Showing papers in "Biotechnology and Bioengineering in 1990"

Plasmid‐encoded protein: The principal factor in the “metabolic burden” associated with recombinant bacteria

Abstract: Experimental elucidation of the metabolic load placed on bacteria by the expression of foreign protein is presented. The host/vector system is Escherichia coli RR1/pBR329 (amp(r), cam(r), and let(r)). Plasmid content results, which indicate that the plasmid copy number monotonically increases with decreasing growth rate, are consistent with the literature on ColE1-like plasmids. More significantly, we have experimentally quantified the reduction in growth rate brought about by the expression of chloramphenicol-acetyl-transferase (CAT) and beta-lactamase. Results indicate a nearly linear decrease in growth rate with increasing foreign protein content. Also, the change in growth rate due to foreign protein expression depends on the growth rate of the cells. The observed linear relationship is media independent and, to our knowledge, previously undocumented. Furthermore, the induction of CAT, mediated by the presence of chloramphenicol, is shown to occur only at low growth rates, which further increases the metabolic load.Results are vdelineated with the aid of a structured kinetic model representing the metabolism of recombinant E. coli. In this article, several previous hypotheses and model predictions are justified and validated. This work provides an important step in the development of comprehensive, methabolically-structured, kinetic models capable of prediciting optimal conditions for maximizing product yield.

Inhibition of Trichoderma reesei cellulase by sugars and solvents.

Abstract: Inhibition of Trichoderma reesei cellulase by sugars (glucose, delta-gluconolactone, and cellobiose) and solvents (ethanol, butanol, and acetone) was studied using cellulose azure. Glucose, cellobiose, ethanol, and butanol were noncompetitive inhibitors, delta-gluconolactone was a mixed inhibitor, and acetone was a noncompetitive activator. Converting cellobiose to glucose reduces the effective inhibitor binding constant by 6 times and converting cellobiose to ethanol reduces it by 16 times.

Prediction of diffusion coefficients of proteins.

Abstract: A correlation for predicting the diffusion coefficients of proteins at standard conditions is proposed by adapting the Stokes-Einstein equation to a model for the equivalent hydrodynamic sphere. The radius of gyration, which accounts for the size and shape of protein molecules in solution, was used in deriving the new correlation. The correlation successfully predicted the diffusion coefficients of proteins, for which experimental data were available, with a higher degree of accuracy than achieved by previous correlation methods.

Simple constrained-optimization view of acetate overflow in E. coli

Abstract: The production of acetate by aerobically growing E. coli is examined. The problem is formulated in terms of a flow network that has as its objective maximal ATP synthesis. It is found that when loads are imposed and flux constraints exist either at the level of NADH turnover rate or the activity of a key Krebs cycle enzyme, switching to acetate overflow is predicted. Moreover, the result found for the latter constraint can be shown to be formally equivalent to a correlation experimentally determined for the specific rate of acetate production by E. coli K-12.

Production of recombinant human growth hormone in Escherichia coli: expression of different precursors and physiological effects of glucose, acetate, and salts.

Abstract: The constitutive cytoplasmic expression in E. coli of human growth hormone (hGH) with different N-terminal extensions (3 or 4 amino acids) has been studied. These hGH precursors were used for in vitro cleavage to obtain the mature, authentic hormone. Small changes in the amino acid extensions of the hGH precursors led to three-fold differences in specific expression rates. The specific expression rate of the hGH precursors was inversely proportional to the ratios of the specific growth rates of plasmid containing and plasmid free cells (micro(+)/micro(-)) and also to the genetic stability. To ensure a satisfactory genetic stability in production fermentors, an hGH precursor with a moderate expression efficiency was chosen.The medium composition and growth conditions were studied, resulting in the choice of a glucose fed batch fermentation process using a complex medium. In this process a yield of 2000 mg/L of met-ala-glu-hGH (MAE-hGH) was obtained. The fermentation process comprised a glucose-limited growth phase followed by a second phase with increased glucose feed and exhaustion of phosphate from the medium. The second phase is characterized by an MAE-hGH production, whereas further biomass formation is blocked. High concentrations of glucose led to reduced specific expression of MAE-hGH--the specific and total yield in batch glucose fermentations is only about 30% of the yield in optimized fed batch fermentations. The physiological background for this was investigated. Chemostat experiments showed that the glucose concentration and the metabolic condition of the cells--i.e. with or without formation of acetate--was not critical per se in order to obtain a high specific yield of MAE-hGH. Therefore it is unlikely that formation of MAE-hGH is catabolite repressed by glucose. Furthermore it was shown that the specific production rate of MAE-hGH was independent of the specific growth rate and it was further demonstrated that the decrease in expression efficiency in glucose batch fermentation was a result of an inhibitory effect of acetic acid. In batch fermentations this inhibitory effect was enhanced by a salt effect caused by increased consumption of acid and base used to control pH. The identity of the acid and the base used are not important in this context. From studies of the expression of other proteins in E. coli. with constitutive as well as inducible promoters we conclude that glucose fed batch processes are often superior to batch processes in the production of heterologous proteins E. coli.

