform for biomolecule purification; (ii) weaknesses of ATPS partitioning in regard to intrinsic problems and possible solutions; (iii) opportunities related to biotechnological challenges that ATPS partitioning may solve; and (iv) threats related to alternative techniques that may compete with ATPS in performance, economic benefits, scale up and reliability. This approach provides insight into the current status of ATPS as a bioprocessing technique and it can be concluded that most of the perceived weakness towards industrial implementation have now been largely overcome, thus paving the way for opportunities in fermentation feed clarification, integration in multi-stage operations and in single-step purification processes. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Excavation of attractor modules for nasopharyngeal carcinoma via integrating systemic module inference with attract method.

PubMed

Jiang, T; Jiang, C-Y; Shu, J-H; Xu, Y-J

2017-07-10

The molecular mechanism of nasopharyngeal carcinoma (NPC) is poorly understood and effective therapeutic approaches are needed. This research aimed to excavate the attractor modules involved in the progression of NPC and provide further understanding of the underlying mechanism of NPC. Based on the gene expression data of NPC, two specific protein-protein interaction networks for NPC and control conditions were re-weighted using Pearson correlation coefficient. Then, a systematic tracking of candidate modules was conducted on the re-weighted networks via cliques algorithm, and a total of 19 and 38 modules were separately identified from NPC and control networks, respectively. Among them, 8 pairs of modules with similar gene composition were selected, and 2 attractor modules were identified via the attract method. Functional analysis indicated that these two attractor modules participate in one common bioprocess of cell division. Based on the strategy of integrating systemic module inference with the attract method, we successfully identified 2 attractor modules. These attractor modules might play important roles in the molecular pathogenesis of NPC via affecting the bioprocess of cell division in a conjunct way. Further research is needed to explore the correlations between cell division and NPC.

Thermophilic lignocellulose deconstruction.

PubMed

Blumer-Schuette, Sara E; Brown, Steven D; Sander, Kyle B; Bayer, Edward A; Kataeva, Irina; Zurawski, Jeffrey V; Conway, Jonathan M; Adams, Michael W W; Kelly, Robert M

2014-05-01

Thermophilic microorganisms are attractive candidates for conversion of lignocellulose to biofuels because they produce robust, effective, carbohydrate-degrading enzymes and survive under harsh bioprocessing conditions that reflect their natural biotopes. However, no naturally occurring thermophile is known that can convert plant biomass into a liquid biofuel at rates, yields and titers that meet current bioprocessing and economic targets. Meeting those targets requires either metabolically engineering solventogenic thermophiles with additional biomass-deconstruction enzymes or engineering plant biomass degraders to produce a liquid biofuel. Thermostable enzymes from microorganisms isolated from diverse environments can serve as genetic reservoirs for both efforts. Because of the sheer number of enzymes that are required to hydrolyze plant biomass to fermentable oligosaccharides, the latter strategy appears to be the preferred route and thus has received the most attention to date. Thermophilic plant biomass degraders fall into one of two categories: cellulosomal (i.e. multienzyme complexes) and noncellulosomal (i.e. 'free' enzyme systems). Plant-biomass-deconstructing thermophilic bacteria from the genera Clostridium (cellulosomal) and Caldicellulosiruptor (noncellulosomal), which have potential as metabolic engineering platforms for producing biofuels, are compared and contrasted from a systems biology perspective. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

The emergence of Clostridium thermocellum as a high utility candidate for consolidated bioprocessing applications

PubMed Central

Akinosho, Hannah; Yee, Kelsey; Close, Dan; Ragauskas, Arthur

2014-01-01

First isolated in 1926, Clostridium thermocellum has recently received increased attention as a high utility candidate for use in consolidated bioprocessing (CBP) applications. These applications, which seek to process lignocellulosic biomass directly into useful products such as ethanol, are gaining traction as economically feasible routes toward the production of fuel and other high value chemical compounds as the shortcomings of fossil fuels become evident. This review evaluates C. thermocellum's role in this transitory process by highlighting recent discoveries relating to its genomic, transcriptomic, proteomic, and metabolomic responses to varying biomass sources, with a special emphasis placed on providing an overview of its unique, multivariate enzyme cellulosome complex and the role that this structure performs during biomass degradation. Both naturally evolved and genetically engineered strains are examined in light of their unique attributes and responses to various biomass treatment conditions, and the genetic tools that have been employed for their creation are presented. Several future routes for potential industrial usage are presented, and it is concluded that, although there have been many advances to significantly improve C. thermocellum's amenability to industrial use, several hurdles still remain to be overcome as this unique organism enjoys increased attention within the scientific community. PMID:25207268

Rational design of polymer-based absorbents: application to the fermentation inhibitor furfural.

