Showing papers on "Bioprocess published in 2011"

Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered Escherichia coli

Abstract: One approach to reducing the costs of advanced biofuel production from cellulosic biomass is to engineer a single microorganism to both digest plant biomass and produce hydrocarbons that have the properties of petrochemical fuels. Such an organism would require pathways for hydrocarbon production and the capacity to secrete sufficient enzymes to efficiently hydrolyze cellulose and hemicellulose. To demonstrate how one might engineer and coordinate all of the necessary components for a biomass-degrading, hydrocarbon-producing microorganism, we engineered a microorganism naive to both processes, Escherichia coli, to grow using both the cellulose and hemicellulose fractions of several types of plant biomass pretreated with ionic liquids. Our engineered strains express cellulase, xylanase, beta-glucosidase, and xylobiosidase enzymes under control of native E. coli promoters selected to optimize growth on model cellulosic and hemicellulosic substrates. Furthermore, our strains grow using either the cellulose or hemicellulose components of ionic liquid-pretreated biomass or on both components when combined as a coculture. Both cellulolytic and hemicellulolytic strains were further engineered with three biofuel synthesis pathways to demonstrate the production of fuel substitutes or precursors suitable for gasoline, diesel, and jet engines directly from ionic liquid-treated switchgrass without externally supplied hydrolase enzymes. This demonstration represents a major advance toward realizing a consolidated bioprocess. With improvements in both biofuel synthesis pathways and biomass digestion capabilities, our approach could provide an economical route to production of advanced biofuels.

High level secretion of cellobiohydrolases by Saccharomyces cerevisiae.

Abstract: Background The main technological impediment to widespread utilization of lignocellulose for the production of fuels and chemicals is the lack of low-cost technologies to overcome its recalcitrance. Organisms that hydrolyze lignocellulose and produce a valuable product such as ethanol at a high rate and titer could significantly reduce the costs of biomass conversion technologies, and will allow separate conversion steps to be combined in a consolidated bioprocess (CBP). Development of Saccharomyces cerevisiae for CBP requires the high level secretion of cellulases, particularly cellobiohydrolases.

An industrial perspective on bioreactor scale-down: What we can learn from combined large-scale bioprocess and model fluid studies

Abstract: For industrial bioreactor design, operation, control and optimization, the scale-down approach is often advocated to efficiently generate data on a small scale, and effectively apply suggested improvements to the industrial scale. In all cases it is important to ensure that the scale-down conditions are representative of the real large-scale bioprocess. Progress is hampered by limited detailed and local information from large-scale bioprocesses. Complementary to real fermentation studies, physical aspects of model fluids such as air-water in large bioreactors provide useful information with limited effort and cost. Still, in industrial practice, investments of time, capital and resources often prohibit systematic work, although, in the end, savings obtained in this way are trivial compared to the expenses that result from real process disturbances, batch failures, and non-flyers with loss of business opportunity. Here we try to highlight what can be learned from real large-scale bioprocess in combination with model fluid studies, and to provide suitable computation tools to overcome data restrictions. Focus is on a specific well-documented case for a 30-m(3) bioreactor. Areas for further research from an industrial perspective are also indicated.

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

Abstract: 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.

Overcoming substrate inhibition during biological treatment of monoaromatics: recent advances in bioprocess design

Abstract: The biological removal of monoaromatic compounds from contaminated environments, usually arising from industrial activity, is challenging because of the inherent toxicity of these compounds to microorganisms, particularly at the concentrations that can be encountered in industrial waste streams. A wide range of bioprocess designs have been proposed and tested with the aim of achieving high removal efficiencies, with varying degrees of technical success, and potential for practical implementation. This review reports on the progress on variations of well-known themes made in the last 3–4 years, as well as new bioprocess technologies that address the cytotoxicity of monoaromatics directly. Areas for further research are also proposed.

Assessment of pretreatments and enzymatic hydrolysis of wheat straw as a sugar source for bioprocess industry.

