Effects of dissolved oxygen tension and agitation rate on the production of heat-shock protein glycoprotein 96 by MethA tumor cell suspension culture in stirred-tank bioreactors

Heat-shock protein glycoprotein (gp96) serves as a natural adjuvant for chaperoning antigenic peptide into the immune surveillance pathway. In our laboratory, MethA tumor cell suspension culture process has been recently developed for gp96 production in spinner flask. In this work, effects of dissolved oxygen tension (DOT) and agitation rate on this process were studied in stirred-tank bioreactor. The optimal conditions for gp96 production were different with those for MethA tumor cell growth. MethA tumor cell growth pattern was not much changed by various levels of DOT and agitation rate, while gp96 biosynthesis was more sensitive to DOT and agitation rate. Compared with 50% of DOT, the production and specific productivity of gp96 was increased by 27 and 66% at 10% of DOT, respectively. Compared with the agitation rate of 100 rpm, the production and volumetric productivity of gp96 was increased by 48 and 144% at the agitation rate of 200 rpm, respectively. Low DOT (i.e., 10% of air saturation) and high agitation rate (i.e., 200 rpm) were identified to be favorable for gp96 biosynthesis. The results of this work might be useful to scale-up the bioprocess into the pilot scale.     

Effect of various process parameters on morphology, rheology, and polygalacturonase production by Aspergillus sojae in a batch bioreactor

The effects of pH, agitation speed, and dissolved oxygen tension (DOT), significant in common fungal fermentations, on the production of polygalacturonase (PG) enzyme and their relation to morphology and broth rheology were investigated using Aspergillus sojae in a batch bioreactor. All three factors were effective on the response parameters under study. An uncontrolled pH increased biomass and PG activity by 27% and 38%, respectively, compared to controlled pH (pH 6) with an average pellet size of 1.69 +/- 0.48 mm. pH did not significantly affect the broth rheology but created an impact on the pellet morphology. Similarly, at constant agitation speed the maximum biomass obtained at 500 rpm and at 30 h was 3.27 and 3.67 times more than at 200 and 350 rpm, respectively, with an average pellet size of 1.08 +/- 0.42 mm. The maximum enzyme productivity of 0.149 U mL-1 h-1 was obtained at 200 rpm with an average pellet size of 0.71 +/- 0.35 mm. Non-Newtonian and pseudoplastic broth rheology was observed at 500 rpm agitation speed, broth rheology exhibited dilatant behavior at the lower agitation rate (200 rpm), and at the medium agitation speed (350 rpm) the broth was close to Newtonian. Furthermore, a DOT range of 30-50% was essential for maximum biomass formation, whereas only 10% DOT was required for maximum PG synthesis. Non-Newtonian shear thickening behavior (n > 1.0) was depicted at DOT levels of 10% and 30%, whereas non-Newtonian shear thinning behavior (n < 1.0) was dominant at 50% DOT. The overall fermentation duration (50-70 h) was considerably shorter compared to common fungal fermentations, revealing the economic feasibility of this particular process. As a result this study not only introduced a new strain with a potential of producing a highly commercially significant enzyme but also provided certain parameters significant in the design and mathematical modeling of fungal bioprocesses.     

Effect of cycling dissolved oxygen concentrations on product formation in penicillin fermentations

