Effects of agitation and aeration on the production of extracellular glucose oxidase from a recombinant Saccharomycescerevisiae

A recombinant strain of Saccharomyces cerevisiae, GAL-GO2, was developed to facilitate the production of extracellular glucose oxidase (GOD). The recombinant strain secreted 85% (8.7?U/ml) of the total GOD (10.3?U/ml) produced in shake flask culture. For further enhancement of GOD production, optimization of the speed of agitation and the rate of aeration in a stirred tank fermentor was carried out. Response surface methodology with appropriate statistical experimental design was employed for this purpose. The maximal level of extracellular GOD was achieved when the speed of agitation and the rate of aeration were 420?rpm and 0.25?vvm, respectively. The enzyme production was increased by 74% compared to the level obtained under unoptimized conditions.     

Effect of dissolved oxygen concentration and impeller tip speed on itaconic acid production by Aspergillus terreus

Summary Effect of aeration rate and impeller tip speed on mycelium growth and itaconic acid production was investigated in a batch culture of Aspergillus terreus IFO-6365. When impeller tip speed was 94.2 cm/sec at a fixed aeration rate of 0.5 vvm, itaconic acid concentration was 3.6 and 1.6 times higher than those in the impeller tip speed of 62.8 and 125.7 cm/sec, respectively. When an oxygen-enriched air was supplied at a fixed impeller tip speed of 94.2 cm/sec and dissolved oxygen concentration was maintained in the 20–60 % range, both itaconic acid concentration and mycelium growth were not affected by the dissolved oxygen concentration.     

Effect of oxygen transfer on lipase production by Acinetobacter radioresistens

The influence of oxygen on alkaline lipase production by Acinetobacter radioresistens was studied under two operating modes: controlled dissolved oxygen (DO) concentration and controlled aeration rate. Compared with cell growth, the lipase production depended more extensively on oxygen. The intrinsic factor determining cell growth and lipase production was oxygen transfer rate (OTR) rather than DO concentration. Improvements in OTR, either by aeration or agitation, resulted in an increase in lipase yield and/or a reduction in fermentation time. The formation of A. radioresistens lipase could be described by a mixed-growth-associated model, and the enzyme was mainly a growth-associated product. The overall productivity for the lipase, which depended more strongly on agitation than aeration, could be related with kLa. DO concentration could not be employed in this correlation, though it has been useful as a criterion for ensuring no oxygen limitation in an aerobic fermentation.     

Production of carbonyl reductase by Geotrichum candidum in a laboratory scale bioreactor

Fungal fermentation is very complex in nature due to its nonlinear relationship with the time, especially in batch culture. Growth and production of carbonyl reductase by Geotrichum candidum NCIM 980 have been studied in a laboratory scale stirred tank bioreactor at different pH (uncontrolled and controlled), agitation, aeration and dissolved oxygen concentration. The yield of the process has been calculated in terms of glucose consumed. Initial studies showed that fermenter grown cells have more than 15 times higher activity than that of the shake flask grown cells. The medium pH was found to have unspecific but significant influence on the enzyme productivity. However, at controlled pH 5.5 the specific enzyme activity was highest (306U/mg). Higher agitation had detrimental effect on the cell mass production. Dissolved oxygen concentration was maintained by automatic control of the agitation speed at an aeration rate of 0.6 volume per volume per minute (vvm). Optimization of glucose concentration yielded 21g/l cell mass with and 9.77x10(3)U carbonyl reductase activity/g glucose. Adaptation of different strategies for glucose feeding in the fermenter broth was helpful in increasing the process yield. Feeding of glucose at a continuous rate after 3h of cultivation yielded 0.97g cell mass/g glucose corresponding to 29.1g/l cell mass. Volumetric oxygen transfer coefficient (K(L)a) increased with the increasing of agitation rate.     

A novel centrifugal impeller bioreactor. II. Oxygen transfer and power consumption

The effects of the impeller configuration, aeration rate, and agitation speed on oxygen transfer coefficient K(L)a were studied in a newly designed centrifugal impeller bioreactor (5-L). The oxygen transfer rates in the novel bioreactor were also compared with those in a cell-lift bioreactor with comparable dimensions. The cell-lift impeller produced much higher surface oxygen transfer rates than the centrifugal one at an agitation speed over 200 rpm. This result was in good agreement with our observation that the cell-lift impeller produced much higher unfavorable turbulence. In addition, the experiments using granulated agar particles as pseudo plant cells indicated that the K(L)a value decreased steadily with an increase in agar particle concentration, and the centrifugal impeller still demonstrated a larger K(L)a than the cell lift up to a high pseudo cell concentration of 19.5 g dry weight (DW)/L (under 150 rpm and 0.20 vvm) or 22.3 g DW/L (under 200 rpm and 0.20 vvm). Furthermore, the correlation between power number and impeller Reynolds number for both the centrifugal and the cell-lift impellers was successfully obtained, which could be used for predicting the power input required by each impeller. From the results obtained, the centrifugal impeller bioreactor is expected to have great potential in its application to shear-sensitive biological systems.     

