Oxygen uptake rate regulation during cell growth phase for improving avermectin B<Subscript>1a</Subscript> batch fermentation on a pilot scale (2 m<Superscript>3</Superscript>)

Avermectin B_1a batch fermentation of Streptomyces avermitilis in a 2 m³ fermentor was investigated by oxygen uptake rate (OUR) regulation during cell growth phase. OUR was controlled by adjusting of aeration and agitation. Result showed that OUR strongly affected cell growth and antibiotics production. Avermectin B_1a biosynthesis could be effectively enhanced when OUR was stably regulated at an appropriate level in batch fermentation of S. avermitilis. Avermectin B_1a yield reached 5568 ± 111 mg/l by controlling maximal OUR between 15 and 20 mmol/l/h during cell growth phase, which was increased by 21.8% compared with the control (maximal OUR above 20 mmol/l/h). The stimulation effect on avermectin B_1a production could be attributed to the improved supply of propionic acid and acetic acid, the precursors of avermectin B_1a, in the cells. Hence, this OUR control method during cell growth phase may be a simple and applicable way to improve industrial production of avermectin.     

Citric acid production from glucose. I. Growth and excretion kinetics in a stirred fermentor

The growth and citric acid production kinetics of Saccharomycopsis lipolytica on glucose are investigated in an aerated stirred fermentor. Cellular growth first proceeds exponentially until exhaustion of ammonia in the fermentation medium. Cells then continue to grow at a reduced rate with a concomitant decrease in intracellular nitrogen content. Citric and isocitric acid production starts at the end of the growth phase. During about 80 hr excretion proceeds at a constant rate of 0.7 g/liter/hr for citric acid and 0.1 g/liter/hr for isocitric acid. The final citric and isocitric acid concentrations are 95 and 10g/liter, respectively. During acid excretion cellular respiration accounts for 60 and 35% of consumed oxygen and glucose. Both acid and CO₂ production rates follow a Michaelis–Menten-type dependence on oxygen concentration with Michaelis–Menten constants of 0.9 and 0.15 mg/liter for acid and CO₂ productions, respectively.     

Optimization of process parameters for the production of carbonyl reductase by <Emphasis Type="Italic">Candida viswanathii</Emphasis> in a laboratory-scale fermentor

The effect of pH, aeration and mixing on the growth and production of carbonyl reductase by Candida viswanathii was investigated in a 6.6-l fermentor. Controlling the pH at 8.0 had a very significant effect on the enzyme production. Aeration and agitation influenced the dissolved oxygen concentration which in turn affected growth as well as enzyme production. A maximum carbonyl reductase activity (53 Umg⁻¹) was attained in 24 h under the optimal cultivation conditions of controlled pH at 8.0, aeration rate 1 vvm and an agitation speed of 250 rpm at 25°C. The enzyme activity was twice as high (56 Umg⁻¹) in the fermentor as compared to a shake flask. Further, the duration of growth and enzyme production in the fermentor was shortened. Cells cultivated under the optimized conditions were used for the preparative scale reduction of N, N-dimethyl-(3-keto)-2-thienyl-propanamine to (S)-N, N-dimethyl-(3-hydroxy)-2-thienyl-propanamine, a key intermediate in the production of the important antidepressant drug (S)-duloxetine.     

Hydroxyethylation of Comenic Acid

A small jar fermentor was developed in order to investigate the effect of oxygen supply on hydrocarbon fermentation. Several indices to oxygen transfer were examined with this small jar fermentor. Conditions for suitable oxygen supply were examined in l-glutamic acid fermentation from hydrocarbon by use of shaking flasks and these small jar fermentors. The data indicated that the rate of oxygen transfer ought to be more than 14.3 × 10^?7 mole/ml·min in order to obtain satisfactory results. The coefficient of oxygen transfer rate (K_La/H) decreased as the fermentation went on, so the supply of oxygen enriched gas mixture was effective to increase the production of l-glutamic acid.     

Acetic acid production by Acetobacter aceti in a silicone tube bioreactor

Summary Continuous acetic acid fermentation was carried out using a column reactor, in which 20 to 200 thin silicone tubes (0.33 mm in outer diameter) were packed to supply oxygen by permeation. The highest value of volumetric oxygen transfer coefficient determined by the sulfite oxidation method was 2,860 h^–1, which was comparable to that of a well agitated and aerated fermentor. The maximum production rate of acetic acid by the bacterial films of Acetobacter aceti M7 grown on the shell-side surface of the tubes was 38.0 g/lh at an acetic acid concentration of 44.5 g/l. This was 29 times that of a continuous culture using a jar fermentor.     

