Kinetic studies of phenol degradation by Rhodococcus sp. P1 I. Batch cultivation

Rhodococcus sp. P1 utilizes phenol as the sole carbon and energy source via the beta-ketoadipate pathway. In batch cultivation, concentrations up to 2.8 g.l-1 phenol were degraded. The highest values for the specific growth rate of 0.32 h-1 were obtained at concentrations near 0.25 g.l-1. At higher concentrations, substrate inhibition was observed, characterized by increases in lag phase and decreasing growth rates. A mathematical expression was proposed to fit the kinetic pattern of phenol inhibition on the specific growth rate mu: [formula: see text] Nomenclature: K- Exponent of the inhibition function, Ks- Monod saturation constant, g.l-1, KI- Inhibition constant, g.l-1, S- Substrate concentration in culture broth, g.l-1, So- Initial substrate concentration, g.l-1, Y- Yield constant, g cell dry mass.g substrate-1, mu- Specific growth rate, h-1, mu max- Maximum growth rate, h-1.     

D,L-Hydantoinase activity of an Ochrobactrum anthropi strain

AIMS: A microorganism with the ability to release methionine from D,L-(2-methylthioethyl) hydantoin (strain 245) was isolated from soil. The aim of this study was the identification of the strain and the adjustment of the conditions of growth and of the enzymatic reaction, in order to achieve high specific activities of bioconversion of the hydantoin. METHODS AND RESULTS: Strain 245 was identified as Ochrobactrum anthropi. The strain grew at alkaline pH (up to 10.0) and its hydantoinase activity was found to be inducible by the substrate D,L-(2-methylthioethyl) hydantoin. The enzyme is also alkalostable, with a pH optimum of 9.0. Under these conditions, hydantoinase activity was significantly enhanced and its half life prolonged when 200 mmol l-1 ammonium and phosphate were added. The addition of Ca2+, Na+, Cu2+, Co2+, Mg2+, Zn2+ or Fe3+ (0.5 mmol l-1) to the reaction mixture increased the hydantoinase activity of strain 245 up to tenfold after 24 h of incubation, compared with unamended controls. CONCLUSION: The adequate adjustment of some environmental parameters (pH, addition of inducer, presence of ammonium, phosphate, heavy metals and other ions) can considerably increase the D, L-hydantoinase activity of strain 245. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings reported here set up the initial conditions for a further application of strain 245 in the production of methionine from hydantoine.     

Volumetric productivity improvement for endoglucanase of Trichoderma pseudokoingii S-38

AIMS: The objective of this study was to design an economically feasible process for endoglucanase (EG) production. METHODS AND RESULTS: Trichoderma pseudokoingii S-38 EG synthesis was studied. Initially, either glucose at 2.5, 5 or 10 g l-1, or cellulose powder (CF11) at 5 g l-1 was used as the sole carbon source. The results showed that enzyme synthesis and biomass formation were closely correlated, and both were affected by the carbon source. To improve EG volumetric product efficiency, a new technique was developed. Glucose and CF11 (2.5 and 5 g l-1, respectively) were used as initial carbon source, and glucose was added at 2.5 g l-1 day-1. EG activity, volumetric and specific EG productivities were 6.17 IU l-1, 53 IU l-1 h-1 and 114.3 IU (g cell protein)-1 h-1, respectively. Batch production in a 2-l laboratory fermenter confirmed the advantage of the technique. The product contained 10.86 IU ml-1 EG activity in 88 h. The volumetric and specific EG productivities were 123.4 IU l-1 h-1 and 177.8 IU (g cell protein)-1 h-1, respectively. CONCLUSIONS: These results suggest that optimization of the ratio of glucose to CF11 for balancing the induction and growth rate in the production of EG may lead to technical and economical benefits. SIGNIFICANCE AND IMPACT OF THE STUDY: A new technique was developed for the production of EG which improves both the volumetric product efficiency and the specific activity.     

Large-scale (20 l) culture of surface-immobilized Catharanthus roseus cells.

