Hyperproduction of cordycepin by two-stage dissolved oxygen control in submerged cultivation of medicinal mushroom Cordyceps militaris in bioreactors

Effect of oxygen supply on cordycepin production was investigated in submerged cultivation of Cordyceps militaris, a famous traditional Chinese medicinal mushroom, in a 5-L turbine-agitated bioreactor (TAB). Initial volumetric oxygen transfer coefficient (kLa) within the range of 11.5-113.8 h(-1) had significant influence on cordycepin production. The highest cordycepin concentration of 167.5 mg/L was obtained at an initial kLa value of 54.5 h(-1), where a moderate dissolved oxygen (DO) pattern was observed throughout cultivation. The possible correlation between cordycepin production and DO level was explored by DO control experiments, and the results showed that DO within the range of 10-80% of air saturation greatly affected the cultivation process. To obtain a high specific cordycepin formation rate (rho) throughout cultivation, a two-stage DO control strategy was developed based on the analysis of the relationship of rho and DO. That is, DO was controlled at 60% from the beginning of cultivation and then shifted to a lower control level of 30% when rho started to decrease. As a result, a high cordycepin production of 201.1 mg/L and a high productivity of 15.5 mg/(L.d) were achieved, which was enhanced by about 15% and 30% compared to the highest titers obtained in conventional DO control experiments, respectively. The proposed DO control strategy was also applied to a recently developed 5-L centrifugal impeller bioreactor (CIB) with cordycepin production and productivity titers of 188.3 mg/L and 14.5 mg/(L.d). Furthermore, the scale-up of the two-stage DO control process from 5-L CIB to 30-L CIB was successfully demonstrated. The work is useful for the efficient large-scale production of bioactive metabolites by mushroom cultures.     

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。     

Comparative study on the influence of dissolved oxygen control approaches on the microbial hyaluronic acid production of <Emphasis Type="Italic">Streptococcus zooepidemicus</Emphasis>

Three different dissolved oxygen (DO) control approaches were proposed to improve hyaluronic acid (HA) production: a three-stage agitation speed control approach, a two-stage DO control approach, and an oxygen vector perfluorodecalin (PFC) applied approach. In the three-stage agitation speed control approach, agitation speed was 200 rpm during 0–8 h, 400 rpm during 8–12 h, and 600 rpm during 12–20 h. In the two-stage DO control strategy, DO was controlled at above 10% during 0–8 h and at 5% during 8–20 h. In the PFC applied approach, PFC (3% v/v) was added at 8 h. HA production reached 5.5 g/L in the three-stage agitation speed control culture model, and 6.3 g/L in two-stage DO control culture model, and 6.6 g/L in the PFC applied culture model. Compared with the other two DO control approaches, the PFC applied approach had a lower shear stress and thus a higher HA production was achieved.     

Combined dissolved oxygen and pH control strategy to improve the fermentative production of l-isoleucine by Brevibacterium lactofermentum

The effect of both dissolved oxygen (DO) and pH on l-isoleucine production by batch culture of Brevibacterium lactofermentum was investigated. A two-stage agitation speed control strategy was developed, and the isoleucine production reached 23.3 g L⁻¹ in a relative short time (52 h), increased by 11.6% compared to the results obtained in the single agitation speed control process. In order to make sure whether the combination of DO and pH control can boost the production by a mutual effect, different control modes were conducted, based on the data obtained from the two-stage agitation speed control strategy and the analysis of kinetics parameters at different pH values. The results showed that the mode of combining two-stage DO with two-stage pH control strategy was the optimal for isoleucine production. The isoleucine production can reach 26.6 g L⁻¹ at 56 h, increased by 14.3% comparing to that obtained by the single two-stage DO control strategy.     

