Showing papers on "Bioprocess published in 2010"

Encyclopedia of industrial biotechnology : bioprocess, bioseparation, and cell technology

Abstract: The Encyclopedia of Industrial Biotechnology combines Wiley’s acclaimed Encyclopedia of Bioprocess Technology and the Encyclopedia of Cell Technology in order to create a single resource and gateway to the many areas of industrial biotechnology for students, researchers, and technologists. In addition to revising and updating existing articles, the new Encyclopedia of Industrial Biotechnology has been greatly expanded to cover important areas of pharmaceutical and biologics bioprocess technology, including: • Production of vaccines

Engineering cellulolytic ability into bioprocessing organisms

Abstract: Lignocellulosic biomass is an abundant renewable feedstock for sustainable production of commodities such as biofuels. The main technological barrier that prevents widespread utilization of this resource for production of commodity products is the lack of low-cost technologies to overcome the recalcitrance of lignocellulose. Organisms that hydrolyse the cellulose and hemicelluloses in biomass and produce a valuable product such as ethanol at a high rate and titre would significantly reduce the costs of current biomass conversion technologies. This would allow steps that are currently accomplished in different reactors, often by different organisms, to be combined in a consolidated bioprocess (CBP). The development of such organisms has focused on engineering naturally cellulolytic microorganisms to improve product-related properties or engineering non-cellulolytic organisms with high product yields to become cellulolytic. The latter is the focus of this review. While there is still no ideal organism to use in one-step biomass conversion, several candidates have been identified. These candidates are in various stages of development for establishment of a cellulolytic system or improvement of product-forming attributes. This review assesses the current state of the art for enabling non-cellulolytic organisms to grow on cellulosic substrates.

On-line infrared spectroscopy for bioprocess monitoring

Abstract: One of the major aims of bioprocess engineering is the real-time monitoring of important process variables. This is the basis of precise process control and is essential for high productivity as well as the exact documentation of the overall production process. Infrared spectroscopy is a powerful analytical technique to analyze a wide variety of organic compounds. Thus, infrared sensors are ideal instruments for bioprocess monitoring. The sensors are non-invasive, have no time delay due to sensor response times, and have no influence on the bioprocess itself. No sampling is necessary, and several components can be analyzed simultaneously. In general, the direct monitoring of substrates, products, metabolites, as well as the biomass itself is possible. In this review article, insights are provided into the different applications of infrared spectroscopy for bioprocess monitoring and the complex data interpretation. Different analytical techniques are presented as well as example applications in different areas.

Escherichia coli Binary Culture Engineered for Direct Fermentation of Hemicellulose to a Biofuel

Abstract: Metabolic engineering has created several Escherichia coli biocatalysts for production of biofuels and other useful molecules. However, the inability of these biocatalysts to directly use polymeric substrates necessitates costly pretreatment and enzymatic hydrolysis prior to fermentation. Consolidated bioprocessing has the potential to simplify the process by combining enzyme production, hydrolysis, and fermentation into a single step but requires a fermenting organism to multitask by producing both necessary enzymes and target molecules. We demonstrate here a binary strategy for consolidated bioprocessing of xylan, a complex substrate requiring six hemicellulases for complete hydrolysis. An integrated modular approach was used to design the two strains to function cooperatively in the process of transforming xylan into ethanol. The first strain was engineered to coexpress two hemicellulases. Recombinant enzymes were secreted to the growth medium by a method of lpp deletion with over 90% efficiency. Secreted enzymes hydrolyzed xylan into xylooligosaccharides, which were taken in by the second strain, designed to use the xylooligosaccharides for ethanol production. Cocultivation of the two strains converted xylan hemicellulose to ethanol with a yield about 55% of the theoretical value. Inclusion of other three hemicellulases improved the ethanol yield to 70%. Analysis of the culture broth showed that xylooligosaccharides with four or more xylose units were not utilized, suggesting that improving the use of higher xyloogligomers should be the focus in future efforts. This is the first demonstration of an engineered binary culture for consolidated bioprocessing of xylan. The modular design should allow the strategy to be adopted for a broad range of biofuel and biorefinery products.