Carbon dioxide inhibition of yeast growth in biomass production

Saccharomyces cerevisiae was grown under aerobic and substrate-limiting conditions for efficient biomass production. Under these conditions, where the sugar substrate was fed incrementally, the growth pattern of the yeast cells was found to be uniform, as indicated by a constant respiratory quotient during the entire growing period. The effect of carbon dioxide was investigated by replacing portions of the nitrogen in the air stream with carbon dioxide, while maintaining the oxygen content at the normal 20% level, so that identical oxygen transfer rate and atmospheric pressure were maintained for all experiments with different partial pressures of carbon dioxide. Inhibition of yeast growth was negligible below 20% CO₂ in the aeration mixture. Slight inhibition was noted at the 40% CO₂ level and significant inhibition was noted above the 50% CO₂, level, corresponding to 1.6 × 10^?2M of dissolved CO₂ in the fermentor broth. High carbon dioxide content in the gas phase also inhibited the fermentation activity of baker's yeast.     

DAILY FLUCTUATIONS OF EXOPOLYMERS IN CULTURES OF THE BENTHIC DIATOMS CYLINDROTHECA CLOSTERIUM AND NITZSCHIA SP. (BACILLARIOPHYCEAE)1

Dynamics in the production of extracellular polymeric substances (EPS) were investigated for the benthic diatoms Cylindrotheca closterium (Ehrenberg) and Nitzschia sp. The effect of growth phase and light:dark conditions were examined using axenic cultures. Two EPS fractions were distinguished. Soluble EPS was recovered from the culture supernatant and represented polysaccharides that were only loosely associated with the cells. Bound EPS was extracted from the cells using warm (30° C) water and was more closely associated with the diatom aggregates. Concentrations of EPS exceeded internal concentrations of sugar throughout growth, indicating that EPS production is important in these organisms. Soluble and bound EPS revealed distinct differences in daily dynamics during the course of growth. Soluble EPS was produced continuously once cultures entered the stationary phase. During the stationary phase, chl a‐normalized EPS production rates equaled 6.4 and 3.4 d ^{? 1} for C. closterium and Nitzschia sp., respectively. In contrast, production of bound EPS occurred only in the light and was highest during the exponential phase. Up to 90% of the attached EPS that was produced in the light was degraded during the subsequent dark period. The monosaccharide distribution of EPS was constant during the course of the experiment. The soluble EPS consisted of high amounts of galactose and glucuronic acid, relative to rhamnose, glucose, xylose/mannose, and galacturonic acid. In contrast, glucose was the dominant monosaccharide present in the bound EPS. These differences suggest that the production of the two distinct EPS fractions is under different metabolic controls and probably serves different cellular functions.     

Production of nisin with continuous adsorption to Amberlite XAD-4 resin using<Emphasis Type="Italic"> Lactococcus lactis</Emphasis> N8 and<Emphasis Type="Italic"> L. lactis</Emphasis> LAC48

The production of nisin, biomass and lactic acid in pH-controlled and uncontrolled batch fermentation and batch fermentation (pH 5.5) with continuous removal of nisin was examined in the parent strain Lactococcus lactis N8 and LAC48. Strain LAC48 in batch fermentor (pH not controlled) gave a maximum nisin concentration of 2.5×10⁶ IU g dcw^–1. The nisin concentration remained high (2.0×10⁶ IU g dcw^–1) after the logarithmic growth phase (10–22 h), whereas nisin production of strain N8 decreased after the logarithmic growth phase. The maximum nisin production of strain LAC48 was not directly related to the biomass formation and not associated with growth. In order to study end product inhibition in nisin production, a system was built for adsorption of nisin during fermentation. The adsorbent Amberlite XAD-4 was found to have an effective binding capacity for nisin. Cells of LAC48 and N8 compensated for the removal of nisin, indicating that nisin production also occurs in the stationary phase.     

Kinetic models of ribonucleic acid fermentation and continuous culture by <Emphasis Type="Italic">Candida tropicalis</Emphasis> no.121

During ribonucleic acid fermentation, the fermentative processes were researched at pH controlled at 4.0 and under natural conditions. Unstructured models in a 50-L airlift fermentor were established for batch RNA production at pH 4.0 using the Verhulst equation for microbial growth, the Luedeking–Piret equation for product formation and a Luedeking–Piret-like equation for substrate uptake. Parameters of the kinetic models were determined using origin 7.5. Based on the models estimated above, another batch fermentation experiment was conducted in a 300-L airlift fermentor, which demonstrated that the models could be useful for RNA production on an industrial scale. Additionally, continuous fermentation based on kinetic models was proposed to make full use of substrates and reduce the cost of waste water treatment. As a result, although the DCW and RNA concentration were 11.5 and 1.68 g L⁻¹, which were lower than that of batch fermentation, the sugar utilization increased by 14.3%, while the waste water decreased by more than 90%.     