Summary Limitations in mass and momentum transfer coupled with high hydrostatic pressures create significant spatial variations in dissolved gas concentrations in large fermenters. Microorganisms are subjected to fluctuating environmental conditions as they pass through the zones in a stirred vessel or along a closed loop fermenter.A 7-litre fermenter was modified to simulate the dissolved gas and hydrostatic pressure gradients in large vessels.The effect of cycling dissolved oxygen tension (DOT) on penicillin production by Penicillium chrysogenum P1 was investigated. The fermentation was affected by evironmental conditions such as medium composition, pH, size of inoculum, stirrer speed and DOT. Inoculum size below 10% (v/v) and stirrer speeds above 850 rpm caused significant reductions in specific prenicillin production rates (q_pen). q_pen values were measured at different constant DOT levels. Below 30% air saturation q_pen decreased sharply and no production was observed at 10%. Penicillin synthesis was impaired irreversibly below 10% DOT. The same profile was observed at higher stirrer speeds and air flow rates indicating that the effect was a physiological one. Oxygen uptake of the culture was affected significantly below 7% DOT, demonstrating that the critical DOT values for penicillin production and oxygen uptake are two distinct parameters. Carrying out the fermentation at one atmosphere over pressure was found to have no effect. When the dissolved oxygen concentration of the culture medium was cycled around the critical DOT for penicillin production, a considerable decrease in the specific penicillin production rate was observed. The effect was reversible but not transient, indicating a shift in cell metabolism.These results demonstrate the unfavourable effect of fluctuating environmental conditions on culture performance in stirred tanks. They suggest that these effects should be accounted for during strain selection, process development and scale up stages of an industrial process if the productivities in small scale vessels are to be obtained.     

Improvement in Production and Quality of Gellan Gum by Sphingomonas paucimobilis Under High Dissolved Oxygen Tension Levels

The effect of agitation rate and dissolved oxygen tension (DOT) on growth and gellan production by Sphingomonas paucimobilis was studied. Higher cell growth of 5.4 g l⁻¹ was␣obtained at 700 rpm but maximum gellan (15 g l⁻¹) was produced at 500 rpm. DOT levels above 20% had no effect on cell growth but gellan yield was increased to 23 g l⁻¹ with increase in DOT level to 100%. Higher DOT levels improved the viscosity and molecular weight of the polymer with change in acetate and glycerate content of the polymer.     

The production of hemicellulases by Thermomyces lanuginosus strain SSBP: influence of agitation and dissolved oxygen tension

Shake-flask cultivation of T. lanuginosus strain SSBP on coarse corn cobs yielded β-xylanase levels of 56,500 nkat/ml at 50 °C, whereas other hemicellulases (β-xylosidase, β-glucosidase, and α-l-arabinofuranosidase) were produced at levels less than 7 nkat/ml. Cultivation on d-xylose yielded much lower levels of xylanase (350 nkat/ml), although other hemicellulase levels were similar to those produced on corn cobs. The influence of agitation rate and dissolved oxygen tension (DOT) on hemicellulase production was studied further in a bioreactor. On xylose, xylanase activities of 4,330 nkat/ml and 4,900 nkat/ml were obtained at stirrer speeds up to 1,400 rpm to control DOT. At a constant stirrer speed of 400 rpm, xylanase activities of 10,930 nkat/ml and 15,630 nkat/ml were obtained when cultivated on xylose and beechwood xylan respectively, despite DOT levels below 5% for the duration of fermentation. The results indicate that there is an interaction between agitation rate and DOT, impacting on xylanase and accessory enzyme production. Higher agitation rates favoured the production of xylosidase, arabinofuranosidase and glucosidase by T. lanuginosus strain SSBP, whereas the lower agitation rates favoured xylanase production. Rheological difficulties precluded cultivation on corn cobs in the bioreactor. Volumetric xylanase productivities of 1,060,000 nkat/l · h and 589,000 nkat/l · h obtained on beechwood xylan and xylose indicate that T. lanuginosus strain SSBP is a hyper-xylanase producer with considerable industrial potential. Received: 5 May 1999 / Received revision: 31 May 2000 / Accepted: 3 June 2000     

Influence of dissolved oxygen tension and agitation speed on alginate production and its molecular weight in cultures of Azotobacter vinelandii*