Surface-immobilized C. roseus cell cultures were grown in a 20-l modified airlift bioreactor operated at 0.51 vvm (kLa approximately 8 h-1) under various gassing regimes [air, 2% (v/v) and 5% CO2]. Extracellular ammonium, phosphate, and nitrate ions as well as carbohydrate uptake and pH value of the medium were monitored together with on-line dissolved oxygen concentration, conductivity of the medium, and carbon dioxide production rate (CPR) of the cultures. Cultures supplemented with 2% CO2 showed higher nitrate (5.0-7.0 mM d-1) and carbohydrate (3.3 g l-1 d-1) uptake rates and biomass production (mu approximately 0.24 d-1, yield approximately 0.33 g dw g CHO-1 and 7.4 g dw L-1) as compared to air (3.6 mM d-1, 2.1 g l-1 d-1; 0.20 d-1, 0.25 g dw g CHO-1 and 5 g dw l-1) and 5% CO2 (2.0-3.6 mM d-1, 2.0 g l-1 d-1; 0.11 d-1, 0.20 g dw g CHO-1 and 5 g dw l-1) cultures and as reported previously for suspension cultures. In addition, air and 5% CO2 cultures displayed incomplete carbohydrate uptake and, more important, phosphate and ammonium ion release into the medium at the end, which was ascribed to loss of viability. This was not observed for 2% CO2 immobilized bioreactor as well as shake flask control suspension cultures, which suggests that sparged C. roseus surface-immobilized cell cultures require 2% CO2 supplementation of the gas phase for both maximum growth and retained viability. The maximum CPRs of all cultures were in the same range (2.1-2.8 mM CO2 l-1 h-1). However, the estimated maximum specific CO2 production rates of 2% CO2 and 5% CO2 immobilized cultures (0.6 mM g dw-1 h-1) were lower than those found for air-sparged immobilized cultures (1.0-1.3 mM g dw-1 h-1). These rates are significantly higher than those reported in the literature for C. roseus cell suspension cultures performed in bioreactors gassed with air (approximately 0.2-0.55 mM g dw-1 h-1).     

Effect of the nitrogen source on caffeine degradation by Aspergillus tamarii

AIMS: To evaluate caffeine degradation and nitrogen requirements during Aspergillus tamarii growth in submerged culture. METHODS AND RESULTS: Aspergillus tamarii spores produced on a coffee infusion agar medium added with sucrose were used. Several caffeine and ammonium sulphate concentrations (0-1 and 0-1.36 g l-1, respectively) were tested simultaneously on fungal biomass production and caffeine degradation. An additional caffeine pulse (4 g l-1) was added for all experiments after 48 h of fermentation. Results revealed that when using 0.90 g l-1 of caffeine and 0.14 g l-1 of ammonium sulphate, biomass production and caffeine degradation were enhanced. Highest biomass production (Xmax = 9.87 g l-1) with a specific growth rate (micro) of 0.073 h-1 and caffeine degradation rate of 0.033 g l-1 h-1, was observed under these conditions. CONCLUSIONS: Caffeine degradation as well as biomass production were characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: These studies set the stage for future characterization studies of intracellular enzymes involved in caffeine degradation. Moreover, results observed may help in the biotreatment of residues from the coffee agroindustry.     

Factors affecting the biosynthesis of L-tryptophan by genetically modified strains of Escherichia coli

Derivatives of Escherichia coli strain W3110 with increased tryptophan synthase (TS) activity were constructed. The biosynthesis of serine was shown to limit tryptophan production in minimal medium with indole as precursor. In the presence of serine and indole we obtained a correlation between the specific activity of TS and the specific productivity (qp) of tryptophan. Supplementation of the growth medium with glycine enhanced qp two-fold. In a strain with high serine hydroxymethyltransferase (SHMT) activity no such increase of tryptophan productivity was observed, although crude extracts from these cells were shown to produce tryptophan with indole, one-carbon units and glycine as precursors. Growth of the strain with high SHMT activity was inhibited in a medium with high glycine concentration. This inhibition could not be released by addition of isoleucine and valine. In a buffer system with permeabilized cells high in specific TS and SHMT activities we did not obtain any tryptophan production in presence of indole, glycine, one-carbon units and cofactors. On the other hand, in a buffer system with indole and serine as precursors we obtained high qp of tryptophan [13.3 g tryptophan (g dry wt cells)-1 h-1], which was correlated to the TS specific activity.     

Influence of medium composition on the cephalosporin C production with a highly productive strain Cephalosporium acremonium.