Increase of xylitol production rate by controlling redox potential in Candida parapsilosis

The effect of redox potential on xylitol production by Candida parapsilosis was investigated. The redox potential was found to be useful for monitoring the dissolved oxygen (DO) level in culture media, especially when the DO level was low. An increase in the agitation speed in a 5 L fermentor resulted in an increased culture redox potential as well as enhanced cell growth. Production of xylitol was maximized at a redox potential of 100 mV. As the initial cell concentration increased from 8 g/L to 30 g/L, the volumetric productivity of xylitol increased from 1.38 g/L. h to 4.62 g/L. h. A two-stage xylitol production strategy was devised, with stage 1 involving rapid production of cells under well-aerated conditions, and stage 2 involving cultivation with reduced aeration such that the culture redox potential was 100 mV. Using this technique, a final xylitol concentration of 180 g/L was obtained from a culture medium totally containing 254.5 g/L xylose in a 3,000 L pilot scale fermentor after 77 h fermentation. The volumetric productivity of xylitol during the fermentation was 2.34 g/L. h.     

Enhancement of butanol production and reducing power using a two-stage controlled-pH strategy in batch culture of Clostridium acetobutylicum XY16

The effects of pH control on the process of acetone/butanol/ethanol (ABE) production in batch cultures of Clostridium acetobutylicum XY16 have been investigated. Based on the specific acid- and solvent-forming rates in batch fermentation at different pH values (from 4.3 to 6.0), a two-stage controlled-pH strategy was developed in which the pH was shifted from 5.5 to 4.9 after a dry cell weight of 0.5 g L(-1) was achieved. By applying this strategy, the maximum ABE concentration and productivity reached 20.3 g L(-1) and 0.63 g L(-1) h (-1), and were significantly improved by 12.2 and 40.1 %, respectively, compared with the process with no pH control. In addition, reducing power capability was significantly enhanced by this strategy. The two-stage controlled-pH strategy was a convenient and rapid method for high intensity ABE production.     

Production of somatic embryos in a helical ribbon impeller bioreactor

Embryogenic cultures of a transformed Eschscholtzia californica cell line were carried out in a 11-L helical ribbon impeller bioreactor operated under various conditions to evaluate the performance of this equipment for somatic embryo (SE) production. All bioreactor cultures produced SE suspensions with maximum concentrations at least comparable to those obtained from flask control cultures ( approximately 8-13 SE . mL(-;1)). However, an increase of the mixingspeed, from 60 to 100 rpm, and low sparging rate ( approximately 0.05 VVM, k(L) a approximately 6.1 h(-;1)) for dissolved oxygen concentration (DO) control yielded poorer quality embryogenic cultures. The negative effects on SE production were attributed mainly to the low but excessive shear experienced by the embryogenic cells and/or embryoforming aggregates. High DO ( approximately 60% of air saturation) conditions favored undifferentrated biomass production and high nutrient uptake rates at the expense of the slower SE differentiation process in both flask and bioreactor cultures. Too low DO (-5-10%) inhibited biomass and SE production. The best production of SE ( approximately 44 SE . mL(-1) or approximately 757 SE . g dw(-1) . d(-1)) was achieved by operating the bioreactor at 60 rpm while controlling DO at approximately 20%by surface oxygenation only (0.05 VVM, k(L) a approximately 1.4 h(-;1)). This production was found to be a biomass production/growth-associated process and was mainly limited by the availability of extracellular phosphate, magnesium, nitrogen salts, and carbohydrates. (c) 1994 John Wiley & Sons, Inc.     

Dissolved-oxygen-stat fed-batch fermentation of 1,3-dihydroxyacetone from glycerol by Gluconobacter oxydans ZJB09112

This study investigated the effects of DO concentration on DHA fermentation and of DO-stat fed-batch fermentation using a pH control strategy, on 1,3-dihydroxyacetone (DHA) production. The results showed that DO-stat fed-batch fermentation with pH-shift control was the optimal bioprocess for DHA production. DO-stat fed-batch fermentation was carried out at 30% air saturation, and the culture pH was automatically maintained at pH 6.0 during the first 20 h and then shifted to pH 5.0 until the end of the fermentation. An optimal DHA concentration of 175.9 ± 6.7 g/L, with a production yield to glycerol of 0.87 ± 0.04 g/g, was obtained at 72 h of DO-stat fed-batch fermentation at 30°C in a 15 L fermenter.     