Novel approach of high cell density recombinant bioprocess development: Optimisation and scale-up from microlitre to pilot scales while maintaining the fed-batch cultivation mode of E. coli cultures

Abstract: Bioprocess development of recombinant proteins is time consuming and laborious as many factors influence the accumulation of the product in the soluble and active form. Currently, in most cases the developmental line is characterised by a screening stage which is performed under batch conditions followed by the development of the fed-batch process. Performing the screening already under fed-batch conditions would limit the amount of work and guarantee that the selected favoured conditions also work in the production scale. Here, for the first time, high throughput multifactorial screening of a cloning library is combined with the fed-batch technique in 96-well plates, and a strategy is directly derived for scaling to bioreactor scale. At the example of a difficult to express protein, an RNase inhibitor, it is demonstrated that screening of various vector constructs and growth conditions can be performed in a coherent line by (i) applying a vector library with promoters and ribosome binding sites of different strength and various fusion partners together with (ii) an early stage use of the fed-batch technology. It is shown that the EnBase® technology provides an easy solution for controlled cultivation conditions in the microwell scale. Additionally the high cell densities obtained provide material for various analyses from the small culture volumes. Crucial factors for a high yield of the target protein in the actual case were (i) the fusion partner, (ii) the use of of a mineral salt medium together with the fed-batch technique, and (iii) the preinduction growth rate. Finally, it is shown that the favorable conditions selected in the microwell plate and shake flask scales also work in the bioreactor. Cultivation media and culture conditions have a major impact on the success of a screening procedure. Therefore the application of controlled cultivation conditions is pivotal. The consequent use of fed-batch conditons from the first screening phase not only shortens the developmental line by guarantying that the selected conditions are relevant for the scale up, but in our case also standard batch cultures failed to select the right clone or conditions at all.

Bioprocess Control in Microscale: Scalable Fermentations in Disposable and User-Friendly Microfluidic Systems

Abstract: Background The efficiency of biotechnological production processes depends on selecting the best performing microbial strain and the optimal cultivation conditions. Thus, many experiments have to be conducted, which conflicts with the demand to speed up drug development processes. Consequently, there is a great need for high-throughput devices that allow rapid and reliable bioprocess development. This need is addressed, for example, by the fiber-optic online-monitoring system BioLector which utilizes the wells of shaken microtiter plates (MTPs) as small-scale fermenters. To further improve the application of MTPs as microbioreactors, in this paper, the BioLector technology is combined with microfluidic bioprocess control in MTPs. To realize a user-friendly system for routine laboratory work, disposable microfluidic MTPs are utilized which are actuated by a user-friendly pneumatic hardware.

Advancements in the Production of Secondary Metabolites

Abstract: Plant cell culture systems represent a potential renewable source of valuable medicinal compounds, flavours, fragrances, and colorants, which cannot be produced by microbial cells or chemical synthesis. The evolving commercial importance of the secondary metabolites has in recent years resulted in a great interest, in secondary metabolism, and particularly in the possibility to alter the production of bioactive metabolites by means of cell culture technology. The principle advantage of this technology is that it may provide continuous, reliable source of plant pharmaceuticals and could be used for the large scale culture of plant cells from which these metabolite can be extracted. Plant cell and tissue cultures hold great promise for controlled production of myriad of useful secondary metabolites on demand. The current yield and productivity cannot fulfill the commercial goal of plant cell-based bioprocess for the production of most secondary metabolites. In order to stretch the boundary, recent advances, new directions and opportunities in plant cell based processes are being critically examined.

Bioprocessing Data for the Production of Marine Enzymes

Abstract: 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.

Optimized bioprocess for production of fructofuranosidase by recombinant Aspergillus niger

Abstract: A comprehensive approach of bioprocess design at various levels was used to optimize microbial production of extracellular fructofuranosidase, important as biocatalyst to derive fructooligosaccharides with broad application in food or pharmaceutical industry. For production, the recombinant strain Aspergillus niger SKAn1015 was used, which expresses the fructofuranosidase encoding gene suc1 under control of a strong constitutive promoter. In a first screening towards an optimized medium, glucose, nitrate, Fe2+, and Mn2+ were identified as beneficial for production. A minimal medium with optimized concentration of these key nutrients, obtained by central composite design experiments and quadratic modelling, provided a threefold increased fructofuranosidase activity in the culture supernatant (400 U/mL) as compared to the originally described medium. Utilizing the optimized medium, the process was then transferred from shake flask into a fed-batch-operated bioreactor. Hereby, the intended addition of talc microparticles allowed engineering the morphology of A. niger into a highly active mycelial form, which strongly boosted production. Fructofuranosidase production was highly specific as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The secreted enzyme activity of 2,800 U/mL, corresponding to about 3 g/L of fructofuranosidase, achieved by the microparticle-enhanced fed-batch process, is tenfold higher than that of any other process reported so far, so that the presented bioprocess strategy appears as a milestone towards future industrial fructofuranosidase production.

