Effect of <Emphasis Type="Italic">m</Emphasis>-carbonyl cyanide 3-chlorophenylhydrazone on inorganic polyphosphates synthesis in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> under different growth conditions

The effects of different concentrations of the protonophore uncoupler m-carbonyl cyanide 3-hchlorophenylhydrazone (CCCP) on the synthesis of inorganic polyphosphates (polyP) during the first 0.5 h of hypercompensation in the yeast Saccharomyces cerevisiae VKM Y-1173 growing on media with 2% glucose under low (hypoxia) or high aeration or with 1% (vol/vol) ethanol under high aeration were studied. It was shown that the yeast growth on ethanol was completely inhibited by 5 μM CCCP, while growth on glucose was inhibited by 25 μM CCCP, independently of aeration of the medium. The maximum rate of H2 absorption was shown at 2, 5, and 25 μM CCCP for the cells grown on ethanol, on glucose under high aeration, and on glucose under hypoxia, respectively. Against the decrease of total ATP level and total polyP, CCCP had a nonuniform effect on the synthesis of individual polyP fractions. CCCP maximally inhibited synthesis of the most actively formed fractions: polyPI during growth on glucose under hypoxia, polyPIII during growth on glucose under aeration, and polyPIII and polyPV during growth on ethanol. CCCP had no substantial effect on the synthesis of polyPII and polyPIV fractions, the formation of which seems to be less related to the electrochemical potential gradient of H⁺ ions.     

Continuous production of gluconic acid by immobilized Gluconobacter oxydans cell bioreactor

Summary Living Gluconobacter oxydans cells were attached on fibrous nylon carrier. Free gluconic acid was directly continuously produced in an aerated tubular immobilized-cell bioreactor for at least 6 months, with a volumetric productivity of at least 5 g/lh at 100 g/l substrate glucose and about 80 g/l product gluconic acid concentrations. The highest volumetric productivity in respect to glucose concentration was obtained with 175 g/l glucose, with about 120 g/l product gluconic acid level. With self-directing optimization procedure in respect to maximum product gluconic acid level, productivities as high as about 12–15 g/lh were obtained at relatively high substrate feed rate of 0.166 l/lh and relatively low aeration rate of 0.5 l/lmin. The highest glucose conversion of about 96% was obtained with a long residence time, at the lowest substrate feed rate used at a relatively low aeration rate, resulting however in a significant increase in ketogluconic acid production.     

Enhancement of glucose oxidase production in batch cultivation of recombinant Saccharomyces cerevisiae: optimization of oxygen transfer condition

AIMS: To obtain an optimal combination of agitation speed and aeration rate for maximization of specific glucose oxidase (GOD) production in recombinant Saccharomyces cerevisiae, and to establish a correlation between kLa vis-à-vis oxygen transfer condition and specific glucose oxidase production. METHODS AND RESULTS: The oxygen transfer condition was manifested indirectly by manipulating the impeller speed and aeration rate in accordance with a Central Composite Rotatory Design (CCRD). The dissolved oxygen concentration and the volumetric oxygen transfer coefficient (kLa) were determined at corresponding combinations of impeller speed and aeration rate. The maximal specific extracellular glucose oxidase production (3.17 U mg-1 dry cell mass) was achieved when the initial dissolved oxygen concentration was 6.83 mg l-1 at the impeller speed of 420 rev min-1 and at the rate of aeration of 0.25 vvm. It was found out that while impeller speed had a direct effect on the production of enzyme, a correlation between kLa and specific GOD production could not be established. CONCLUSION: At the agitation speed of 420 rev min-1 and at 0.25 vvm aeration rate, the degree of turbulence and the dissolved oxygen concentration were thought to be optimal both for cellular growth and production of enzyme. SIGNIFICANCE AND IMPACT OF THE STUDY: The combined effect of agitation and aeration on recombinant glucose oxidase production in batch cultivation has not yet been reported in the literature. Therefore, this study gives an insight into the effect of these two important physical parameters on recombinant protein production. It also suggests that since there is no correlation between kLa and specific production of GOD, kLa should not be used as one of the scale-up parameters.     

