Influence of culture modes on the microbial production of hyaluronic acid by <Emphasis Type="Italic">Streptococcus zooepidemicus</Emphasis>

The principal objective of this study was to assess the effects of culture modes including batch culture, pulse fed-batch culture, constant feeding rate fed-batch culture, and exponential fed-batch culture on the production of hyaluronic acid (HA) by Streptococcus zooepidemicus. Batch cultures had the highest levels of HA productivity, whereas fed-batch cultures were more favorable with regard to cell growth, and exponential fed-batch cultures evidenced the highest cell concentrations. A two-step culture model was proposed to enhance HA production: an exponential fed-batch culture was conducted prior to 8 h and then sucrose supplementation was applied for 8 h to start the batch fermentation of S. zooepidemicus. HA production and productivity were increased by 36 and 37% in the proposed two-step culture process as compared with that observed in the batch culture, respectively. The proposed two-step culture model can be applied in the production of secondary metabolites, and particularly of the exopolysaccharides.     

Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 in fed-batch fermentation: Effects of sucrose concentration in a complex medium and process modeling

Production of pediocin SM-1 by Pediococcus pentosaceus Mees 1934 was investigated in semi-aerobic, pH-controlled, batch and fed-batch fermentations using a complex medium containing sucrose as the main source of carbon. The effects of sucrose concentration were studied in fed-batch fermentations in which a sucrose solution was added at stable feeding rates (5, 7, 9 and 10 g/l/h). The results showed that pediocin is produced as a product of the primary metabolism and its titer could be greatly improved by adjusting the sucrose feeding rate in fed-batch fermentation. The maximum titer of pediocin of 145 AU/ml was obtained in the fed-batch culture with 7 g/l/h feeding rate and that was 119% higher compared to the titer obtained in batch culture. Higher feeding rates (9 and 10 g/l/h) resulted in decreased pediocin yields while biomass levels appeared to be rather unaffected. The specific rate of pediocin formation was also sensitive to sucrose concentration levels. A mathematical model developed on the basis of well-known rate equations for batch and fed-batch cultures and growth associated production, described successfully cell growth, sucrose assimilation, lactate production and pediocin production in fed-batch culture.     

Production of a desulfurization biocatalyst by two-stage fermentation and its application for the treatment of model and diesel oils

For the production of oil-desulfurizing biocatalyst, a two-stage fermentation strategy was adopted, in which the cell growth stage and desulfurization activity induction stage were separated. Sucrose was found to be the optimal carbon source for the growth of Gordonia nitida CYKS1. Magnesium sulfate was selected to be the sulfur source in the cell growth stage. The optimal ranges of sucrose and magnesium sulfate were 10-50 and 1-2.5 g x L(-1), respectively. Such a broad optimal concentration of sucrose made the fed-batch culture easy, while the sucrose concentration was maintained between 10-20 g x L(-1) in the actual operation. As a result, 92.6 g x L(-1) of cell mass was acquired by 120 h of fed-batch culture. This cell mass was over three times higher than a previously reported result, though the strain used was different. The desulfurization activity of the harvested cells from the first stage culture was induced by batch cultivation with dibenzothiophene as the sole sulfur source. The optimal induction time was found to be about 4 h. The resting-cell biocatalyst made from the induced cells was applied for the deep desulfurization of a diesel oil. It was observed that the sulfur content of the diesel oil decreased from 250 mg-sulfur x L-oil(-1) to as low as 61 mg-sulfur x L-oil(-1) in 20 h. It implied that the biocatalyst developed in this study had a good potential to be applied to a deep desulfurization process to produce ultra-low-sulfur fuel oils.     

流加发酵对重组Bacillus subtilis发酵生产角质酶的影响

为实现基因工程菌Bacillus subtilis WSHB06-07生产角质酶的高产,在3L发酵罐中考察了不同初糖浓度对菌体生长和产酶的影响,并在选择38 g/L初始蔗糖浓度的基础上,进行碳源的分批流加和恒速流加,结果表明发酵16 h开始流加碳源,采用总补糖量60g/L,蔗糖平均流速为4g/(L·h)的恒速补料方式,角质酶酶活在31h可达到最大545.87U/ml,比分批发酵酶活提高67.8%,并获得较高的角质酶生产强度,满足工业化生产要求。     

Bioreactor operating strategy inThalictrum rugosum plant cell culture for the production of berberine

