Showing papers on "Bioprocess published in 2009"

Bioprocessing of plant cell cultures for mass production of targeted compounds.

Abstract: More than a century has passed since the first attempt to cultivate plant cells in vitro. During this time, plant cell cultures have become increasingly attractive and cost-effective alternatives to classical approaches for the mass production of plant-derived metabolites. Furthermore, plant cell culture is the only economically feasible way of producing some high-value metabolites (e.g., paclitaxel) from rare and/or threatened plants. This review summarizes recent advances in bioprocessing aspects of plant cell cultures, from callus culture to product formation, with particular emphasis on the development of suitable bioreactor configurations (e.g., disposable reactors) for plant cell culture-based processes; the optimization of bioreactor culture environments as a powerful means to improve yields; bioreactor operational modes (fed-batch, continuous, and perfusion); and biomonitoring approaches. Recent trends in downstream processing are also considered.

Microbiological and engineering aspects of biohydrogen production.

Abstract: Dramatically rising oil prices and increasing awareness of the dire environmental consequences of fossil fuel use, including startling effects of climate change, are refocusing attention worldwide on the search for alternative fuels. Hydrogen is poised to become an important future energy carrier. Renewable hydrogen production is pivotal in making it a truly sustainable replacement for fossil fuels, and for realizing its full potential in reducing greenhouse gas emissions. One attractive option is to produce hydrogen through microbial fermentation. This process would use readily available wastes as well as presently unutilized bioresources, including enormous supplies of agricultural and forestry wastes. These potential energy sources are currently not well exploited, and in addition, pose environmental problems. However, fuels are relatively low value products, placing severe constraints on any production process. Therefore, means must be sought to maximize yields and rates of hydrogen production while at the same time minimizing energy and capital inputs to the bioprocess. Here we review the various attributes of the characterized hydrogen producing bacteria as well as the preparation and properties of mixed microflora that have been shown to convert various substrates to hydrogen. Factors affecting yields and rates are highlighted and some avenues for increasing these parameters are explored. On the engineering side, we review the potential waste pre-treatment technologies and discuss the relevant bioprocess parameters, possible reactor configurations, including emerging technologies, and how engineering design-directed research might provide insight into the exploitation of the significant energy potential of biomass resources.

Ultra scale-down prediction using microwell technology of the industrial scale clarification characteristics by centrifugation of mammalian cell broths.

Abstract: This article describes how a combination of an ultra scale-down (USD) shear device feeding a microwell centrifugation plate may be used to provide a prediction of how mammalian cell broth will clarify at scale. In particular a method is described that is inherently adaptable to a robotic platform and may be used to predict how the flow rate and capacity (equivalent settling area) of a centrifuge and the choice of feed zone configuration may affect the solids carry over in the supernatant. This is an important consideration as the extent of solids carry over will determine the required size and lifetime of a subsequent filtration stage or the passage of fine particulates and colloidal material affecting the performance and lifetime of chromatography stages. The extent of solids removal observed in individual wells of a microwell plate during centrifugation is shown to correlate with the vertical and horizontal location of the well on the plate. Geometric adjustments to the evaluation of the equivalent settling area of individual wells (Sigma(M)) results in an improved prediction of solids removal as a function of centrifuge capacity. The USD centrifuge settling characteristics need to be as for a range of equivalent flow rates as may be experienced at an industrial scale for a machine of different shear characteristics in the entry feed zone. This was shown to be achievable with two microwell-plate based measurements and the use of varying fill volumes in the microwells to allow the rapid study of a fivefold range of equivalent flow rates (i.e., at full scale for a particular industrial centrifuge) and the effect of a range of feed configurations. The microwell based USD method was used to examine the recovery of CHO-S cells, prepared in a 5 L reactor, at different points of growth and for different levels of exposure to shear post reactor. The combination of particle size distribution measurements of the cells before and after shear and the effect of shear on the solids remaining after centrifugation rate provide insight into the state of the cells throughout the fermentation and the ease with which they and accumulated debris may be removed by continuous centrifugation. Hence bioprocess data are more readily available to help better integrate cell culture and cell removal stages and resolve key bioprocess design issues such as choice of time of harvesting and the impact on product yield and contaminant carry over. Operation at microwell scale allows data acquisition and bioprocess understanding over a wide range of operating conditions that might not normally be achieved during bioprocess development.

Nuclear based expression of genes for production of biofuels and process co-products in algae

Abstract: Various embodiments provide, for example, vectors, expression cassettes, and cells useful for transgenic expression of nucleic acid sequences In various embodiments, vectors can contain nuclear-based sequences of unicellular photosynthetic bioprocess organisms for the production of food- and feed-stuffs, oils, biofuels, starches, raw materials, pharmaceuticals or fine chemicals

Squalene versus ergosterol formation using Saccharomyces cerevisiae: combined effect of oxygen supply, inoculum size, and fermentation time on yield and selectivity of the bioprocess.