PubMed

Nwaneshiudu, Ikechukwu C; Schwartz, Daniel T

2015-01-01

Reducing the amount of water-soluble fermentation inhibitors like furfural is critical for downstream bio-processing steps to biofuels. A theoretical approach for tailoring absorption polymers to reduce these pretreatment contaminants would be useful for optimal bioprocess design. Experiments were performed to measure aqueous furfural partitioning into polymer resins of 5 bisphenol A diglycidyl ether (epoxy) and polydimethylsiloxane (PDMS). Experimentally measured partitioning of furfural between water and PDMS, the more hydrophobic polymer, showed poor performance, with the logarithm of PDMS-to-water partition coefficient falling between -0.62 and -0.24 (95% confidence). In contrast, the fast setting epoxy was found to effectively partition furfural with the logarithm of the epoxy-to-water partition coefficient falling between 0.41 and 0.81 (95% confidence). Flory-Huggins theory is used to predict the partitioning of furfural into diverse polymer absorbents and is useful for predicting these results. We show that Flory-Huggins theory can be adapted to guide the selection of polymer adsorbents for the separation of low molecular weight organic species from aqueous solutions. This work lays the groundwork for the general design of polymers for the separation of a wide range of inhibitory compounds in biomass pretreatment streams.

The emergence of Clostridium thermocellum as a high utility candidate for consolidated bioprocessing applications

NASA Astrophysics Data System (ADS)

Ragauskas, Arthur; Akinosho, Hannah; Yee, Kelsey; Close, Dan

2014-08-01

First isolated in 1926, Clostridium thermocellum has recently received increased attention as a high utility candidate for use in consolidated bioprocessing applications. These applications, which seek to process lignocellulosic biomass directly into useful products such as ethanol, are gaining traction as economically feasible routes towards the production of fuel and other high value chemical compounds as the shortcomings of fossil fuels become evident. This review evaluates C. thermocellum’s role in this transitory process by highlighting recent discoveries relating to its genomic, transcriptomic, proteomic, and metabolomic responses to varying biomass sources, with a special emphasis placed on providing an overview of its unique, multivariate enzyme cellulosome complex and the role that this structure performs during biomass degradation. Both naturally evolved and genetically engineered strains are examined in light of their unique attributes and responses to various biomass treatment conditions, and the genetic tools that have been employed for their creation are presented. Several future routes for potential industrial usage are presented, and it is concluded that, although there have been many advances to significantly improve C. thermocellum’s amenability to industrial use, several hurdles still remain to be overcome as this unique organism enjoys increased attention within the scientific community.

Dual-emitting biosensors for glucose and glutamine from genertically engineered E. coli binding proteins

NASA Astrophysics Data System (ADS)

Tolosa, Leah; Ge, Xudong; Kostov, Yordan; Lakowicz, Joseph R.; Rao, Govind

2003-07-01

Glucose is the major source of carbon, and glutamine is the major source of nitrogen in cell culture media. Thus, glucose and glutamine monitoring are important in maintaining optimal conditions in industrial bioprocesses. Here we report reagentless glucose and glutamine sensors using the E. coli glucose binding protein (GBP) and the glutamine binding protein (GlnBP). Both of these proteins are derived from the permease system of the gram-negative bacteria. The Q26C variant of GBP was labeled at the 26-position with anilino-naphthalene sulfonate (ANS), while the S179C variant of GlnBP was labeled at the 179-position with acrylodan. The ANS and acrylodan emissions are quenched in the presence of glucose and glutamine, respectively. The acrylodan-labeled GlnBP was labeled at the N-terminal with ruthenium bis-(2,2"-bipyridyl)-1,10-phenanthroline-9-isothiocyanate. The ruthenium acts as a non-responsive long-lived reference. The apparent binding constant, Kd", of 8.0 μM glucose was obtained from the decrease in intensity of ANS in GBP. The reliability of the method in monitoring glucose during yeast fermentation was determined by comparison with the YSI Biochemistry Analyzer. The apparent binding constant, Kd", of 0.72 μM glutamine was calculated from the ratio of emission intensities of acrylodan and ruthenium (I515/I610) in GlnBP. The presence of the long-lived ruthenium allowed for modulation sensing at lower frequencies (1-10 MHz) approaching an accuracy of +/- 0.02 μM. The conversion of the GBP into a similar ratiometric sensor was described.

