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.     

The effect of culture conditions on the production of erythromycin by Saccharopolyspora erythraea in batch culture

Summary Saccharopolyspora erythraea growth is inhibited when grown at a low constant dissolved oxygen tension (DOT) of 10% air saturation. However, the specific erythromycin production is virtually identical to that of a culture where the DOT did not fall below 65%. In addition, at constant DOT (10%) a stirrer speed of 750 rpm in a 7 litre causes mechanical damage to the mycelia in comparison with result at 500 rpm.     

Influence of a propeller on Saccharomyces cerevisiae fermentations in a pilot scale airlift bioreactor

The hydrodynamics (sectional gas holdup and liquid velocities) and oxygen transfer performance of a conventionally operated multiconfigurable pilot scale (0.25?m³) concentric airlift bioreactor containing baker's yeast were significantly improved by operating a marine propeller to draw liquid down the draft tube and aid recirculation at the base of the vessel. Propeller operation reduced the severe DOT heterogeneity of the reactor, which gave DOT values below 1% air saturation in the riser, by producing DOTs above 40% around the vessel at maximum energy dissipation rate. As a consequence the overall oxygen uptake rate (OUR) of the baker's yeast increased up to 3 fold with the total energy dissipation rate into the reactor until the lowest DOTs of the vessel were at or above 10%. The different degrees of heterogeneity generated by the two reactor configurations enabled the reactor to be used as a scale down tool to study the impact of heterogeneity on the physiology of fermentation broths. Comparison of the hydrodynamics and oxygen transfer between tall and short reactor heights revealed that the faster circulation times of the short reactor produced a greater improvement in the OUR with propeller operation even though similar DOT changes occurred around both sizes of reactor. This indicated that the yeast cells were responding to the rapid DOT changes around the vessel.     

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.     

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.     

Hexadecane mineralization in oxygen-controlled sediment-seawater cultivations with autochthonous microorganisms.

Laboratory studies investigated the influence of dissolved oxygen tension (DOT) on microbial degradation of hexadecane in cultures with sediment-seawater suspensions. With a fermentor system, it was possible to adjust and regulate different oxic conditions (DOTs between 0.4 and 80% of oxygen saturation) as well as anoxia. The effects of DOT reduction on the amount and rate of hexadecane degraded and on the degree of mineralization and on the production of biomass were investigated. When the DOT was reduced from 80% to 5%, no dependence of the investigated parameters on the oxygen concentration was found. The amount of hexadecane degraded was constant, with an average value of 86% of the initially applied amount. The degradation rate was constant even down to 1% DOT, with an average value of 0.15 mg of hexadecane per g of sediment per h (16.2 mg liter-1 h-1). The mean degree of mineralization was 70% of the initially applied hexadecane, and biomass production reached a value of about 1.5 g per g of hexadecane consumed. A significant influence on the degradation process was detected only with DOTs below 1%. The degree of mineralization and the amount of degraded hexadecane decreased, whereas the degradation rate was still unaffected. Under anoxic conditions, no hexadecane degradation occurred within 190 h. The fact that the hexadecane biodegradation rate was constant down to at least 0.04% DOT shows that the actual oxygen concentration is of minor importance as long as the oxygen supply is high enough to guarantee the oxygen-dependent degradation step.     

Hexadecane mineralization in oxygen-controlled sediment-seawater cultivations with autochthonous microorganisms.

Laboratory studies investigated the influence of dissolved oxygen tension (DOT) on microbial degradation of hexadecane in cultures with sediment-seawater suspensions. With a fermentor system, it was possible to adjust and regulate different oxic conditions (DOTs between 0.4 and 80% of oxygen saturation) as well as anoxia. The effects of DOT reduction on the amount and rate of hexadecane degraded and on the degree of mineralization and on the production of biomass were investigated. When the DOT was reduced from 80% to 5%, no dependence of the investigated parameters on the oxygen concentration was found. The amount of hexadecane degraded was constant, with an average value of 86% of the initially applied amount. The degradation rate was constant even down to 1% DOT, with an average value of 0.15 mg of hexadecane per g of sediment per h (16.2 mg liter-1 h-1). The mean degree of mineralization was 70% of the initially applied hexadecane, and biomass production reached a value of about 1.5 g per g of hexadecane consumed. A significant influence on the degradation process was detected only with DOTs below 1%. The degree of mineralization and the amount of degraded hexadecane decreased, whereas the degradation rate was still unaffected. Under anoxic conditions, no hexadecane degradation occurred within 190 h. The fact that the hexadecane biodegradation rate was constant down to at least 0.04% DOT shows that the actual oxygen concentration is of minor importance as long as the oxygen supply is high enough to guarantee the oxygen-dependent degradation step.     

