Factors Influencing the Production of a Novel Compound, 7,10-Dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic Acid,by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3 (NRRL B-18602) in Batch Cultures

Pseudomonas aeruginosa PR3 (NRRL B-18602) converts oleic acid to a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD). Parameters that included medium volume, cell growth time, gyration speed, pH, substrate concentration, and dissolved oxygen concentration were evaluated for a scale-up production of DOD in batch cultures using Fernbach flasks and a bench-top bioreactor. Maximum production of about 2 g DOD (38% yield) was attained in Fernbach flasks containing 500 ml medium when cells were grown at 28°C and 300 rpm for 16–20 h and the culture was adjusted to pH 7 prior to substrate addition. Increases of medium volume and substrate concentration failed to enhance yield. When batch cultures were initially conducted in a reactor, excessive foaming occurred that made the bioconversion process inoperable. This was overcome by a new aeration mechanism that provided adequate dissolved oxygen to the fermentation culture. Under the optimal conditions of 650 rpm, 28°C, and 40–60% dissolved oxygen concentration, DOD production reached about 40 g (40% yield) in 4.5 L culture medium using a 7-L reactor vessel. This is the first report on a successful scale-up production of DOD. Received: 26 September 2002 / Accepted: 24 October 2002     

Scale-up from shake flasks to fermenters in batch and continuous mode with Corynebacterium glutamicum on lactic acid based on oxygen transfer and pH

Scale-up from shake flasks to fermenters has been hampered by the lack of knowledge concerning the influence of operating conditions on mass transfer, hydromechanics, and power input. However, in recent years the properties of shake flasks have been described with empirical models. A practical scale-up strategy for everyday use is introduced for the scale-up of aerobic cultures from shake flasks to fermenters in batch and continuous mode. The strategy is based on empirical correlations of the volumetric mass transfer coefficient (k(L) a) and the pH. The accuracy of the empirical k(L) a correlations and the assumptions required to use these correlations for an arbitrary biological medium are discussed. To determine the optimal pH of the culture medium a simple laboratory method based on titration curves of the medium and a mechanistic pH model, which is solely based on the medium composition, is applied. The effectiveness of the scale-up strategy is demonstrated by comparing the behavior of Corynebacterium glutamicum on lactic acid in shake flasks and fermenters in batch and continuous mode. The maximum growth rate (micro(max) = 0.32 h(-1)) and the oxygen substrate coefficient (Y O2 /S= 0.0174 mol/l) of C. glutamicum on lactic acid were equal for shake flask, fermenter, batch, and continuous cultures. The biomass substrate yield was independent of the scale, but was lower in batch cultures (Y(X/S) = 0.36 g/g) than in continuous cultures (Y(X/S) = 0.45 g/g). The experimental data (biomass, respiration, pH) could be described with a simple biological model combined with a mechanistic pH model.     

Modeling of growth and laccase production by Pycnoporus sanguineus

Production of extracellular laccase by the white-rot fungus Pycnoporus sanguineus was examined in batch submerged cultures in shake flasks, baffled shake flasks and a stirred tank bioreactor. The biomass growth in the various culture systems closely followed a logistic growth model. The production of laccase followed a Luedeking-Piret model. A modified Luedeking-Piret model incorporating logistic growth effectively described the consumption of glucose. Biomass productivity, enzyme productivity and substrate consumption were enhanced in baffled shake flasks relative to the cases for the conventional shake flasks. This was associated with improved oxygen transfer in the presence of the baffles. The best results were obtained in the stirred tank bioreactor. At 28 °C, pH 4.5, an agitation speed of 600 rpm and a dissolved oxygen concentration of ~25 % of air saturation, the laccase productivity in the bioreactor exceeded 19 U L^?1 days^?1, or 1.5-fold better than the best case for the baffled shake flask. The final concentration of the enzyme was about 325 U L^?1.     