Cephalosporin production by a highly productive Cephalosporium acremonium strain was carried out and optimized by fed-batch operation in a 40 l stirred tank reactor using a complex medium containing 30-120 g l-1 peanut flour. The concentrations of cephalosporin C (CPC) and its precursors: penicillin N (PEN N), deacetoxy cephalosporin C (DAOC), and deacetyl cephalosporin C (DAC) were monitored with an on-line HPLC. The concentrations of amino acids valine (VAL), cysteine (CYS), alpha-amino adipic acid (alpha-AAA), the dipeptide alpha-amino-adipyl-cysteine (AC), and the tripeptide alpha-amino-adipyl-cysteinyl-valine (ACV), were determined off-line by HPLC. The RNA content and dry weight of the sediment as well as the oxygen transfer rate (OTR) and the CO2 production rate (CPR) were used to calculate the cell mass concentration (X). The influences of peanut flour (PF) and the on-line monitored and controlled medium components: glucose (GLU), phosphate, methionine (MET) as well as the dissolved oxygen (DOC) on the cell growth, the product formation, and the pathway of cephalosporin C biosynthesis were investigated and evaluated. When the glucose fed-batch cycle was optimized and oxygen transfer limitation was avoided (DOC greater than 20% of the saturation value), high process performance (103.5 g l-1 X, 11.84 g l-1 CPC, a maximum CPC productivity of 118 mg l-1 h-1, and the whole concentration of the beta-lactam antibiotics CPC, DAC, DAOC, PEN N 17.34 g l-1) was achieved by using 100 g l-1 PF in the medium with the optimum concentration of phosphate (260-270 mg l-1) and a low glucose concentration (less than 0.5 g l-1). The cultivations with different medium concentrations demonstrated that the product formation was directly proportional to the cell mass concentration. On the average, the cell mass-based yield coefficient of CPC: YCPC/X amounted to 0.115 g CPC per g cell mass.     

Physiology and metabolic fluxes of wild-type and riboflavin-producing Bacillus subtilis.

Continuous cultivation in a glucose-limited chemostat was used to determine the growth parameters of wild-type Bacillus subtilis and of a recombinant, riboflavin-producing strain. Maintenance coefficients of 0.45 and 0.66 mmol of glucose g-1 h-1 were determined for the wild-type and recombinant strains, respectively. However, the maximum molar growth yield of 82 to 85 g (cell dry weight)/mol of glucose was found to be almost identical in both strains. A nonlinear relationship between the specific riboflavin production rate and the dilution rate was observed, revealing a coupling of product formation and growth under strict substrate-limited conditions. Most prominently, riboflavin formation completely ceased at specific growth rates below 0.15 h-1. For molecular characterization of B. subtilis, the total amino acid composition of the wild type was experimentally determined and the complete building block requirements for biomass formation were derived. In particular, the murein sacculus was found to constitute approximately 9% of B. subtilis biomass, three- to fivefold more than in Escherichia coli. Estimation of intracellular metabolic fluxes by a refined mass balance approach revealed a substantial, growth rate-dependent flux through the oxidative branch of the pentose phosphate pathway. Furthermore, this flux is indicated to be increased in the strain engineered for riboflavin formation. Glucose catabolism at low growth rates with reduced biomass yields was supported mainly by the tricarboxylic acid cycle.     

Continuous cultivation of a human intestinal microflora at high cell concentration via controlled culture recycle

A bioreactor system equipped with a hollow fiber cross-filtration module was used for continuous cultivation of Lactobacillus acidophilus at high cell concentrations. The growth rate did not correlate with the lactate concentration if the residual glucose concentration was kept nearly zero in the culture broth. To achieve this, an effective control method of medium feed rate was developed on the basis of the correlation between the specific glucose consumption rate (nu) and the specific cell growth rate (mu), i.e. nu = 52.90 mu + 0.39. Growth up to 50 g dry wt l-1 was achieved without glucose accumulation under the total cell recycle. Via the partial cell recycle, continuous biomass production was achieved with a steady-state L. acidophilus concentration and dilution rate being 40 gl-1 and 0.09 h-1. © Rapid Science Ltd. 1998     

High cell density cultivation of Escherichia coli at controlled specific growth rate.