Influence of sugar source (lactose,glucose, galactose) on 2,3-butanediol production by Klebsiella oxytoca NRRL-B199

The ability of Klebsiella oxytoca NRRL-B199 to use either lactose or the mixture of glucose and galactose as substrate for the production of 2,3-butanediol was studied in batch fermentations with different conditions of aeration and pH. 2,3-butanediol was undetected, or present in minute concentration in the fermentation broths with lactose, while it was the main product from glucose+galactose with final concentrations of up to 18.8 g/l in media at pH 6.0. Under conditions optimal for 2,3-butanediol synthesis, when aeration limited growth, the rate of biomass growth was more tightly related to the aeration rate in lactose medium than in glucose+galactose medium. These relations suggest that the growth rate is very low on lactose but still considerable on glucose+galactose when aeration rate tends toward zero. Correspondingly, the metabolism is more oxidative in the former medium, yielding mainly acetate as product.Abbreviations CDW cell dry weight     

Development of a stepwise aeration control strategy for efficient docosahexaenoic acid production by Schizochytrium sp.

The effect of aeration on the performance of docosahexaenoic acid (DHA) production by Schizochytrium sp. was investigated in a 1,500-L bioreactor using fed-batch fermentation. Six parameters, including specific growth rate, specific glucose consumption rate, specific lipid accumulation rate, cell yield coefficient, lipid yield coefficient, and DHA yield coefficient, were used to understand the relationship between aeration and the fermentation characteristics. Based on the information obtained from the parameters, a stepwise aeration control strategy was proposed. The aeration rate was controlled at 0.4 volume of air per volume of liquid per minute (vvm) for the first 24 h, then shifted to 0.6 vvm until 96 h, and then switched back to 0.4 vvm until the end of the fermentation. High cell density (71 g/L), high lipid content (35.75 g/L), and high DHA percentage (48.95%) were achieved by using this strategy, and DHA productivity reached 119 mg/L h, which was 11.21% over the best results obtained by constant aeration rate.     

Production of human epidermal growth factor by an ampicillin resistant recombinant Escherichia coli strain

Summary The effect of medium composition and initial glucose concentration on production of hEGF by recombinant E. coli cells was investigated. Optimum hEGF production was observed in a yeast extract/acid hydrolysed casein/salts media containing an initial glucose concentration of 10 g.l^-1. A maximum hEGF titer of 250 mg.l^-1 was obtained in this medium after 32 h in laboratory fermenters with pH, temperature, agitation and aeration set at 6.8, 30°C, 500 rpm and 2 vvm, respectively.     

Continuous cultivation of the yeastSaccharomyces cerevisiae at different dilution rates and glucose concentrations in nutrient media

The influence of glucose concentration in nutrient media on the specific growth rate and biomass yield in the course of continuous fermentation ofSaccharomyces cerevisiae was investigated. An increase of glucose content in media decreased the specific growth rate and the biomass yield. Glucose concentration had significant effects on protein and phosphate contents of cells. However, an increased glucose concentration increased the fermentative power ofS. cerevisiae (SJA-method). An increase of the dilution rate decreased the cell concentration in the fermentor. Specific growth rate approached the values of the dilution rate. The best agreement has been obtained at a dilution rate of 0.20/h. This dilution rate proved to be most convenient for the investigated microorganism and cultivation conditions (media composition, pH, aeration intensity and temperature). Biomass yield proved to be decreased by an increase of the dilution rate.     

Effect of the cultivation conditions on alkaloid biosynthesis by Penicillium gorlenkoanum

The effect of a carbohydrate component of the medium, trace elements and aeration on biosynthesis of the alkaloids costaclavine and epicostaclavine was studied with Penicillium gorlenkoanum. Alkaloid biosynthesis was shown to depend on the nature of a carbohydrate component: virtually no alkaloids were accumulated in media with glucose and fructose although these were synthesized at a high rate in a medium with mannitol. The quantity of synthesized alkaloids and the dynamics of the biosynthesis depended on carbohydrate concentration. The growth and alkaloid synthesis were influenced by traces of zinc, iron, copper and manganese. A more intensive aeration stimulated biomass accumulation but suppressed alkaloid biosynthesis.     