The alginate production by Azotobacter vinelandii, as well as the molecular weight of the polymer, are strongly influenced by the dissolved oxygen tension (DOT) and stirring speed of the culture. Under high DOT (5% of air saturation), the bacteria produced more alginate (4.5 g/l) than that obtained at low (0.5%) oxygen tension (1.0 g/l) in cultures conducted at 300 rpm. On the other hand, under constant DOT (3%), the higher the stirring speed (from 300 to 700 rev./min), the higher the specific growth rate and the alginate production rate. However, low agitation speed (300 rev./min) lead the culture to produce a polymer of high molecular weight (680 000 g/g mol) whereas a low molecular weight (352 000 g/g mol) alginate was isolated from cultures conducted at high (700 rev./min) stirring speed. At 700 rev./min, the MMW increased to a plateau between 1 and 3% DOT and then decreased to a minimum of 0.11 x 10(6) g/g mol at 7%. Microscopic observations revealed the presence of cell aggregates (one order of magnitude larger than individual cells) when the culture was conducted at 300 rev./min. Oxygen gradients occurring within the aggregates could be responsible of this phenomenon. At high agitation rate, the MMW of the alginate dropped towards the end of the culture in all conditions evaluated. Alginase activity was detected, which would be responsible for this phenomenon.     

Engineering Escherichia coli to improve culture performance and reduce formation of by-products during recombinant protein production under transient intermittent anaerobic conditions

Three E. coli strains, named VAL22, VAL23, and VAL24, were engineered at the level of mixed-acid fermentation pathways to improve culture performance under transient anaerobic conditions. VAL22 is a single mutant with an inactivated poxB gene that codes for pyruvate oxidase which converts pyruvate to acetate. VAL23 is a double mutant unable to produce lactate and formate due to deletions of the ldhA and pflB genes that code for lactate dehydrogenase and pyruvate-formate lyase, respectively. VAL24 is a triple mutant with ldhA and pflB deleted and poxB inactivated. Engineered strains were cultured under oscillating dissolved oxygen tension (DOT) in a scale-down system, to simulate gradients occurring in large-scale bioreactors. Kinetic and stoichiometric parameters of constant (10%) and oscillating DOT cultures of the engineered strains were compared with those of the parental strain, W3110. All strains expressed recombinant green fluorescent protein (GFP) as a protein model. Mutant strains showed improved specific growth rate, reduced by-product formation, and reduced specific glucose uptake rate compared to the parental strain, when cultured under oscillating DOT. In particular, lactate and formate production was abolished and acetate accumulation was reduced by 9-12%s. VAL24 showed the best performance, as specific growth and GFP production rates, and maximum GFP concentration were not affected by DOT gradients and were at least twofold higher than those of W3110 under constant DOT. Under oscillating DOT, VAL24 wasted about 40% less carbon into fermentation by-products than W3110. It was demonstrated that, although E. coli responds rapidly to DOT fluctuations by deviating to fermentative metabolism, such pathways can be eliminated as they are not necessary for bacterial survival during the short circulation times typical of large-scale cultures. The approach shown here opens new possibilities for designing metabolically engineered strains, with reduced sensitivity to DOT gradients and improved performance under typical conditions of large-scale cultures.     

Evaluation of the effects and interactions of mixing and oxygen transfer on the production of Fab’ antibody fragments in Escherichia coli fermentation with gas blending

Fermentations carried out at 450-L and 20-L scale to produce Fab’ antibody fragments indicated a serious problem to control levels of dissolved oxygen in the broth due to the large oxygen demand at high cell densities. Dissolved oxygen tension (DOT) dropped to zero during the induction phase and it was hypothesised that this could limit product formation due to inadequate oxygen supply. A gas blending system at 20-L scale was employed to address this problem and a factorial 2² experimental design was executed to evaluate independently the effects and interaction of two main engineering factors: agitation rate and DOT level (both related to mixing and oxygen transfer in the broth) on Fab’ yields. By comparison to the non-gas blending system, results in the gas blending system at same scale showed an increase in the production of Fab’ by 77% independent of the DOT level when using an agitation rate of 500 rpm level and by 50% at an agitation rate of 1,000 rpm with 30% DOT. Product localisation in the cell periplasm of >90% was obtained in all fermentations. Results obtained encourage further studies at 450-L scale initially, to evaluate the potential of gas blending for the industrial production of Fab’ antibody fragments.     

