Showing papers in "Biotechnology Progress in 1995"

In pursuit of the optimal fed-batch process for monoclonal antibody production

Abstract: Fed-batch culture currently represents the most attractive choice for large scale production for monoclonal antibodies (MAbs), due to its operational simplicity, reliability, and flexibility for implementation in multipurpose facilities. Development of highly productive cell lines, maximization of cell culture longevity, and maintenance of high specific antibody secretion rates through genetic engineering techniques, nutrition supplementation, waste product minimization, and control of environmental conditions are important for the design of high-yield fed-batch processes. Initially simple supplementation protocols have evolved into sophisticated serum-free multi-nutrient feeds that result in MAb titers on the order of 1-2 g/L. Limited research has been published to date on the effect of various culture parameters on potentially important quality issues, such as MAb glycosylation and stability. Although most fed-batch protocols to date have relied on relatively simple control schemes, increasingly sophisticated algorithms must be applied in order to take full advantage of the potentially additive effects of manipulating nutrient and environmental parameters to maximize fed-batch process productivity.

Membrane Chromatography—An Integrative Concept in the Downstream Processing of Proteins

Abstract: Membrane chromatography was introduced as an integrative technology for the purification of proteins several years ago. The main feature of chromatographic separations based on membranes is the absence of pore diffusion, which is the main transport resistance in conventional column chromatography using porous particles. This is achieved by attaching the active ligands to the inner surface of the through-pores of microfiltration membranes, where mass transport takes place mainly by convective flow, thus reducing the transport limitations from pore to film diffusion. In combination with a low pressure drop across a membrane, very high volumetric flows are possible in a membrane-based purification step; thus, the time requirement of a complete chromatographic cycle is reduced. The method is characterized by fast processing at preserved or even increased resolution compared to standard chromatography on particulate materials. Since its introduction, many successful applications of membrane chromatography have been described. Additionally, theoretical analysis has been performed, which has significantly helped to understand present and visualize future applications of this technology. In this review, the fundamental theoretical considerations from the actual literature are described, and then the applications of membrane-based separation processes are presented. We have attempted to demonstrate the usefulness of this new technology in the purification of many interesting proteins.

Potential for Bioremediation of Xenobiotic Compounds by the White-Rot Fungus Phanerochaete chrysosporium

Abstract: The white-rot fungi produce an unusual enzyme system, characterized by a specialized group of peroxidases, that catalyzes the degradation of the complex plant polymer lignin. This ligninolytic system shows a high degree of nonspecificity and oxidizes a very large variety of compounds in addition to lignin. Among these compounds are numerous environmental pollutants. Thus, the white-rot fungi show considerable promise as bioremediation agents for use in the restoration of environments contaminated by xenobiotic molecules. One white-rot fungus, Phanerochaete chrysosporium, has been studied in great detail with regard to ligninolytic enzymes and the degradation of anthropogenic chemicals. It has been widely promoted as a bioremediation agent. This article examines literature concerning the degradation of xenobiotic compounds by Phanerochaete chrysosporium and attempts to critically assess this organism's real potential as a bioremediation tool.

Protein—Polyelectrolyte Phase Boundaries

Abstract: The separation of proteins by polyelectrolyte coacervation or precipitation is based on electrostatically-driven complex formation. We have investigated complexation between the globular protein BSA and the polyelectrolyte poly(dimethyldially1ammonium chloride) (PDMDAAC) using light-scattering techniques to monitor solution turbidity. Turbidimetric pH titrations were used to determine the specific pH values where soluble complex formation is initiated (pH,) and where phase separation occurs (pH& These values, collected at different ionic strengths, can be presented as phase boundaries. The effects of macromolecular concentration, protein:polymer concentration ratio (r), and polymer molecular weight upon the phase boundary are examined. The macromolecular concentration and polymer molecular weight have little or no effect on the phase boundary. While pH, is independent of r, pH$ varies inversely with r. The use of phase boundaries in the selection of optimal pH and ionic strength for maximum yield and purity in protein separations is discussed.