Abstract: The dynamics of two wild type strains of Saccharomyces cerevisiae (BY4741 and EGY48) that vary in the ability to produce sterols were compared in batch cultures under different aeration conditions. Poor supply of oxygen enhanced selectivity of the bioprocess in favor of squalene formation. Optimization of inoculum size and fermentation time arranged according to a central composite statistical design revealed significant differences between the strains in terms of yield and productivity. Experimental verification showed that an optimized bioprocess under semianaerobic conditions is competitive with regard to those reported in the literature. Maximum squalene yield and productivity were, respectively, 2967.6 ± 118.7 μg/L of culture medium and 104 ± 4.2 μg/Lh for BY4741 and 3129 ± 109.5 μg/L of culture medium and 155.9 ± 5.5 μg/Lh for EGY48. The prospect of developing high-purity squalene preparations that meet food safety regulation demands is expected to attract the interest of the food industry.

Step change in the efficiency of centrifugation through cell engineering: co-expression of Staphylococcal nuclease to reduce the viscosity of the bioprocess feedstock.

Abstract: Cell engineering to enable step change improvements in bioprocessing can be directed at targets other than increasing product titer. The physical properties of the process suspension such as viscosity, for example, have a major impact on various downstream processing unit operations. The release of chromosomal DNA during homogenization of Escherichia coli and its influence on viscosity is well-recognized. In this current article we demonstrate co-expression of Staphylococcus aureus nuclease in E. coli to reduce viscosity through auto-hydrolysis of nucleic acids. Viscosity reduction of up to 75% was achieved while the particle size distribution of cell debris was maintained approximately constant (d(50) = 0.5-0.6 mu m). Critically, resultant step change improvements to the clarification performance under disc-stack centrifugation conditions are shown. The cell-engineered nuclease matched or exceeded the viscosity reduction performance seen with the addition of exogenous nuclease removing the expense and validation issues associated with such additions to a bioprocess. The resultant material dramatically altered performance in scale-down mimics of continuous disc-stack centrifugation. Laboratory scale data indicated that a fourfold reduction in the settling area of a disc-stack centrifuge can be expected due to a less viscous process stream achieved through nuclease co-expression with a recombinant protein.

Optical Instrumentation for Bioprocess Monitoring

Abstract: In this chapter the optical sensors for oxygen, pH, carbondioxide and optical density (OD) which are essential for bioprocess monitoring are introduced, their measurement principles are explained and their realization and applications are shown. In addition sensors for ethanol and GFP are presented. With the exception of the optical density sensor all others employ certain fluorophores that are sensitive to the designated parameter. These fluorophores along with their optical properties, the sensing mechanisms and their mathematical formulations are described. An important part of this chapter covers the development of the optoelectronic hardware for low cost systems that are able to measure the fluorescence lifetime and fluorescence intensity ratio. The employment of these probes in the bioprocess monitoring is demonstrated in different fermentation examples.

Using DNA as a drug-Bioprocessing and delivery strategies

Abstract: DNA may take a leading role in a future generation of blockbuster therapeutics. DNA has inherent advantages over other biomolecules such as protein, RNA and virus-like particles including safety, production simplicity and higher stability at ambient temperatures. Vaccination is the principal measure for preventing influenza and reducing the impact of pandemics; however, vaccines take up to 8-9 months to produce, and the global production capacity is woefully low. With production times as short as 2 weeks, improved safety and stability, bioprocess engineering developments, and the ability to perform numerous therapeutic roles, DNA has the potential to meet the demands of emerging and existing diseases. DNA is experiencing sharp growths in demand as indicated by its use in gene therapy trials and DNA vaccine related patents. Of particular interest for therapeutic use is plasmid DNA (pDNA), a form of non-genomic DNA that makes use of cellular machinery to express proteins or antigens. The production stages of fermentation and downstream purification are considered in this article. Forward looking approaches to purifying and delivering DNA are reported, including affinity chromatography and nasal inhalation. The place that pDNA may take in the preparation for and protection against pandemics is considered. If DNA therapeutics and vaccines prove to be effective, the ultimate scale of production will be huge which shall require associated bioprocess engineering research and development for purification of this large, unique biomolecule.

Computational Intelligence Techniques for Bioprocess Modelling, Supervision and Control

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Yeast cell factory: fishing for the best one or engineering it?