Damage mechanisms of suspended animal cells in agitated bioreactors with and without bubble entrainment.

Abstract: We show that when freely suspended hybridoma cells are cultured in an agitated bioreactor, two fluid-mechanical mechanisms can cause cell damage and growth retardation. The first is present only when there is a gas phase, and is associated with vortex formation accompanied by bubble entrainment and breakup. In the absence of a vortex and bubble entrainment, cells can be damaged only at very high agitation rates, above approximately 700 rpm, by stresses in the bulk turbulent liquid. Cell damage then correlates with Kolmogorov eddy sizes similar to or smaller than the cell size. In the absence of a vortex, the entrainment and motion of very fine bubbles cause no growth retardation even at agitation rates as high as 600 rpm.

Adsorption of cellulase from Trichoderma reesei on cellulose and lignacious residue in wood pretreated by dilute sulfuric acid with explosive decompression

Abstract: The adsorption of cellulase on cellulose and a lignacious residue was examined by using cellulase from Trichoderma reesei, hardwood pretreated by dilute sulfuric acid under high pressure, and a lignacious residue prepared by a complete enzymatic hydrolysis of the pretreated wood. A significant amount of cellulase was found to adsorb on the lignacious residue during the hydrolysis of the pretreated wood. Hence, the adsorption of enzyme on the lignacious residue as well as cellulose must be taken into account in the development of the hydrolysis kinetics. It was found that the adsorption of enzyme on cellulose and on the lignacious residue could be represented by Langmuir type isotherms. The data show that the pretreatment at a higher temperature results in more enzyme adsorption on the cellulose fraction and less on the lignacious residue fraction. The relationship between the hydrolysis rate and the amount of enzyme adsorbed is discussed.

Group contributions for estimating standard gibbs energies of formation of biochemical compounds in aqueous solution

Abstract: A method is presented for the estimation of the standard Gibbs energies of formation of biochemical compounds (and hence the Gibbs energies and equilibrium constants of biochemical reactions) from the contributions of groups. The method employs a large set of groups and special corrections. The contributions were estimated via multiple linear regression, using screened and weighted literature data. For most of the data employed, the error is less than 2 kcal/mol. The method provides a useful first approximation to Gibbs energies and equilibrium constants in biochemical systems.

Diffusion of phenol through a biofilm grown on activated carbon particles in a draft-tube three-phase fluidized-bed bioreactor

Abstract: Diffusion of phenol through a biofilm attached to activated carbon particles was investigated. The biofilm was grown on activated carbon particles in a draft-tube three-phase fluidized-bed bioreactor operating in a fed-batch mode. It was found that phenol did not adsorb on the biofilm and that the diffusion coefficient of phenol within the biofilm varied from 13 to 39% of its corresponding value in water. The diffusion coefficient of phenol within the biofilm was reduced by increasing the biofilm density. An extensive literature review of diffusion of substrates through biofilms indicated that this conclusion could be extended to biofilms grown on flat surfaces, rotating cylinders, and even bioflocs.

Structure of activated sludge floes.