A high cell density cultivation (HCDC) for growth of Escherichia coli in an especially designed glucose/mineral salt medium is proposed. The HCDC essentially starts as a batch process which is followed by a two-phase fed-batch cultivation. After unlimited growth at mu max = 0.45 h-1 in the batch part, growth was controlled at a reduced specific growth rate (mu = 0.11 h-1 less than mu max) over a period of 3 doubling times in which the biomass concentration increased from 12 to 95 g 1(-1) (phase 1 of fed-batch cultivation). Control of growth (mu) was realized by a PO2 control loop (by variation of glucose feeding) and a mu control loop (by variation of agitation speed N) while the actual mu was calculated from the off-gas composition. If the agitation rate cannot be increased anymore the mu controller is switched off (end of phase 1). In the following phase 2, mu declines, however, the still acting pO2 (glucose) controller guarantees sufficient O2 supply till the end of the cultivation with a biomass concentration of 110 g 1(-1) (dry mass). The proposed HCDC suppresses generation of inhibitory by-products and the high yield coefficients indicate the economy of the process.     

Production of recombinant human interferon-alpha 1 by Escherichia coli using a computer-controlled cultivation process.

Genetically engineered E. coli K12 BMH-71-18 with plasmid PBV-867 was used for constitutive expression of human interferon-alpha 1 (IFN) with a defined medium. A manual, time-based, fed-batch cultivation process produced a cell density of 26.3 g l-1 (OD550 89), an IFN activity of 1.55 x 10(8) IU l-1 and a specific IFN productivity of 0.65 x 10(6) IU g-1. An analysis was conducted to characterize the problems involved in the high density microbial processes of recombinant protein production. The strategy suggested by the analysis is to establish a nutrient feeding profile that improves both the plasmid stability and the overall productivity of IFN. The nutrient feeding procedure developed here was based on the growth dynamics and a glucose consumption model. By using this procedure to continuously supply nutrients during cultivations, cell density reached 58 to 80 g l-1 and the specific IFN productivities of these runs were increased over that of the manual process. Nutrient feeding rates were found to affect the specific IFN productivity substantially. The optimized process achieved an IFN activity of 1.26 x 10(9) IU l-1, a cell density of 58 g l-1 and a specific IFN productivity of 2.2 x 10(7) IU g-1. More significantly, the overall productivity IU l-1 h-1 of the optimized, computer-controlled cultivation process was increased 12.9-fold over that of the manual cultivation process.     

Use of a novel air separation system in a fed-batch fermentative culture of Escherichia coli.

A novel air separation system based on permeable membrane gas separation technology was used to cultivate Escherichia coli. The system fulfilled the dissolved oxygen requirements of a culture of E. coli grown on a glucose synthetic medium at a high and constant growth rate of 0.55 h-1. A biomass yield of 45 g (dry weight) per liter was achieved, and no by-product inhibition by acetate or CO2 was observed.     

High production of 1,3-propanediol from industrial glycerol by a newly isolated Clostridium butyricum strain

Batch and continuous cultures of a newly isolated Clostridium butyricum strain were carried out on industrial glycerol, the major by-product of the bio-diesel production process. For both types of cultures, the conversion yield obtained was around 0.55 g of 1,3-propanediol formed per 1 g of glycerol consumed whereas the highest 1,3-propanediol concentration, achieved during the single-stage continuous cultures was 35-48 g l-1. Moreover, the strain presented a strong tolerance at the inhibitory effect of the 1,3-propanediol, even at high concentrations of this substance at the chemostat (e.g. 80 g l-1). 1,3-Propanediol was associated with cell growth whereas acetate and butyrate seemed non growth-associated products. At low and medium dilution rates (until 0.1 h-1), butyrate production was favoured, whereas at higher rates acetate production increased. The maximum 1,3-propanediol volumetric productivity obtained was 5.5 g l-1 h-1. A two-stage continuous fermentation was also carried out. The first stage presented high 1,3-propanediol volumetric productivity, whereas the second stage (with a lower dilution rate) served to further increase the final product concentration. High 1,3-propanediol concentrations were achieved (41-46 g l-1), with a maximum volumetric productivity of 3.4 g l-1 h-1. A cell concentration decrease was reported between the second and the first fermentor.     