Optimal substrate feeding strategy in bioreactor operation was investigated to increase the production of secondary metabolite in a high density culture of plant cell. It was accomplished by the previously proposed structured kinetic model that describes the cell growth and synthesis of the secondary metabolite, berberine, in a batch suspension culture ofThalictrum rugosum. Four types of operation strategies for sugar feeding intoT. rugosum culture were proposed based on the model, which were the periodic fedbatch operations to maintain the cell activity, the cell viability, and the specific production rate, and the perfusion operation to maintain the specific production rate. From the simulation results of these strategies, it could be found that the periodic fed-batch operation and the perfusion operation could achieve the higher volumetric production of berberine (mg berberine/L) and specific production yield (mg berberine/g dry cell weight) than those of batch cultures. Although the highest productivity (mg berberine/day) of berberine could be achieved by the periodic fed-batch operation to maintain the cell activity compared with the other strategies in the periodic fed-batch operations, the specific production yield was low due to the higher maximum dry cell weight than other cases. The periodic fed-batch operation to maintain cell viability resulted in the highest volumetric production of berberine and specific production yield compared with the other strategies. In the cases of maintaining the specific production rate, the per-formance of the periodic fed-batch operation was better than that of the perfusion operation in the respect of the volumetric production and productivity of berberine. In order to increase the volumetric production of berberine and to get the highest specific production yield, the periodic fed-batch operation to maintain cell viability could be chosen as the optimal operating strategy in high density, culture ofT. rugosum plant cell.     

Production of flavonoids and polysaccharide by adding elicitor in different cellular cultivation processes of Glycyrrhiza uralensis Fisch

In this study, callus and cell suspension were induced from seedlings of licorice (G. uralensis). In addition, it was revealed that the appropriate concentration of sucrose could promote the callus growth and increase the content of polysaccharide. The methyl jasmonate (MJ) and phenylalanine (PHE) could enhance the callus growth and content of flavonoids for G. uralensis. For producing more flavonoids and polysaccharide, two-stage cultivation was performed. In the first step, 30 g L^?1 sucrose was fed into a 5-L balloon-type bubble bioreactor on 8th day of culture to enhance cell production and metabolite production. In a two-stage cultivation process, PHE (2 mM) and MJ (5 mg L^?1) were added into a 5-L balloon-type bubble bioreactor after 10 days of culture. Using a fed-batch cultivation strategy (30 g L^?1 sucrose was fed into a 5-L balloon-type bubble bioreactor on 8th day), polysaccharide production was enhanced to 1.19 g L^?1, which was 2.12-fold greater than that in batch cultivation. The flavonoids yield (55.42 mg L^?1) which was about 22 % higher than that in batch cultivation was obtained on 21st day. In a two-stage cultivation process, the polysaccharide content was increased by 1.14- and 2.12-fold compared with fed-batch cultivation and batch cultivation on 15th day. Meanwhile, total flavonoids yield (132.36 mg L^?1) on 15th day, was increased by 2.26- and 2.67-fold compared with fed-batch cultivation and batch cultivation. In conclusion, two-stage cultivation process combined with the sucrose and elicitor treatment could promote both the callus growth and the secondary metabolites accumulation.     

Development and optimization of two-stage cyclic fed-batch culture for hG-CSF production using L-arabinose promoter of Escherichia coli

The long-term process for producing human granulocyte-colony stimulating factor (hG-CSF) was developed using two-stage cyclic fed-batch culture, in which hG-CSF expressing-recombinant Escherichia coli was directed by an L-arabinose promoter system. For the optimization, the preinduction growth rate during the growth stage and the feeding strategy during the production stage were investigated. The maximum harvest volume during the production stage was predicted before long-term cyclic operation. Based on those optimized strategies, the two-stage cyclic fed-batch culture was performed for 12 cycles (86 h). The cell growths in both stages were maintained at 45-50 g/L and 71-77 g/L, respectively. hG-CSF was stably produced at a level of 8-9 g/L and the plasmid stability was maintained at more than 90%. Volumetric productivity by the two-stage cyclic fed-batch culture was 0.643 g/L/h, which was about 280% higher than that of conventional DO-stat fed-batch culture.     

