Effect of ammonium sulphate concentration and agitation speed on the kinetics of alginate production by Azotobacter vinelandii DSM576 in batch fermentation

Growth and alginate production by Azotobacter vinelandii DSM576 as a function of initial ammonium sulphate concentration (0.45–1.05 g l⁻¹) and agitation speed (300–700 rpm) were studied in batch fermentations at controlled pH. The time course of growth, alginate production and substrate consumption and the effect of nitrogen concentration and agitation speed on kinetic parameters and on maximum alginate molecular weight (MW) was modelled using empirical equations. The kinetics of growth, alginate production and polymerization were deeply affected by agitation speed and, to a lesser extent, by inorganic nitrogen concentration. Average and maximum specific growth rate and maximum alginate MW all increased with agitation speed, and were higher at intermediate ammonium sulphate concentration. Maximum alginate MW (>250,000) was obtained at high agitation speed (600–700 rpm) and alginate depolymerization was limited or did not occur at all when the agitation speed was higher than 500 rpm, while at 400 rpm depolymerization significantly reduced the alginate. However, alginate yield was negatively affected by increasing agitation speed. A good compromise between alginate yield (>2 g l⁻¹) and quality (MW>250,000) was obtained with agitation speed of 500–600 rpm and 0.75–0.90 g l⁻¹ of ammonium sulphate. Journal of Industrial Microbiology & Biotechnology (2000) 25, 242–248. Received 23 February 2000/ Accepted in revised form 04 August 2000     

发酵法生产L-异亮氨酸的溶氧控制策略

研究了溶氧对Brewibacterium lactofermentation分批发酵生产L-异亮氨酸（Ile）的影响,提出了前10h恒700d/min以维持溶氧在35％以上,10h后调至600r／min以维持溶氧在15％～20％的两阶段供氧控制模式。与对照相比,获得了较高的产率（0．094g／g）和糖耗速度（4．76/L·h）,在较短时间内（52h）获得较高的Ile产量（23．3g／L）,比结果最好的单一搅拌转速（600r/min）提高11．6％。生产强度（0．448d/L·h）比恒定搅拌转速（500、600、700、800r/min）控制下的过程分别提高了83．6％、28．7％、44．9％、35．7％。最后采用代谢通量分析对该结果产生的原因进行了定量解释。     

Biohydrogen production in a continuous stirred tank bioreactor from synthesis gas by anaerobic photosynthetic bacterium: Rhodopirillum rubrum

Hydrogen may be considered a potential fuel for the future since it is carbon-free and oxidized to water as a combustion product. Bioconversion of synthesis gas (syngas) to hydrogen was demonstrated in continuous stirred tank bioreactor (CSTBR) utilizing acetate as a carbon source. An anaerobic photosynthetic bacterium, Rhodospirillum rubrum catalyzed water-gas shift reaction which was applied for the bioconversion of syngas to hydrogen. The continuous fermentation of syngas in the bioreactor was continuously operated at various gas flow rates and agitation speeds, for the period of two months. The gas flow rates were varied from 5 to 14 ml/min. The agitation speeds were increasingly altered in the range of 150-500 rpm. The pH and temperature of the bioreactor was set at 6.5 and 30 degrees C. The liquid flow rate was kept constant at 0.65 ml/min for the duration of 60 days. The inlet acetate concentration was fed at 4 g/l into the bioreactor. The hydrogen production rate and yield were 16+/-1.1 mmol g(-1)cell h(-1) and 87+/-2.4% at fixed agitation speed of 500 rpm and syngas flow rate of 14 ml/min, respectively. The mass transfer coefficient (KLa) at this condition was approximately 72.8h(-1). This new approach, using a biocatalyst was considered as an alternative method of conventional Fischer-Tropsch synthetic reactions, which were able to convert syngas into hydrogen.     

Enhanced welan gum production using a two-stage agitation speed control strategy in Alcaligenes sp. CGMCC2428

Batch fermentative production of welan gum by Alcaligenes sp. CGMCC2428 was investigated under various oxygen supply conditions using regulating agitation speed. Based on a three kinetic parameters analysis that includes specific cell growth rate (μ), specific glucose consumption rate (q _s), and specific welan formation rate (q _p), a two-stage agitation speed control strategy was proposed to achieve high concentration, high yield, and high viscosity of welan. During the first 22 h, the agitation speed in 7.5 L fermenter was controlled at 800 rpm to maintain high μ for cell growth. The agitation was then reduced step-wise to 600 rpm to maintain a changing profile with stable dissolved oxygen levels and obtain high qp for high welan accumulation. Finally, the maximum concentration of welan was reached at 26.3 ± 0.89 g L⁻¹ with a yield of 0.53 ± 0.003 g g⁻¹ and the welan gum viscosity of 3.05 ± 0.10 Pa s, which increased by an average of 15.4, 15.2, and 20.1% over the best results controlled by constant agitation speeds.     