Effect of the aeration rate and agitation speed on β-carotene production and morphology of Blakeslea trispora in a stirred tank reactor: mathematical modeling

The effect of aeration rate and agitation speed on β-carotene production and morphology of Blakeslea trispora in a stirred tank reactor was investigated. B. trispora formed hyphae, zygophores and zygospores during the fermentation. The zygospores were the morphological form responsible for β-carotene production. Both aeration and agitation significantly affected β-carotene concentration, productivity, biomass and the volumetric mass transfer coefficient (K_La). The highest β-carotene concentration (1.5 kg m⁻³) and the highest productivity (0.08 kg m⁻³ per day) were obtained at low impeller speed (150 rpm) and high aeration rate (1.5 vvm). Also, maximum productivity (0.08 kg m⁻³ per day) and biomass dry weight (26.4 kg m⁻³) were achieved at high agitation speed (500 rpm) and moderate aeration rate (1.0 vvm). Conversely, the highest value of K_La (0.33 s⁻¹) was observed at high agitation speed (500 rpm) and high aeration rate (1.5 vvm). The experiments were arranged according to a central composite statistical design. Response surface methodology was used to describe the effect of impeller speed and aeration rate on the most important fermentation parameters. In all cases, the fit of the model was found to be good. All fermentation parameters (except biomass concentration) were strongly affected by the interactions among the operation variables. β-Carotene concentration and productivity were significantly influenced by the aeration, agitation, and by the positive or negative quadratic effect of the aeration rate. Biomass concentration was principally related to the aeration rate, agitation speed, and the positive or negative quadratic effect of the impeller speed and aeration rate, respectively. Finally, the volumetric mass transfer coefficient was characterized by the significant effect of the agitation speed, while the aeration rate had a small effect on K_La.     

Utilization of an external loop bioreactor for the isolation of a flocculating strain ofKluyveromyces marxianus

A continuous open loop bioreactor was used to induce flocculation in an originally nonflocculent strain ofKluyveromyces marxianus. The sedimentation capacity of the isolated strain was of such a magnitude that the cell concentration inside the fermentor was 50 times larger than in the effluent. Also, a batch system was used with the same objective, but no flocculation was obtained.The kinetic parameters of the flocculent strain were compared with those of the mother strain. It was shown that both maximum specific growth rate and maximum specific ethanol production rate were lower in the flocculent strain. Ethanol had a larger inhibitory effect on the kinetic parameters of the isolated strain. Also, the batch fermentations with this strain presented a larger final biomass concentration and a reduced ethanol yield.     

Must deacidification with an induced flocculant yeast strain of Schizosaccharomyces pombe

The use of flocculant cells of the yeast strain Schizosaccharomyces pombe for the deacidification of grape musts in continuous culture was developed. An external loop reactor was used to induce flocculation. The flocs obtained were stable in the pH range 3.0–6.0 and in the presence of several sugars. Some inhibition was observed for high (above 6.0) and low (below 3.0) pH values. Once induced, flocculation could no longer be completely inhibited. Vinho Verde, a typical Portuguese wine, has a relatively low ethanol content and a high acid concentration. The external loop reactor loaded with the flocculant cells was used to deacidify a synthetic medium with sugar and malic acid concentrations similar to the ones found in Vinho Verde grape must. A desirable malic acid decrease with moderate glucose consumption was obtained at a dilution rate of 0.7 H^–1. Improved results were obtained when the synthetic medium was replaced by Vinho Verde grape must. Correspondence to: M. Mota     

Effect of stirrer speed,aeration rate and cell mass concentration on volumetric oxygen transfer coefficients (KLa) in the cultivation of Curvularia lunata in a batch reactor

Summary Curvularia lunata was grown in a stirred and aerated reactor for the production of extracellular rifamycin oxidase. Volumetric oxygen transfer coefficients (K_La) were measured for various stirrer speeds, rates of aeration and cell mass concentrations in the reactor. Stirrer speed and aeration rate were optimized and it was found that stirrer speeds of 400–500 rpm and aeration rates of 0.75–1 vvm were optimum for the maximum amount of enzyme production. It was noticed that the increase in cell mass decreased the oxygen transfer coefficient. It was also noticed that the organism formed pellets rather than mycelia when grown on glucose and with an increase in the concentration of glucose in the reactor, there was heavy pellet formation.     