Enhancement of pyruvate production by Torulopsis glabrata using a two-stage oxygen supply control strategy

The effect of agitation speeds on the performance of producing pyruvate by a multi-vitamin auxotrophic yeast, Torulopsis glabrata, was investigated in batch fermentation. High pyruvate yield on glucose (0.797 g g(-1)) was achieved under high agitation speed (700 rpm), but the glucose consumption rate was rather low (1.14 g l(-1) h(-1)). Glucose consumption was enhanced under low agitation speed (500 rpm), but the pyruvate yield on glucose decreased to 0.483 g g(-1). Glycerol production was observed under low agitation speed and decreased with increasing agitation speed. Based on process analysis and carbon flux distribution calculation, a two-stage oxygen supply control strategy was proposed, in which the agitation speed was controlled at 700 rpm in the first 16 h and then switched to 500 rpm. This was experimentally proven to be successful. Relatively high concentration of pyruvate (69.4 g l(-1)), high pyruvate yield on glucose (0.636 g g(-1)), and high glucose consumption rate (1.95 g l(-1)h(-1)) were achieved by applying this strategy. The productivity (1.24 g l(-1) h(-1)) was improved by 36%, 23% and 31%, respectively, compared with fermentations in which agitation speeds were kept constant at 700 rpm, 600 rpm, and 500 rpm. Experimental results indicate that the difference between the performances for producing pyruvate under a favorable state of oxygen supply (dissolved oxygen concentration >50%) was caused by the different regeneration pathways of NADH generated from glycolysis.     

Four‐stage dissolved oxygen strategy based on multi‐scale analysis for improving spinosad yield by Saccharopolyspora spinosa ATCC49460

Dissolved oxygen (DO) is an important influencing factor in the process of aerobic microbial fermentation. Spinosad is an aerobic microbial‐derived secondary metabolite. In our study, spinosad was used as an example to establish a DO strategy by multi‐scale analysis, which included a reactor, cell and gene scales. We changed DO conditions that are related to the characteristics of cell metabolism (glucose consumption rate, biomass accumulation and spinosad production). Consequently, cell growth was promoted by maintaining DO at 40% in the first 24 h and subsequently increasing DO to 50% in 24 h to 96 h. In an in‐depth analysis of the key enzyme genes (gtt, spn A, spn K and spn O), expression of spinosad and specific Adenosine Triphosphate (ATP), the spinosad yield was increased by regulating DO to 30% within 96 h to 192 h and then changing it to 25% in 192 h to 240 h. Under the four‐phase DO strategy, spinosad yield increased by 652.1%, 326.1%, 546.8%, and 781.4% compared with the yield obtained under constant DO control at 50%, 40%, 30%, and 20% respectively. The proposed method provides a novel way to develop a precise DO strategy for fermentation.     

Polygalacturonase enzyme production from bacterial isolated from raw milk and green and black olives

Forty microbial strains isolated from raw milk samples and black and green olives were grown in MP5 (mineral pectin 5) medium containing 0.5% lemon pectin. All strains synthesized an extracellular polygalacturonase. Rhodotorula sp. ONRh9 (0.44 U x mL(-1)) and Leuconostoc sp. LLn1 (0.16 U x mL(-1)), which had a more active polygalacturonase in MP5 medium, were studied in MAPG5 medium containing polygalacturonic acid. Highest biomass and polygalacturonase production by these two strains were observed for polygalacturonic acid concentrations of 10 g x L(-1) (Rhodotorula sp. ONRh9) and 5 g x L(-1) (Leuconostoc sp. LLn1) and for initial pH values of 6 (Rhodotorula sp. ONRh9) and 5.5 (Leuconostoc sp. LLn1). The two strains grown in fermenters in MAPG5 medium generated the following results: with controlled initial pH, Rhodotorula sp. produced maximum biomass (DO) and polygalacturonase (PG) after 20 h (DO, 3.86; PG, 0.24 U x mL(-1)) of growth, and this level was sustained until the end of the culture; Leuconostoc sp. LLn1 synthesized more cells and polygalacturonase between 4 h (DO, 1.80; PG, 0.17 U x mL(-1)) and 24 h (DO, 3.90; PG, 0.27 U x mL(-1)) of culture. With uncontrolled initial pH, the cultures produced maximum biomass and polygalacturonase after 20 h (DO, 3.30; PG, 0.26 U x mL(-1)) for Rhodotorula sp. ONRh9 and 10 h (DO, 2.84; PG, 0.17 U x mL(-1)) for Leuconostoc sp. LLn1.     