A facile reactor process for producing 7,10-dihydroxy-8(E)-octadecenoic acid from oleic acid conversion by Pseudomonas aeruginosa

Pseudomonas aeruginosa strain PR3 (NRRL B-18602) converts oleic acid to a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD). The bioconversion was scaled up in a 7-l bench-top, stirred-batch reactor to produce DOD for testing of potential industrial uses. Aeration was supplied continuously from the top through two ports on the headplate and periodically through a bottom sparger, in conjunction with the use of marine impellers for agitation. This unique aeration arrangement maintained the dissolved O₂ concentration in the 40–60% range during the period of maximal bioconversion and it also avoided excessive medium foaming during the reaction. Furthermore, the level of dissolved O₂ in the first 24 h of reaction played an important role in the initial rate of DOD production. DOD production reached a plateau after 72 h with a yield up to 100 g (or 50% recovery) from a total of 9 l medium from two reactors run simultaneously. The final culture broth was processed using newly adapted procedures in the pilot plant that included crystallization of DOD from ethyl acetate solution at –15°C. The newly developed bioprocess will serve as a platform for the scale-up production of other value-added products derived from vegetable oils and their component fatty acids.     

Recombinant antibody production by perfusion cultures of rCHO cells in a depth filter perfusion system

Recombinant Chinese hamster ovary cells, producing recombinant antibody against the human platelet, were cultivated in a depth filter perfusion system (DFPS). When perfusion cultures with working volume of 1 L were operated at perfusion rates of 5/d and 6/d, volumetric antibody productivities reached values 28 and 34 times higher than that of batch suspension culture in Erlenmeyer flasks and 43 and 53 times higher than that of batch culture in a controlled stirred tank reactor, respectively. Perfusion cultures in the DFPS showed stable antibody production over the whole culture period of up to 20 days. In the DFPS, inoculated cells in suspension were entrapped in a few hours within the depth filter matrix by medium circulation and retained there until the void space of the filter matrix was saturated by the cultured cells. After cells in the depth filter matrix reached saturation, overgrown viable cells at a perfusion rate of 5/d or 6/d were continuously collected into waste medium at a density of 2-4 x 10(5) cells/mL, which resulted in stable operation at high perfusion rates, maintaining values of process parameters such as glucose/lactate concentration, pH, and dissolved oxygen concentration. Because the DFPS overcomes most drawbacks observed with conventional perfusion systems, it is preferable to be used as a key culture system to produce monoclonal antibody stably for a long culture period.     

Mass production of aphicidal Beauveria bassiana SFB-205 supernatant with the parameter of chitinase

Beauveria bassiana SFB-205 supernatant can effectively control cotton aphid populations, which is closely associated with its chitinase activity. The present work extends to optimizing a culture medium to produce more efficacious supernatant in flask conditions, followed by scale-up in 7 L, 300 L and 1.2 KL fermentors with the parameter of chitinase. In flask conditions, a combination of soluble starch and yeast extract produced the greatest amount of chitinase (5.1 units/ml) and its supernatant had the highest aphicidal activity. An optimal quantitative combination of the two substrates, estimated by a response surface method, enabled the supernatant to have 15.7 units/ml of chitinase activity and 3.7 ml/l of median lethal concentration (LC50) of toxicity against cotton aphid adults in laboratory conditions. In the scale-up conditions, overall supernatant had 25-28 units/ml of chitinase activity. Decrease in pH and limitation of dissolved oxygen (DO) during cultures were significantly related to the yield of chitinase. These results suggest that the substrate-dependent chitinase production can be background information for optimizing a culture medium, and pH and DO are critical factors in maximizing the production in scale-up conditions.     

Multiple microfermentor battery: a versatile tool for use with automated parallel cultures of microorganisms producing recombinant proteins and for optimization of cultivation protocols

A multiple microfermentor battery was designed for high-throughput recombinant protein production in Escherichia coli. This novel system comprises eight aerated glass reactors with a working volume of 80 ml and a moving external optical sensor for measuring optical densities at 600 nm (OD600) ranging from 0.05 to 100 online. Each reactor can be fitted with miniature probes to monitor temperature, dissolved oxygen (DO), and pH. Independent temperature regulation for each vessel is obtained with heating/cooling Peltier devices. Data from pH, DO, and turbidity sensors are collected on a FieldPoint (National Instruments) I/O interface and are processed and recorded by a LabVIEW program on a personal computer, which enables feedback control of the culture parameters. A high-density medium formulation was designed, which enabled us to grow E. coli to OD600 up to 100 in batch cultures with oxygen-enriched aeration. Accordingly, the biomass and the amount of recombinant protein produced in a 70-ml culture were at least equivalent to the biomass and the amount of recombinant protein obtained in a Fernbach flask with 1 liter of conventional medium. Thus, the microfermentor battery appears to be well suited for automated parallel cultures and process optimization, such as that needed for structural genomics projects.     