Systems biotechnology – Rational whole-cell biocatalyst and bioprocess design

Abstract: Microbial biocatalysis has emerged to a standard technology in the food, feed, pharmaceutical, and fine chemical industries. Since microorganisms are optimized by nature to maximize survival and typically not for the high-level accumulation of any sort of product, effective engineering strategies are required to satisfy the growing demand of new, economically competitive, and environmentally friendly products and processes. Random mutagenesis and subsequent selection used to be successful strateges, despite the fact that genetic changes were often not clear and include potentially unwanted detrimental alterations. The recent advances in systems biology research allow a genome-scale characterization of the complex composition and function of cells. We consider it important to recognize that the comprehensive understanding of a bioprocess is not restricted to the quantitative description of cellular functions but, in addition, includes analyses and evaluations on the reaction and process level. Using today's unprecedented toolbox, new whole-cell catalyst design targets can be systematically identified and the properties of microbial hosts can be specifically fine-tuned according to the requirements of a given bioprocess. This review proposes an integrated strategy as a holistic knowledge-driven approach for systems biotechnology and summarizes recent advances on the basis of selected examples.

Recent advances in bioreactor engineering

Abstract: A bioreactor is the core of biological processes. To design an appropriate bioreactor system for a particular bioprocess, intensive studies on the biological system, such as cell growth, metabolism, genetic manipulation, and protein or other product expression are needed to understand the cells’ requirement on their physical and chemical environments. It is also necessary to control and optimize the bioreactor environment via operating variables in order to favor the desired functions of cells and achieve cost-effective large-scale manufacture. This article briefly describes fundamental design principles and new types of bioreactors such as centrifugal impeller and wave bioreactors. Bioreactor operation factors and modes including mixing, oxygen supply, shear force, fed-batch, and perfusion cultures are discussed. The trends in bioreactor engineering are also briefly shown.

Milliliter-scale stirred tank reactors for the cultivation of microorganisms.

Abstract: This review focuses on recent developments in the field of miniaturized stirred tank bioreactors for application in high-throughput bioprocess development. Different reactor concepts and their potential for parallel bioprocess development are discussed. A detailed description of important engineering state variables, their measurement at small-scale and their implication for scale-up and scale-down of bioprocesses are given. Examples of two different parallel cultivations at small-scale are presented: one with Escherichia coli and the other one with the filamentous microorganism Streptomyces tendae. It is shown that results obtained in parallelized milliliter-scale stirred tank reactors can be scaled up to the laboratory- and/or pilot-scale in a highly reliable manner. This helps to reduce development times for bioprocesses significantly. Finally, directions for future research are presented.

Rapid whole monoclonal antibody analysis by mass spectrometry: An ultra scale-down study of the effect of harvesting by centrifugation on the post-translational modification profile.

Abstract: With the trend towards the generation and production of increasing numbers of complex biopharmaceutical (protein based) products, there is an increased need and requirement to characterize both the product and production process in terms of robustness and reproducibility. This is of particular importance for products from mammalian cell culture which have large molecular structures and more often than not complex post-translational modifications (PTMs) that can impact the efficacy, stability and ultimately the safety of the final product. It is therefore vital to understand how the operating conditions of a bioprocess affect the distribution and make up of these PTMs to ensure a consistent quality and activity in the final product. Here we have characterized a typical bioprocess and determined (a) how the time of harvest from a mammalian cell culture and, (b) through the use of an ultra scale-down mimic how the nature of the primary recovery stages, affect the distribution and make up of the PTMs observed on a recombinant IgG(4) monoclonal antibody. In particular we describe the use of rapid whole antibody analysis by mass spectrometry to analyze simultaneously the changes that occur to the cleavage of heavy chain C-terminal lysine residues and the glycosylation pattern, as well as the presence of HL dimers. The time of harvest was found to have a large impact upon the range of glycosylation patterns observed, but not upon C-terminal lysine cleavage. The culture age had a profound impact on the ratio of different glycan moieties found on antibody molecules. The proportion of short glycans increased (e.g., (G0F)(2) 20-35%), with an associated decrease in the proportion of long glycans with culture age (e.g., (G2F)(2) 7-4%, and G1F/G2F from 15.2% to 7.8%). Ultra scale-down mimics showed that subsequent processing of these cultures did not change the post-translational modifications investigated, but did increase the proportion of half antibodies present in the process stream. The combination of ultra scale-down methodology and whole antibody analysis by mass spectrometry has demonstrated that the effects of processing on the detailed molecular structure of a monoclonal antibody can be rapidly determined early in the development process. In this study we have demonstrated this analysis to be applicable to critical process design decisions (e.g., time of harvest) in terms of achieving a desired molecular structure, but this approach could also be applied as a selection criterion as to the suitability of a platform process for the preparation of a new drug candidate. Also the methodology provides means for bioprocess engineers to predict at the discovery phase how a bioprocess will impact upon the quality of the final product.

Extremophile-inspired strategies for enzymatic biomass saccharification.