Effect of agitation and aeration on the citric acid production by Yarrowia lipolytica grown on glycerol

The effects of agitation rates from 400 to 900 rpm and aeration rates ranging from 0.18 to 0.6 vvm on biomass and citric acid production on glycerol media by acetate-negative mutants of Yarrowia lipolytica, Wratislavia 1.31 and Wratislavia AWG7, in batch culture were studied. The agitation rates of 800 and 900 rpm (at a constant aeration rate of 0.36 vvm) and aeration rates within the range of 0.24-0.48 vvm (at a constant agitation rate of 800 rpm), which generated dissolved oxygen concentration (DO) higher than 40%, were found the best for citric acid biosynthesis from glycerol. An increase in agitation rate (higher than 800 rpm) and aeration rate (higher than 0.36 vvm) had no impact on DO and citric acid production. The highest citric acid concentration (92.8 g/L) and yield (0.63 g/g) were obtained with Wratislavia 1.31 strain at 0.24 vvm. The highest volumetric citric acid production rate (1.15 g/Lh) and specific citric acid production rate (0.071 g/gh) were reached at 0.48 vvm.     

Modeling and optimization of poly(3hydroxybutyrate-co-3hydroxyvalerate) production from cane molasses by Azohydromonas lata MTCC 2311 in a stirred-tank reactor: effect of agitation and aeration regimes

The effects of agitation and aeration rates on copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] production by Azohydromonas lata MTCC 2311 using cane molasses supplemented with propionic acid in a bioreactor were investigated. The experiments were conducted in a three-level factorial design by varying the impeller (150-500 rev min(-1)) and aeration (0.5-1.5 vvm) rates. Further, the data were fitted to mathematical models [quadratic polynomial equation and artificial neural network (ANN)] and process variables were optimized by genetic algorithm-coupled models. ANN and hybrid ANN-GA were found superior for modeling and optimization of process variables, respectively. The maximum copolymer concentration of 7.45 g l(-1) with 21.50 mol% of 3HV was predicted at process variables: agitation speed, 287 rev min(-1); and aeration rate, 0.85 vvm, which upon validation gave 7.20 g l(-1) of P(3HB-co-3HV) with 21 mol% of 3HV with the prediction error (%) of 3.38 and 2.32, respectively. Agitation speed established a relative high importance of 72.19% than of aeration rate (27.80%) for copolymer accumulation. The volumetric gas-liquid mass transfer coefficient (k (L) a) was strongly affected by agitation and aeration rates. The highest P(3HB-co-3HV) productivity of 0.163 g l(-1) h(-1) was achieved at 0.17 s(-1) of k (L) a value. During the early phase of copolymer production process, 3HB monomers were accumulated, which were shifted to 3HV units (9-21%) during the cultivation period of 24-42 h. The enhancement of 7.5 and 34% were reported for P(3HB-co-3HV) production and 3HV content, respectively, by hybrid ANN-GA paradigm, which revealed the significant utilization of cane molasses for improved copolymer production.     

The role of aeration and agitation in the production of glucose oxidase in submerged culture. II

The main purpose of the work reported here was to establish the effectiveness of aeration and agitation, and to determine the best conditions of aeration for the growth and production of glucose oxidase of Aspergillus niger, on a semi-industrial scale. Concentration of dissolved O₂, O₂ consumption and CO₂ production were measured. It was found that the rate of growth and the activity of glucose oxidase per gram mycelium increased with the increase of speed of agitation. The concentration of dissolved oxygen of the fermentation broth, as well as the rate of respiration (O₂ consumption and CO₂ production) increased in direct proportion to the increase of speed of agitation, while assimilation of sugars was accelerated. The values of the respiratory ratio showed a fluctuation according to the presence or absence of sugar in the medium.     

Effect of aeration and agitation on the production of mycelial biomass and exopolysaccharides in an enthomopathogenic fungus Paecilomyces sinclairii

AIMS: The objective of the present study was to investigate the influence of aeration rate and agitation intensity on the production of mycelial biomass and exopolysaccharide (EPS) in Paecilomyces sinclairii. METHODS AND RESULTS: The P. sinclairii was cultivated under various aeration and agitation conditions in a 5 l stirred-tank bioreactor. The highest mycelial biomass (30.5 g l-1) and EPS production (11.5 g l-1) were obtained at a high aeration rate (3.5 v.v.m.) and at a high agitation speed (250 rev min-1). The apparent viscosities (6000-8000 cP) of fermentation broth increased rapidly towards the end of fermentations at high aeration and agitation conditions. CONCLUSIONS: The high level of dissolved oxygen achieved at a high aeration rate (3.5 v.v.m.) associated with higher hyphal density eventually resulted in enhanced EPS production. Agitation intensity was also proved to be a critical factor influencing on both the mycelial biomass and EPS production: high agitation speeds up to 250 rev min-1 were preferred to the yields of biomass and EPS production. SIGNIFICANCE AND IMPACT OF THE STUDY: The critical effects of aeration and agitation in the culture process of P. sinclairii were found, which is widely applicable to other kinds of basidiomycetes or ascomycetes in their submerged culture processes.     