Scale-up and optimization of culture conditions of a human heterohybridoma producing serotype-specific antibodies to Pseudomonas aeruginosa

Summary Three different stirred bioreactors of 0.5 to 12 l volume were used to scale up the production of a human monoclonal antibody. Inoculation density and stirrer speed were evaluated in batch cultures, whereas dilution rate and pH were optimized in chemostat cultures with respect to high specific antibody production rate and high antibody yield per time and reactor volume. The cell line used for the experiments was a heterohybridoma, producing immunoglobulin M (IgM) against lipopolysaccharide of Pseudomonas aeruginosa. Cells were cultured in spinner flasks of 500 ml liquid volume for adaptation to stirred culture conditions. Subsequently cells were transferred to the 1.5-1 KLF 2000 bioreactor and to the 12-1 NLF 22 bioreactor for pilot-scale cultures. Chemostat experiments were done in the 1.5-1 KLF bioreactor. Cell density, viability, glucose and lactate and antibody concentration were measured during culture experiments. In batch cultures in all three stirred bioreactors, comparable maximal cell densities and specific growth rates were achieved. Chemostat experiments showed that at a pH of 6.9 and a dilution rate of 0.57 per day the specific antibody production rate was threefold higher than similar experiments done at pH 7.2 with a dilution rate of 0.36 per day. By optimizing pH and dilution rate in chemostat cultures the daily yield of human IgM increased nearly threefold from 6 to 16 mg/day and per litre of reactor volume. The yield per litre of medium increased twofold. Correspondence to: U. Schürch     

Multiple Microfermentor Battery: a Versatile Tool for Use with Automated Parallel Cultures of Microorganisms Producing Recombinant Proteins and for Optimization of Cultivation Protocols

A multiple microfermentor battery was designed for high-throughput recombinant protein production in Escherichia coli. This novel system comprises eight aerated glass reactors with a working volume of 80 ml and a moving external optical sensor for measuring optical densities at 600 nm (OD₆₀₀) ranging from 0.05 to 100 online. Each reactor can be fitted with miniature probes to monitor temperature, dissolved oxygen (DO), and pH. Independent temperature regulation for each vessel is obtained with heating/cooling Peltier devices. Data from pH, DO, and turbidity sensors are collected on a FieldPoint (National Instruments) I/O interface and are processed and recorded by a LabVIEW program on a personal computer, which enables feedback control of the culture parameters. A high-density medium formulation was designed, which enabled us to grow E. coli to OD₆₀₀ up to 100 in batch cultures with oxygen-enriched aeration. Accordingly, the biomass and the amount of recombinant protein produced in a 70-ml culture were at least equivalent to the biomass and the amount of recombinant protein obtained in a Fernbach flask with 1 liter of conventional medium. Thus, the microfermentor battery appears to be well suited for automated parallel cultures and process optimization, such as that needed for structural genomics projects.     

Isomaltulose production using free cells: optimisation of a culture medium containing agricultural wastes and conversion in repeated-batch processes

The enzyme glucosyltransferase is an industrially important enzyme since it produces non-cariogenic isomaltulose (6-O-alpha-D-glucopyronosyl-1-6-D-fructofuranose) from sucrose by intramolecular transglucosylation. The experimental designs and response surface methodology (RSM) were applied for the optimisation of the nutrient concentrations in the culture medium for the production of glucosyltransferase by Erwinia sp. D12 in shaken flasks at 200 rpm and 30 degrees C. A statistical analysis of the results showed that, in the range studied, the factors had a significant effect (P < 0.05) on glucosyltransferase production and the highest enzyme activity (10.84 U/ml) was observed in culture medium containing sugar cane molasses (150 g l(-1)), corn steep liquor (20 g l(-1)), yeast extract Prodex Lac SD (15 g l(-1)) and K2HPO4 (0.5 g l(-1)) after 8 h at 30 degrees C. The production of cell biomass by the strain of Erwinia sp. D12 was carried out in a 6.6-l fermenter with a mixing rate of 200 rpm and an aeration rate of 1 vvm. Fermentation time, cellular growth, medium pH and glucosyltransferase production were observed. The greatest glucosyltransferase activity was 22.49 U/ml, obtained after 8 h of fermentation. The isomaltulose production from sucrose was performed using free Erwinia sp. D12 cells in a batch process using an orbital shaker. The influence of the parameters sucrose concentration, temperature, pH, and cell concentration on the conversion of sucrose into isomaltulose was studied. The free cells showed a high conversion rate of sucrose into isomaltulose using batch fermentation, obtaining an isomaltulose yield of 72.11% from sucrose solution 35% at 35 degrees C.     