Effective production of biologically active water-soluble β-1,3-glucan by a coupled system of Agrobacterium sp. and Trichoderma harzianum

Water-soluble β-1,3-glucan (w-glucan) prepared from curdlan is reported to possess various bioactive and medicinal properties. To develop an efficient and cost-effective microbial fermentation method for the direct production of w-glucan, a coupled fermentation system of Agrobacterium sp. and Trichoderma harzianum (CFS-AT) was established. The effects of Tween-80, glucose flow rate, and the use of a dissolved oxygen (DO) control strategy on w-glucan production were assessed. The addition of 10?g?L^?1 Tween-80 to the CFS-AT enhanced w-glucan production, presumably by loosening the curdlan ultrastructure and increasing the efficiency of curdlan hydrolysis. A two-stage glucose and DO control strategy was optimal for w-glucan production. At the T. harzianum cell growth stage, the optimal glucose flow rate and agitation speed were 2.0?g?L^?1 hr^?1 and 600?rpm, respectively, and at the w-glucan production stage, they were 0.5?g?L^?1 hr^?1 and 400?rpm, respectively. W-glucan production reached 17.31?g?L^?1, with a degree of polymerization of 19–25. Furthermore, w-glucan at high concentrations exhibited anti-tumor activity against MCF-7, HepG2, and Hela cancer cells in vitro. This study provides a novel, cost-effective, eco-friendly, and efficient microbial fermentation method for the direct production of biologically active w-glucan.     

枯草芽孢杆菌(Bacillus subtilis)发酵生产乙偶姻的pH调控策略

【目的】为了提高Bacillus subtilis CCTCC M 208157发酵生产乙偶姻的效率。【方法】在7 L发酵罐水平上考察不同pH条件对菌株生长及乙偶姻合成的影响。【结果】pH对菌株合成乙偶姻有显著影响,pH 4.5有利于细胞合成乙偶姻,但是延迟期较长;pH 5.5时菌株生长较快,但乙偶姻的产量偏低。因此提出了两阶段pH控制策略:发酵前期(0 16 h),控制pH 5.5;发酵中后期(16 72 h),控制pH 4.5。【结论】通过此策略,菌株合成乙偶姻的能力得到进一步提高,乙偶姻的产量、产率和生产强度分别为32.7 g/L、0.41 g/g和0.91 g/(L.h),分别比初始发酵条件下提高了41%、42%和69%。     

丙酮酸发酵过程中光滑球拟酵母过程功能的强化

对不同葡萄糖浓度下光滑球拟酵母分批发酵生产丙酮酸的动力学模型分析发现, 葡萄糖浓度是影响光滑球拟酵母发酵生产丙酮酸过程功能的关键因素。在发酵初始阶段, 低浓度葡萄糖可维持较高的菌体比生长速率; 对数生长中前期, 葡萄糖快速进料使菌体浓度接近最大值, 并实现碳流从菌体生长转向丙酮酸积累; 对数生长后期葡萄糖浓度控制在33.4 g/L以维持高丙酮酸对葡萄糖产率系数 (0.71 g/g)。采用奇异控制的葡萄糖流加方式, 在7 L发酵罐上控制不同发酵阶段葡萄糖浓度处于最佳水平以强化光滑球拟酵母过程功能, 丙酮酸产量 (83.1 g/L)、产率 (0.621 g/g)、生产强度[1.00 g/(L·h)]与分批发酵对比, 分别提高了21.3%、21.6%和29.9%。     

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.     

Enhanced hyaluronic acid production by a two-stage culture strategy based on the modeling of batch and fed-batch cultivation of Streptococcus zooepidemicus

This study aimed to enhance hyaluronic acid (HA) production by a two-stage culture strategy based on the modeling of batch and fed-batch culture of Streptococcus zooepidemicus. Batch culture had higher specific HA synthesis rate while fed-batch culture had higher specific cell growth rate. The lower specific HA synthesis rate in fed-batch culture resulted from the competition of cell growth for the common precursors at a low substrate concentration. Based on the modeling of batch and fed-batch culture of S. zooepidemicus, a two-stage culture strategy was proposed to enhance HA production. S. zooepidemicus were cultured in a fed-batch mode with sucrose concentration maintained at 1.0+/-0.2g/L during 0-8h and then batch culture was performed during 8-20h with an initial sucrose concentration of 15g/L. With the proposed two-stage culture strategy, HA production was increased to 6.6g/L compared with 5.0g/L in batch culture with the same total sucrose. The enhanced HA production by the proposed two-stage culture strategy resulted from the decreased inhibition of cell growth and the increased transformation rate of sucrose to HA.     