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.     

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.     

Improved poly-ε-lysine biosynthesis using Streptomyces noursei NRRL 5126 by controlling dissolved oxygen during fermentation

The growth kinetics of Streptomyces noursei NRRL 5126 was investigated under different aeration and agitation combinations in a 5.0 l stirred tank fermenter. Poly-epsilon-lysine biosynthesis, cell mass formation, and glycerol utilization rates were affected markedly by both aeration and agitation. An agitation speed of 300 rpm and aeration rate at 2.0 vvm supported better yields of 1,622.81 mg/l with highest specific productivity of 15 mg/l.h. Fermentation kinetics performed under different aeration and agitation conditions showed poly- epsilon-lysine fermentation to be a growth-associated production. A constant DO at 40% in the growth phase and 20% in the production phase increased the poly-epsilon-lysine yield as well as cell mass to their maximum values of 1,992.35 mg/l and 20.73 g/l, respectively. The oxygen transfer rate (OTR), oxygen utilization rate (OUR), and specific oxygen uptake rates (qO2) in the fermentation broth increased in the growth phase and remained unchanged in the stationary phase.     

Influence of the intensity of oxygen mass transfer on the biosynthesis of amphotericin B

Influence of oxygen mass transfer intensity characterized by the rate of oxygen dissolution (S) and the agitation rate (n), as well as influence of dissolved oxygen concentration on the process of amphotericin B biosynthesis was studied. It was shown that S = 40 and 110 mg/l. min and n = 450 and 800 min-1 were respectively the lower and the upper levels of the optimal conditions by oxygen mass transfer during amphotericin B biosynthesis. When biosynthesis of amphotericin B was conducted under conditions of the optimal oxygen mass transfer, the dissolved oxygen concentration of about 12 to 15 per cent of the saturation level was critical for the culture respiration. Inhibition of the culture respiration and antibiotic synthesis was induced under conditions of increased oxygen mass transfer intensity (S greater than 110 mg/l. min and n greater than 800 min-1) by high intensity mechanical agitation of the fermentation broth. Under conditions of decreased oxygen mass transfer (S less than 40 mg/l. min and n = less than 450 min-1) it was induced by insufficient supply of oxygen to the culture. On the basis of the results it was shown possible to control the aeration and agitation conditions by the rate of oxygen uptake and dissolved oxygen concentration. The data should be considered in optimization of aeration and agitation conditions in biosynthesis of amphotericin B in large fermenters.     

High density cultivation of Anchusa officinalis in a stirred-tank bioreactor with in situ filtration

Previously, Su et al. [Biotechnol Bioeng 42: 884–890 (1993)] reported improved production of rosmarinic acid by Anchusa officinalis in shake-flask cultures using a cultivation strategy that involved intermittent medium exchange. Implementation of this cultivation strategy in 2.5-1 stirred-tank bioreactor cultures is investigated in the present study. Intermittent cell/medium separation in the bioreactor was accomplished by means of automated in situ culture filtration. In the bioreactor culture, rosmarinic acid production was found very sensitive to agitation and aeration conditions as well as dissolved oxygen concentration. A maximum cell density of 35 g dry weight/l and a rosmarinic acid concentration of 3.7 g/l were obtained by maintaining the dissolved oxygen concentration above 30% air saturation, gradually raising the impeller tip speed from 34 cm/s to 72 cm/s, and keeping the aeration rate at 0.44 vvm while increasing the O₂: air ratio in the gas feed stream to 4:1. This result is comparable with the data obtained from shake-flask cultures using the same culture strategy.     

Oxygen transfer kinetics of riboflavin fermentation by Ashbya gossypii in agitated fermentors

Effects of agitation and aeration rates on volumetric oxygen transfer coefficient and oxygen uptake rate of a riboflavin broth containing Ashbya gossypii were investigated in three batch, sparged, and agitated fermentors having the working volumes of 0.42, 0.85, and 2.5 l. The change of oxygen uptake rate with time at 250 rev min⁻¹ stirring and vvm aeration rates was shown. The volumetric oxygen transfer coefficients and maximum oxygen uptake rates obtained have been correlated to mechanical power inputs per unit volume of the fermentation broth and the superficial air velocities.     

