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

The antibiotic, difficidin, and its hydroxylated derivative oxydifficidin, were synthesized by cultures of Bacillus subtilis grown on a complex medium. Maximum titers of about 200 and 130 mg/L, respectively, were obtained. In fermentations where the dissolved oxygen tension (DOT) was controlled, the maximum specific growth rate was only reduced below 5% air saturation. DOT had little effect on the volumetric rateof synthesis of oxydifficidin but greatly influenced the rate for difficidin, which was reduced at DOT values below 40% air saturation. (c) 1994 John Wiley & Sons, Inc.     

Control of the production of individual fusicoccins at different dissolved oxygen concentrations

The regulatory effect of different concentrations of dissolved oxygen on the production of fusicoccins by the fungus Fusicoccum amygdali Del. was studied. The maximum output of total fusicoccins was obtained by using a profiled dissolved oxygen tension (DOT) regime, in which the DOT was maintained at 15–20% during the biomass growth phase and at 5–8% during the fusicoccins production phase. In comparison with the profiled regime, the maintenance of DOT at 15–20% during the whole fermentation shortened the fusicoccins production phase. The fermentation performance at a low DOT (5–8%) inhibited both the accumulation of biomass and the production of fusicoccins. At high DOT (40–50%), an accelerated accumulation of the biomass with an expressed autolysis of mycelia took place, and the production of fusicoccins was lowered. The qualitative composition of individual fusicoccins varied substantially at different DOTs. Fusicoccins, A, C, D, J, H, 16-O-demethyl-J, detretpentenylfusicoccin and some minor fusicoccin metabolites were found in the fermentation broth using the method of liquid secondary ion mass spectrometry. It was established that the profiled DOT regime (15–20% to 5–8%) provided both the maximum concentration of fusicoccins and an enhanced accumulation of the main metabolite – fusicoccin A (FC A). The performance of the fermentation at a DOT of 15–20% decreased the content of FC A by 2–6% in comparison with the profiled DOT regime, and increased the content of fusicoccin C to 14–20% of the total fusicoccins. Fermentation at DOT of 5–8% was characterized by the highest content of the precursors of FC A, the less oxidized fusicoccins H and J, the contents of which were in range 7–12% and 16–17% of total fusicoccins, respectively.     

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.     

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.     

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.     

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.     

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.     

The effect of neutral resins on the fermentation production of rubradirin

Summary Rubradirin is an antibiotic of complex chemical structure which is activevs. methicillin resistant staphylococci. Its development has been limited due to inadequate production yields. The incorporation of neutral resins into fermentations ofStreptomyces achromogenes v.rubradiris, UC^® 8051 resulted in the enhanced production of rubradirin. Resins HP-20, HP-21, XAD-2, XAD-7 and XAD-16 were employed in flask and tank fermentations. The incorporation of these resins promoted 2- to 4-fold enhancements of the rubradirin activity produced in flask fermentations, and the incorporation of XAD-16 and HP-21 into tank fermentations promoted production titer increases >5 fold.     

Studies related to the scale-up of high-cell-density E. coli fed-batch fermentations using multiparameter flow cytometry: effect of a changing microenvironment with respect to glucose and dissolved oxygen concentration

Multiparameter flow cytometric techniques developed in our laboratories have been used for the "at-line" study of fed-batch bacterial fermentations. These fermentations were done at two scales, production (20 m(3)) and bench (5 x 10(-3) m(3)). In addition, at the bench scale, experiments were undertaken where the difficulty of achieving good mixing (broth homogeneity), similar to that found at the production scale, was simulated by using a two-compartment model. Flow cytometric analysis of cells in broth samples, based on a dual-staining protocol, has revealed, for the first time, that a progressive change in cell physiological state generally occurs throughout the course of such fermentations. The technique has demonstrated that a changing microenvironment with respect to substrate concentration (glucose and dissolved oxygen tension [DOT]) has a profound effect on cell physiology and hence on viable biomass yield. The relatively poorly mixed conditions in the large-scale fermentor were found to lead to a low biomass yield, but, surprisingly, were associated with a high cell viability (with respect to cytoplasmic membrane permeability) throughout the fermentation. The small-scale fermentation that most clearly mimicked the large-scale heterogeneity (i.e., a region of high glucose concentration and low DOT analogous to a feed zone) gave similar results. On the other hand, the small-scale well-mixed fermentation gave the highest biomass yield, but again, surprisingly, the lowest cell viability. The scaled-down simulations with high DOT throughout and locally low or high glucose gave biomass and viabilities between. Reasons for these results are examined in terms of environmental stress associated with an ever-increasing glucose limitation in the well-mixed case. On the other hand, at the large scale, and to differing degrees in scale-down simulations, cells periodically encounter regions of relatively higher glucose concentration.     