Abstract: Relatively large activated sludge floes (larger than about 100 microm) were stabilized, using a histological tissue specimen preparation procedure, and then were sliced into sections of 3 to 6 microm thick. The study of these sections, after staining, revealed the internal structure of the activated sludge floes. No uniformity of this structure was found. The distribution of microorganisms and of extracellular polymers (EPs) in the floes varied randomly on the plane of the sections and along the dimension perpendicular to the plane, leaving large water channels and reservoirs in some of the floes. The lack of a characteristic size for the water gaps in the floes and a general self-similar appearance of the sections suggested that the activated sludge floes might be characterized by the fractal concept within a certain size limit. Direct observation of the interior of the floes indicated an abundant presence of extracellular polymers (EPs) in amorphous forms, surrounding microorganisms in most of the floes.

Heat transfer simulation in solid substrate fermentation

Abstract: A mathematical model was developed and tested to simulate the generation and transfer of heat in solid substrate fermentation (SSF). The experimental studies were realized in a 1-L static bioreactor packed with cassava wet meal and inoculated with Aspergillus niger. A simplified pseudohomogeneous monodimensional dynamic model was used for the energy balance. Kinetic equations taking into account biomass formation (logistic), sugar consumption (with maintenance), and carbon dioxide formation were used. Model verification was achieved by comparison of calculated and experimental temperatures. Heat transfer was evaluated by the estimation of Biot and Peclet heat dimensionless numbers 5-10 and 2550-2750, respectively. It was shown that conduction through the fermentation fixed bed was the main heat transfer resistance. This model intends to reach a better understanding of transport phenomena in SSF, a fact which could be used to evaluate various alternatives for temperature control of SSF, i.e., changing air flow rates and increasing water content. Dimensionless numbers could be used as scale-up criteria of large fermentors, since in those ratios are described the operating conditions, geometry, and size of the bioreactor. It could lead to improved solid reactor systems. The model can be used as a basis for automatic control of SSF for the production of valuable metabolites in static fermentors.

Optimization of sulphur production in a biotechnological sulphide-removing reactor.

Abstract: A new biotechnological process for sulphide removal is proposed. The principle of this process is that sulphide is converted into elemental sulphur, which can be removed by sedimentation. In this article, investigations on the optimization of the sulphur production are reported. It seems that less than 10% sulphate is produced at low oxygen concentration, when the sulphide concentration in the reactor exceeds 10 mg/L. At sulphide concentrations higher than 20 mg/L only 5% of the incoming sulphide is converted to sulphate even at high oxygen concentrations. An immobilized biomass on recticulated polyurethane produced more sulphate than a free cell suspension at the same oxygen and sulphide concentration.

On-line prediction of fermentation variables using neural networks

Abstract: This article presents an introduction to the use of neural network computational algorithms for the dynamic modeling of bioprocesses. The dynamic neural model is used for the prediction of key fermentation variables. This relatively hew method is compared with a more traditional prediction technique to judge its performance for prediction. Illustrative simulation results of a continuous stirred tank fermentor are used for this comparison. It is shown that neural network models are accurate with a certain degree of noise immunity. They offer the distinctive ability over more traditional methods to learn very naturally complex relationships without requiring the knowledge of the model structure.

The effect of agitation on the morphology and penicillin production of Penicillium chrysogenum

Abstract: Penicillium chrysogenum strain P1 was grown on complex media in 10 and 100 L agitated fermenters at various aeration rates and stirrer speeds. Samples were removed at intervals for measurements of the culture morphology. At high stirrer speeds (1000 and 1200 rpm) in 10-L fermentations the rate of decrease in the mean effective hyphal length was faster and the rate of penicillin production was lower than fermentations done at 800 rpm. At similar power inputs per unit volume in 100-L fermentations, the change in mean effective hyphal length was less and higher penicillin production rates were observed. This work comparing the results at two scales has shown that neither of the concepts of impeller tip speed or the dissipation rate of turbulence have general validity as a measure of hyphal damage. Our results are reasonaby well correlated by groups similar to circulation rate (ND(i) (3)/V) with lower circulation rates being beneficial. An adaptation of the van Suijdam and Metz relationship, expressed as P/D(i) (3)t(c), was most successful. Our data are insufficient to demonstrate the generality of the relationship but do support the concept of a dispersion zone around the impellers in which mycelia may be damaged. The greater the frequency of circulation of mycelia through the zone the greater the damage and the lower the rate of penicillin synthesis by the culture.