The effect of dissolved oxygen tension and the utility of oxygen uptake rate in insect cell culture

Dissolved oxygen tension and oxygen uptake rate are critical parameters in animal cell culture. However, only scarce information of such variables is available for insect cell culture. In this work, the effect of dissolved oxygen tension (DOT) and the utility of on-line oxygen uptake rate (OUR) measurements in monitoring Spodoptera frugiperda (Sf9) cultures were determined. Sf9 cells were grown at constant dissolved oxygen tensions in the range of 0 to 30%. Sf9 metabolism was affected only at DOT below 10%, as no significant differences on specific growth rate, cell concentration, amino acid consumption/production nor carbohydrates consumption rates were found at DOT between 10 and 30%. The specific growth rate and specific oxygen uptake rate followed typical Monod kinetics with respect to DOT. The calculated _max and max were 0.033 h^-1 and 3.82×10^-10 mole cell^-1h^-1, respectively, and the corresponding saturation constants were 1.91 and 1.57%, respectively. In all aerated cultures, lactate was consumed only after glucose and fructose had been exhausted. The yield of lactate increased with decreasing DOT. It is proposed, that an apparent DOT in non-instrumented cultures can be inferred from the lactate yield of bioreactors as a function of DOT. Such a concept, can be a useful and important tool for determining the average dissolved oxygen tension in non-instrumented cultures. It was shown that the dynamic behavior of OUR can be correlated with monosaccharide (fructose and glucose) depletion and viable cell concentration. Accordingly, OUR can have two important applications in insect cell culture: for on-line estimation of viable cells, and as a possible feed-back control variable in automatic strategies of nutrient addition.Abbreviations DOT Dissolved oxygen tension - OUR Oxygen uptake rate - specific oxygen uptake rate - specific growth rate - X_v viable cell concentration - C_L, C^*, and oxygen concentrations in liquid phase, in equilibrium with gas phase, and medium molar concentration, respectively - H Henry's constant - K_La volumetric oxygen transfer coefficient - P_T total pressure - oxygen partial pressure - oxygen molar fraction - i discrete element     

Heterogeneous conditions in dissolved oxygen affect N-glycosylation but not productivity of a monoclonal antibody in hybridoma cultures

It is known that heterogeneous conditions exist in large-scale animal cell cultures. However, little is known about how heterogeneities affect cells, productivities, and product quality. To study the effect of non-constant dissolved oxygen tension (DOT), hybridomas were subjected to sinusoidal DOT oscillations in a one-compartment scale-down simulator. Oscillations were forced by manipulating the inlet oxygen partial pressure through a feedback control algorithm in a 220-mL bioreactor maintained at a constant agitation. Such temporal DOT oscillations simulate spatial DOT gradients that can occur in large scales. Different oscillation periods, in the range of 800 to 12,800 s (axis of 7% (air saturation) and amplitude of 7%), were tested and compared to constant DOT (10%) control cultures. Oscillating DOT decreased maximum cell concentrations, cell growth rates, and viability indexes. Cultures at oscillating DOT had an increased glycolytic metabolism that was evidenced by a decrease in yield of cells on glucose and an increase in lactate yield. DOT gradients, even several orders of magnitude higher than those expected under practical large-scale conditions, did not significantly affect the maximum concentration of an IgG(1) monoclonal antibody (MAb). The glycosylation profile of the MAb produced at a constant DOT of 10% was similar to that reported in the literature. However, MAb produced under oscillating culture conditions had a higher amount of triantennary and sialylated glycans, which can interfere with effector functions of the antibody. It was shown that transient excursions of hybridomas to limiting DOT, as occurs in deficiently mixed large-scale bioreactors, is important to culture performance as the oscillation period, and thus the time cells spent at low DOT, affected cell growth, metabolism, and the glycosylation pattern of MAb. Such results underline the importance of monitoring protein characteristics for the development of large-scale processes.     