Effects of phosphate, glucose, and ammonium on cell growth and lincomycin production by Streptomyces lincolnensis in chemically defined media

Cell growth and lincomycin production were measured in batch cultures of Streptomyces lincolnensis in chemically defined media. In these fermentations the specific rate of antibiotic production was maximal during growth and always declined at the end of the growth phase. It was found that both phosphate and ammonium salts, while required for cell growth, had negative effects on antibiotic production. By increasing the concentration of magnesium sulfate, it was possible to increase both the production rates and final titers of lincomycin. The mechanism for this effect was found to be the reduction of soluble phosphate in the medium through the precipitation of ammonium magnesium phosphate. Lincomycin production rates were not inhibited by glucose at concentrations of up to 30 g/L.     

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.     

Culture of Escherichia coli under dissolved oxygen gradients simulated in a two-compartment scale-down system: metabolic response and production of recombinant protein

A significant problem of large-scale cultures, but scarcely studied for recombinant E. coli, is the presence of gradients in dissolved oxygen tension (DOT). In this study, the effect of DOT gradients on the metabolic response of E. coli and production of recombinant pre-proinsulin, accumulated as inclusion bodies, was determined. DOT gradients were simulated in a two-compartment scale-down system consisting of two interconnected stirred-tank bioreactors, one maintained at anoxic conditions and the other at a DOT of at least 6%. Cells were continuously circulated between both vessels to simulate circulation times (tc) of 20, 50, 90, and 180 sec. A complete kinetic and stoichiometric characterization was performed in the scale-down system as well as in control cultures maintained at constant DOT in the range of 0-20%. The performance of E. coli cultured under oscillating DOT was significantly affected, even at a tc of 20 sec corresponding to transient exposures of only 13.3 sec to anaerobic conditions. Specific growth rate decreased linearly with tc to a maximum reduction of 30% at the highest tc tested. The negative effect of DOT gradients was even more pronounced for the overall biomass yield on glucose and the maximum concentration and yield of pre-proinsulin. In these cases, the losses were 9%, 27%, and 20%, respectively, at tc of 20 sec and 65%, 94%, and 87%, respectively, at tc of 180 sec. Acetic, lactic, formic, and succinic acids accumulated during oscillatory DOT cultures, indicating that deviation of carbon flow to anaerobic metabolism was responsible for the observed losses. The results of this study indicate that even very short exposures to anaerobic conditions, typical of large-scale operations, can substantially reduce recombinant protein productivity. The information presented here is useful for establishing improved rational scale-up strategies and understanding the behavior of recombinant E. coli exposed to DOT gradients.     

Maintaining rapid growth in moderate-density Escherichia coli fermentations

A novel feeding strategy that prolongs rapid growth rates for Escherichia coli fermentations to moderately high cell density is presented. High-density fermentations are a common and successful means of producing biological products. However, acetate accumulation can be a substantial problem in these procedures. To avoid this problem, many feeding strategies and host modifications have been developed, but all result in relatively low growth rates. If a faster growth rate could be maintained, the growth phase of the process would be shortened, leading to increased productivity. It is also possible that the subsequent specific production rate could be enhanced by growing the early culture at a faster rate. We have developed a procedure to enable rapid growth to a cell density of 20 g/L and have used cell-free protein synthesis to evaluate the relative potential of the resulting cells for producing recombinant proteins. The method uses glucose pulses and the duration of the dissolved oxygen response to calculate the appropriate glucose feed rate based on the glucose demand of the culture. Amino acids and vitamins were supplied in the medium to increase the growth rate. We were able to sustain a growth rate of 0.8/h up to 20 g/L dry cell weight without significant acetate accumulation. Analysis of amino acid consumption indicates that cell composition is an accurate predictor of amino acid demand for most amino acids. Cell-free protein synthesis was used to compare the protein production potential of the high-density cultures with that of cells grown in complex medium and harvested at low cell density and maximum growth rate. Protein production for the extract from the controlled, high-density fermentations was 950 mg/L compared with 860 mg/L for the low-density control. Therefore, the new control procedure has promising potential for developing rapid and productive industrial fermentations.     

The effects of temperature on growth and production of the antibiotic granaticin by a thermotolerant streptomycete

The synthesis of granaticin, a polyketide-derived antibiotic synthesized as a secondary metabolite by Streptomyces thermoviolaceus strain NCIB 10076, was studied at different growth temperatures. Quantitative measurements of the antibiotic made during batch fermentations showed that the yield was greatest at 45 degrees C, whereas the rate of synthesis was most rapid at 37 degrees C. The timing of the appearance of granaticin in culture could not be assigned to any particular phase of growth or to de-repression due to depletion of any particular nutrient. However, at all temperatures, appearance of the antibiotic coincided with a rise in ammoniacal nitrogen presumably due to deamination of glutamate, the carbon source for growth. We have previously shown that production of the antibiotic is pH sensitive and that some carbon sources result in higher titres than others. This paper examines the effect of temperature on the physiology of growth and on antibiotic production in more detail under conditions that also allow an exact measurement of granaticin yield.     