Induction of microcin B17 formation in Escherichia coli ZK650 by limitation of oxygen and glucose is independent of glucose consumption rate

We examined the consumption of glucose from the media in which Escherichia coli ZK650 was grown. This organism, which produces the polypeptide antibiotic microcin B17 best under conditions of limiting supplies of glucose and air, was grown with a low level of glucose (0.5 mg/ml) as well as a high level (5.0 mg/ml) under both high and low aeration. Glucose consumption rates were virtually identical under both high and low aeration. Thus, glucose consumption rate is not a regulating factor in microcin B17 formation. Journal of Industrial Microbiology & Biotechnology (2001) 26, 341–344. Received 25 September 2000/ Accepted in revised form 16 April 2001     

Factors affecting growth physiology of Bacillus thuringiensis

Summary Physiological studies on Bacillus thuringiensis var. entomocidus revealed the failure of the organism to survive or sporulate under low aeration levels, notably in the presence of high sugar concentrations. Cell counts, sporulation titers and potency of resulting endotoxin were found to vary with the level of aeration. The incremental feeding of glucose with continuous pH adjustment prevented cell injury and death which results from prolonged exposure to acidity liberated at the high sugar concentrations which occur when glucose is added batchwise. Increasing of dipotassium phosphate concentration in growth medium increased the potency of the resulting endotoxin.     

Effect of aeration and agitation on synthesis of poly (γ-glutamic acid) in batch cultures of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> NCIM 2324

The effects of aeration and agitation on the production and molecular weight of poly (γ-glutamic acid) (PGA) were systematically investigated in batch fermentor cultures of Bacillus licheniformis NCIM 2324. A high aeration rate and agitation speed enhanced the growth of B. licheniformis NCIM 2324, but did not always lead to high PGA production. Additionally, PGA production actually decreased at very high aeration rates and agitation speeds. The maximum PGA concentration was obtained at 750 rpm and 1 vvm. Rheological studies revealed that fermentation broth during production of PGA exhibited pseudoplastic behavior. The effects of aeration and agitation on the molecular weight of PGA were also studied, and the rate and extent of the decrease in the molecular weight of PGA as a function of time were found to be much greater at high aeration than low aeration. The PGA production of 46.34 g/L with a specific productivity of 0.17 g-PGA/g-biomass/ h and a PGA yield of 0.48 with respect to total substrate observed in the present study are much higher than the values reported in previously conducted studies.     

Effect of montmorillonite and the capsular polysaccharide on the flocculation of Klebsiella aerogenes

A study was undertaken on the effect of colloidal montmorillonite and exocellular polysaccharide produced by Klebsiella aerogenes on the flocculation process of the bacterium.The addition of a low concentration of K-montmorillonite (350 g/ml) led to the flocculation of the non-capsulated strain K₅₄A₃ (0) of K. aerogenes. The volume of the sediment was dependent on the relative concentration of the bacteria and the clay. In contrast with its non-capsulated counterpart, the encapsulated strain K₅₄A₃ was more stable in the presence of a low concentration of K-montmorillonite. The flocculation of the cells was affected by the composition of the growth medium, the suspension being more stable when the bacteria were grown on a rich sugar agar. The addition of capsular polysaccharide to the non-capsulated strain reduced or prevented the flocculation process. These results suggest that the capsular polysaccharide, which contain COO- as sole ionogenic groups, probably attach to and neutralize the positive charges at the edges of the clay platelets. Consequently, K-montmorillonite did not cause any flocculation of the cells when the capsular polysaccharide was present.     

Effect of cycloheximide, iodoacetamide, and antimycin A on inorganic phosphate synthesis in Saccharomyces cerevisiae VKM Y-1173