Lipid formation in the oleaginous mould Entomophthora exitalis grown in continuous culture: effects of growth rate,temperature and dissolved oxygen tension on polyunsaturated fatty acids

Summary The oleaginous fungus Entomophthora exitalis was grown in continuous culture at a constant dilution rate (0.04 h^–1) and over a range of temperatures (20–30° C). As the growth temperature was decreased from 30 to 20° C the percentage of polyunsaturated fatty acids (PUFA) increased proportionally from 18 to 27% (w/w) of the total fatty acids. The increase in unsaturation was as a result of an increased proportion of n-6 PUFA (particularly arachidonic acid) in the phospholipid and sphingo- plus glycolipid fractions. The triacylglycerol fraction of lipids displayed a negligible change. The proportion of phospholipids within the extracted lipid increased between 26 and 20° C without any change in the lipid content of the fungus. Although the changes in lipid unsaturation correlated, at first inspection, to the culture dissolved O₂ tension (DOT), growth of the fungus at a constant dilution rate and temperature (22° C) over a range of DOT values failed to influence lipid unsaturation. Thus temperature is the principal regulation factor of the degree of unsaturation in the lipids of this organism. Offprint requests to: C. Ratledge     

The Impact of Oxygen Tension on Cell Density and Metabolic Diversity of Microbial Communities in Alkane Degrading Continuous-Flow Cultures

Abstract The impact of the dissolved O₂ tension (DOT) and the dilution rate on the metabolic diversity of an autochthonous hexadecane-degrading community in continuous-flow cultures containing hexadecane-coated intertidal sediment was determined in a set of experiments. The DOT was kept constant within each culture at values of 80% (168 μmol O₂L⁻¹) or 0.4% (0.84 μmol O₂ L⁻¹). The dilution rate was increased from D= 0.012 h⁻¹ to D= 0.06 h⁻¹. To determine the culture activity, we analyzed the hexadecane degradation rate, the protein production rate, and the oxygen consumption rate. The cell concentration of different metabolic groups was determined by colony forming units (CFU), and by most probable number (MPN). The metabolic diversity was determined by the substrate utilization spectrum in Biolog GN microtiter plates. The substrate utilization pattern of the cultures decreased considerably as D increased. This effect was more pronounced at 0.4% of DOT than at 80% of DOT. The MPN and CFU revealed that as D increased, only minor changes occurred in the community structure. The hexadecane degradation rate, the protein production rate, and the oxygen consumption rate increased parallel to D independently of the DOT. This means that the biocenosis at 0.4% of DOT was different from the biocenosis at 80% of DOT, although the metabolic activity of the cultures was unaffected by a 200-factor difference in the oxygen tension and revealed a considerable buffer capacity with respect to changes in DOT. Received: 23 May 1998; Accepted: 24 August 1998     

Effect of oscillating dissolved oxygen tension on the production of alginate by Azotobacter vinelandii.

The effect of oscillating dissolved oxygen tension (DOT) on the metabolism of an exopolysaccharide-producing bacteria (Azotobacter vinelandii) was investigated, particularly on the mean molecular weight (MMW) of the alginate produced. Sinusoidal DOT oscillations were attained by manipulating the oxygen and nitrogen partial pressures at the inlet of a 1.0 L working volume bioreactor. Periods of 1200, 2400, and 4000 s and average amplitudes between 1.0% and 2.2% DOT, with an oscillation axis fixed at 3% DOT, were tested. A culture carried out at constant 3% DOT was used as comparison. The average wave amplitude had an important effect on the maximum mean molecular weight (MMW(max)) of the alginate produced. The higher the amplitude, the lower the MMW(max). As the average wave amplitudes decreased from 2.2% to 1.0%, the MMW(max) increased from 64 to 240 KDa, respectively. Furthermore, at 3% constant DOT (0.0% of amplitude), a MMW(max) of 350 KDa was obtained. No important effect of the oscillating DOT on kinetics of biomass growth, alginate production, and sucrose consumption was observed, compared with constant DOT. The findings of this study point out that accurate DOT control is crucial if a particular molecular weight species of alginate needs to be produced, particularly in large fermentors, where bacteria are exposed to an oscillatory environment as a result of DOT gradients caused by the high viscosity of the broth and insufficient mixing.     