Effect of oxygen and carbon dioxide on germination and growth ofRhizopus oligosporus on model media and soya beans

 The microcolony technique enables the effects of several atmospheric conditions on fungal growth to be studied by measuring the radius of the colony, while excluding effects of those conditions on germination of the sporangiospores. Various concentrations of oxygen and carbon dioxide in the gas environment were found to influence growth of Rhizopus oligosporus on malt extract/soya peptone/agar. The maximum radial growth rate was 1.48 mm/h and the maximum specific growth rate was 0.109 h^-1 at 30 °C. Oxygen became limiting below 1% (v/v), but growth remained possible at levels of 0.001% oxygen. Carbon dioxide stimulated growth at limiting oxygen levels. The specific growth rate increased from 0.043 h^-1 at 0.5% (v/v) oxygen and 0% (v/v) carbon dioxide to 0.096 h^-1 at 0.5% (v/v) oxygen and 5% (v/v) carbon dioxide. A mixture of 0.5% (v/v) oxygen and 35% (v/v) carbon dioxide inhibited growth. Delay of sporangiospore germination due to low (less than 0.001%) amounts of oxygen was not observed with the techniques used. Fungal activity in a rotating drum fermentor was more strongly affected by low levels of oxygen than was biomass formation on model media. High concentrations of carbon dioxide inhibited growth in the rotating drum fermentor at non-limiting levels of oxygen. It is concluded that aeration and heat removal are both essential aspects of optimization of large-scale solid-substrate bioreactors with Rh. oligosporus. Received: 5 August 1994/Received revision: 14 November 1994/Accepted: 5 December 1994     

Production of biomass and lutein by Chlorella protothecoides at various glucose concentrations in heterotrophic cultures

The influence of initial glucose concentrations on the production of biomass and lutein by Chlorella protothecoides CS-41 was investigated in batch cultures using both shake flasks and fermentors. The maximum biomass concentration increased from 4·9 to 31·2 g litre⁻¹ dry cells with an increase in initial glucose concentration from 10 to 80 g litre⁻¹. An even higher initial glucose concentration (100 g litre⁻¹) resulted in a lower biomass concentration, a lower specific growth rate, a lower growth yield coefficient and a considerably longer lag phase, which might be due to substrate inhibition. The initial glucose level also had a significant effect on the production of lutein. In a 3·7-litre fermentor an increase in lutein production from 19·39 to 76·56 mg litre⁻¹ was obtained with an increase in initial glucose concentration from 10 to 40 g litre⁻¹, within which range, lutein yield coefficient was constant (Y_Itn=1·90±0·02 mg g⁻¹). A simple substrate inhibition model was developed, which fitted the experimental data better than the classical Haldane model. A group of time-dependent kinetic models for algal cultivation in fermentors were also constructed, which were in good agreement with the experimental results and could be employed to predict the production of biomass and lutein, and the consumption of glucose in fermentor cultures.     

Citrate production from hydrocarbons by use of a nonsterile,semicontinuous cell recycle system

In order to minimize product toxicity, decrease fermentation cost, prolong the effective production phase, and shorten the lag phase before production in the citrate-hydrocarbon fermentation by Candida lipolytica ATCC 8661, the use of a nonsterile semicontinuous cell recycle system was investigated. Model experiments demonstrated that portions of the fermentor broth could periodically be removed and centrifuged under nonaseptic conditions with the cells being added to fresh medium and returned to the fermentor. Citrate production continued, however with repeated semicontinuous cell recycle, acid production gradually decreased. It was postulated that this decrease could be attributed largely to physiological trauma and that a truly continuous cell recycle system would minimize these effects and permit maintenance of higher citrate production rates.     