Adsorption of Copper and Chromium by Aspergillus carbonarius

Abstract: Aspergillus carbonarius NRC 401121 adsorbs some copper and chromium from their solutions. The amount of the adsorbed metal per unit of biomass increased with a decrease in the biomass concentration. The increases in the initial concentrations of metals and pH of the solutions resulted in an increase in copper and chromium uptake. The optimum temperature for copper uptake was 25 °C. Heating of the biomass prior to utilizing it in the adsorption tests decreased its metal adsorption capacity. Preincubation of the biomass with glucose enhanced the metal adsorption. The optimum glucose concentration in this process was 0.1%.

A Kinetic Model for Substrate and Energy Consumption of Microbial Growth under Substrate-Sufficient Conditions

Abstract: The growth of heterotrophic microorganisms can be classified into substrate-limited and substrate-sufficient growth according to the relative availability of the substrate (carbon and energy source) and other nutrients. It is generally observed that the consumption rates of substrate and energy (ATP) are higher under substrate-sufficient conditions than under conditions of substrate limitation. The excess substrate and ATP consumption is often influenced by the residual concentration of substrate in a. relatively wide range. To account for these effects, a kinetic model is proposed to describe substrate and ATP consumption rates of microbial growth under substrate-sufficient conditions. According to the model, the specific substrate consumption rate of a substrate-sufficient culture can be expressed as the sum of the substrate consumption rate under substrate-limited conditions at the corresponding specific growth rate and an additional consumption rate due to excess substrate. The same kinetic form also applies to the specific ATP consumption rate and to the specific oxygen consumption rate of an aerobic culture, respectively. The linear equations for substrate and ATP consumption rates of Pirt and of Stouthamer and Bettenhausen can be used for substrate-limited growth. The excess of substrate and ATP consumption rates at carbon surplus can be described in a form similar to that of Michaelis&β;Menten kinetics. The proposed kinetic model has been verified with experimental data from three continuous cultures representing both anaerobic and aerobic microbial growth on substrates with low and high degrees of reductance. Using this model, the parameters maximum growth yield and maintenance requirement (both in terms of substrate and ATP) of a culture under different growth limitations can be better defined and quantified. The range of residual substrate concentrations in which the specific rates of substrate and ATP consumption are affected can also be assessed. This information should be helpful in designing medium and reactor operating conditions and in interpreting experimental results obtained under different limiting conditions.

Development of Metabolically Engineered Saccharomyces cerevisiae Cells for the Production of Lactic Acid

Abstract: Interesting challenges from metabolically engineered Saccharomyces cerevisiae cells arise from the opportunity to obtain yeast strains useful for the production of chemical(s). In this paper, we describe the accumulation of lactic acid in the culture medium of growing, engineered yeast cells expressing a mammalian lactate dehydrogenase gene (LDH-A). High and reproducible productions (20 g/L) and productivities (up to 11 g/L/h) of lactic acid have been obtained by modulating the physiological growth conditions. Since yeast cells are acid tolerant and survive at very low pH values, the production of lactate can be avoided. In perspective, the approaches described could be useful for the production of lactic acid, outflanking the problems related to the synthesis from bacteria cells. In fact, during industrial productions, there is an inhibitory effect on the metabolic activities of the growing bacteria (i.e., Lactobacillus spp.) caused by the acid produced and by the low pH value. Thus, strategies to prevent the lowering of pH are conventional operations. These processes allow the production of lactate(s) and require the purification of the acid from its salt. The biotechnological implications of this study are also discussed.

Pellet formation and fragmentation in submerged cultures of Penicillium chrysogenum and its relation to penicillin production.

Abstract: The spores of Penicillium chrysogenum are of the noncoagulating type, and after spore germination a culture of disperse mycelia is obtained. In this study, it is shown that when the hyphal elements increase in size, they may agglomerate, and depending on the operating conditions, these agglomerates may develop into pellets with a dense core. The influence of initial spore concentration and agitation rate on agglomeration, leading to pellet formation, was studied. For a low concentration of spores in the inoculum, only a few hyphal elements agglomerate and pellets with a small diameter are obtained. At higher spore concentrations, many hyphal elements agglomerate and develop into large diameter pellets. Finally, at a very high spore concentration in the inoculum, the final hyphal element size is small and agglomerates therefore are not formed. With a high agitation rate, the agglomeration of hyphal elements is reduced. In a repeated fed-batch cultivation, where there was a shift from pellet morphology to disperse mycelia, it was found that there is no relation between macroscopic morphology and penicillin production by P. chrysogenum. The morphology was quantified throughout the repeated fed-batch cultivation, and both the pellet diameter and the concentration of pellets were affected by the agitation rate.