Renaissance of protein crystallization and precipitation in biopharmaceuticals purification.

PubMed

Dos Santos, Raquel; Carvalho, Ana Luísa; Roque, A Cecília A

The current chromatographic approaches used in protein purification are not keeping pace with the increasing biopharmaceutical market demand. With the upstream improvements, the bottleneck shifted towards the downstream process. New approaches rely in Anything But Chromatography methodologies and revisiting former techniques with a bioprocess perspective. Protein crystallization and precipitation methods are already implemented in the downstream process of diverse therapeutic biological macromolecules, overcoming the current chromatographic bottlenecks. Promising work is being developed in order to implement crystallization and precipitation in the purification pipeline of high value therapeutic molecules. This review focuses in the role of these two methodologies in current industrial purification processes, and highlights their potential implementation in the purification pipeline of high value therapeutic molecules, overcoming chromatographic holdups. Copyright © 2016 Elsevier Inc. All rights reserved.

Phosphoketolase pathway engineering for carbon-efficient biocatalysis

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

Henard, Calvin Andrew; Freed, Emily Frances; Guarnieri, Michael Thomas

2015-12-01

Recent advances in metabolic engineering have facilitated the development of microbial biocatalysts capable of producing an array of bio-products, ranging from fuels to drug molecules. These bio-products are commonly generated through an acetyl-CoA intermediate, which serves as a key precursor in the biological conversion of carbon substrates. Moreover, conventional biocatalytic upgrading strategies proceeding through this route are limited by low carbon efficiencies, in large part due to carbon losses associated with pyruvate decarboxylation to acetyl-CoA. Bypass of pyruvate decarboxylation offers a means to dramatically enhance carbon yields and, in turn, bioprocess economics. Here, we discuss recent advances and prospects formore » employing the phosphoketolase pathway for direct biosynthesis of acetyl-CoA from carbon substrates, and phosphoketolase-based metabolic engineering strategies for carbon efficient biocatalysis.« less

Advanced modelling, monitoring, and process control of bioconversion systems

NASA Astrophysics Data System (ADS)

Schmitt, Elliott C.

Production of fuels and chemicals from lignocellulosic biomass is an increasingly important area of research and industrialization throughout the world. In order to be competitive with fossil-based fuels and chemicals, maintaining cost-effectiveness is critical. Advanced process control (APC) and optimization methods could significantly reduce operating costs in the biorefining industry. Two reasons APC has previously proven challenging to implement for bioprocesses include: lack of suitable online sensor technology of key system components, and strongly nonlinear first principal models required to predict bioconversion behavior. To overcome these challenges batch fermentations with the acetogen Moorella thermoacetica were monitored with Raman spectroscopy for the conversion of real lignocellulosic hydrolysates and a kinetic model for the conversion of synthetic sugars was developed. Raman spectroscopy was shown to be effective in monitoring the fermentation of sugarcane bagasse and sugarcane straw hydrolysate, where univariate models predicted acetate concentrations with a root mean square error of prediction (RMSEP) of 1.9 and 1.0 g L-1 for bagasse and straw, respectively. Multivariate partial least squares (PLS) models were employed to predict acetate, xylose, glucose, and total sugar concentrations for both hydrolysate fermentations. The PLS models were more robust than univariate models, and yielded a percent error of approximately 5% for both sugarcane bagasse and sugarcane straw. In addition, a screening technique was discussed for improving Raman spectra of hydrolysate samples prior to collecting fermentation data. Furthermore, a mechanistic model was developed to predict batch fermentation of synthetic glucose, xylose, and a mixture of the two sugars to acetate. The models accurately described the bioconversion process with an RMSEP of approximately 1 g L-1 for each model and provided insights into how kinetic parameters changed during dual substrate fermentation with diauxic growth. Model predictive control (MPC), an advanced process control strategy, is capable of utilizing nonlinear models and sensor feedback to provide optimal input while ensuring critical process constraints are met. Using the microorganism Saccharomyces cerevisiae, a commonly used microorganism for biofuel production, and work performed with M. thermoacetica, a nonlinear MPC was implemented on a continuous membrane cell-recycle bioreactor (MCRB) for the conversion of glucose to ethanol. The dilution rate was used to control the ethanol productivity of the system will maintaining total substrate conversion above the constraint of 98%. PLS multivariate models for glucose (RMSEP 1.5 g L-1) and ethanol (RMSEP 0.4 g L-1) were robust in predicting concentrations and a mechanistic kinetic model built accurately predicted continuous fermentation behavior. A setpoint trajectory, ranging from 2 - 4.5 g L-1 h-1 for productivity was closely tracked by the fermentation system using Raman measurements and an extended Kalman filter to estimate biomass concentrations. Overall, this work was able to demonstrate an effective approach for real-time monitoring and control of a complex fermentation system.