Mechanism of enhanced oxygen transfer in fermentation using emulsified oxygen-vectors.

Abstract: Limitations of oxygen transfer in fermentation can be solved using auxiliary liquids immiscible in the aqueous phase The liquids (called oxygen-vectors) used in this study were hydrocarbon (n-dodecane) and perfluorocarbon (forane F66E) in which oxygen is highly soluble (549 mg/L in n-dodecane and 118 mg/L in forane F66E at 35 degrees C in contact with air at atmospheric pressure) It has been demonstrated that the use of n-dodecane emulsion in a culture of Aerobacter aerogenes enabled a 3 5-fold increase of the volumetric oxygen transfer coefficient(k(L)a) calculated on a per-liter aqueous phase basis The droplet size of the vector played a crucial role in the phenomena When a static contact between gas bubble and vector droplet was established in water, the vector covered the bubble, in agreement with positive values of the spreading coefficient for these fluids The determination of the oxygen transfer coefficients (k(L)) in a reactor with a definite interfacial area enabled the main resistance to be located in the boundary layer of the waterside either for a gas-water or a vector-water interface Because oxygen consumption by weakly hydrophobic cells can only occur in the aqueous phase, the oxygen transfer is achieved according to the following pathway: gas-vector-water-cell Finally, a mechanism for oxygen transfer within this four-phased system is proposed

Morphological measurements on filamentous microorganisms by fully automatic image analysis.

Abstract: Characterization of mycelial morphology is important for physiological and engineering studies of filamentous fermentations, and in the design and operation of such fermentations. Image analysis has been developed as a method for this characterization, and has been shown to be faster and generally more accurate than previous methods. A fully automatic system has been developed, in which speed is gained, but with loss of accuracy in some cases. The method has been tested on Streptomyces clavuligerus and Penicillium chrysogenum P1 batch fermentations. It has also been tested on a fed-batch Penicillium chrysogenum P2 fermentation, in which the medium contained solid ingredients. Fully automatic image analysis for morphological characterization of filamentous microorganisms is an important development which will make practical many engineering and physiological studies of such fermentations that have so far not been completely satisfactory.

Mechanism of biosorption of copper(II) by Ganoderma iucidum.

Abstract: In the present study, the mechanism of biosorption of copper (II) by the wood rotting fungi Ganoderma lucidum is being investigated, with specific reference to the mechanisms already proposed

Cell cycle‐ and growth phase‐dependent variations in size distribution, antibody productivity, and oxygen demand in hybridoma cultures

Abstract: Simultaneous determination of cell size and DNA content of hybridomas (HB-32) revealed a direct correlation between average cell volume and progression through the cell cycle. Pseudocontinuous experiments showed that G(1) cells, as estimated from cell size measurements, secreted monoclonal antibody at rates higher than those of cells in other stages of interphase and mitosis. Similarly, fed-batch and batch experiments suggested that specific oxygen uptake rate (qO(2)) is also a function of cell cycle, being minimum for cells in G(0) and G(1) phase. In batch cultures, HB-32 showed a rapid decrease in oxygen uptake rate (OUR) just prior to reaching maximum cell concentration. The OUR steadily increased from 0.01-0.05 to 0.5-0.7 mmol O(2)/L h as the cells went from the lag to the midexponential phase. The qO(2) increased from 0.3 x 10(-10)-0.9 x 10(-10) mmol O(2)/cell h at inoculation to 3.3 x 10(-10)-3.7 x 10(-10) mmol O(2)/cell h during the early exponential phase where it remained relatively constant. Several hours before maximum cell concentration was reached, OUR and qO(2) rapidly decreased to levels below those observed at inoculation. The time at which the shift in OUR and qO(2) occurred and the onset of decrease in the average cell size corresponded to the time of glutamine depletion. Based on monitoring OUR on-line in batch cultures, glutamine was supplemented, resulting in increased cell concentration, extension of culture viability, and increased MAb concentration.