Production of exopolysaccharides by submerged culture of an enthomopathogenic fungus, Paecilomyces tenuipes C240 in stirred-tank and airlift reactors

The objective of this study was to investigate the effect of shearing effect on the production of exopolysaccharides (EPS) from an enthomopathogenic fungus, Paecilomyces tenuipes C240 in a stirred-tank reactor (STR) and in an airlift reactor (AR). The optimal agitation rate for the production of EPS in the STR was 150 rpm with the mycelial morphology of hairy pellets, where the final concentration and the specific production rate of EPS were 2.33 g l(-1) and 0.312 gg(-1) h(-1), respectively. However, the maximum concentration of biomass (21.06 g l(-1)) in the STR was obtained at a high agitation speed of 300 rpm. The specific production rate of EPS (0.456 gg(-1) h(-1)) in the AR was significantly higher than that achieved in the STR, in which the typical morphological form of mycelium was a loose clump. The three EPS groups in the STR (designated as STR-I, -II, and -III) and two groups of EPS in the AR (designated as AR-I and -II) were obtained from the culture filtrates by a gel filtration chromatography on Sepharose CL-6B. The molecular weights of STR-I, STR-II, STR-III, AR-I, and AR-II were determined to be 1,820, 25, 1.8, 1,160, and 6.7 kDa, respectively. An agitation rate of 150 rpm in the STR was selected as the optimal culture condition for maximum EPS production (2.33 g l(-1)), which was similar to the level achieved in the AR (2.30 g l(-1)). The carbohydrate composition in each EPS was quite different from each other: the major component was glucose (in STR-I, -III, and AR-I), mannose (in STR-II), and arabinose (in AR-II). In contrast, no significant difference in amino acid composition was observed.     

Beta-glucan production by Botryosphaeria rhodina in different bench-top bioreactors

AIMS: Evaluation of the technical feasibility of transferring beta-glucan production by Botryosphaeria rhodina DABAC-P82 from shaken flasks to bench-top bioreactors. METHODS AND RESULTS: Three different bioreactors were used: 3 l stirred tank reactor (STR-1) equipped with two different six-blade turbines; STR as above but equipped with a three-blade marine propeller plus draft-tube (STR-2); 2 l air-lift column reactor (ALR) equipped with an external loop. STR-1, tested at three different stirrer speeds (300, 500 and 700 rev min(-1)) appeared to be less suitable for beta-glucan production by the fungus, being maximum production (19.4 g l(-1)), productivity (0.42 g l(-1) h(-1)) and yield (0.48 g g(-1) of glucose consumed) markedly lower than those obtained in shaken culture (29.7 g l(-1), 1.23 g l(-1) h(-1) and 0.61 g g(-1), respectively). Better performances were obtained with both STR-2 and ALR. With the latter, in particular, the increase of production was accompanied by reduced fermentation time (25.7 g l(-1) after only 22 h); productivity and yield were highest (1.17 g l(-1) h(-1) and 0.62 g g(-1) of glucose consumed, respectively). CONCLUSION: Using an air-lift reactor with external loop, the scaling up from shaken flasks to bench-top bioreactor of the beta-glucan production by B. rhodina DABAC-P82 is technically feasible. SIGNIFICANCE AND IMPACT OF THE STUDY: Although culture conditions are still to be optimized, the results obtained using the ARL are highly promising.     

Enhanced 2,3-butanediol production by Klebsiella oxytoca using a two-stage agitation speed control strategy

Batch fermentative production of 2,3-butanediol by Klebsiella oxytoca was investigated using various oxygen supply methods though varying agitation speed. Based on the analysis of three kinetic parameters including specific cell growth rate (μ), specific glucose consumption rate (q_s) and specific 2,3-butanediol formation rate (q_p), a two-stage agitation speed control strategy, aimed at achieving high concentration, high yield and high productivity of 2,3-butanediol, was proposed. At the first 15 h, agitation speed was controlled at 300 rpm to obtain high μ for cell growth, subsequently agitation speed was controlled at 200 rpm to maintain high q_p for high 2,3-butanediol accumulation. Finally, the maximum concentration of 2,3-butanediol reached 95.5 g l⁻¹ with the yield of 0.478 g g⁻¹ and the productivity of 1.71 g l⁻¹ h⁻¹, which were 6.23%, 6.22% and 22.14% over the best results controlled by constant agitation speeds.     