Abstract: When today scientists and bioprocess engineers look at a Microbial Cell Factory for the production of a protein or a metabolite of commercial or research interest, they think at the microorganism of choice first from a (i) molecular, then from a (ii) metabolic and finally from a (iii) process point of view. Analyses and manipulations of the pathway(s) involved in the synthesis of the desired product and how this pathway(s) interacts with the overall cell functions and activities are indeed steps required to obtain high yield of the product (g of compound per g of substrate), high production (g/l) and high productivity (g/l/h). Conceptually, the whole set of biochemical reactions that take place in the microbial cell factory should be considered. This is valid for processes involving natural and/or recombinant products in wild type and/or engineered hosts.

In situ magnetic separation for extracellular protein production

Abstract: A new approach for in situ product removal from bioreactors is presented in which high-gradient magnetic separation is used. This separation process was used for the adsorptive removal of proteases secreted by Bacillus licheniformis. Small, non-porous bacitracin linked magnetic adsorbents were employed directly in the broth during the fermentation, followed by in situ magnetic separation. Proof of the concept was first demonstrated in shake flask culture, then scaled up and applied during a fed batch cultivation in a 3.7 L bioreactor. It could be demonstrated that growth of B. licheniformis was not influenced by the in situ product removal step. Protease production also remained the same after the separation step. Furthermore, degradation of the protease, which followed first order kinetics, was reduced by using the method. Using a theoretical modeling approach, we could show that protease yield in total was enhanced by using in situ magnetic separation. The process described here is a promising technique to improve overall yield in bio production processes which are often limited due to weak downstream operations. Potential limitations encountered during a bioprocess can be overcome such as product inhibition or degradation. We also discuss the key points where research is needed to implement in situ magnetic separation in industrial production. Biotechnol. Bioeng. 2009;102: 535–545. © 2008 Wiley Periodicals, Inc.

Edible Oil Cakes

Abstract: The very sustainability of the growing bioprocess industry depends on the progressive reduction of expensive nutrient inputs into fermentation media. The use of cheap agricultural and food-processing by-products such as oil cakes, as feedstock is highly favored so as to improve the commercial feasibility of bioprocess technology. Due to stringent nutritional requirements of these edible oil cakes as animal feed, there is considerable interest in using them as substrates in the fermentation industry. This chapter will provide an impetus to further research in this area enabling better utilization of edible oil cakes as sources of protein and carbohydrates for economic viability of the bioprocess industry.

Preservation and composition of bioprocess algae for production of lipids, seedstock, and feed

Abstract: The present invention relates to compositions and uses of a novel Dunaliella salina HT04 microorganism. In addition, the present invention relates to novel methods for culturing harvesting, preservation, production of algae seedstock and uses thereof.

Utilisation of glycerol to platform chemicals within the biorefinery concept: a case for succinate production

Abstract: In this study, a biorefinery concept is introduced for the production of platform chemicals by utilising the by-products of the biodiesel industry. An unstructured kinetic model for the bacterial growth of Actinobacillus succinogenes, which is our chosen biocatalyst, is proposed. The model describes cell growth and considers both substrate and product inhibition. The main product chosen here is succinic acid and by-products like acetate, formate and ethanol have insignificant low concentrations. Experiments on different initial glycerol concentrations at the same environmental conditions are carried out and simulation studies are conducted using the proposed model. Parametric values are estimated based on experimental results. Prior to that, the main environmental factors that affect the bioprocess are examined and beneficial conditions in terms of yield, final succinic acid concentration and productivity are assessed by a factorial experimental procedure.

The use of plant cell biotechnology for the production of phytochemicals

Abstract: In this chapter, we bring together up-to-date information concerning plant cell biotechnology and its applications. Because plants contain many valuable secondary metabolites that are useful as drug sources (pharmaceuticals), natural fungicides and insecticides (agrochemicals), natural food flavorings and coloring agents (nutrition), and natural fragrances and oils (cosmetics), the production of these phytochemicals through plant cell factories is an alternative and concurrent approach to chemical synthesis. It also provides an alternative to extraction of these metabolites from overcollected plant species. While plant cell cultures provide a viable system for the production of these compounds in laboratories, its application in industry is still limited due to frequently low yields of the metabolites of interest or the feasibility of the bioprocess. A number of factors may contribute to the efficiency of plant cells to produce desired compounds. Genetic stability of cell lines, optimization of culture condition, tissue-diverse vs. tissue-specific site-specific localization and biosynthesis of metabolites, organelle targeting, and inducible vs. constitutive expression of specific genes should all be taken into consideration when designing a plant-based production system. The major aims for engineering secondary metabolism in plant cells are to increase the content of desired secondary compounds, to lower the levels of undesirable compounds, and to introduce novel compound production into specific plants. Recent achievements have also been made in altering various metabolic pathways by use of specific genes encoding biosynthetic enzymes or genes that encode regulatory proteins. Gene and metabolic engineering approaches are now being used to successfully achieve highest possible levels of value-added natural products in plant cell cultures. Applications through functional genomics and systems biology make plant cell biotechnology much more straightforward and more attractive than through previous, more traditional approaches.