Tryptophan over-producing cell suspensions of Catharanthus roseus (L) G. Don and their up-scaling in stirred tank bioreactor: detection of a phenolic compound with antioxidant potential

Five cell suspension lines of Catharanthus roseus resistant to 5-methyl tryptophan (5-MT; an analogue of tryptophan) were selected and characterized for growth, free tryptophan content and terpenoid indole alkaloid accumulation. These lines showed differential tolerance to analogue-induced growth inhibition by 30 to 70 mg/l 5-MT supplementation (LD₅₀?=?7–15 mg/l). Lines P40, D40, N30, D50 and P70 recorded growth indices (i.e. percent increment over the initial inoculum weight) of 840.9, 765.0, 643.9, 585.7 and 356.5 in the absence and, 656.7, 573.9, 705.8, 489.0 and 236.0 in the presence of 5-MT after 40 days of culture, respectively. A corresponding increment in the free tryptophan level ranging from 46.7 to 160.0 μg/g dry weight in the absence and 168.0 to 468.0 μg/g dry weight in the presence was noted in the variant lines. Higher tryptophan accumulation of 368.0 and 468.0 g/g dry weight in lines N30 and P40 in 5-MT presence also resulted in higher alkaloid accumulation (0.65 to 0.90 % dry weight) in them. High-performance liquid chromatography (HPLC) analysis of the crude alkaloid extracts of the selected lines did not show the presence of any pharmaceutically important monomeric or dimeric alkaloids except catharanthine in traces in the N30 line that was also unique in terms of a chlorophyllous green phenotype. The N30 line under optimized up-scaling conditions in a 7-l stirred tank bioreactor using Murashige and Skoog medium containing 2 mg/l α-naphthalene acetic acid and 0.2 mg/l kinetin attained 18-folds biomass accumulation within 8 weeks. Interestingly, the cell biomass yield was enhanced to 30-folds if 30 mg/l 5-MT was added in the bioreactor vessel one week prior to harvest. Crude alkaloid extract of the cells grown in shake flask and this bioreactor batch also showed the formation of yellow-coloured crystals which upon ¹HNMR and ESI-MS analysis indicated a phenolic identity. This crude alkaloid extract of bioreactor-harvested cells containing this compound at 50 μg/ml concentration registered 65.21, 17.75, 97.0, 100 % more total antioxidant capacity, reducing power, total phenolic content, and ferric-reducing antioxidant power, respectively, when compared with that of extracts of cells grown in shake flask cultures. The latter, however, showed 57.47 % better radical scavenging activity (DPPH) than the bioreactor-harvested cells.     

营养条件对兽疫链球菌发酵生产透明质酸的影响

透明质酸 (Ｈｙａｌｕｒｏｎｉｃａｃｉｄ ,简称ＨＡ)是Ｎ 乙酰氨基葡萄糖胺和葡萄糖醛酸以 β 1 3糖苷键和 β 1 4糖苷键连接而成的二糖单体重复构建而成的杂多糖 ,广泛存在于高等动物的结缔组织内。由于结构上的特点 ,ＨＡ具有很高的粘弹性和极强的保水性等特征 ,已被大量用于医学医药、化妆品工业[1,2 ] 。1937年Ｋｅｎｄａｌｌ[3 ] 等发现用溶血性链球菌 (Ｓｔｒｅｐｔｏｃｏｃｃｕｓｈａｅｍｏｌｙｔｉｃｕｓ)可以产生ＨＡ。其后 ,陆续发现许多能产生ＨＡ的微生物菌种 ,逐渐开发出一条可替代传统的动物组织提取法[4 ] 生产ＨＡ的新途径…     

Heterotrophic high cell-density fed-batch cultures of the phycocyanin-producing red alga Galdieria sulphuraria

Growth and phycocyanin production in batch and fed-batch cultures of the microalga Galdieria sulphuraria 074G, which was grown heterotrophically in darkness on glucose, fructose, sucrose, and sugar beet molasses, was investigated. In batch cultures, specific growth rates and yields of biomass dry weight on the pure sugars were 1.08-1.15 day-1 and 0.48-0.50 g g-1, respectively. They were slightly higher when molasses was the carbon source. Cellular phycocyanin contents during the exponential growth phase were 3-4 mg g-1 in dry weight. G. sulphuraria was able to tolerate concentrations of glucose and fructose of up to 166 g L-1 (0.9 M) and an ammonium sulfate concentration of 22 g L-1 (0.17 M) without negative effects on the specific growth rate. When the total concentration of dissolved substances in the growth medium exceeded 1-2 M, growth was completely inhibited. In carbon-limited fed-batch cultures, biomass dry weight concentrations of 80-120 g L-1 were obtained while phycocyanin accumulated to concentrations between 250 and 400 mg L-1. These results demonstrate that G. sulphuraria is well suited for growth in heterotrophic cultures at very high cell densities, and that such cultures produce significant amounts of phycocyanin. Furthermore, the productivity of phycocyanin in the heterotrophic fed-batch cultures of G. sulphuraria was higher than is attained in outdoor cultures of Spirulina platensis, where phycocyanin is presently obtained.     