Computer-aided synthesis of biochemical pathways

Abstract: The synthesis of biochemical pathways satisfying stoichiometric constraints is discussed. Stoichiometric constraints arise primarily from designating compounds as required or allowed reactants, and required or allowed products of the pathways; they also arise from similar restrictions on intermediate metabolites and bioreactions participating in the pathways. An algorithm for the complete and correct solution of the problem is presented; the algorithm satisfies each constraint by recursively transforming a base-set of pathways. The algorithm is applied to the problem of lysine synthesis from glucose and ammonia. In addition to the established synthesis routes, the algorithm constructs several alternative pathways that bypass key enzymes, such as malate dehydrogenase and pyruvate dehydrogenase. Apart from the construction of pathways with desired characteristics, the systematic synthesis of pathways can also uncover fundamental constraints in a particular problem, by demonstrating that no pathways exist to meet certain sets of specifications. In the case of lysine, the algorithm shows that oxaloacetate is a necessary intermediate in all pathways leading to lysine from glucose, and that the yield of lysine over glucose cannot exceed 67% in the absence of enzymatic recovery of carbon dioxide.

Lipolytic activities of a lipase immobilized on six selected supporting materials

Abstract: Six different types of materials including PVC, chitosan, chitin, agarose, Sepharose, and Trisacryl were evaluated for their lipase-coupling efficiencies. Among those tested, chitosan yielded the highest amount of lipase (79 mg/mL packed gel) immobilized but with lowest oil hydrolytic activity (0.03 mg eq/mL gel). The amount of lipase immobilized was affected by the length of the hydrocarbon chain attached to the PVC matrix but not by the pore size of the supports used. On the other hand, the specific activity of the immobilized lipase was affected by the pore size but not by the chain length of the hydrocarbon attached to the support. After immobilization, the optimal reaction pH was shifted from 7.5 to 8.5 and the optimal reaction temperature from 35 to 45-55 degrees C. Lipase immobilized on PVC exhibited higher thermal stability than that on agarose. The half-life of the PVC immobilized lipase operating at 30 degrees C in a packed-bed reactor was estimated to be about 400 h.

Growing Saccharomyces cerevisiae in calcium‐alginate beads induces cell alterations which accelerate glucose conversion to ethanol

Abstract: Nongrowing Saccharomyces cerevisiae cells previously grown in alginate exhibit ethanol production rates 1.5 times greater than cells previously grown in suspension. Analysis of glucose, ethanol, and glycerol formation data using quasi-steady-state pathway stoichiometry shows that alginate-grown cells possess phosphofructokinase (PFK), ATPase, and polysaccharide synthesis maximum activities which are approximately two-, two-, and ninefold larger, respectively, than in suspension-grown cells. The estimated change in PFK maximum velocity is consistent with in vitro assays of PFK activity in extracts of suspension- and alginate-grown yeast. Estimation of ethanol production flux control coefficients using in vivo nuclear magnetic resonance (NMR) spectroscopy measurements of intracellular metabolite concentrations and a previously proposed detailed kinetic model of ethanol fermentation in yeast shows that glucose uptake dominates flux control in alginate-grown cells in suspension while earlier research revealed that PFK and ATPase exert significant flux control in suspension-grown cells. When placed in a calcium alginate matrix, alginate-grown cells produced ethanol 1.8 times more rapidly and accumulated substantially more polyphosphate than suspension-grown cells placed in alginate. Cells growing in alginate elicit responses at the genetic level which substantially alter pathway rates and flux control when these cells are used as either a suspended or an immobilized biocatalyst. These responses in gene expression to growth in alginate serve to reconfigure flux controls in alginate to a pattern which is similar to that obtained for suspended-grown cells in suspension.

A liquid emulsion membrane process for the separation of amino acids.