Perfusion strategy for rosmarinic acid production by Anchusa officinalis

The production of an intracellular secondary metabolite rosmarinic acid (RA) by plant cell suspensions of Anchusa officinalis cultivated with intermittent medium exchange is investigated. Initially, a two-stage perfusion culture method was employed. After being cultured in the batch mode for ca. 6 days in B5 medium plus 3% sucrose, 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.1 mg/L kinetin (2,4-D B5 medium), Anchusa culture was cultivated to high cell density by perfusion during the growth stage using a hormone-free Gamborg B5 medium supplemented with 6% sucrose. This was followed by a production stage, in which a complete medium exchange into B5 medium plus 3% sucrose and 0.25 mg/L naphthleneacetic acid (NAA) was conducted. The two-stage perfusion culture had a higher maximum culture RA concentration but a lower RA content per cell than the batch stock culture maintained in the 2,4-D B5 medium. Higher culture RA concentration was due primarily to high cell density. The high packed cell volume, however, seemed to reduce the synergistic effect of NAA on RA synthesis. Subsequently, a single-stage perfusion culture method was investigated. The best result was obtained by growing the culture in the batch mode for ca. 10 days using B5 medium supplemented with 3% sucrose and 0.25 mg/L NAA, followed by perfusing the culture with B5 medium plus 6% sucrose and 0.25 mg/L NAA at a constant perfusion rate of 0.1/day. A maximum cell dry weight of 35 g/L and a RA concentration of almost 4 g/L were achieved. This is the highest RA concentration ever reported in the Anchusa culture. (c) 1993 John Wiley & Sons, Inc.     

Enhanced cutinase production of Thermobifida fusca by a two-stage batch and fed-batch cultivation strategy

In this study, cutinase production by Thermobifida fusca WSH03-11 was investigated with mixed short-chain organic acids as co-carbon sources to demonstrate the possibility of producing high value-added products from organic wastes. T. fusca WSH03-11 was cultured with different combinations of butyrate, acetate, and lactate with a purpose of increasing cutinase activity. The optimum proportion of butyrate, acetate, and lactate was 4:1:3. In batch cultivation, acetate and lactate were consumed quickly, while the consumption of butyrate was depressed in the presence of acetate with a concentration higher than 0.5 g/L. Based on these results, a two-stage batch and fed-batch cultivation strategy was proposed: a batch culture with acetate and lactate as the co-carbon sources in the first 10 h, and then a fed-batch culture with a constant butyrate feeding rate of 12 mL/h during 11∼20 h. By this two-stage cultivation strategy, cutinase activity, dry cell weight, and consumption rate of butyrate were increased by 70%, 103.4%, and 4.3-fold, respectively, compared to those of the batch cultivation. These results provided a novel and efficient way to produce high value-added products from organic wastes.     

High cell density cultivation of <Emphasis Type="Italic">Escherichia coli</Emphasis> with surface anchored transglucosidase for use as whole-cell biocatalyst for α-arbutin synthesis

A fed-batch culture strategy for the production of recombinant Escherichia coli cells anchoring surface-displayed transglucosidase for use as a whole-cell biocatalyst for α-arbutin synthesis was developed. Lactose was used as an inducer of the recombinant protein. In fed-batch cultures, dissolved oxygen was used as the feed indicator for glucose, thus accumulation of glucose and acetate that affected the cell growth and recombinant protein production was avoided. Fed-batch fermentation with lactose induction yielded a biomass of 18 g/L, and the cells possessed very high transglucosylation activity. In the synthesis of α-arbutin by hydroquinone glucosylation, the whole-cell biocatalysts showed a specific activity of 501 nkat/g cell and produced 21 g/L of arbutin, which corresponded to 76% molar conversion. A sixfold increased productivity of whole cell biocatalysts was obtained in the fed-batch culture with lactose induction, as compared to batch culture induced by IPTG.     

营养条件对兽疫链球菌发酵生产透明质酸的影响

透明质酸 (Ｈｙａｌｕｒｏｎｉｃａｃｉｄ ,简称ＨＡ)是Ｎ 乙酰氨基葡萄糖胺和葡萄糖醛酸以 β 1 3糖苷键和 β 1 4糖苷键连接而成的二糖单体重复构建而成的杂多糖 ,广泛存在于高等动物的结缔组织内。由于结构上的特点 ,ＨＡ具有很高的粘弹性和极强的保水性等特征 ,已被大量用于医学医药、化妆品工业[1,2 ] 。1937年Ｋｅｎｄａｌｌ[3 ] 等发现用溶血性链球菌 (Ｓｔｒｅｐｔｏｃｏｃｃｕｓｈａｅｍｏｌｙｔｉｃｕｓ)可以产生ＨＡ。其后 ,陆续发现许多能产生ＨＡ的微生物菌种 ,逐渐开发出一条可替代传统的动物组织提取法[4 ] 生产ＨＡ的新途径…     