过氧化氢酶发酵生产条件优化及在染整清洁生产中的应用研究

在以嗜热子囊菌生产过氧化氢酶的摇瓶发酵研究中,获得了适合工业化生产的碳源,即以26g/L的糊化玉米淀粉和1%(v/v)乙醇为混合碳源,过氧化氢酶的酶活达到了1996 u/mL,比优化前提高了25%。在此基础上,重点研究了发酵罐上的主要影响因素溶解氧对发酵的影响,通过分段控制搅拌转速,在52h将搅拌转速从200r/min提高到350r/min,过氧化氢酶最高酶活达到4505 u/mL,与不使用控制溶氧水平策略相比,酶活力提高了2.4倍。此外,还考察了该酶去除过氧化氢的效率,并在实际纺织生产中进行了应用实验,取得了良好的效果。     

Effects of glucose, vitamins, and DO concentrations on pyruvate fermentation using Torulopsis glabrata IFO 0005 with metabolic flux analysis

The effects of glucose, vitamins, and DO concentrations on efficient pyruvic acid fermentation were investigated using Torulopsis glabrata IFO 0005, and a novel biphasic culture method was developed on the basis of the metabolic flux analysis. T. glabrata requires the four vitamins nicotinic acid (NA), thiamine hydrochloride (B(1)), pyridoxine hydrochloride, and biotin for cell growth. The deficiency of these vitamins plays an essential role in pyruvate fermentation. In the present study, we considered the effects of the first two vitamins on the pyruvate fermentation. On the basis of several batch and fed-batch experiments, it was found that, as a result of glucose inhibition of cell growth, the initial glucose concentration should be around 30-40 g/L, and the glucose concentration during fermentation should be controlled at high level around 30 g/L. On the basis of an analysis of carbon flux distribution, a biphasic fermentation method was developed where the cultivation started with a high DO (at 40-50% of air saturation) for efficient cell growth and then was reduced to 5-10% for efficient pyruvate production. Since a fair amount of ethanol was formed when the DO concentration was decreased, the addition of NA turned out to be effective in reducing the ethanol formation. This may be due to a relaxing of the requirement for NADH oxidation by the alcohol dehydrogenase pathway. Since B(1) affects both the pyruvate dehydrogenase complex and pyruvate decarboxylase, its initial concentration must be carefully determined by considering both the cell growth and pyruvate production phases.     

Improved curdlan fermentation process based on optimization of dissolved oxygen combined with pH control and metabolic characterization of <Emphasis Type="Italic">Agrobacterium</Emphasis> sp. ATCC 31749

A significant problem in scale-down cultures, rarely studied for metabolic characterization and curdlan-producing Agrobacterium sp. ATCC 31749, is the presence of dissolved oxygen (DO) gradients combined with pH control. Constant DO, between 5% and 75%, was maintained during batch fermentations by manipulating the agitation with PID system. Fermentation, metabolic and kinetic characterization studies were conducted in a scale-down system. The curdlan yield, intracellular nucleotide levels and glucose conversion efficiency into curdlan were significantly affected by DO concentrations. The optimum DO concentrations for curdlan production were 45–60%. The average curdlan yield, curdlan productivity and glucose conversion efficiency into curdlan were enhanced by 80%, 66% and 32%, respectively, compared to that at 15% DO. No apparent difference in the gel strength of the resulting curdlan was detected. The comparison of curdlan biosynthesis and cellular nucleotide levels showed that curdlan production had positive relationship with intracellular levels of UTP, ADP, AMP, NAD⁺, NADH and UDP-glucose. The curdlan productivity under 45% DO and 60% DO was different during 20–50 h. However, after 60 h curdlan productivity of both conditions was similar. On that basis, a simple and reproducible two-stage DO control process for curdlan production was developed. Curdlan production yield reached 42.8 g/l, an increase of 30% compared to that of the single agitation speed control process.