Abstract: The method of using liquid emulsion membranes featuring the cation carrier D2EHPA [di-(2-ethylhexyl) phosphoric acid] for the separation of L-phenylalanine is examined. Results from experiments performed under various conditions are discussed and an optimal condition for separation is determined. The selectivity of the liquid emulsion membrane system is discussed. The effects of impurities such as sodium chloride, glucose, lactic acid, and L-tryptophan on the transport of L-phenylalanine are evaluated. It is shown that the liquid emulsion membrane system is a potential operation not only to separate L-phenylalanine but also concentrate it with great efficiency.

Esterification reactions of lipase in reverse micelles

Abstract: The activities of lipase from Candida cylindracea and Rhizopus delemar have been investigated in water/AOT/iso-octane reverse micellar media through the use of two esterification reactions: fatty acid-alcohol esterification and glyceride synthesis. Such media promotes the occurrence of these two lipase-catalyzed reactions due to its low water content. The effect of various parameters on the activity of lipase from C. cylindracea in reverse micelles was determined and compared to results where alternate media were employed. It was observed that the structure of the media, as dictated by the type and concentration of the substrates and products and by the water/AOT ratio, w0, had a strong impact on enzyme activity. Strong deactivation of both typase types occurred in reverse micelles, especially in the absence of substrates and for w0 values greater than 3.0. Glyceride synthesis was realized with lipase from R. delemar, but not with that from C. cylindracea; the temperature and concentration of substrates and water strongly dictated the reaction rate and the percent conversion.

The role of the plasma membrane fluidity on the shear sensitivity of hybridomas grown under hydrodynamic stress

Abstract: The role of the plasma membrane fluidity (PMF) on the shear sensitivity of HB-32 hybridomas to laminar fluid shear was investigated. Steady-state fluorescence anisotropy (r(s)) of the cationic fluorescent probe 1-[4-(trimethylamino) phenyl]-6-phenylhexa-1,3,5-triene, was used to evaluate the PMF of whole hybridoma cells. The PMF was manipulated by the addition of the anesthetic benzyl alcohol, by temperature changes and by cholesterol enrichment. The effect of these PMF modifying procedures on the shear sensitivity of HB-32 was assessed by exposing the cells to defined levels of laminar shear stress in a Couette flow device. Conditions that resulted in lower r(s) values (indicating higher PMF) yielded a more fragile cell. Batch cultivations supplemented with the shear protective agent Pluronic(R) F-68 showed higher values of r(s) compared to control experiments during various growth phases, suggesting that the protective mechanism of Pluronic F-68 relies on its ability to decrease the PMF through direct interaction with the plasma membrane. The protective mechanism of serum against turbulent fluid shear is also discussed from analysis of growth and death kinetics of agitated and static cultures at increasing serum levels. The results of this study show that the fluid state of the plasma membrane is important in determining the integrity of hybridomas when exposed to lethal shear levels. It is concluded that increasing membrane fluidity correlates with increasing shear sensitivity.

Production of glycerides from glycerol and fatty acid by immobilized lipases in non-aqueous media

Abstract: Immobilized Mucor miehei lipase catalyzes synthesis reactions between glycerol and oleic acid. No organic solvent is necessary to solubilize the substrates, which allows for the use of a reaction medium solely composed of the necessary substrates. Water produced in the reaction evaporates due to the high temperature used for the process. A conversion of 86% of oleic acid into triolein is obtained when using the substrates in stoichiometric amounts. Varying the ratio of glycerol over oleic acid allows for the preferential synthesis of one of the glycerides. Some batch reactors have been set up using different means of removing the water: spontaneous evaporation, molecular sieves, vacuum, and dry air bubbling.

High-level recombinant protein production in bioreactors using the baculovirus-insect cell expression system.

Abstract: In order to develop an efficient process for large-scale production of recombinant protein, various factors were studied which affect the productivity of Sf-9 (Spodoptera frugiperda) insect cells when using the baculovirus expression system. It was shown that upon infection with the Bac-BRV6L recombinant baculovirus, the level per cell of VP6 (a bovine rotavirus nucleocapsid protein) would drop 10-fold when host cell density at the time of infection increased from 2 x 10(6) to 3 x 10(6) cells/mL. The decrease was found to be totally reversible by culture medium renewal after infection, even when cells were infected at the stationary phase. Recombinant protein production was 4-6 times higher using TNMFH medium supplemented with 10% fetal bovine serum (FBS) than in IPL/41 serum-free medium. Fine-tuning of infection parameters in a 4-L surface-aerated bioreactor resulted in the production of typically 350 mg/L of VP6 protein, representing more than 25% of total cell proteins.