Co-immobilized aerobic/anaerobic mixed cultures in stirred tank and gaslift loop reactors

Co-immobilized Aspergillus awamori and Zymomonas mobilis cultures were investigated in a stirred tank reactor on synthetic medium with starch as substrate at various dissolved oxygen concentrations. In a gaslift loop reactor, freely suspended and immobilized A. awamori were cultivated on synthetic medium and soluble potato starch. In the same reactor, the growth and ethanol production of freely suspended and immobilized Z. mobilis cultures were studied on synthetic medium and glucose. Co-immobilized A. awamori and Z. mobilis were cultivated in batch and continuous operations in the gaslift loop reactor on synthetic medium with starch substrate at different dissolved oxygen concentrations. The interrelations between the different process variables are discussed.     

溶氧对Blakeslea trispora分批发酵生产β-胡萝卜素的影响

在摇瓶和5 L发酵罐中研究了溶氧 (DO) 对Blakeslea trispora分批发酵生产β-胡萝卜素的影响,总结了5 L发酵罐中β-胡萝卜素发酵过程中溶氧的变化规律.结果表明,当500 mL摇瓶装液量为50 mL,转速为240 r/min条件下发酵生产β-胡萝卜素产量最大,达到3.416 g/L; 5 L发酵罐中,在搅拌转速为1 000 r/min,通气量为1.5 vvm的条件下,β-胡萝卜素的产量可达到3.712 g/L,略高于摇瓶,这可能是由于5 L发酵罐中的气液传递和混合状况好于摇瓶,促进了产物的合成.     

The pH mediated effects of initial glucose concentration on the transitory occurrence of extracellular metabolites, gas exchange and growth yields of aerobic batch cultures of Klebsiella pneumoniae

The changes in growth kinetics in aerobic batch cultures of Klebsiella pneumoniae were followed by measurements of extracellular metabolites, rates of gas exchange, dissolved oxygen tension, pH, and carbon balance at all stages of growth. When the initial growth-limiting glucose concentration in media without pH control was increased from 1.0 g carbon L(-1) to 2.2 g carbon L(-1), the number of different, mainly acidic, extracellular metabolites of glucose at the end of exponential growth increased, while the proportion of acetate decreased. During the postexponential growth phase, the extracellular metabolites were oxidized, resulting in an increasing complexity of changes in pH, gas exchange, and dissolved oxygen tension with increasing initial substrate concentration. All these parameters showed concomitant stepwise changes. This pattern was independent of the dissolved oxygen tension in the range 30-200 muM. When pH was kept constant, the number, slope, and relative magnitude of the steps in gas exchange and dissolved oxygen tension were pH-dependent, being most complex at low pH. Detailed carbon balances showed that 20% of the initial glucose was converted into extracellular metabolites at the end of exponential growth at neutral pH. In the postexponential phase, pyruvate (2%) was reoxidized first followed by acetate (13%). The observed molar growth yield coefficient (Y(ATP)) was 8.4 if the transitory occurrence of pyruvate and acetate was accounted for, and 6.4 if it was neglected. The corrected observed molar growth yield coefficient (Y'(ATP)) was 9.4 and compared well with the true molar growth yield coefficient (Y(Max) (ATP)), which was found to be 11.0. Specific in situ respiration rates of the exponential growth phase of cultures grown at different controlled pH values compared well with in situ values for energy-limited chemostat grown cells at the same growth rates, suggesting that growth in the batch culture was energy-limited throughout the exponential growth phase. This view was supported by low levels of intracellular glycogen and exopolysaccharides of all cultures, by the value of Y'(ATP) of 9.4, and by a constant specific production rate of the extracellular metabolites throughout exponential growth. It was concluded that even under strictly aerobic conditions, control of pH is as important as control of dissolved oxygen tension during growth of enterobacteriaceae in batch cultures.     