Metabolic Engineering of Escherichia coli To Enhance Recombinant Protein Production through Acetate Reduction

Abstract: Genetic and metabolic engineering provide powerful and effective tools for the systematic manipulation and fine tuning of cellular metabolic activities. In this study, successful application of such techniques to enhance recombinant protein production by reducing acetate accumulation in Escherichia coli is presented. The alsS gene from Bacillus subtilis encoding the enzyme acetolactate synthase was introduced into E. coli cells using a multicopy plasmid. This newly introduced heterologous enzyme modifies the glycolytic fluxes by redirecting excess pyruvate away from acetate to acetolactate. Acetolactate is then converted to a nonacidic and less harmful byproduct acetoin, which appears in the broth. Furthermore, comparative fermentation studies show that the reduction in acetate accumulation leads to a significant improvement of recombinant protein production. The expression of a model recombinant CadA/beta-galactosidase fusion protein, under the control of a strong pH-regulated promoter, was found to increase by about 60% for the specific protein activity (to a level of 30% of total cellular protein) and 50% in terms of the volumetric activity in a batch fermenter. In fed-batch cultivation, the engineered strain achieved a volumetric recombinant protein yield of 1.6 million units/mL (about 1.1 g/L of beta-galactosidase), which represented a 220% enhancement over the control strain. In the meantime, acetate excretion was maintained below 20 mM compared with 80 mM for the control, and the final cell density was improved by 35%.

Microbial polysaccharides of applied interest: ongoing research activities in Europe.

Abstract: After an introductory note on the main reasons underlying the interest in microbial polysaccharides as subjects of fundamental structural/physicochemical investigations, as well as of applied studies, and after a brief historical account, a survey is offered of ongoing research activities in Europe in the broad field of carbohydrate polymers of microbial origin. Attention is drawn to glycopolymers of actual or potential industrial relevance.

Tissue Engineering of a Bioartificial Pancreas: Modeling the Cell Environment and Device Function

Abstract: Cell-based implantable artificial tissues are most promising for the long-term treatment of endocrine diseases, such as diabetes. One type of a bioartificial pancreas device consists of calcium alginate microbeads containing insulin-secreting cells and is surrounded by a poly(L-lysine) (PLL) membrane. The membrane is semipermeable, allowing cellular nutrients and metabolites to diffuse through but excluding the antibodies and cytotoxic cells of the host, thus immunoprotecting the cells. The device can be modeled by writing the equations for diffusion of nutrients and metabolites through the polymer and for consumption of the former and production of the latter by the cells. In this paper, we describe the construction and analysis of such a model for alginate/PLL microbeads with insulin-secreting recombinant mouse pituitary AtT-20 and mouse insulinoma beta TC3 cells. Entrapped AtT-20 cells are a simplified model system, whereas microbeads with beta TC3 cells constitute a realistic artificial pancreatic device. Effective diffusivities of key compounds through the polymer with entrapped, inactivated AtT-20 spheroids were measured first. The kinetics of glucose and oxygen consumption and insulin secretion were modeled next, and the equations for diffusion and reaction were then combined to describe the entire system. The model was used to compute nutrient and metabolite concentration profiles in beads and the bead secretory response for different bead sizes and cell loadings. The size and loading necessary for the cells to be well nourished and for the beads to be rapidly responsive to step-ups and step-downs of secretion stimuli were evaluated. It was shown that if the cells are hypersensitive to glucose, i.e., they do not shut off secretion at the physiological glucose threshold but at a lower one, so are the microbeads. This work demonstrates the usefulness of mechanistic models with representative parameter values in optimizing the design of artificial tissues and in characterizing aspects of their behavior that are of importance for restoring in vivo function.

Batch and semicontinuous aggregation and sedimentation of hybridoma cells by acoustic resonance fields.