Surface characterization of pretreated and microbial-treated populus cross-sections

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

Tolbert, Allison K.

The first objective of this thesis is to illustrate the advantages of surface characterization in biomass utilization studies. The second objective is to gain insight into the workings of potential consolidated bioprocessing microorganisms on the surface of poplar samples. The third objective is to determine the impact biomass recalcitrance has on enzymatic hydrolysis and microbial fermentation in relation to the surface chemistry.

A polysaccharide isolated from the liquid culture of Lentinus edodes (shiitake) mushroom mycelia containing black rice bran protects mice against Salmonellosis through up-regulation of the Th1 immune reaction

USDA-ARS?s Scientific Manuscript database

The present study investigated the antibacterial effect of a bioprocessed polysaccharide (BPP) isolated from Lentinus edodes liquid mycelial culture supplemented with black rice bran against murine salmonellosis. BPP was not bactericidal in vitro, but did, however stimulate uptake of the bacteria i...

Wolfiporia cocos : a potential agent for composting of bioprocessing Douglas-fir wood treated with copper-based preservatives

Treesearch

Rodney. De Groot; Bessie. Woodward

1998-01-01

In laboratory experiments, Douglas-fir wood blocks that were treated with copper- based wood preservatives were challenged with two wood decay fungi known to be tolerant of copper. Factors influencing the amount of decay, as determined by loss of weight in the test blocks, were preservative, then fungus. Within those combinations, the relative importance of...

Design and validation of a consistent and reproducible manufacture process for the production of clinical-grade bone marrow-derived multipotent mesenchymal stromal cells.

PubMed

Codinach, Margarita; Blanco, Margarita; Ortega, Isabel; Lloret, Mireia; Reales, Laura; Coca, Maria Isabel; Torrents, Sílvia; Doral, Manel; Oliver-Vila, Irene; Requena-Montero, Miriam; Vives, Joaquim; Garcia-López, Joan

2016-09-01

Multipotent mesenchymal stromal cells (MSC) have achieved a notable prominence in the field of regenerative medicine, despite the lack of common standards in the production processes and suitable quality controls compatible with Good Manufacturing Practice (GMP). Herein we describe the design of a bioprocess for bone marrow (BM)-derived MSC isolation and expansion, its validation and production of 48 consecutive batches for clinical use. BM samples were collected from the iliac crest of patients for autologous therapy. Manufacturing procedures included: (i) isolation of nucleated cells (NC) by automated density-gradient centrifugation and plating; (ii) trypsinization and expansion of secondary cultures; and (iii) harvest and formulation of a suspension containing 40 ± 10 × 10(6) viable cells. Quality controls were defined as: (i) cell count and viability assessment; (ii) immunophenotype; and (iii) sterility tests, Mycoplasma detection, endotoxin test and Gram staining. A 3-week manufacturing bioprocess was first designed and then validated in 3 consecutive mock productions, prior to producing 48 batches of BM-MSC for clinical use. Validation included the assessment of MSC identity and genetic stability. Regarding production, 139.0 ± 17.8 mL of BM containing 2.53 ± 0.92 × 10(9) viable NC were used as starting material, yielding 38.8 ± 5.3 × 10(6) viable cells in the final product. Surface antigen expression was consistent with the expected phenotype for MSC, displaying high levels of CD73, CD90 and CD105, lack of expression of CD31 and CD45 and low levels of HLA-DR. Tests for sterility, Mycoplasma, Gram staining and endotoxin had negative results in all cases. Herein we demonstrated the establishment of a feasible, consistent and reproducible bioprocess for the production of safe BM-derived MSC for clinical use. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Production of EV71 vaccine candidates.