Application of agent-based system for bioprocess description and process improvement

Abstract: Modeling plays an important role in bioprocess development for design and scale-up. Predictive models can also be used in biopharmaceutical manufacturing to assist decision-making either to maintain process consistency or to identify optimal operating conditions. To predict the whole bioprocess performance, the strong interactions present in a processing sequence must be adequately modeled. Traditionally, bioprocess modeling considers process units separately, which makes it difficult to capture the interactions between units. In this work, a systematic framework is developed to analyze the bioprocesses based on a whole process understanding and considering the interactions between process operations. An agent-based approach is adopted to provide a flexible infrastructure for the necessary integration of process models. This enables the prediction of overall process behavior, which can then be applied during process development or once manufacturing has commenced, in both cases leading to the capacity for fast evaluation of process improvement options. The multi-agent system comprises a process knowledge base, process models, and a group of functional agents. In this system, agent components co-operate with each other in performing their tasks. These include the description of the whole process behavior, evaluating process operating conditions, monitoring of the operating processes, predicting critical process performance, and providing guidance to decision-making when coping with process deviations. During process development, the system can be used to evaluate the design space for process operation. During manufacture, the system can be applied to identify abnormal process operation events and then to provide suggestions as to how best to cope with the deviations. In all cases, the function of the system is to ensure an efficient manufacturing process. The implementation of the agent-based approach is illustrated via selected application scenarios, which demonstrate how such a framework may enable the better integration of process operations by providing a plant-wide process description to facilitate process improvement.

The Potential of Mid- and Near-infrared Spectroscopy for Reliable Monitoring of Bioprocesses

Abstract: The processes involved in bioreactors are complex and a detailed monitoring of the chemical composition of gaseous, liquid and solid matter involved in the bioprocess is of crucial importance for efficient operation. However, only a few parameters can be reliably monitored in real time with commercially available instrumentation, but there is a growing demand and need for effective and efficient process monitoring tools. Infrared (IR) spectroscopy has proved to be a powerful method for the study of various biomedical and bioanalytical applications. In this paper, applications of this analytical method for bioprocess monitoring have been discussed by reviewing the relevant literature from the last ten years. Furthermore, recent advances in our laboratory in the field of bio-reactor monitoring by use of mid-IR attenuated total reflection (ATR) spectroscopy and the development of mid-IR sensor systems for continuous biofluid monitoring based on transmission spectroscopy have also been addressed.

High-gain observers for estimation of kinetics in a nonlinear bioprocess

Abstract: The paper addresses the problem of on-line estimation of kinetic rates inside a nonlinear bioprocess. The analyzed biotechnological process is a lipase production process that takes place into a Fed-batch Bioreactor. The lipase production process is highly nonlinear and, furthermore, the available on-line measurements are lack and the reaction kinetics is not perfectly known. The unknown kinetic parameters of the bioprocess are estimated by using a nonlinear observer, based on high-gain approach. The estimation scheme does not require any model for the kinetic rates. The tuning of the proposed observers is reduced to the calibration of a single parameter. Numerical simulations are included in order to test the behavior and the performance of the proposed observers.

A systems approach to plant bioprocess optimization

Abstract: A dynamic model for plant cell metabolism was used as a basis for a rational analysis of plant production potential in in vitro cultures. The model was calibrated with data from 3-L bioreactor cultures. A dynamic sensitivity analysis framework was developed to analyse the response curves of secondary metabolite production to metabolic and medium perturbations. Simulation results suggest that a straightforward engineering of cell metabolism or medium composition might only have a limited effect on productivity. To circumvent the problem of the dynamic allocation of resources between growth and production pathways, the sensitivity analysis framework was used to assess the effect of stabilizing intracellular nutrient concentrations. Simulations showed that a stabilization of intracellular glucose and nitrogen reserves could lead to a 116% increase in the specific production of secondary metabolites compared with standard culture protocol. This culture strategy was implemented experimentally using a perfusion bioreactor. To stabilize intracellular concentrations, adaptive medium feeding was performed using model mass balances and estimations. This allowed for a completely automated culture, with controlled conditions and pre-defined decision making algorithm. The proposed culture strategy leads to a 73% increase in specific production and a 129% increase in total production, as compared with a standard batch culture protocol. The sensitivity analysis on a mathematical model of plant metabolism thus allowed producing new insights on the links between intracellular nutritional management and cell productivity. The experimental implementation was also a significant improvement on current plant bioprocess strategies.