Abstract: Ultrasound was used to enhance the sedimentation of hybridoma cells from medium in a 75 mL resonator chamber. Forces in the acoustic standing waves aggregated the cells, and the aggregates were then rapidly sedimented by gravity. Cell separation increased with acoustic treatment time and cell concentration. The separation efficiency was over 97% for cell concentrations between 10(6) and 10(7) cells/mL. During acoustic treatment at 180 W/L, the medium temperature increased at a rate of 1.3 degrees C/min. Ultrasonic exposures up to 220 W/L did not influence the viability or subsequent growth and antibody production of the cells. A decrease in cell viability was observed at a power level of 260 W/L. Batch separation efficiencies were as high as 98%. Acoustic separation was tested under semicontinuous operation, and above 90% separation efficiency was achieved at a flow rate of 0.7 L/h.

Evaluation of the Cd, Cu, and Zn Biosorption in Two‐Metal Systems Using an Algal Biosorbent

Abstract: The equilibrium sorption capacity of the formaldehyde-cross-linked brown seaweed Ascophyllum nodosum biomass was studied using two-metal systems comprising either (Cu + Zn), (Cu + Cd), or (Zn + Cd). Three-dimensional sorption isotherm surfaces were used in the evaluation of sorption performance, replacing simple isotherm curves. While each of the metals tested can inhibit the sorption of the others, at low total residual metal concentrations the total metal sorption uptake increases. At higher total metal concentrations, the total metal sorption uptake either remains constant, or there may be a slight decrease compared to single-metal uptakes. This is an indication of a mutual interference in the sorption uptake of the two metals tested in each system.

Tissue Plasminogen Activator Coexpressed in Chinese Hamster Ovary Cells with .alpha.(2,6)-Sialyltransferase Contains NeuAc.alpha.(2,6)Gal.beta.(1,4)Glc-N-AcR Linkages

Abstract: Genetic alteration of the set of oligosaccharide biosynthesis enzymes expressed in a genetically engineered host cell line is a plausible strategy for manipulating the oligosaccharides on a cloned glycoprotein coexpressed in that cell line. This hypothesis was verified for the particular case of sialylation of recombinant human tissue plasminogen activator (tPA) expressed by an engineered Chinese hamster ovary (CHO) cell line. The gene for rat liver beta-galactoside alpha(2,6)-sialyltransferase (2,6-ST) was cloned behind the MMTV promoter in the vector pMSG and transfected into a tPA-expressing CHO cell line. Selected and screened transfectants exhibited significantly greater surface fluorescence than controls in flow cytometric analyses of cells labeled with Sambacus nigra agglutinin (SNA)-biotin and streptavidin-R-phycoerythrin; SNA specifically binds to NeuAc alpha(2,6)Gal beta(1,4)Glc-N-AcR linkages, which are synthesized by 2,6-ST and which are not normally found on CHO cells. SNA blots of partially purified tPA from the culture supernatant demonstrated that tPA synthesized in the 2,6-ST transfectants possessed terminal NeuAc alpha(2,6)Gal beta(1,4)Glc-N-AcR linkages, while tPA from the original recombinant CHO cell line did not. Besides possibly allowing the production of glycoproteins in cell culture with glycosylation more closely resembling that in humans, extensions of this strategy have the potential to tailor the pharmacokinetics, targeting, and antigenic properties of cloned glycoproteins.

Fluidized-bed bioreactors

Abstract: Fluidized-bed reactors present a number of advantages that make them an attractive alternative in processes involving biocatalysts. However, fluidized-bed bioreactors are also realtively complex, basically for two reasons. First, their use requires the biocatalyst, commonly cells or enzymes, to be immobilized into or onto a solid support. Second, the hydrodynamic characterization is difficult, especially in those systems where three phases (gas—liquid—solid) are involved. The mathematical model of a fluidized-bed bioreactor needs to take into account those hydrodynamic aspects that will determine the flux model in the reactor. Moreover, the description of other aspects is also required: the mechanisms of transport between the different phases, the kinetic equations for the phenomena taking place in the biocatalytic particles, such as cell growth, product formation, substrate consumption, enzyme deactivation, and the mass balance equations in the reactor. In addition to these aspects, the application of fluidized-bed bioreactors to different kind of processes is also discussed. The potential of this type of bioreactor is also emphasized from the point of view of the different number of possible modifications in the design both of the bioreactor and the biocatalyst particles, in order to enhance its operation.