Inhibition of glucose oxidation in Erwinia herbicola by a high concentration of dissolved O2

Oxidation of glucose to 2,5-diketogluconic acid by Erwinia herbicola was inhibited at 100% dissolved O₂ tension (DOT) relative to air at 1 atm. Gluconic acid accumulation, however, increased under this condition. The negative influence of the high DOT is attributed to a 10-fold decrease in 2-ketogluconate dehydrogenase activity.The authors are with the Department of Biotechnology, Regional Research Laboratory, Canal Road, Jammu Tawi-180001, India     

D-xylose fermentation by Candida shehatae and pichia stipitis at low dissolved oxygen levels in fed-batch cultures

Summary The fermentation of D-xylose byCandida shehatae andPichia stipitis was studied in fed-batch fermentations using dissolved oxygen tension (DOT) control in the range of 0.2 to 1.4% air saturation. The response of these two yeasts to DOT was significantly different. Whereas the ethanol yield withC. shehatae was 0.35 to 0.38 g.g^–1 at all DOT levels, that ofP. stipitis decreased from 0.44 at a zero DOT reading to 0.19 g.g^–1 at 1.4% DOT.     

The effect of cycling dissolved oxygen tension on the synthesis of the antibiotic difficidin by bacillus subtilis

The antibiotic, difficidin, and its hydroxylated derivative, oxydifficidin, were synthesised by cultures of Bacillus subtilis grown on a complex medium in batch culture at dissolved oxygen tensions (DOT) of 15, 20 and 40% air saturation. During part of the growth phase the DOT was cycled about the control value and the effect on growth and antibiotic production observed. In fermentations with cycling at 15 and 20% DOT the growth yields were lower than for the fermentations done at constant DOT throughout. There appears to be a complex interaction between growth rate and difficidin production rate which led to a reduced specific production rate at 15% DOT as a result of cycling.UCL is the Biotechnology and Biological Sciences Research Council Interdisciplinary Research Centre for Biochemical Engineering and the Council's support is gratefully acknowledged. The authors wish to thank Merck & Co. for provision of the difficidin and oxydifficidin used to calibrate the HPLC assay.     

Optimization of the production of progesterone 11α-hydroxylase by Rhizopus nigricans

The progesterone 11α-hydroxylase of Rhizopus nigricans ATCC 6227b is an inducible enzyme system that is primarily induced by its substrate progesterone. Maximum induction was found at a progesterone concentration of 0.5 g/liter or above. Oxygen is the other substrate for the hydroxylation and this was found to have a major effect on the amounts of hydroxylase synthesized. Optimum induction of the hydroxylase in a fermentation with a 3.1 m/sec impeller tip speed was found to occur at a dissolved oxygen tension (DOT) of 10% of air saturation. The agitation rate also effects the amount of hydroxylase synthesized with an apparent maximum at 3.1 m/sec impeller tip speed. The DOT for a maximum hydroxylation rate was much higher than for enzyme synthesis so that it was preferable to increase the DOT after induction was completed.     