Abstract: Environmental concerns and rising oil prices have led to development of biofuels from crop residue lignocelluloses, among which wheat straw is an important feedstock used in leading commercial bioethanol processes. Lignocellulose is structured in a way that makes direct bioconversion of biomass into sugars by hydrolytic enzymes difficult and unfeasible, requiring a pretreatment step. Common biomass pretreatment technologies are assessed for potential application in obtaining fermentable sugars of wheat straw. Current outlook, challenges and opportunities on enzymatic hydrolysis of lignocellulose are also presented. Copyright © 2011 International Energy and Environment Foundation All rights reserved.

Metabolic Engineering of Bacteria

Abstract: Yield and productivity are critical for the economics and viability of a bioprocess. In metabolic engineering the main objective is the increase of a target metabolite production through genetic engineering. Metabolic engineering is the practice of optimizing genetic and regulatory processes within cells to increase the production of a certain substance. In the last years, the development of recombinant DNA technology and other related technologies has provided new tools for approaching yields improvement by means of genetic manipulation of biosynthetic pathway. Industrial microorganisms like Escherichia coli, Actinomycetes, etc. have been developed as biocatalysts to provide new or to optimize existing processes for the biotechnological production of chemicals from renewable plant biomass. The factors like oxygenation, temperature and pH have been traditionally controlled and optimized in industrial fermentation in order to enhance metabolite production. Metabolic engineering of bacteria shows a great scope in industrial application as well as such technique may also have good potential to solve certain metabolic disease and environmental problems in near future.

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.

Heterologous Protein Expression

Abstract: Heterologous protein expression has been the foundation of the industrial and pharmaceutical biotechnology industries over the past 30 years. There are a wide variety of production platforms now commercially available and an increasing number under development encompassing different host cells, expression systems, and methods for utilizing the genetic instructions, as well as bioprocessing strategies that take advantage of the particular combinations to maximize volumetric productivity and product quality. It is generally accepted that there is no single, universal heterologous expression system and bioprocess strategy that will work for all proteins and that each protein product needs to be considered individually, based on the specific protein product, unique quality specifications, and intended application. Here, we describe some of the different cell-based and cell-free protein expression platforms for bioreactor-based production of heterologous proteins, an overview of how they work and how they have been used, and the potential advantages and disadvantages when compared with alternative approaches.

Fully automated production of potential Malaria vaccines with Pichia pastoris in integrated processing

Abstract: Here, we have studied the setup of an integrated bioprocess for the production of artificial Malaria vaccine candidates with Pichia pastoris. Production of pharmaceutically relevant proteins such as vaccines has high demands regarding protein processing in the bioreactor and for subsequent purification. To implement this challenging protein expression process, a highly instrumented bioreactor was configured for repeated fed batch cultivations and supplemented with an at-line monitoring of the target protein production via HPLC. The integration of a fast in situ purification of the sensitive products using an expanded bed adsorption for a sequential integrated bioprocess allows cyclic product separation. Thus, a fully automated production of artificial malaria vaccines was achieved.

Raman spectroscopy for bioprocess operations

Abstract: A method of characterizing a multi-component mixture for use in a bioprocess operation that includes providing a multi-component mixture standard with pre-determined amounts of known components; performing a Raman Spectroscopy analysis on the multi-component mixture standard; providing a multi- component test mixture from the bioprocess operation; performing a Raman Spectroscopy analysis on the multi-component test mixture; and comparing the analysis of the multi-component mixture standard and the multi-component test mixture to characterize the multi-component test mixture. In one embodiment, the multi-component mixture standard and the multi-component test mixture both comprise one or more of, at least two, at least three of, or each of, a polysaccharide (e.g. sucrose or mannitol), an amino acid (e.g., L-arginine, L-histidine or L-omithine), a surfactant (e.g. polysorbate 80) and a pH buffer (e.g., a citrate formulation).