Effect of dissolved oxygen concentration on the metabolism of glucose inPseudomonas putida BM014

The effect of dissolved oxygen concentration on the metabolism of glucose inPseudomonas putida BM014 was investigated. Glucose was completely converted to 2-ketogluconatevia extracellular oxdative pathway and then taken up for cell growth under the condition of sufficient dissolved oxygen concentration. On the other hand, oxygen limitation below dissolved oxygen tension (DOT) value of 20% of air saturation caused the shift of glucose metabolism from the extracellular oxidative pathway to the intracellular phosphorylative pathway. Specific activities of hexokinase and gluconate kinase in intracellular phosphorylation pathway decreased as the DOT increased, while 2-ketogluconokinase activity in extracellular oxidative pathway increased under the same condition. This result can be usefully applied to microbial transformation of glucose to 2-ketogluconate, the synthetic precursor for iso-vitamine C, with almost 100% yieldvia extracellular oxidation by simple DOT control.     

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.     

Effects of dissolved oxygen concentration on hybridoma growth and metabolism in continuous culture

Oxygen transport is a major limitation in large-scale mammalian cell culture. The effects of the dissolved oxygen concentration (DO; from 0.1 to 100% saturation with air) on Sp2/0-derived mouse hybridomas were investigated using continuous culture. The steady-state concentration of viable cells increased with decreasing DO until a critical dissolved oxygen concentration of 0.5% of air saturation was reached. The cell concentration declined at lower DO because of incomplete glutamine oxidation, and the specific lactate production from glucose increased to offset the reduced energy production from glutamine. Cell viability increased as the DO was decreased; the viability continued to increase even when the DO was reduced below 0.5%. The specific oxygen uptake rate was essentially constant for DO greater than or equal to 10% of air saturation and then decreased with decreasing DO. The P/O ratio (ATP molecules produced per O atom consumed) appears to change from 2 to 3 between 10 and 0.5% DO. The specific ATP production rate calculated using this assumption decreases only slightly with decreasing DO. The optimum DO of 50% for antibody production is different than the optimum (approximately 0.5% DO) for cell growth.     

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.     

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 dissolved oxygen and aeration rate on antibiotic production of Streptomyces fradiae

Different dissolved oxygen concentrations and aeration rates were imposed on a stable mutant of Streptomyces fradiae during the antibiotic-producing phase. At high aeration rate (1 vvm), the tylosin yield in the fermentor broth with dissolved oxygen (DO) concentrations controlled close to 100% saturation (6-8 ppm) increased 10% as against uncontrolled. The rates of cellular growth, oil consumption, and tylosin production were severely reduced when DO concentration fell below 25% saturation, but all resumed to their initial rates when DO was raised to saturation level again. The DO concentration in combination with air flow rate affected the pattern of the antibiotics produced. At high DO levels, an additional macrolide antibiotic, macrocin, was synthesized to more than one-third the amount of tylosin at high aeration rate (1 vvm). On the other hand, tylosin production rate remained constant and no significant amount of macrocin was produced at low aeration rate (0.2 vvm).     

Temperature and dissolved oxygen concentration as parameters of Azotobacter chroococcum cultivation for use in biofertilizers

Summary Azotobacter chroococcum was grown in continuous culture at two temperatures (30 °C and 20 °C) and different dissolved oxygen tensions (DOT) (30 % to 40 % and 70 % to 80 % of air saturation), respectively. At the temperature of 30 °C and low DOT a relatively high volumetric productivity and efficiency of nitrogen fixation were obtained. After lowering the temperature to 20 °C, an intensive formation of cysts was observed associated with a drastic decrease of the bacterial growth. Bacteria in the form of cysts kept their physiological activity for a long period of time depending on temperature and preparation.     