Fate of virginiamycin through the fuel ethanol production process

Antibiotics are frequently used to prevent and treat bacterial contamination of commercial fuel ethanol fermentations, but there is concern that antibiotic residues may persist in the distillers grains coproducts. A study to evaluate the fate of virginiamycin during the ethanol production process was conducted in the pilot plant facilities at the National Corn to Ethanol Research Center, Edwardsville, IL. Three 15,000-liter fermentor runs were performed: one with no antibiotic (F1), one dosed with 2 parts per million (ppm) of a commercial virginiamycin product (F2), and one dosed at 20 ppm of virginiamycin product (F3). Fermentor samples, distillers dried grains with solubles (DDGS), and process intermediates (whole stillage, thin stillage, syrup, and wet cake) were collected from each run and analyzed for virginiamycin M and virginiamycin S using a liquid chromatography-mass spectrometry method. Virginiamycin M was detected in all process intermediates of the F3 run. On a dry-weight basis, virginiamycin M concentrations decreased approximately 97 %, from 41 μg/g in the fermentor to 1.4 μg/g in the DDGS. Using a disc plate bioassay, antibiotic activity was detected in DDGS from both the F2 and F3 runs, with values of 0.69 μg virginiamycin equivalent/g sample and 8.9 μg/g, respectively. No antibiotic activity (<0.6 μg/g) was detected in any of the F1 samples or in the fermentor and process intermediate samples from the F2 run. These results demonstrate that low concentrations of biologically active antibiotic may persist in distillers grains coproducts produced from fermentations treated with virginiamycin.     

林肯链霉菌合成林可霉素代谢调节的研究

在摇瓶条件下研究了葡萄糖、铵盐、磷酸盐对林可霉素产生菌林肯链霉菌的生长及林可霉素生物合成的影响。发酵过程中林可霉素的合成主要发生在菌体生长期,逐渐下降。使用6%的葡萄糖未发现通常所说的“葡萄糖效应”。0.2%铵盐有利于细胞生长,但0.8%NH+4对林可霉素的生物合成具有抑制作用。发酵48h后补加0.6% NH^＋_４,对林可霉素的生成没有显著影响。0.05%～0.1%磷酸盐对林可霉素合成具有较强的抑制作用。并就磷酸盐对菌体由初级代谢转向次级代谢的作用作了初步考察。     

Production of the antibiotic gallidermin by Staphylococcus gallinarum – development of a scale-up procedure

A scale-up strategy into 200 l pilot-scale for the production of the antibiotic gallidermin by Staphylococcus gallinarum Tü 3928 was developed. Large-scale fermentations were simulated by consecutive liquid cultures of smaller scale. Afterwards, optimised cultivation conditions were transferred to pilot-scale. Best results were achieved by addition of Maltose during the late production phase leading to a final concentration of 330 mg gallidermin per litre. Compared to the concentrations found in a non-pulsed pilot-scale fermentations this is an increase of 20–30%.     

Improved production of teicoplanin using adsorbent resin in fermentations

AIMS: To use adsorbent resins in fermentations to eliminate toxic effects on growth, reduce feedback repression of production and assist in recovery of teicoplanin. METHODS AND RESULTS: An adsorbent resin was added to the culture broth for the adsorption of teicoplanin. Amberlite XAD-16, Diaion HP-20, charcoal and silica gel were investigated as adsorbent resins. The adsorbed teicoplanin was extracted from the resin by 80% methanol after fermentation. Antibiotic activity was quantified by the disc-agar diffusion assay against Bacillus substilis, and qualitative evaluations were based on HPLC using YMC-Pack ODS-A column. Diaion HP-20 was the most effective adsorbent resin when added at a concentration of 5% (w/v) in the inoculation stage. CONCLUSIONS: Addition of Diaion HP-20 in fermentations eliminated toxic effects on growth and reduced feedback repression of teicoplanin by adsorption. There was a 4.2-fold increase in the quantities of teicoplanin. Addition of adsorbent assisted in the recovery of teicoplanin by reducing the recovery steps. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study provide useful information for the production of teicoplanin, a glycopeptide antibiotic produced by Actinoplanes teicomyceticus. Addition of adsorbent in fermentation increased productivity of teicoplanin by more than five times.     

Influence of the phosphorus and nitrogen source on nisin production in Lactococcus lactis subsp. lactis batch fermentations using a complex medium

The influence of different phosphorus and nitrogen sources on Lactococcus lactis subsp. lactis NIZO 22186 growth and nisin production was studied in batch fermentations using a complex medium. KH₂PO₄ was found to be the best phosphorus source for nisin production. Increasing initial phosphate concentrations from 0 to 5% KH₂PO₄ exerted a double effect, creating favourable pH conditions and particularly stimulating the nisin production levels, which were highest at 5% KH₂PO₄. Up to now, no such high initial phosphate concentrations have been reported for the production of other antibiotics or bacteriocins. Nisin, a lanthionine-containing peptide antibiotic with bacteriocin properties, clearly behaved as a primary metabolite, since its formation was linked with active growth and was not suppressed by phosphate concentrations up to 5%. A complex medium supplemented with cotton seed meal as nitrogen source also gave very high nisin yields. Correspondence to: L. De Vuyst     

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.