The effect of inhibitors of protein synthesis (cycloheximide, CHI), glycolysis (iodoacetamide, IAA), and oxidative phosphorylation (antimycin A, ANM) on inorganic phosphate (polyP) synthesis during the first 0.5 h of their hypercompensation in Saccharomyces cerevisiae VKM Y-l173 grown on 2% glucose-containing media at low (hypoxia) or high aeration rates or in the presence of 1 vol % ethanol under high aeration conditions was studied. PolyP accumulation was highest in the medium with glucose under hypoxia; lower, with glucose at high aeration; and lowest, in the medium with ethanol. CHI had a small effect on the total polyP level but significantly stimulated ATP accumulation, irrespective of the culture growth conditions. The low-polymer acid-soluble polyP1 were synthesized most actively by the cells grown on glucose under hypoxia, alkali-soluble polyP3 were synthesized at en hanced aeration, and the most hig-molecular fraction, polyP5, was actively accumulated along with polyP3 at cultivation on ethanol. Regardless of the growth conditions, CHI inhibited accumulation of polyP4, the synthesis of which is associated with the synthesis of mannoproteins. IAA and ANM largely inhibited synthesis of all fractions at yeast growth under hypoxia and on ethanol, respectively. The results as a whole demonstrate the dependence of polyP formation on the main energy-generating cell processes and, at the same time, the absence of direct dependence of their synthesis on ATP concentration in Saccharomyces cerevisiae VKM Y-l 173.     

Effect of agitation and aeration on the production of nitrile hydratase by Rhodococcus erythropolis MTCC 1526 in a stirred tank reactor

Aims: To evaluate the effect of different physicochemical parameters such as agitation, aeration and pH on the growth and nitrile hydratase production by Rhodococcus erythropolis MTCC 1526 in a stirred tank reactor. Methods and Results: Rhodococcus erythropolis MTCC 1526 was grown in 7‐l reactor at different agitation, aeration and controlled pH. The optimum conditions for batch cultivation in the reactor were an agitation rate of 200 rev min^?1, aeration 0·5 v/v/m at controlled pH 8. In this condition, the increase in nitrile hydratase activity was almost threefold compared to that in the shake flask. Conclusion: Agitation and aeration rate affected the dissolved‐oxygen concentration in the reactor which in turn affected the growth and enzyme production. Significance and Impact of the Study: Cultivation of R. erythropolis MTCC 1526 in the reactor was found to have significant effect on the growth and nitrile hydratase production when compared to the shake flask.     

Formation of ethyl acetate by Kluyveromyces marxianus on whey: Influence of aeration and inhibition of yeast growth by ethyl acetate

The ability of the yeast Kluyveromyces marxianus to convert lactose into ethyl acetate offers good opportunities for the economical reuse of whey. The formation of ethyl acetate as a bulk product depends on aerobic conditions. Aeration of the bioreactor results in discharge of the volatile ester with the exhaust gas that allows its process‐integrated recovery. The influence of aeration (varied from 10 to 50 L/h) was investigated during batch cultivation of K. marxianus DSM 5422 in 0.6 L whey‐borne medium using a stirred reactor. With lower aeration rates, the ester accumulated in the bioreactor and reached higher concentrations in the culture medium and the off gas. A high ester concentration in the gas phase is considered beneficial for ester recovery from the gas, while a high ester concentration in the medium inhibited yeast growth and slowed down the process. To further investigate this effect, the inhibition of growth by ethyl acetate was studied in a sealed cultivation system. Here, increasing ester concentrations caused a nearly linear decrease of the growth rate with complete inhibition at concentrations greater than 17 g/L ethyl acetate. Both the cultivation process and the growth rate depending on ethyl acetate were described by mathematical models. The simulated processes agreed well with the measured data.     

Biocalorimetric Studies of the Metabolic Activity of Pseudomonas aeruginosa Aerobically Grown in a Glucose-Limited Complex Growth Medium

Biocalorimetric experiments were performed to investigate the aerobic growth of Pseudomonas aeruginosa, isolated from tannery saline wastewater. Growth factors (pH, Inoculum size, carbon source, temperature, aeration rate, and agitation rate) were optimized in shaker and calorimeter based on the growth of P. aeruginosa and heat generation rates. A limiting value of 0.2% glucose concentration was found to be optimum for the growth of P. aeruginosa in a complex growth medium, and the heat flux (q_r) profiles resulting from the metabolic activity of P. aeruginosa further confirmed this observation. The bacterial growth profile was found to correlate well with the metabolic heat generated. Heat-yield values were calculated for both glucose consumption and the growth of P. aeruginosa from the calorimetric results. Metabolic shifts in substrate uptake from glucose to peptone present in growth medium was observed by the variations in heat-flux profile. The calorimetric data presented in this study should be useful in understanding the behavior of the isolated bacterial strain in degrading complex and mixed substrates commonly observed in tannery saline waste stream, and further to extend the results for scale-up studies.