Antibody production in Bacillus megaterium: Strategies and physiological implications of scaling from microtiter plates to industrial bioreactors

Abstract: Bacillus megaterium was used as an alternative high potential microbial production system for the production of antibody fragment D1.3 scFv. The aim of the study was to follow a holistic optimization approach from medium screening in small scale microtiter platforms, gaining deeper process understanding in the bioreactor scale and implementing advanced process strategies at larger scales (5–100 L). Screening and optimization procedures were supported by statistical design of experiments and a genetic algorithm approach. The process control relied on a soft-sensor for biomass estimation to establish a μ-oscillating time-dependent fed-batch strategy. Several cycles of growth phases and production phases, equal to starving phases, were performed in one production. Flow cytometry was used to monitor and characterize the dynamics of secretion and cell viability. Besides the biosynthesis of the product, secretion was optimized by an appropriate medium design considering different carbon sources, metal ions, (NH 4 ) 2 SO 4 , and inductor concentrations. For bioprocess design, an adapted oscillating fed-batch strategy was conceived and successfully implemented at an industrially relevant scale of 100 L. In comparison to common methods for controlling fed-batch profiles, the developed process delivered increased overall productivities. Thereby measured process parameters such as growth stagnation or productivity fluctuations were directly linked to single cell or population behavior leading to a more detailed process understanding. Above all, the importance of single cell analysis as key scale-free tool to characterize and optimize recombinant protein production is highlighted, since this can be applied to all development stages independently of the cultivation platform.

Optimization of biomass production of a mutant of Yarrowia lipolytica with an increased lipase activity using raw glycerol

Abstract: The yeast Yarrowia lipolytica accumulates oils and is able to produce extracellular lipases when growing in different carbon sources including glycerol, the principal by-product of the biodiesel industry. In this study, biomass production of a novel mutant strain of Y. lipolytica was statistically optimized by Response Surface Methodology in media containing biodiesel-derived glycerol as main carbon source. This strain exhibited distinctive morphological and fatty acid profile characteristics, and showed an increased extracellular lipase activity. An organic source of nitrogen and the addition of 1.0 g/l olive oil were necessary for significant lipase production. Plackett-Burman and Central Composite Statistical Designs were employed for screening and optimization of fermentation in shaken flasks cultures, and the maximum values obtained were 16.1 g/l for biomass and 12.2 Units/ml for lipase, respectively. Optimized batch bioprocess was thereafter scaled in aerated bioreactors and the values reached for lipase specific activity after 95 % of the glycerol had been consumed, were three-fold higher than those obtained in shaken flasks cultures. A sustainable bioprocess to obtain biomass and extracellular lipase activity was attained by maximizing the use of the by-products of biodiesel industry.

Single cell analysis applied to antibody fragment production with Bacillus megaterium: development of advanced physiology and bioprocess state estimation tools.

Abstract: Single cell analysis for bioprocess monitoring is an important tool to gain deeper insights into particular cell behavior and population dynamics of production processes and can be very useful for discrimination of the real bottleneck between product biosynthesis and secretion, respectively. Here different dyes for viability estimation considering membrane potential (DiOC2(3), DiBAC4(3), DiOC6(3)) and cell integrity (DiBAC4(3)/PI, Syto9/PI) were successfully evaluated for Bacillus megaterium cell characterization. It was possible to establish an appropriate assay to measure the production intensities of single cells revealing certain product secretion dynamics. Methods were tested regarding their sensitivity by evaluating fluorescence surface density and fluorescent specific concentration in relation to the electronic cell volume. The assays established were applied at different stages of a bioprocess where the antibody fragment D1.3 scFv production and secretion by B. megaterium was studied. It was possible to distinguish between live, metabolic active, depolarized, dormant, and dead cells and to discriminate between high and low productive cells. The methods were shown to be suitable tools for process monitoring at single cell level allowing a better process understanding, increasing robustness and forming a firm basis for physiology-based analysis and optimization with the general application for bioprocess development.