Surfactant‐Coated Lipase Suitable for the Enzymatic Resolution of Menthol as a Biocatalyst in Organic Media

Abstract: Enantioselective esterification of menthol with fatty acids using a surfactant-coated lipase was carried out in organic media. The surfactant-coated lipase originating from Candida cylindracea appeared to be highly enantioselective and good biocatalyst for the resolution of racemic menthol. The enzymatic activity of the lipase in organic media was significantly increased by a coating with a nonionic surfactant. The reaction rate of the coated lipase was more than 100 times that of the powder lipase. In order to investigate the effect of the organic solvent on enantioselectivity, 19 kinds of solvents were employed. The nature of the organic solvent strongly affected the efficiency of the biocatalyst and the enantioselectivity. Among them, isooctane was the best organic solvent from the viewpoint of reaction rate and enantioselectivity. The effect of reaction temperature on esterification was also investigated. The optimal reaction temperature was around 35 °C. The enzymatic activities using n-saturated fatty acids with different alkyl chain lengths were compared, and long-chain fatty acids were found to be better substrates than shorter ones. The relationship between the initial rate of the esterification and the carbon number of the fatty acid was not linear. These results suggest that there are inherent Km values for each fatty acid.

Metabolic Control Analysis of the Penicillin Biosynthetic Pathway in a High-Yielding Strain of Penicillium chrysogenum

Abstract: Metabolic control analysis is used to identify the rate-limiting step in the penicillin biosynthetic pathway in Penicillium chrysogenum. The analysis is carried out using a kinetic model for the first two steps in the pathway, i.e., the ACV synthetase (ACVS) and the isopenicillin N synthetase (IPNS). The kinetic model is based on Michaelis-Menten type kinetics, with noncompetitive inhibition of the ACVS by ACV and competitive inhibition of the IPNS by glutathione. From measurements of the enzyme activities and intracellular metabolites during a fed-batch cultivation, the kinetic model is used to predict the flux through the pathway. The model prediction corresponds well with the measured rate of penicillin biosynthesis. From measurement of the activity of the acyl-CoA:isopenicillin acyltransferase, which catalyzes the third and last reaction in the pathway, it is concluded that the rate-limiting step is either at the ACVS or at the IPNS. From the kinetic model, the elasticity coefficients and the flux control coefficients are calculated throughout the fed-batch cultivations, and it is found that there is a shift in the flux control from the ACVS to the IPNS during the cultivation.

Continuous fermentation and stripping of ethanol.

Abstract: Recycling the contents of a continuous fermentor through a stripping column is proposed as a means of reducing product inhibition and lowering the cost of fuel ethanol production. A 2-L fermentor and 10-cm packed column were continuously operated for 150 days without contamination. Some fouling of the packing with attached yeast cells was observed which partially blocked the column. Cell yield was lower than in a simple continuous fermentor. Complete conversion of 200 g/L glucose feed and 90% conversion of 600 g/L glucose feed were achieved. Data were analyzed by computerized process simulation. Cost analysis indicated that, with heat recovery to reduce heating and cooling costs, the continuous fermentor/stripper is possibly a lower-cost alternative to conventional fermentation and distillation.

Kinetic and Stoichiometric Analysis of Hairy Roots in a Segmented Bubble Column Reactor

Abstract: Hairy roots of Atropa belladonna were cultured in a modified 2.5-L multicompartment bubble column for analysis of growth kinetics, stoichiometry, and atropine production. Average biomass density reached 9.9 g L -1 dry weight after 43 days of batch culture ; local root densities in some parts of the vessel were considerably higher, up to 17 g L -1 . Bulk mixing in the reactor was very poor : after 14 days of culture, the time taken to reach 95% of the equilibrium value after a concentration pulse in the vessel was 12 min. Growth and specific sugar uptake rates declined continuously throughout the culture even though adequate sugar and nitrogen remained in the medium. The observed biomass yield from sugar was approximately constant at 0.35 g g -1 ; biomass yields from ammonia and nitrate were 0.44 and 0.35 g mmol -1 , respectively. Specific atropine content in the roots varied from 4.1 mg g -1 dry weight at the beginning of the culture to 1.4 mg g -1 after 28 days ; 35 mg or 14 mg L -1 atropine was produced over the 43-day culture period. Biomass composition was represented by the elemental formula CH 1.63 O 0.80 N 0.13 , plus 9.8% (w/w) ash. A balanced stoichiometric equation was developed for hairy root growth ; this indicated that 8.3% of carbon supplied to the culture was excreted into the medium as byproducts.