Optimization of the aeration and agitation speed of Aeribacillus palidus 418 exopolysaccharide production and the emulsifying properties of the product

The specific properties of exopolysaccharides (EPS) from thermophilic microorganisms have attracted interest in their optimized production. In this study, the ability of Aeribacillus pallidus 418 to grow and produce polysaccharide in a 5-l stirred tank bioreactor was investigated. Agitation rates of 100, 200, 600, 900, and 1100 revolutions per minute (rpm), at an air flow rate of 0.5 gas volumes per unit medium volume per minute (vvm), and aeration rates of 0.25, 0.5, 1.0, and 1.5 vvm, at an agitation rate of 900 rpm, were examined. A maximum EPS yield of 170 μg/ml has been registered in a single impeller bioreactor equipped with an original Narcissus impeller at agitation speed of 900 rpm, with an aeration rate of 0.5 vvm. The bioprocess oxygen uptake rate (OUR) and oxygen mass transfer coefficient (K_La) were evaluated. The emulsifying properties of the specific EPS produced by A. pallidus 418 were determined. Stable oil-in-water emulsions, a low level of separated water phase and high dispersion stability were found, which together demonstrate the prospects for the industrial exploration of EPS production. Enhanced synergism between the A. pallidus 418 synthesized EPS and various commercially used hydrocolloids was observed; superior synergy was achieved in combination with xanthan gum.     

Enhancement of production of cloned glucoamylase under conditions of low aeration from recombinant yeast using a SUC2 promoter

The effect of aeration rate on the production of cloned glucoamylase in a recombinant yeast was investigated. This system consisted of Saccharomyces cerevisiae transformed with the 2 μ-based plasmid YEpSUCSTA which contains the SUC2 promoter, the STA signal sequence, and the STA structural gene. In contrast to typical yeast expression reports, high production of cloned glucoamylase was achieved at low aeration level (0·3 vvm). The recombinant yeast grown at 0·3 vvm aeration produced more glucoamylase (0·94 units/ml) than when grown at 0·0 vvm, 0·6 vvm, or 0·9 vvm (9·4, 1·4, and 3·1 times more, respectively). A high dissolved oxygen level early in the cultivation was important for cell growth and a low dissolved oxygen level during the production stage was important for glucoamylase production. In large scale processes for the production of recombinant proteins, the maintenance of aeration and dissolved oxygen at high levels is difficult and expensive. In this work, we have evaluated the coordination of oxygen level with growth and protein production and developed optimal conditions. Since a low aeration rate was optimal, our results demonstrate that the method described at the laboratory scale should be successfully applied at an industrial scale.     

The role of aeration and agitation in the production of glucose oxidase in submerged culture

The influence of agitation and aeration on growth and on production of glucose oxidase of Asp. niger has been studied. It was found that both rate of growth and glucose oxidase production was higher at an agitation speed of 700 rpm than at 460 rpm. Further increase in speed of agitation resulted in neither a higher rate of growth nor a higher glucose oxidase activity. Total glucose oxidase activity was highest in a medium containing 5% sugar (at an agitation speed of 700 rpm) and did not get higher when the sugar concentration of the medium was increased to 7%. When pure oxygen was bubbled through the culture the rate of growth of the culture (in the linear phase) was 95 mg. mycelial dry wt./100 ml./hr., and only 61 mg. when air was applied. The glucose oxidase activity of oxygenated culture was double the activity of aerated culture. Viscosity of the homogenized culture became higher with higher concentration of mycelia. The viscosity of oxygenated culture was found to be lower than that of aerated culture.     

Influence of Dissolved Oxygen Levels on Production of l-Asparaginase and Prodigiosin by Serratia marcescens

The effect of dissolved oxygen concentrations on the behavior of Serratia marcescens and on yields of asparaginase and prodigiosin produced in shaken cultures and in a 55-liter stainless-steel fermentor was studied. A range of oxygen transfer rates was obtained in 500-ml Erlenmeyer flasks by using internal, stainless-steel baffles and by varying the volume of medium per flask, and in the fermentor by high speed agitation (375 rev/min) or low rates of aeration (1.5 volumes of air per volume of broth per min), or both. Dissolved oxygen levels in the fermentation medium were measured with a membrane-type electrode. Peak yields of asparaginase were obtained in unbaffled flasks (3.0 to 3.8 IU/ml) and in the fermentor (2.7 IU/ml) when the level of dissolved oxygen in the culture medium reached zero. A low rate of oxygen transfer was accomplished by limited aeration. Production of prodigiosin required a supply of dissolved oxygen that was obtainable in baffled flasks with a high rate of oxygen transfer and in the fermentor with a combination of high-speed agitation and low-rate aeration. The fermentation proceeded at a more rapid rate and changes in pH and cell populations were accelerated by maintaining high levels of dissolved oxygen in the growth medium.