Bioprocess and Bioreactor: Next Generation Technology for Production of Potential Plant-based Antidiabetic and Antioxidant Molecules

Abstract: Globally, diabetes and obesity are two of the most common metabolic diseases of the 21st century. Increasingly, not only adults but children and adolescents are being affected. New approaches are needed to prevent and treat these disorders and to reduce the impact of associated disease-related complications. Industrial-scale production using plant-root cultures can produce quantities and quality of inexpensive bioactive small molecules with nutraceutical and pharmaceutical properties. Using this approach, and targeting these diseases, a next generation approach to tackling this emerging global health crisis may be developed. Adventitious roots cultured in bioreactors under controlled and reproducible conditions have been shown effective for production of natural products. The liquid-phase airlift bioreactor in particular has been used successfully for culturing roots on an industrial-scale and thus may provide an economical production platform for expressing promising plant-based antidiabetic and antioxidant molecules. This review focuses on a next-generation, scalable, bioprocessing approach for adventitious and hairy root cultures that are a pesticide-free, seasonally-independent, plant-based source of three molecules that have shown promise for the therapeutic management of diabetes and obesity: corosolic acid, resveratrol and ginsenosides.

Bioprocess and microbe engineering for total carbon utilization in biofuel production

Abstract: Some aspects of this invention provide methods and bioreactors for converting a carbon source into a lipid. In some embodiments, lipid production is carried out in an aerobic fermentor and carbon dioxide generated during lipid production is converted into a carbon substrate by CO2 fixation in an anaerobic fermentor. In some embodiments, the carbon substrate generated by CO2 fixation is used as the carbon source for lipid production, thus achieving total carbon utilization in lipid production.

Sustainable Bioprocess Evaluation for Xylanase Production by Isolated Aspergillus terreus and Aspergillus fumigatus Under Solid - State Fermentation Using Oil Palm Empty Fruit Bunch Fiber

Abstract: Xylanase bioprocess by isolated Aspergillus terreus and Aspergillus fumigatus strains under solid-state fermentation using oil palm empty fruit bunch fiber as substrate was investigated. The productions of xylanase enzyme in these fungal strains were influenced by bioprocess parameters. Though enzyme production was noticed in the wide range of pH (3.0 – 8.0), effective xylanase production observed at pH 6.0 and 7.0 by A. terreus and A. fumigatus respectively. Similarly, enzyme titer values improved with increase in moisture content (70%) and inoculum concentration of 2.0 and 1.5 ml (1 x 106 spore solution per ml) for A. terreus and A. fumigatus respectively. Particle size mediated variation also noticed where 2.0 0.7 and 2.8- 2.0 mm (15,990 and 14,563 U/g) was effective for xylanase production by A. terreus and A. fumigatus, respectively. Whereas, supplementation of xylose and fructose to A. terreus and A. fumigatus were enhanced the xylanase production to 32,074 and 25,038 U/g, respectively. Furthermore, addition of 0.6 g of sodium nitrate and 0.4 g of ammonium chloride had resulted 39,136 and 35,380 U/g of xylanase titers by using A. terreus and A. fumigatus respectively. Over all xylanase enzyme productivity was improved to the tune of 3.0 and 2.8 folds with A. terreus and A. fumigatus after bioprocess optimization, respectively.

An integrated scale‐down plant for optimal recombinant enzyme production by Pichia pastoris

Abstract: An integrated bioprocess was created in a scale-down production plant by developing a two-stage enzyme production process with Pichia pastoris, containing a cell-breeding reactor and a production reactor in combination with a three-stage downstream process. To harvest the secreted enzymes, a disc separator and a cross-flow microfiltration clear the broth from the cells. Purification with hydrophobic interaction chromatography removes other proteins, concentrates the product, and prepares the enzyme solution for lyophilization. Fully automated and broad observable multi-stage parallel process courses have been developed using industrial process control systems and at-line measurements for enzyme concentration and enzyme activity. Optimal process conditions were found by application of Design of Experiments (DoE) for the production process.