Specificity of a Lipase in Ester Synthesis: Effect of Alcohol

Abstract: Ester synthesis by the Mucor miehei lipase has been studied for various alcohol substrates: n-propanol, n-butanol, isoamyl alcohol, n-hexanol, n-octanol, 2-ethylhex-anol, n-decanol, and lauryl alcohol. The effects of temperature, the nature of the acid, and immobilization of the lipase on its substrate specficity have been elucidated by carrying out esterifications at 29 and 50 °C with lauric and oleic acids and by using both the soluble and immobilized (resin-adsorbed) forms of the lipase as catalysts. Higher synthesis rates were obtained with oleic acid than with lauric acid. A bimodal distribution pattern was observed for the reaction rate as a function of alcohol chain length. Two superimposed "bells" were obtained with maxima at C4 (butanol) and C10 (decanol) at 29 °C. Whereas immobilization of the lipase did not influence this substrate specificity, an increase in temperature to 50 °C caused a shift in the first peak from C4 to C6 (hexanol), while the second peak position was not affected. The minimum, in all cases, was found to be at C8 (octanol).

Biomass Estimation in Plant Cell Cultures: A Neural Network Approach

Abstract: The special characteristics of plant cell cultures make it difficult to use conventional analytical techniques for on-line biomass monitoring. Meanwhile, promising results have been obtained using mathematical models and recursive estimation algorithms. However, in this case, additional experimental effort is necessary to obtain a reasonable description of the process. Recently, techniques using more empirical approaches have been proposed to describe complex processes, minimizing the experimental work needed for their application. In this paper, we report on the use of artificial neural networks to monitor biomass evolution in plant cell cultures. The results obtained with a three-layered network are presented. Method requirements and capabilities are compared with the method based on the extended Kalman filter used in previous work.

Investigation of the ethanol tolerance of Clostridium thermosaccharolyticum in continuous culture

Abstract: The ethanol tolerance of Clostridium thermosaccharolyticum HG8 has been studied in continuous culture using a new technique that requires knowledge of the kinetic constants and measurement of substrate concentrations at various concentrations of the inhibitor. Endogenously produced ethanol was supplemented with exogenously supplied ethanol to achieve various inhibitor concentrations. The ethanol tolerance of C. thermosaccharolyticum was significantly greater than expected on the basis of most previous reports, which may be explained in part by acclimation occurring over time periods exceeding those typical of batch systems studied previously. An ethanol concentration of 40 g/L is required for 50% growth inhibition of C. thermosaccharolyticum at 55 °C. Process considerations suggest that the ethanol tolerance of C. thermosaccharolyticum is unlikely to significantly constrain its use for ethanol production from cellulosic biomass. Ester-linked phospholipid membrane analyses (ELPFA) revealed that growth in the presence of high concentrations of ethanol (33 g/L) resulted in a membrane profile having increased fluidity and molecular diversity. Ethanol-induced changes included a significant increase in shorter chain unsaturated fatty acids (C15 :0) at the expense of longer chain unsaturated fatty acids (C17 :0) and a slight increase in the amount of mono-unsaturated fatty acids.

Constitutive overexpression of secreted heterologous proteins decreases extractable BiP and protein disulfide isomerase levels in Saccharomyces cerevisiae.

Abstract: High-level gene expression does not always lead to corresponding high-level secretion of heterologous proteins in yeast. The rate-limiting step in many cases has been shown to exit from the endoplasmic reticulum (ER). Within the ER, the correct folding of secreted proteins is required for export competence; hence, the cellular proteins involved in these events are likely to be important for efficient secretion. We have found that the extractable levels of two ER-resident proteins involved in folding—heavy chain binding protein (BiP) and protein disulfide isomerase (PDI)--are significantly reduced by prolonged constitutive overexpression of human granulocyte colony stimulating factor (GCSF), human erythropoietin, or Schizosaccharomyces pombe acid phosphatase. However, the rate of BiP synthesis measured in pulse-chase radiolabeling experiments is not reduced by GCSF overexpression, and galactose-directed transcription of the BiP gene does not restore normal BiP protein levels once they have been depleted. The observed loss of lumenal resident proteins, either by proteolysis or irreversible aggregation, is expected to contribute significantly to the inefficiency of foreign protein secretion in yeast.