PubMed

Chong, Pele; Hsieh, Shih-Yang; Liu, Chia-Chyi; Chou, Ai-Hsiang; Chang, Jui-Yuan; Wu, Suh-Chin; Liu, Shih-Jen; Chow, Yen-Hung; Su, Ih-Jen; Klein, Michel

2012-12-01

Enterovirus 71 (EV71) is now recognized as an emerging neurotropic virus in Asia and with Coxsackie virus (CV) it is the other major causative agent of hand-foot-mouth diseases (HFMD). Effective medications and/or prophylactic vaccines against HFMD are urgently needed. From a scientific (the feasibility of bioprocess, immunological responses and potency in animal challenge model) and business development (cost of goods) points of view, we in this review address and discuss the pros and cons of different EV71 vaccine candidates that have been produced and evaluated in animal models. Epitope-based synthetic peptide vaccine candidates containing residues 211-225 of VP1 formulated with Freund's adjuvant (CFA/IFA) elicited low EV71 virus neutralizing antibody responses, but were protective in the suckling mouse challenge model. Among recombinant EV71 subunits (rVP1, rVP2 and rVP3) expressed in E. coli, purified and formulated with CFA/IFA, only VP1 elicited mouse antibody responses with measurable EV71-specific virus neutralization titers. Immunization of mice with either a DNA plasmid containing VP1 gene or VP1 expressed in Salmonella typhimurium also generated neutralizing antibody responses and protected animals against a live EV71 challenge. Recombinant EV71 virus-like particles (rVLP) produced from baculovirus formulated either with CFA/IFA or alum elicited good virus neutralization titers in both mice and non-human primates, and were found to be protective in the suckling mouse EV71 challenge model. Synthetic peptides or recombinant EV71 subunit vaccines (rVP1 and rVLP) formulated in alum were found to be poorly immunogenic in rabbits. Only formalin-inactivated (FI) EV71 virions formulated in alum elicited cross-neutralizing antibodies against different EV71 genotypes in mice, rabbits and non-human primates but induced weak neutralizing responses against CAV16. From a regulatory, economic and market acceptability standpoint, FI-EV71 virion vaccines are the most promising candidates and are currently being evaluated in human clinical trials. We further describe and analyze some new bioprocesses technologies that have great potential applications in EV71 vaccine development. This review also demonstrates the opportunities and challenges that the Asian vaccine industry faces today.

A carbon dioxide stripping model for mammalian cell culture in manufacturing scale bioreactors.

PubMed

Xing, Zizhuo; Lewis, Amanda M; Borys, Michael C; Li, Zheng Jian

2017-06-01

Control of carbon dioxide within the optimum range is important in mammalian bioprocesses at the manufacturing scale in order to ensure robust cell growth, high protein yields, and consistent quality attributes. The majority of bioprocess development work is done in laboratory bioreactors, in which carbon dioxide levels are more easily controlled. Some challenges in carbon dioxide control can present themselves when cell culture processes are scaled up, because carbon dioxide accumulation is a common feature due to longer gas-residence time of mammalian cell culture in large scale bioreactors. A carbon dioxide stripping model can be used to better understand and optimize parameters that are critical to cell culture processes at the manufacturing scale. The prevailing carbon dioxide stripping models in literature depend on mass transfer coefficients and were applicable to cell culture processes with low cell density or at stationary/cell death phase. However, it was reported that gas bubbles are saturated with carbon dioxide before leaving the culture, which makes carbon dioxide stripping no longer depend on a mass transfer coefficient in the new generation cell culture processes characterized by longer exponential growth phase, higher peak viable cell densities, and higher specific production rate. Here, we present a new carbon dioxide stripping model for manufacturing scale bioreactors, which is independent of carbon dioxide mass transfer coefficient, but takes into account the gas-residence time and gas CO 2 saturation time. The model was verified by CHO cell culture processes with different peak viable cell densities (7 to 12 × 10 6  cells mL -1 ) for two products in 5,000-L and 25,000-L bioreactors. The model was also applied to a next generation cell culture process to optimize cell culture conditions and reduce carbon dioxide levels at manufacturing scale. The model provides a useful tool to understand and better control cell culture carbon dioxide profiles for process development, scale up, and characterization. Biotechnol. Bioeng. 2017;114: 1184-1194. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.