Enhancement of ribitol production during fermentation of <Emphasis Type="Italic">Trichosporonoides oedocephalis</Emphasis> ATCC 16958 by optimizing the medium and altering agitation strategies

To maximize the productivity of ribitol, which is an important starting material for the production of one expensive rare sugar, L-ribose, the effects of culture medium and agitation speed on cell growth as well as on the productivity of ribitol were thoroughly investigated in a 7 L fermentor. The maximum volumetric productivity, 0.322 g/L/h of ribitol, were obtained at an initial glucose concentration of 200 g/L in a batch culture. Based on the optimum glucose concentration, the ribitol yield conversed from glucose was up to 0.193 g/g when 1% yeast extract was used as a nitrogen source. When the agitation speed was maintained at 200 rpm, the ribitol concentration of 38.60 g/L was collected after 120 h of cultivation time. Additionally, the scheme of two-phase agitation and glucose infusion was employed. To begin, in the first 24 h of fermentation, a high agitation rate at 350 rpm and the initial glucose concentration of 50 g/L were applied, and the biomass concentration of 25.50 g/L was achieved at 36 h of incubation; whereas this value was observed until 60 h in the former batch fermentation methods. Then, in the second phase, with the agitation speed reduced to 150 rpm and the infusion amount of glucose controlled at 150 g/L, the yield of ribitol reached to 65.00 g/L in two-phase agitation fermentation and was 1.68 fold of that obtained in one-stage batch fermentation. To our knowledge, this study first demonstrates its significant effectiveness in improving ribitol production with the application of Trichosporonoides oedocephalis ATCC 16958.     

溶氧对Blakeslea trispora分批发酵生产β-胡萝卜素的影响

在摇瓶和5 L发酵罐中研究了溶氧 (DO) 对Blakeslea trispora分批发酵生产β-胡萝卜素的影响,总结了5 L发酵罐中β-胡萝卜素发酵过程中溶氧的变化规律.结果表明,当500 mL摇瓶装液量为50 mL,转速为240 r/min条件下发酵生产β-胡萝卜素产量最大,达到3.416 g/L; 5 L发酵罐中,在搅拌转速为1 000 r/min,通气量为1.5 vvm的条件下,β-胡萝卜素的产量可达到3.712 g/L,略高于摇瓶,这可能是由于5 L发酵罐中的气液传递和混合状况好于摇瓶,促进了产物的合成.     

Enhanced acetoin production by Serratia marcescens H32 using statistical optimization and a two-stage agitation speed control strategy

Enhanced acetoin production was carried out by Serratia marcescens H32. First, medium compositions were optimized statistically for shake flask fermentations to produce acetoin. Sucrose and corn steep liquor powder (CSLP) were identified as the most significant factors by Plackett-Burman design. The path of steepest ascent and response surface methodology were then employed to determine the optimal concentrations of the two factors. Acetoin yield was up to 41.5 g/L in flask fermentations using the optimized medium. Furthermore, the optimal medium was used to conduct fermentation experiments in a 3.7-L bioreactor. The influences of different agitation speeds on acetoin production were investigated. Based on a process analysis, a two-stage agitation speed control strategy was proposed, in which the agitation speed was controlled at 700 rpm during the first 8 h and then switched to 600 rpm. A relatively high acetoin concentration (44.9 g/L) and high acetoin productivity (1.73 g/L/h) were achieved by applying this strategy. Fed-batch fermentation based on the two-stage agitation speed control strategy was performed, and a maximum acetoin concentration of 60.5 g/L with productivity of 1.44 g/L/h was achieved.     