Studies of High Solidity Ratio Hydrofoil Impellers for Aerated Bioreactors. 1. Review

Abstract: This first part of a four-part series of papers presents a review of the literature on high solidity ratio axial flow hydrofoil impellers. Interest in the application of high-flow, low power number, hydrofoil impellers, such as the Prochem Maxflo T and the Lightnin A315, has developed over recent years and has largely been stimulated by reports of improved fermentation performance relative to the Rushton disc turbine. Initially, the review briefly examines the characteristics of the radial flow Rushton turbine, as traditionally used in fermenters, with particular reference to its weaknesses. It also discusses the use of pitched blade turbines (mixed flow impellers) for air dispersion since these show some characteristics similar to those of the axial flow hydrofoils. The consideration of these two impeller types provides the background to explain the advantages and potential problems associated with high solidity ratio, axial flow, downward pumping hydrofoils. These aspects are discussed in relation to existing literature which is still relatively scarce. Finally, the advantages of retrofitting are introduced. Parts 2–4 report on a recent, detailed, fluid dynamic study of the Prochem Maxflo T and Lightnin A315 hydrofoil impellers which extends our knowledge of these important impeller types.

Effect of viscosity upon hydrodynamically controlled natural aggregates of animal cells grown in stirred vessels.

Abstract: The effect of medium viscosity upon cell growth and aggregate characteristics of baby hamster kidney (BHK) cells cultivated in stirred tanks was evaluated. Two thickening agents were tested, 9300 MW dextran and a low-viscosity sodium (carboxymethyl)-cellulose; both were used in two different sets of experiments: (i) 250 cm3 Wheaton spinner flasks with a ball impeller operated at 45 rpm; (ii) 500 cm3 Corning spinner flasks with a paddle impeller, operated at constant power dissipation (88 cm2 s-3). Aggregate diameter and the fraction of cells in aggregates increased with the increase in viscosity. Power laws were applied to the experimental results. A dependence of aggregate size upon power dissipation of the order of -0.19 and kinematic viscosity of 0.34 and 0.49 for the constant agitation and constant power dissipation tests were obtained, respectively. A model based upon the entire universal-equilibrium range (i.e., the entire spectrum of isotropic eddies) was used to predict theoretical relationships between the variables studied. The model leads to a power dependence of -0.25 for the energy dissipated in the entire universal-equilibrium range and between 0.25 and 0.5 for the kinematic viscosity in the viscous dissipation subrange, depending on the energy correlation used; it also gives a good explanation for the dependence of aggregate size on the hydrodynamics of the vessel.

Characterization of a Bench‐Scale System for Studying the Biodegradation of Organic Solid Wastes

Abstract: A bench-scale system was developed that through its use contributes toward a more fundamental understanding of the composting process. The computer-controlled system maintained temperature and moisture levels within narrow ranges in thin layers of compost positioned on nylon mesh trays within the vessel. This system minimized complications caused by gradients observed in earlier systems. Specific O{sub 2} consumption and CO{sub 2} evolution rates were determined on the basis of dynamic mass balances. Water was introduced intermittently. Microbial biomass (ATP) was monitored at specific time intervals throughout the process. The system allows close observation of decomposition of feed materials at constant temperature and moisture levels throughout the process. 31 refs., 8 figs., 6 tabs.

Isolation of human blood cells, platelets, and plasma proteins by centrifugal SPLITT fractionation

Abstract: Centrifugal SPLITT fractionation, a technique designed for the continuous high-resolution separation of colloids and low-density particles, is applied here to fresh human blood, producing six purified fractions consisting of proteins and lipoproteins, platelets, red blood cells, lymphocytes, monocytes, and neutrophils. Production of the six fractions requires five steps, each yielding two fractions. These five steps are carried out in sequence using a single apparatus, with conditions varying from step to step in accordance with theoretical guidelines in order to achieve the desired cut points. In the first step, a stream of diluted blood is separated into one fraction consisting of platelets and plasma and another containing blood cells. The throughput of diluted blood is 162 mL/h and that of whole blood is about 2 mL/h or approximately 10(10) cells/h; guidelines are given for significantly increasing throughput. The purity of the blood cell fractions ranged from 92 to 98%, and the viability fell in the range 97-99%.