Effect of amino acid supplement on cell yield and gene product in Escherichia coli harboring plasmid

Escherichia coli harboring a recombinant plasmid was cultivated in fed-batch culture to enhance production of a gene product. Expression of the leucine gene from Thermus thermophilus in the recombinant plasmid was examined by the assay of beta-isopropylmalate dehydrogenase activity at 75 degrees C. When E. coli was cultivated in medium without leucine, biomass concentration reached 15 g/L and the specific activity became 0.082 U/mg protein. When leucine was fed in the medium throughout cultivation, although biomass concentration reached 63 g/L, the specific activity decreased to 0.016 U/mg protein. When E. coli was cultivated in medium containing 1 g leucine/L, the specific activity remained virtually constant (about 0.13 U/mg protein) and biomass concentration reached 32 g dry cells/L. In these cultivations, growth yields of several amino acids and glucose were examined. When leucine was not added to the medium, growth yields except for histidine were lowest. When leucine was fed throughout the cultivation, growth yields of glucose and tryptophan were highest. The pH-stat was useful for feeding amino acids.     

Biosynthesis of exopolysaccharide by Pseudomonas aeruginosa.

In batch cultures of Pseudomonas aeruginosa, the maximum rate of exopolysaccharide synthesis occurred during exponential growth. In nitrogen-limited continuous culture, the specific rate of exopolysaccharide synthesis increased from 0.27 g g of cell-1 h-1 at a dilution rate (D) of 0.05 h-1 to 0.44 g g of cells h-1 at D=0.1 H-1. The yield of exopolysaccharide on the basis of glucose used was in the range of 56 to 64%. Exopolysaccharide was also synthesized in carbon-limited cultures at 0.19 g g of cell-1 h-1 at D=0.05 h-1 in a 33% yield. Nonmucoid variants appeared after seven generations in continuous culture and rapidly increased in proportion to the total number of organisms present.     

Production of biomass and beta-D-galactosidase by Candida pseudotropicalis grown in continuous culture on whey

The production of biomass and beta-D-galactosidase by the lactose-utilizing yeast Candida pseudotropicalis NCYC 744 in whey medium was studied. Apparent optimization of growth conditions and medium was done in continuous culture. Optimaql pH and temperature were 2.6 and 36-38 degrees C, respectively, Limitations in Cu, Zn, and possbily Mn were detected in deproteinized whey medium. Additions of tryptophan estimulated growth of the yeast. Under optimal conditions in medium supplemented with excess tryptophan, Cu, Zn, and Mn the maximum values obtained: yeast concentration, 4.6 g/L; yeast productivity, 1.4 g/L h (at D = 0.35 h(-1)); enzyme volumetric productivity, 2100 U/L h (at D = 0.25 h(-1)); maintenance coefficient, 5-10 mg lactose/g cell h; saturation constant (K(s)) for lactose, 4.76mM; maximum specific growth rate, (mu(max)), 0.47 h(-1). No significant increase in specific enzyme activity (U/mg cell) was observed after medium optimiztion evidencing the importance of regulatory controls in enzyme synthesis.     

Production of a new D-amino acid oxidase from the fungus Fusarium oxysporum.

The fungus Fusarium oxysporum produced a D-amino acid oxidase (EC 1. 4.3.3) in a medium containing glucose as the carbon and energy source and ammonium sulfate as the nitrogen source. The specific D-amino acid oxidase activity was increased up to 12.5-fold with various D-amino acids or their corresponding derivatives as inducers. The best inducers were D-alanine (2.7 microkat/g of dry biomass) and D-3-aminobutyric acid (2.6 microkat/g of dry biomass). The addition of zinc ions was necessary to permit the induction of peroxisomal D-amino acid oxidase. Bioreactor cultivations were performed on a 50-liter scale, yielding a volumetric D-amino acid oxidase activity of 17 microkat liter(-1) with D-alanine as an inducer. Under oxygen limitation, the volumetric activity was increased threefold to 54 microkat liter(-1) (3,240 U liter(-1)).