A balanced DO-stat and its application to the control of acetic acid excretion by recombinant Escherichia coli.

Abstract: During fed-batch cultivation of a recombinant Escherichia coli AT2471 harboring plasmid pSY130-14 for phenylalanine production, a large amount of acetic acid was excreted by the cells and accumulated in the culture medium. Acetic acid concentration reached 30-35 g/L at the end of a process conducted without special precautions for the reduction of this excretion. Cell growth stopped when acetic acid concentration was about 15 g/L, resulting in poor growth, 16 g/L cell concentration, and poor production - 8 g/L phenylalanine. A novel control strategy, called a balanced DO-stat. was developed to prevent acetic acid excretion. It represents a model-independent two-loop control structure, which is simple, reliable, and convenient for computer application. Using the balanced DO-stat, implemented in a computer control system, acetic acid concentration was kept at zero during the entire cultivation period. As a result, the cell concentration increased to 36 g/L and phenylalanine concentration reached 24 g/L. Aside from the phenylalanine fermentation, the proposed control approach might be applied to cultivation of other bacterial and yeast strains which have similar mechanism of the excretion of fermentative by-products.

Immobilization of Arthrobacter simplex in a thermally reversible hydrogel: Effect of temperature cycling on steroid conversion

Abstract: Arthrobacter simplex cells, which convert the steroid hydrocortisone to prednisolone, have been entrapped in a thermally reversible hydrogel. Such hydrogels exhibit a lower critical solution temperature (LCST) wherein the gel shrinks and deswells when it is warmed through its LCST, and then reversibly expands and reswells when it is cooled below the LCST. The immobilized cell-hydrogel system has been thermally cycled between two temperatures, each below the LCST. The upper temperature was selected to be just below the LCST, where the gel deswells but does not collapse, as it does at the LCST. The thermal cycling acts like a “hydraulic pump” which enhances mass transfer of the substrate (hydrocortisone) in and the product (prednisolone) out of the gel, thereby increasing steroid conversion dramatically relative to isothermal operation at either the upper or lower temperature. The increased conversion can also be due in part to reduced product inhibition. Mass transfer resistance and product inhibition are among the most serious problems in immobilized biocatalyst technology and thermal cycling of LCST hydrogels is both a novel and useful approach to minimizing these problems.

Pellet formation and cellular aggregation in Streptomyces tendae

Abstract: In submerged cultures, Streptomyces tendae tended to form fluffy spherical pellets of the noncoagulative type. An increase in the average pellet size could be attained by decreasing any of the following: shear rate, pH, temperature, or inoculum size. Conditions leading to oxygen limitation tended to reduce the average pellet size and induced pulpy growth, whereas oxygen sufficiency seemed to induce pellet formation. Factors inducing pellet formation simultaneously increased cell wall hydrophobicity. It is therefore proposed that the main forces inducing cellular aggregation in S. tendae are hydrophobic interactions of cell walls, and these interactions are controlled by availability of dissolved oxygen.

Solvent selection for biocatalysis in mainly organic systems: predictions of effects on equilibrium position.

Abstract: Predictions may be made for the influence of solvent choice on the equilibrium position of biocatalyzed reactions, based on data for the liquid–liquid distribution of the reactants. The most reliable predictions are probably for dilute systems, based on partition coefficients or correlations derived from them. The effective equilibrium constant for esterification reactions is predicted to alter by more than four orders of magnitude on changing between different water-immiscible solvents. The equilibrium constant correlates well with the solubility of water in the solvent, and is most favorable for synthesis in the least polar solvents (aliphatic hydrocarbons). Similar effects seem to apply for other reactions, including oxidation of alcohols and hydrolysis of chlorides. Predictions can be made for nondilute systems using the UNIFAC system of group contributions, but the reliability of these is more questionable.