Suppressed acid formation by cofeeding of glucose and citrate in Bacillus cultures : emergence of pyruvate kinase as a potential metabolic engineering site

Abstract: Microbial cultures typically produce acids when metabolizing the common carbon source, glucose. Acid production not only represents a waste of carbon, but its accumulation can limit cell concentration and culture stability, thereby reducing productivity. On the basis of prior work, acid production was attributed to be due to a mismatch between glycolytic and tricarboxylic acid (TCA) cycle capacities. To suppress acid production, a strategy entailing adding citrate to glucose minimal medium proved extremely effective. The effect of citrate on in-vivo flux distribution was quantified using a detailed flux-model. When the molar glucose-citrate ratio was varied between 3 and 6, a significant reduction in glycolytic flux and essentially complete suppression of acid formation was found as compared to chemostat cultures grown solely on glucose. Adding other biosynthetic precursors such as glutamine did not invoke the same suppression, thus indicating that citrate's effect is at the regulatory level. We hypothesized that the reduction of glycolytic flux in the presence of citrate results from its transport being coupled with the uptake of divalent metal ions. Citrate transport alters the intracellular balance of metal ions which in turn could trigger a sophisticated series of metabolic events leading to reduction of the activities of the pyruvate kinase and phosphofructokinase (PFK), the regulatory enzymes of glycolysis. On the basis of this scenario and other regulatory information, pyruvate kinase has emerged as a potential metabolic engineering site. It's deactivation in Bacillus subtilis or Escherichia coli strains is expected to yield constructs with a much lower tendency for making acid byproducts.

Conversion of Mixed Waste Office Paper to Ethanol by Genetically Engineered Klebsiella oxytoca Strain P2

Abstract: Unsorted, mixed waste office paper (MWOP) is an excellent substrate for conversion into fuel ethanol using a recombinant strain of Klebsiella oxytoca which ferments cellobiose and cellotriose to ethanol at near theoretical yields, eliminating the need for supplemental β-glucosidase. This organism was tested with commercial fungal cellulase in optimized simultaneous saccharification and fermentation experiments (SSF) using MWOP as a substrate (pH 5–15.2, 35 °C). Similar rates and yields were obtained with dilute acid-pulped (hydrolysis of hemicellulose) and water-pulped MWOP on a dry weight basis although viscosity was reduced by the acid pretreatment. In simple batch fermentations, 40 g/L ethanol was produced after 48–72 h with 100 g/L MWOP and 1000 filter paper units (FPU) of cellulase/L, a yield of 550 L of ethanol/metric ton. Cellulase usage was further reduced by recycling SSF residues containing bound enzymes in multistage fermentations. This approach reduced the requirement for fungal cellulase while retaining rapid ethanol production and high ethanol yield. In our optimal design, broths containing an average of 39.6 g/L ethanol were produced in three successive stages with an average fermentation time of 80 h (567 FPU of fungal cellulase/L; 6.1 FPU/g of substrate). This represents a yield of 0.426 g of ethanol/g of substrate, 539 L/metric ton, 129 gal/U.S. ton. MWOP contains approximately 90% carbohydrate. Thus the combined efficiency for saccharification and fermentation to ethanol was 83.3% of the theoretical maximum.

Partial and total cell retention in a filtration-based homogeneous perfusion reactor

Abstract: Suspended mammalian cells can be cultivated in a variety of operational modes (pure chemostat, total cell retention, or partial cell retention) in a homogeneous perfusion bioreactor by varying the cell bleed rate. Hybridomas were grown in the reactor at a perfusion rate of 2.0 day -1 for over 10 weeks at different specific growth rates and viable cell densities achieved by varying the extent of cell retention. Cell metabolism in the reactor was found to vary with the extent of cell retention, which determined both cell density and specific growth rate. With partial cell retention, the nutrient consumption and metabolite production rates decreased with both increasing growth rate and increasing cell density. The specific and volumetric antibody production rates, however, increased dramatically with cell density (and to a lesser extent with decreasing growth rate). The specific MAb production rate was lower with total cell retention than with partial retention at the same growth rate. Since the reactor can be operated over a range of perfusion rates and extents of cell retention, the system can be used to culture cell lines with widely different productivity patterns.