Simultaneous production and partitioning of heterologous polyketide and isoprenoid natural products in an Escherichia coli two-phase bioprocess

Abstract: Natural products have long served as rich sources of drugs possessing a wide range of pharmacological activities. The discovery and development of natural product drug candidates is often hampered by the inability to efficiently scale and produce a molecule of interest, due to inherent qualities of the native producer. Heterologous biosynthesis in an engineering and process-friendly host emerged as an option to produce complex natural products. Escherichia coli has previously been utilized to produce complex precursors to two popular natural product drugs, erythromycin and paclitaxel. These two molecules represent two of the largest classes of natural products, polyketides and isoprenoids, respectively. In this study, we have developed a platform E. coli strain capable of simultaneous production of both product precursors at titers greater than 15 mg l−1. The utilization of a two-phase batch bioreactor allowed for very strong in situ separation (having a partitioning coefficient of greater than 5,000), which would facilitate downstream purification processes. The system developed here could also be used in metagenomic studies to screen environmental DNA for natural product discovery and preliminary production experiments.

Theoretical approach to modelling and analysis of the bioprocess with product inhibition and impulse effect

Abstract: This work presents the first mathematical model of a bioprocess with product inhibition and impulse effect. To begin with, an exemplary mathematical bioprocess model with product inhibition and impulse effect is formulated. Then, according to the model, the analysis of bioprocess stability is presented. The article expresses the product oscillation period, which provides the precise feeding time frame for the regulator bioprocess to achieve an equivalent stable output as that of a bioprocess with impulse effect in the same production environment. Moreover, in this work, the optimization of the production process with respect to the tunable parameters is investigated, and analytical expressions of their optimal values are provided. Numerical simulations using biological data are presented to illustrate the main results.

Bioprocess monitoring by marker gene analysis

Abstract: The optimization and the scale up of industrial fermentation processes require an efficient and possibly comprehensive analysis of the physiology of the production system throughout the process development. Furthermore, to ensure a good quality control of established bioprocesses, on-line analysis techniques for the determination of marker gene expression are of interest to monitor the productivity and the safety of bioprocesses. A prerequisite for such analyses is the knowledge of genes, the expression of which is critical either for the productivity or for the performance of the bioprocess. This work reviews marker genes that are specific indicators for stress- and nutrient-limitation conditions or for the physiological status of the bacterial production hosts Bacillus subtilis, Bacillus licheniformis and Escherichia coli. The suitability of existing gene expression analysis techniques for bioprocess monitoring is discussed. Analytical approaches that enable a robust and sensitive determination of selected marker mRNAs or proteins are presented.

On-line high performance liquid chromatography measurements of extracellular metabolites in an aerobic batch yeast ( Saccharomyces cerevisiae ) culture

Abstract: We constructed a bioprocess environment enabling automatic sampling from a bioreactor combined with a compact on-line high performance liquid chromatography (HPLC) unit. This setup allowed us to measure extracellular glucose, ethanol, glycerol, and acetate concentrations automatically at 5 min intervals during the cultivation. This environment also provides mechanical measurement of the optical density (OD) of cells and enables us to collect and store (−35°C) samples for further off-line analyses. Among the available devices, the performance of the sampling-analysis unit is by far the best with regard to speed and number of analytes. Both the sampling and analysis phases are easily controlled by software; thus, providing a unique environment to perform various bioprocess activity tasks, whether they would be cell line screening or optimisation of conditions for growth and productivity. Complex research set-ups can be created and continuous automated measurements empower long-term cultivations with a time series. We provide evidence for the applicability of this environment by performing three comparable batch cultivations with Saccharomyces cerevisiae yeast and show that both the on-line sampling and analysis modes produce reliable data for further use in the monitoring and controlling of bioprocesses. On-line data provided new insight into the dynamics of the diauxic shift during aerobic glucose batch cultivation. When cell growth and carbon dioxide production ceased for the first time during the diauxic shift, acetate accumulation and consumption of the remaining glucose below 0.15 g/L continued to occur for 1 h. At the same time, glycerol and ethanol began to be consumed. Samples were also collected during cultivation for later analysis of intracellular metabolites and to collect more valuable information about metabolism.