Influence of temperature rise on kinetic parameters during batch propagation of Kluyveromyces fragilis in cheese whey under ambient conditions

Three 5 l working volume fermenters were used to investigate the growth of the yeast Kluyveromyces fragilis in acid cheese whey under ambient temperature in order to assess the specific growth rate and yield, the lactose and oxygen uptake rates during the various phases of batch culture, the effect of increasing temperature on the various kinetic parameters, and the need for a cooling unit for single cell production batch systems. The initial dissolved oxygen in the medium was 5.5 mg l^–1 and the pH was maintained at 4.5. The observed lag phase, specific growth rate and maximum cell number were 4 h, 0.2 h^–1 and 8.4 × 10⁸ cells ml^–1, respectively. About 99% of the lactose in cheese whey was utilized within 20 h, 85% during the exponential growth phase. The specific lactose utilization rates by K. fragilis were 0.20 × 10^–12, 1.457 × 10^–12, 0.286 × 10^–12 and 0.00 g lactose cell^–1 h^–1, for the lag, exponential, stationary and death phases, respectively. The dissolved oxygen concentration in the medium decreased as the cell number increased. The lowest oxygen concentration of 1.2 mg l^–1 was observed during the stationary phase. The volumetric oxygen transfer coefficient was 0.41 h^–1 and the specific oxygen uptake rates were 0.32 × 10^–12, 2.14 × 10^–12, 0.51 × 10^–12 and 0.003 × 10^–12 mg O₂ cell^–1 h^–1, for the lag, exponential, stationary and death phases, respectively. The maximum temperature recorded for the medium was 33 °C, indicating that a cooling unit for batch production of single cell protein at ambient temperature is not needed for this type of bioreactor. The increase in medium temperature affected the cell growth and the lactose and oxygen uptake rates.     

Effect of PO2 on growth and physiological characteristics of Candida utilis in a multistage tower fermentor

The effect of increasing the partial pressure of oxygen in the aeration gas on growth and physiological activity of the yeast Candida utilis in a multistage tower fermentor was studied. The measurements were made at steady states of continuous culture for single values of dilution rate, temperature, and pH in all stages of the fermentor and with one given ethanol concentration in the growth medium feed. The partial pressure of oxygen in the gas phase was changed in the range from 165 to 310 torr. The results revealed the existence of the upper critical value of the partial oxygen pressure in the gas phase. It was demonstrated that the upper critical value of PO ₂ influences not only the growth rate, biomass yield, and productivity but also the cell physiology resulting in changes of respiration activity and activity of alcohol and aldehyde dehydrogenases.     

The effect of aeration and agitation on Claviceps purpurea dimorphism and alkaloid synthesis during submerged fermentation

Summary Varying the air flow rate (vvm) in a fermentor under constant drive speed, Claviceps purpurea dimorphism as well as alkaloid biosynthesis were greatly influenced. At a high flow rate (2.5 vvm) sclerotial growth was favoured in seed and in production media, while at a low air flow rate (1.0 vvm) sphacelial growth dominated. When using high flow rates the oxygen uptake rate was small, but at low flow rates it increased markedly. In both cases the alkaloid production was lower than at the intermediate value of 1.5 vvm of air flow rate, which proved to be optimal. This could be explained by the difference in the air/water interface and two-phase oxygen uptake. At a high air/water interface direct oxygen uptake from the gaseous phase prevails, while at a low air/water interface uptake is due to the oxygen liquid-phase only. Thus for optimal fungal development and alkaloid production a compromise between uptake from the liquid and the gaseous phase has to be established by a defined ratio between aeration and agitation.     

Oxygen transfer and mycelial growth in a tubular loop fermentor

A 22 m long. 20 liter tubular loop fermentor (TLF) has been tested for oxygen transfer characteristics and as a reactor for mycelial growth. Model calculations show that the flow pressure drop has an important influence on the axial oxygen profiles. A design model that accounts for this influence is presented. Using the model, K_L a values are calculated from the results of sulfite oxidation experiments. These are correlated with power consumption and aeration rates. The K_L a dependence on aeration rate was found to be less than found with tank reactors. The growth kinetics of three metabolite-producing mycelial organisms in the TLF are presented: a Streptomyces, a Fusarium, and a Acrophialophora. In order to determine the influence of reactor type on the growth and product formation, these cultures have been grown in tanks and shake flasks. The antibiotic, product spectrum of Streptomyces is compared on the basis of inhibition tests and it is shown that the distribution of products is reactor dependent. The Fusarium culture produced a previously unknown metabolite, whose concentration in the loop fermentor was four times higher than in a shake flask. The Acrophialophora culture grew twice as fast in the loop fermentor, but produced essentially none of the specific product. Power Consumptions of up to 8 kW/m³ in the tubular fermentor did not appear to harm the mycelia.     