Influence of dissolved oxygen tension and agitation speed on alginate production and its molecular weight in cultures of Azotobacter vinelandii*

The alginate production by Azotobacter vinelandii, as well as the molecular weight of the polymer, are strongly influenced by the dissolved oxygen tension (DOT) and stirring speed of the culture. Under high DOT (5% of air saturation), the bacteria produced more alginate (4.5 g/l) than that obtained at low (0.5%) oxygen tension (1.0 g/l) in cultures conducted at 300 rpm. On the other hand, under constant DOT (3%), the higher the stirring speed (from 300 to 700 rev./min), the higher the specific growth rate and the alginate production rate. However, low agitation speed (300 rev./min) lead the culture to produce a polymer of high molecular weight (680 000 g/g mol) whereas a low molecular weight (352 000 g/g mol) alginate was isolated from cultures conducted at high (700 rev./min) stirring speed. At 700 rev./min, the MMW increased to a plateau between 1 and 3% DOT and then decreased to a minimum of 0.11 x 10(6) g/g mol at 7%. Microscopic observations revealed the presence of cell aggregates (one order of magnitude larger than individual cells) when the culture was conducted at 300 rev./min. Oxygen gradients occurring within the aggregates could be responsible of this phenomenon. At high agitation rate, the MMW of the alginate dropped towards the end of the culture in all conditions evaluated. Alginase activity was detected, which would be responsible for this phenomenon.     

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.     

Development of a helical-ribbon impeller bioreactor for high-density plant cell suspension culture

A double helical-ribbon impeller (HRI) bioreactor with a 11-L working volume was developed to grow high-density Catharanthus roseus cell suspensions. The rheological behavior of this suspension was found to be shear-thinning for concentrations higher than 12 to 15 g DW . L(-1). A granulated agar suspension of similar rheological properties was used as a model fluid for these suspensions. Mixing studies revealed that surface baffling and bottom profiling of the bioreactor and impeller speeds of 60 to 150 rpm ensured uniform mixing of suspensions. The HRI power requirement was found to increase singnificantly for agar suspensions higher than 13 g DW . L(-1), in conjunction with the effective viscosity increase. Oxygen transfer studies showed high apparent surface oxygen transfer coefficients (k(L)a approximately 4 to 45 h(-1)) from agar suspensions of 30 g DW . L(-1) to water and for mixing speeds ranging from 120 to 150 rpm. These high surface k(I)a values were ascribed to the flow pattern of this bioreactor configuration combined with surface bubble generation and entrainment in the liquid phase caused by the presence of the surface baffles. High-density C. roseus cell suspension cultures were successfully grown in this bioreactor without gas sparging. Up to 70% oxygen enrichment of the head space was required to ensure sufficient oxygen supply to the cultures so that dissolved oxygen concentration would remain above the critical level (>/=10% air saturation). The best mixing speed was 120 rpm. These cultures grew at the same rate ( approximately 0.4 d(-1)) and attained the same high biomass concentrations ( approximately 25 to 27 g DW . L(-1), 450 to 500 g filtered wet biomass . L(-1), and 92% to 100% settled wet biomass volume) as shake flask cultures. The scale-up potential of this bioreactor configuration is discussed.     

Accumulation of pyruvate by changing the redox status in Escherichia coli

Pyruvate was produced from glucose by Escherichia coli BW25113 that contained formate dehydrogenase (FDH) from Mycobacterium vaccae. In aerobic shake-flask culture (K (L) a?=?4.9?min(-1)), the recombinant strain produced 6.7?g pyruvate?l(-1) after 24?h with 4?g sodium formate?l(-1) and a yield of 0.34?g pyruvate?g?glucose(-1). These values were higher than those of the original strain (0.2?g?l(-1) pyruvate and 0.02?g pyruvate?g?glucose(-1)). Based on the reaction mechanism of FDH, the introduction of FDH into E. coli enhances the accumulation of pyruvate by the regeneration of NADH from NAD(+) since NAD(+) is a shared cosubstrate with the pyruvate dehydrogenase complex, which decarboxylates pyruvate to acetyl-CoA and CO(2). The oxygenation level was enough high to inactivate lactate dehydrogenase, which was of benefit to pyruvate accumulation without lactate as a by-product.     

Multiple substrate growth kinetics of Leptothrix discophora SP-6

The growth parameters of Leptothrix discophora SP-6 were quantified on the basis of the steady-state concentrations and utilization rates of pyruvate, dissolved oxygen, and concentration of microorganisms in a chemostat operated at 25 degrees C, pH 7.2, and an agitation rate of 350 rpm. The results showed that the microbial growth was limited by both pyruvate and dissolved oxygen. A combined growth kinetics model using Monod growth kinetics for pyruvate and Tessier growth kinetics for oxygen showed the best correlation with the experimental data when analyzed using an interactive multiple substrate model. The growth kinetics parameters and the respective confidence limits, estimated using the Monte Carlo simulation, were mu(max) = 0.576 +/- 0.021 h(-1), K(sMp) = 38.81 +/- 4.24 mg L(-1), K(sTo) = 0.39 +/- 0.04 mg L(-1), Y(X/p) = 0.150 (mg microorganism mg(-1) pyruvate), Y(X/o) = 1.24 (mg microorganism mg(-1) oxygen), the maintenance factors for pyruvate and oxygen were m(p) = 0.129 (mg pyruvate consumed mg(-1) microorganism h(-1)) and m(o) = 0.076 (mg oxygen consumed mg(-1) microorganism h(-1)), respectively.     