Abstract: Domestic ethanol production in the USA relies on starch feedstocks using a first generation bioprocess. Enzymes that contribute to this industry remain of critical value in new and established markets as commodity additives and for in planta production. A transition to non‐food feedstocks is both desirable and essential to enable larger scale production. This objective would relieve dependence on foreign oil and strengthen the national economy. Feedstocks derived from corn stover, wheat straw, perennial grasses and timber require pretreatment to increase the accessibility of the cellulosic and hemicellulosic substrates to commodity enzymes for saccharification, which is followed by fermentation‐based conversion of monosaccharides to ethanol. Hot acid pretreatment is the industrial standard method used to achieve deconstruction of lignocellulosic biomass. Therefore, enzymes that tolerate both acid and heat may contribute toward the improvement of lignocellulosic biomass processing. These enzymes are produc...

Bioprocessing of Proximally Analyzed Wheat Straw for Enhanced Cellulase Production through Process Optimization with Trichodermaviride under SSF

Abstract: The purpose of the present work was to study the production and process parameters optimization for the synthesis of cellulase from Trichoderma viride in solid state fermentation (SSF) using an agricultural wheat straw as substrates; as fungal conversion of lignocellulosic biomass for cellulase production is one among the major increasing demand for various biotechnological applications. An optimization of process parameters is a necessary step to get higher yield of product. Several kinetic parameters like pretreatment, extraction solvent, substrate concentration, initial moisture content, pH, incubation temperature and inoculum size were optimized for enhanced production of third most demanded industrially important cellulase. The maximum cellulase enzyme activity 398.10±2.43 μM/mL/min was achieved when proximally analyzed lignocellulosic substrate wheat straw inocubated at 2% HCl as pretreatment tool along with distilled water as extraction solvent, 3% substrate concentration 40% moisture content with optimum pH 5.5 at 45°C incubation temperature and 10% inoculum size. Keywords—Cellulase, Lignocellulosic residue, Process optimization, Proximal analysis, SSF, Trichoderma viride.

Combined Use of Fluorescent Dyes and Flow Cytometry To Quantify the Physiological State of Pichia pastoris during the Production of Heterologous Proteins in High-Cell-Density Fed-Batch Cultures

Abstract: Matching both the construction of a recombinant strain and the process design with the characteristics of the target protein has the potential to significantly enhance bioprocess performance, robustness, and reproducibility. The factors affecting the physiological state of recombinant Pichia pastoris Mut (methanol utilization-positive) strains and their cell membranes were quantified at the individual cell level using a combination of staining with fluorescent dyes and flow cytometric enumeration. Cell vitalities were found to range from 5 to 95% under various process conditions in high-cell-density fed-batch cultures, with strains producing either porcine trypsinogen or horseradish peroxidase extracellularly. Impaired cell vitality was observed to be the combined effect of production of recombinant protein, low pH, and high cell density. Vitality improved when any one of these stress factors was excluded. At a pH value of 4, which is commonly applied to counter proteolysis, recombinant strains exhibited severe physiological stress, whereas strains without heterologous genes were not affected. Physiologically compromised cells were also found to be increasingly sensitive to methanol when it accumulated in the culture broth. The magnitude of the response varied when different reporters were combined with either the native AOX1 promoter or its d6* variant, which differ in both strength and regulation. Finally, the quantitative assessment of the physiology of individual cells enables the implementation of innovative concepts in bioprocess development. Such concepts are in contrast to the frequently used paradigm, which always assumes a uniform cell population, because differentiation between the individual cells is not possible with methods commonly used.

Foam formation and control in bioreactors

Abstract: The generation of foam during the course of a bioprocess remains a major technological challenge to be resolved and needs further investigation. The foaming tendency of the cultivation media used in bioreactors induces various direct,that is microbial cells stripping and contamination, as well as indirect adverse effects, that is modification of the properties of the medium subsequent to the addition of chemical antifoam leading to toxic effects at the level of the microbial metabolism and fouling of the downstream processing equipment. In this work, the mechanisms leading to foam formation are described at the molecular level (adsorption of surface-active molecule at the gas–liquid interface), and also at the process level. In view of the potential foam-associated problems, special attention is given to the level of the compatibility of the intensification of the gas–liquid operation in bioreactors. Due to these considerations, both the chemical and mechanical antifoam techniques have been evolved. Numerous new antifoam formulations and original combinations of chemical antifoams have been especially designed in order to meet the specific requirements of bioprocesses. Original mechanical techniques to prevent foam formation in bioreactors have been elaborated from combined knowledge involving the fluid dynamics of gas–liquid dispersion and interfacial processes (e.g. the “stirring as foam disruption” concept). Keywords: antifoam; mechanical defoamer; gas–liquid dispersion; bioprocess; mixing