High cell density cultivation of Pseudomonas oleovorans: growth and production of poly (3-hydroxyalkanoates) in two-liquid phase batch and fed-batch systems

Pseudomonas oleovorans is able to accumulate poly(3-hydroxyalkanoates) (PHAs) under conditions of excess n-alkanes, which serve as sole energy and carbon source, and limitation of an essential nutrient such as ammonium. In this study we aimed at an efficient production of these PHAs by growing P. oleovorans to high cell densities in fed-batch cultures.To examine the efficiency of our reactor system, P. oleovorans was first grown in batch cultures using n-octane as growth substrate and ammonia water for pH regulation to prevent ammonium limiting conditions. When cell growth ceased due to oxygen limiting conditions, a maximum cell density of 27 g .L(-1) dry weight was obtained. When the growth temperature was decreased from the optimal temperature of 30 degrees -18 degrees C, cell growth continued to a final cell density of 35 g . L(-1) due to a lower oxygen demand of the cells at this lower incubation temperature.To quantify mass transfer rates in our reactor system, the volumetric oxygen transfer coefficient (k(L)a) was determined during growth of P. oleovorans on n-octane. Since the stirrer speed and airflow were increased during growth of the organism, the k(L)a also increased, reaching a constant value of 0.49 s(-1) at maximum airflow and stirrer speed of 2 L . min(-1) and 2500 rpm, respectively. This k(L)a value suggests that oxygen transfer is very efficient in our stirred tank reactor.Using these conditions of high oxygen transfer rates, PHA production by P. oleovorans in fed-batch cultures was studied. The cells were first grown batchwise to a density of 6 g . L(-1), after which a nutrient feed, consisting of (NH(4))(2)SO(4) and MgSO(4), was started. The limiting nutrient ammonium was added at a constant rate of 0.23 g NH(4) (+) per hour, and when after 38 h the feed was stopped, a biomass concentration of 37.1 g . L(-1) was obtained. The Cellular PHA content was 33% (w/w), which is equal to a final PHA yield of 12.1 g . L(-1) and an overall PHA productivity of 0.25 g PHA produced per liter medium per hour. (c) 1993 John Wiley & Sons, Inc.     

Effect of pH, temperature and nutrient limitations on growth and leukotoxin production by Mannheimia haemolytica in batch and continuous culture

AIMS: Quantification of the effects of pH, temperature and nutrient limitations on the growth and leukotoxin (LKT) production parameters of Mannheimia haemolytica in batch and chemostat culture. METHODS AND RESULTS: Mannheimia haemolytica strains OVI-1 and PH12296 were grown aerobically in two semi-defined media. In amino acid-limited cultures, the LKT concentration and yield in terms of biomass (Y(LKT/x)) were up to eightfold greater than in carbon-limited cultures. Supplementing amino acid-limited chemostat cultures with cysteine, glutamine, ferric iron and manganese further enhanced the Y(LKT/x) values up to threefold. Supplementation of an amino acid-limited batch culture of M. haemolytica strain OVI-1 with these nutrients resulted in an LKT concentration of 1.77 g l(-1) that was 45-fold greater than that obtained in RPMI 1640 medium. Aerobiosis enhanced LKT production. High acetic acid concentrations were produced under carbon-sufficient conditions. The highest maximum specific growth rates were recorded in the range of pH 6.8 to 7.8 and 37 to 40 degrees C. CONCLUSIONS: An amino acid-limited culture medium greatly improved LKT production in aerobic batch culture, which could be further enhanced by supplementation with cysteine, glutamine, ferric iron and manganese. SIGNIFICANCE AND IMPACT OF THE STUDY: It was demonstrated that LKT production by M. haemolytica could be dramatically increased through manipulation of the culture medium composition, which could benefit the production of LKT-based vaccines against bovine shipping fever pneumonia.     