Enhanced riboflavin production by recombinant Bacillus subtilis RF1 through the optimization of agitation speed

Dissolved oxygen is one of the most important bioprocess parameters that could affect cell growth and product formation, and it is easy to control by changing agitation speed. In this work, the effects of agitation speed on the performance of riboflavin production by recombinant Bacillus subtilis RF1 was investigated in fed-batch fermentation. The lower agitation speed (600 rpm) was beneficial for cell growth and riboflavin biosynthesis in the initial phase of fermentation process. While, during the later phase, higher agitation speed (900 rpm) was favor for cell growth and riboflavin biosynthesis. Thus, a two-stage agitation speed control strategy was proposed based on kinetic analysis, in which the agitation speed was controlled at 600 rpm in the first 26 h and then switched to 900 rpm to maintain high μ for cell growth and high q _p for riboflavin production during the entire fermentation process. However, it was observed that a sharp increase of agitation speed resulted in an adverse effect on cell growth and riboflavin synthesis within a short time. To avoid this phenomenon, a multi-stage agitation speed control strategy was set up based on the two-stage control strategy, the maximum concentration of riboflavin reached 9.4 g l^?1 in 48 h with the yield of 0.051 g g^?1 by applying this strategy, which were 20.5 and 21.4 % over the best results controlled by constant agitation speeds.     

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.     

环境条件对丙酮酸分批发酵的影响

考察了搅拌转速、pH和温度对丙酮酸分批发酵的影响。高转速（500r／min）下,丙酮酸产率较高（71％）,但葡萄糖消耗速度较慢（1．23g／（L·h））;低转速（300r／min）下,细胞消耗葡萄糖的速度加快（1．95g/（L·h））,而丙酮酸产率（0．48％）却明显下降。将搅拌转速恒定在400r／min可在一定程度上获得较高的丙酮酸产率（0．62％）和葡萄糖消耗速度（1．66g／（L·h））。CaCO3调节pH时,较多碳流从丙酮酸节点转向α-酮戊二酸节点和细胞生长,最终丙酮酸产量比NaOH调节pH时的发酵结果低38．7％;NH3·H2O调节pH时最终细胞浓度和丙酮酸产量仅为NaOH调节时的77．8％和90．9％。pH5．5时最利于丙酮酸的合成。较高的发酵温度加速T.glabrata积累丙酮酸,但同时会导致α-酮戊二酸的提前积累;而较低的温度下甘油和α-酮戊二酸积累较少,丙酮酸发酵的最适温度为28～30℃。     

Cultivation of Methylosinus trichosporium OB3b: III. production of particulate methane monooxygenase in continuous culture

Continous culture experiments with the obligatory methanotroph, Methylosinus trichosporium OB3b, were conducted to study the whole-cell methane monooxygenase (MMO) and nitrogenase activities in a nitrate minimal salts medium under oxygen-limited conditions with methane as the carbone source. The important variables investigated were the feed medium concentrations of copper and nitrate, CO(2) addition, the agitation speed, and the dilution rate. M. trichosporium OB3b required quantitative amounts of copper (2.6 x 10(-4) g Cu/g dry cell Wt) for the exclusive production of particulate MMo during continous culture growth. When the feed medium nitrate concentration was varied in the range of 5-50 mM, the whole-cell specific pMMO activity exhibited a maximum at 40 mM. The elimination of external CO(2) gassing decreased pMMO activity by more than 30%. The steady-state cell density increased continuously over a 300-700 rpm range of agitation speed, whereas, the pMMO activity became maximal at 400 rpm. Also, the pMMO activity increased with the dilution rate up to 0.06 h(-1) and remained constant thereafter. Maximal continuous pMMO productivity was, thus, achieved in Higgin's medium containing 10 muM Cu, 80 muM Fe, and 40 mM nitrate with an agitation speed of 500 rpm and a dilution rate of 0.06 h(-1). Nitrogenase activity, on the other hand, increased over a feed medium copper concentration of 2-15 muM, falling sharply at 20 muM, and it exhibited a minimum at 20 mM when the feed medium nitrate concentration was varied. (c) 1992 John Wiley & Sons, Inc.