Application of fed-batch culture to citric acid production by Aspergillus niger: The effects of dilution rate and dissolved oxygen tension

Studies in conventional batch culture confirmed that the maximum citric acid production rate occurred prior to exhaustion of the growth-limiting nutrient, i.e., when the growth rate was nonzero. The effects of dilution rate and the culture dissolved oxygen tension (DOT) were studied in chemostat culture. Maximum citric acid yield and production rate were observed at low dilution rate (0.017 h(-1)) and high DOT value (90% of saturation). These findings were applied to a nitrogen-limited fed batch culture, and allowed a productivity increase of 100% when compared with conventional batch culture.     

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.     

溶氧水平对瓦氏黄颡鱼幼鱼静止耗氧率和临界游泳运动能力的影响

为了研究瓦氏黄颡鱼(Peltebagrus vachelli)在不同溶氧水平(DO)下的游泳运动能力、限制性机制及能量适应对策, 在25℃分别对不同溶氧水平(125%、100%、75%、50%和25% 空气饱和度)条件下瓦氏黄颡鱼幼鱼静止耗氧率、临界游泳速度(Ucrit)、活跃耗氧率进行了测定。研究发现瓦氏黄颡鱼的静止耗氧率随溶氧水平下降而显著下降(P2crit)为14.52%空气饱和度(1.16 mg/L)。当溶氧水平从100%下降到25%空气饱和度时, 其活跃耗氧率随溶氧的下降而显著下降(PPP<0.05)。研究结果提示: 在低氧条件下, 瓦氏黄颡鱼的临界游泳速度受中心的心鳃系统的限制, 而在常氧条件下,受外周的运动系统(肌肉组织)的限制。       

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.     

Pyruvate relieves the necessity of high induction levels of catalase and enables Campylobacter jejuni to grow under fully aerobic conditions

Aims:  Several cases of campylobacteriosis reported worldwide seemingly conflict with the strict growth requirements and sensitivity to environmental stress of Campylobacter jejuni. In this study, the need for a micro‐aerobic environment [dissolved oxygen tension (DOT): 0·1–90%; 100% air saturation)] and the adaptive responses to oxygen stress were studied. Methods and Results:  The growth of C. jejuni in continuous culture was assessed under different DOT in the presence or absence of pyruvate. In a medium without pyruvate, continuous cultures of C. jejuni showed typically micro‐aerobic behaviour and cells were unable to grow under fully aerobic conditions. However in the presence of pyruvate (25 mmol l^?1), continuous cultures of C. jejuni were able to grow in a broad DOT range, varying from 0·1% to at least 90%, and the catalase activity was decreased. Conclusions:  Addition of pyruvate results in the decrease in the concentration of hydrogen peroxide, which enables C. jejuni to grow aerobically. Significance and Impact of the Study:  New information on the oxidative physiology of C. jejuni and its ability to grow aerobically in media supplemented with pyruvate is presented.     

Structural and functional characterization of three polyketide synthase gene clusters in Bacillus amyloliquefaciens FZB 42

Although bacterial polyketides are of considerable biomedical interest, the molecular biology of polyketide biosynthesis in Bacillus spp., one of the richest bacterial sources of bioactive natural products, remains largely unexplored. Here we assign for the first time complete polyketide synthase (PKS) gene clusters to Bacillus antibiotics. Three giant modular PKS systems of the trans-acyltransferase type were identified in Bacillus amyloliquefaciens FZB 42. One of them, pks1, is an ortholog of the pksX operon with a previously unknown function in the sequenced model strain Bacillus subtilis 168, while the pks2 and pks3 clusters are novel gene clusters. Cassette mutagenesis combined with advanced mass spectrometric techniques such as matrix-assisted laser desorption ionization-time of flight mass spectrometry and liquid chromatography-electrospray ionization mass spectrometry revealed that the pks1 (bae) and pks3 (dif) gene clusters encode the biosynthesis of the polyene antibiotics bacillaene and difficidin or oxydifficidin, respectively. In addition, B. subtilis OKB105 (pheA sfp(0)), a transformant of the B. subtilis 168 derivative JH642, was shown to produce bacillaene, demonstrating that the pksX gene cluster directs the synthesis of that polyketide. The GenBank accession numbers for gene clusters pks1(bae), pks2, and pks3(dif) are AJ 634060.2, AJ 6340601.2, and AJ 6340602.2, respectively.