Exopolysaccharide production by Acremonium persicinum in stirred-tank and air-lift fermentors

Summary Exopolysaccharide production by the fungus Acremonium persicinum was affected by the culture system used. The yields achieved in shake flasks were not obtained in a stirred tank reactor, except at very low stirring speeds (100 rpm). However when grown in an air-lift fermentor, exopolysaccharide levels were similar to those found with shake flask cultures. Results suggest that both dissolved oxygen tension and shear rate may determine the ability of this organism to synthesise this exopolysaccharide. Offprint requests to: R. J. Seviour     

The kinetics of cholesterol oxidase synthesis by Nocardia rhodocrous.

The production of cholesterol oxidase by 3 liter batch cultures of Nocardia rhodocrous growing on a glycerol/yeast extract medium was investigated. Cholesterol was shown to be a good inducer of the enzyme. The optimum time for cholesterol addition and the quantity to be added were determined, resulting in a 15-fold yield increase. Cholesterol oxidase synthesis was influenced by the dissolved oxygen tension. Maximum cholesterol oxidase production was obtained at 30-40% air saturation. The effect of growth conditions on the extraction of cholesterol oxidase by Triton X-100 was investigated. The scale-up of the fermentation to 800 liters in a pilot-plant fermenter is described.     

Biodegradation of di-n-butyl phthalate (DnBP) in bioaugmented bioslurry phase reactor

Bioremediation of di-n-butyl phthalate (DnBP) in soil was studied with various concentrations in a bioslurry phase batch reactor operated in sequenting batch mode (bioaugmented with effluent treatment plant (ETP) microflora) for a total cycle period of 96h. Process performance during the reactor operation was assessed by monitoring DnBP concentration and biochemical process parameters viz., pH, dissolved oxygen (DO), colony forming units (CFU) and oxygen uptake rate (OUR), during the sequence phase operation. The degradation rate was observed to be rapid at lower substrate concentrations and found to be slow as the substrate concentration increased. The potent bacterial strain was also isolated from the slurry phase reactor. Metabolites formed during the degradation of DnBP in the slurry phase reactor were identified. Studies on the kinetics and half-life of the reaction revealed that the degradation process followed zero-order kinetic model.     

A well-mixed, polymer-based microbioreactor with integrated optical measurements

We describe a 150 microL microbioreactor fabricated in poly(methylmethacrylate) (PMMA) and poly(dimethylsiloxane) (PDMS) to cultivate microbial cell cultures. Mixing is achieved by a small magnetic stir bar and fluorescent sensors are integrated for on-line measurement of pH and dissolved oxygen. Optical transmission measurements are used for cell density. The body of the reactor is poly(methylmethacrylate) with a thin layer of poly (dimethylsiloxane) for aeration, oxygen diffuses through this gas-permeable membrane into the microbioreactor to support metabolism of bacterial cells. Mixing in the reactor is characterized by observation of mixing of dyes and computational fluid dynamics simulations. The oxygenation is described in terms of measured K(L)a values for microbioreactor, 20-75/h corresponding to increasing stirring speed 200-800 rpm. Escherichia coli cell growth in the microbioreactor is demonstrated and the growth behavior is benchmarked with conventional bench-scale bioreactors, flasks and tubes. Batch culture experiments with Saccharomyces cerevisiae further demonstrate the reproducibility and flexibility of the microbioreactor system.     

The cultivation of animal cells at controlled dissolved oxygen partial pressure. Reprinted from Biotechnology and Bioengineering Vol. X, Issue 6, Pages 801-814 (1968)

A 3-liter culture vessel has been developed for the growth of animal cells in suspension at controlled pH and dissolved oxygen partial pressure (pO(2)). The culture technique allows metabolically produced CO(2) to be measured; provision can be made to control the dissolved CO(2) partial pressure. In cultures containing a low serum concentration, gas sparging to control pO(2) was found to cause cell damage. This could be prevented by increasing the serum concentration to 10%, or by adding 0.02% of the surface-active polymer Pluronic F68. The growth of mouse LS cells in batch culture without pO(2) control was found to be limited by the availability of oxygen. Maximum viable cell populations were obtained when dissolved pO(2) was controlled at values within the range 40-100 mm Hg.