The roles of oxygen and alginate-lyase in determining the molecular weight of alginate produced by<Emphasis Type="Italic"> Azotobacter vinelandii</Emphasis>

An Azotobacter vinelandii mutant lacking alginate-lyase (SML2) and the wild type (ATCC 9046) were used to discriminate between the roles of the polymerase complex and alginate-lyase in the synthesis of alginate in cultures conducted under controlled dissolved oxygen tension (DOT). To avoid the presence of pre-synthesized alginates, all cultures were inoculated with washed cells. For cultures carried out at 3% DOT using the mutant, a well defined family of alginates of high mean molecular weight (MMW) were obtained (985 kDa). Under 1% and 5% DOT, the mutant produced unique families of alginates with lower MMW (150 and 388 kDa). A similar behavior was observed using the wild type: a production of well defined families of alginates of high MMW at 3% DOT (1,250 kDa) and lower MMW at 1% and 5% DOT (370 and 350 kDa). At the end of the ATCC 9046 fermentations, alginate was depolymerized by the action of lyases. Overall, the evidence indicated that polymerization of alginate is carried out by producing families of polysaccharide in a narrow MMW range, and that it is highly dependent on DOT. The role of alginate-lyase (present in the wild type) is restricted to a post-polymerization step.     

In situ cell retention of a CHO culture by a reverse‐flow diafiltration membrane bioreactor

Heterogeneities occur in various bioreactor designs including cell retention devices. Whereas in external devices changing environmental conditions cannot be prevented, cells are retained in their optimal environment in internal devices. Conventional reverse‐flow diafiltration utilizes an internal membrane device, but pulsed feeding causes temporal heterogeneities. In this study, the influence of conventional reverse‐flow diafiltration on the yeast Hansenula polymorpha is investigated. Alternating 180 s of feeding with 360 s of non‐feeding at a dilution rate of 0.2 h^?1 results in an oscillating DOT signal with an amplitude of 60%. Thereby, induced short‐term oxygen limitations result in the formation of ethanol and a reduced product concentration of 25%. This effect is enforced at increased dilution rate. To overcome this cyclic problem, sequential operation of three membranes is introduced. Thus, quasi‐continuous feeding is achieved reducing the oscillation of the DOT signal to an amplitude of 20% and 40% for a dilution rate of 0.2 h^?1 and 0.5 h^?1, respectively. Fermentation conditions characterized by complete absence of oxygen limitation and without formation of overflow metabolites could be obtained for dilution rates from 0.1 h^?1 – 0.5 h^?1. Thus, sequential operation of three membranes minimizes oscillations in the DOT signal providing a nearly homogenous culture over time. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1348–1355, 2014     

Cultivation of Methylosinus trichosporium OB3b: III. production of particulate methane monooxygenase in continuous culture

Continous culture experiments with the obligatory methanotroph, Methylosinus trichosporium OB3b, were conducted to study the whole-cell methane monooxygenase (MMO) and nitrogenase activities in a nitrate minimal salts medium under oxygen-limited conditions with methane as the carbone source. The important variables investigated were the feed medium concentrations of copper and nitrate, CO(2) addition, the agitation speed, and the dilution rate. M. trichosporium OB3b required quantitative amounts of copper (2.6 x 10(-4) g Cu/g dry cell Wt) for the exclusive production of particulate MMo during continous culture growth. When the feed medium nitrate concentration was varied in the range of 5-50 mM, the whole-cell specific pMMO activity exhibited a maximum at 40 mM. The elimination of external CO(2) gassing decreased pMMO activity by more than 30%. The steady-state cell density increased continuously over a 300-700 rpm range of agitation speed, whereas, the pMMO activity became maximal at 400 rpm. Also, the pMMO activity increased with the dilution rate up to 0.06 h(-1) and remained constant thereafter. Maximal continuous pMMO productivity was, thus, achieved in Higgin's medium containing 10 muM Cu, 80 muM Fe, and 40 mM nitrate with an agitation speed of 500 rpm and a dilution rate of 0.06 h(-1). Nitrogenase activity, on the other hand, increased over a feed medium copper concentration of 2-15 muM, falling sharply at 20 muM, and it exhibited a minimum at 20 mM when the feed medium nitrate concentration was varied. (c) 1992 John Wiley & Sons, Inc.