Continuous high-ethanol fermentation from cane molasses by a flocculating yeast

Repeated-batch fermentation by a flocculating fusant, Saccharomyces cerevisiae HA 2, was done in a molasses medium that contained 20% (w/v) total sugar, at 30°C in an automatically controlled fermentor, and the effects of ethanol concentration on the specific growth rate and the specific production rate of ethanol were studied. Both the specific growth rate and the specific production rate of ethanol fell with increase of ethanol concentration, and there was a linear correlation between each rate and the concentration of thanol. The maximum specific growth rate (μ_max) and the maximum specific production rate of ethanol (q_max) were 0.12 h⁻¹ and 0.1 g ethanol/10⁹ cells·h, respectively. The specific growth rate and the specific production rate of ethanol fell to zero at ethanol concentration of 89 g/l and 95 g/l, respectively. The number of viable cells, calculated from the linear inhibition equation, was 1.3 × 10⁹ cells/ml for production of 85 g/l ethanol at a dilution rate (D₁) of 0.2 h⁻¹. Based on this estimation, a laboratory-scale continuous fermentation, using two fermentors in series, was done. In the second fermentor, 85 g/l ethanol was produced at a dilution rate (D₁) of 0.2 h⁻¹ by the active feedig of the fermented mash from the first fermentor into the second fermentor by pumping (hereafter called active feeding). To maintain the number of viable cells above 10⁹ cells/ml in the second fermentor, a active feeding ratio of more than 23% was required. Under these conditions, 81 g/l ethanol was produced in the second fermentor at a dilution rate (D_t) of 0.25 h⁻¹, and the high ethanol productivity of 20.3 g/l·h could be achieved. A bench-scale continuous fermentation, using two fermentors in series, with a active feeding ratio of 25% was done. An ethanol concentration of 84 g/l in the second fermentor at a dilution rate (D_t) of 0.25 h⁻¹ was achieved, just as it was in the laboratory-scale fermentation test.     

Stoffwechselprodukte von Mikroorganismen

At low iron(III)-concentrations (<10^-5 M) the fungus Aspergillus viridi-nutans Ducker & Thrower excretes desferri-ferricrocin as the main sideramine into the culture medium. While this compound accounts for 95% of the sideramines produced, small amounts of additional sidermines may also be detected. In a search for an inexpensive nutrient medium for optimum production of desferri-ferricrocin, experiments using shake flasks with good aeration were undertaken initially. The best medium conditions were then employed in a fermentor system.In a 20-1 fermentor with intensor system, it was shown that at certain growth rates there was an inverse correlation between rate of growth and rate of sideramine production. A defined nutrient medium of glucose plus acetate as carbon sources, and urea or ammonium acetate as nitrogen sources was used. Two different feeding regimens were used in response to changes of pH or to changes of partial pressure of oxygen in the submerged culture: acetic acid/urea of acetic acid/ammonium acetate additions regulated these conditions. The rate of sideramine production under such feeding achieved a maximum of 20 mg l^-1 h^-1 over a period of several days.

165. Mitteilung: H.-P. Fiedler, J. Sauerbier: Isolation and quantitative determination of siderochromes. Europ. J. Appl. Microbiol. (in press)     

Xanthan batch fermentations: compared performances of a bubble column and a stirred tank fermentor

Fermentations of Xanthomonas campestris, NRRL B-1459, were carried out in a bubble column fermentor (BCF) and in a stirred tank fermentor (STF) to allow comparison of representative variables measured during the microbial growth and the gum production. The microbial growth phase was described by a logistic rate equation where maximum cell concentration was provided by nitrogenous compounds balance. The average value of the maximum specific growth rate was higher in the bubble column (μ _M =0.5 h^?1) than in the stirred reactor (μ _M =0.4 h^?1). The upper values of xanthan yield (Y _g-x =0.65 kg xanthan/kg glucose; Y _{O 2?x} xanthan/kg oxygen) and specific production rate (q _x =0.26 kg xanthan/kg biomass · h) were measured when the oxygen transfer coefficient was kept up above 80 h^?1 in the STF fermentor. In the bubble column the fermentation achieved in the same culture medium lasts two times longer than in the stirred aerated tank; this was attributed to the low value of the oxygen transfer coefficient (K _L a =20 h^?1) at the beginning of the gum synthesis phase. The results obtained in the stirred tank were the basis to estimate the optimal biomass concentration which enables to achieve a culture in non-limiting oxygen transfer conditions. Nevertheless, the transfer characteristics were more homogeneous in the bubble column than in the stirred tank where dead stagnant zones were observed. This is of primary importance when establishing fermentation kinetics models.