A phenomenological model to represent the kinetics of growth by <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> for lysine production

Corynebacterium glutamicum is commonly used for lysine production. In the last decade, several metabolic engineering approaches have been successfully applied to C. glutamicum. However, only few studies have been focused on the kinetics of growth and lysine production. Here, we present a phenomenological model that captures the growth and lysine production during different phases of fermentation at various initial dextrose concentrations. The model invokes control coefficients to capture the dynamics of lysine and trehalose synthesis. The analysis indicated that maximum lysine productivity can be obtained using 72 g/L of initial dextrose concentration in the media, while growth was optimum at 27 g/L of dextrose concentration. The predictive capability was demonstrated through a two-stage fermentation strategy to enhance the productivity of lysine by 1.5 times of the maximum obtained in the batch fermentation. Two-stage fermentation indicated that the kinetic model could be further extended to predict the optimal feeding strategy for fed-batch fermentation.     

A two-stage fed-batch heterotrophic culture of Chlorella protothecoides that combined nitrogen depletion with hyperosmotic stress strategy enhanced lipid yield and productivity

In this study, the influences of major nutrients on cell growth and lipid production were investigated in heterotrophic culture of Chlorella protothecoides. The results demonstrated that phosphorus depletion had no effect on lipid accumulation but restricted cell growth; however, nitrogen depletion could enhance lipid accumulation thus benefiting lipid production. Furthermore, the effects of glucose inhibition were comparatively investigated with osmotic stress, showing that the effects of glucose inhibition were similar to the effect of osmotic stress at equivalent osmotic pressures only if the glucose concentration was less than 100 g/L, otherwise the effects of glucose inhibition became much stronger than osmotic stress. Interestingly, it was found that a specific hyperosmotic stress could significantly enhance lipid accumulation, thus providing a new stress strategy for efficient lipid production. Finally, a novel two-stage fed-batch culture consisting of a growth phase and a lipid accumulation phase with nitrogen depletion and hyperosmotic stress was proposed, yielding a final lipid productivity of 177.3 mg/L/h with a very high lipid yield of 207.0 mg/g glucose and lipid content of 39.2% after 180 h culture, which were 1.60, 1.79 and 1.92-fold of those obtained in one-stage fed-batch culture without stress phase, respectively.     

Enhanced beta-galactosidase production by high cell-density culture of recombinant Bacillus subtilis with glucose concentration control

Cell concentration, recombinant protein (beta-galactosidase) level, and the specific enzyme expression level were increased from 19 to 184 g/L, 18.3 to 129 U/mL, and 3.2 to 5.7 U/mg protein, respectively, in fed-batch culture of recombinant Bacillus subtilis when glucose concentration was controlled at 1 g/L as compared with those of conventional fed-batch culture. Glucose concentration of the culture broth was monitored by an automatic on-line glucose analyzer and controlled with a moving identification combined with optimal control (MICOC) strategy. When glucose concentrations were controlled at 10, 1, and 0.2 g/L, accumulated propionic acid concentrations and specific enzyme activities were 18.5, 4.4, and 0.6 g/L and 2.9, 5.7, and 7.1 U/mg protein, respectively. The addition of various concentrations of sodium propionate to the growth medium in batch cultures resulted in a drastic decrease in the growth rate with respect to propionate concentration. The propionic acid was shown to be responsible for cell growth inhibition and enzyme activity reduction in fed-batch culture. (c) 1992 John Wiley & Sons, Inc.     

在兽疫链球菌中表达vgb基因和HA合成基因提高透明质酸产量

透明质酸(HA)是一种在医药及化妆品领域具有广泛应用的天然粘多糖。兽疫链球菌(Streptococcuszooepidemicus)是工业上生产透明质酸的菌种之一。透明颤菌血红蛋白(VHb)具有增强细胞摄氧的作用。对生产透明质酸的兽疫链球菌进行了基因改造:将兽疫链球菌HA的合成基因hasABC以及合成透明颤菌血红蛋白的vgb基因(Vitreoscillahemoglobingene,vgb)分别或同时插入阳性菌表达质粒pEU308中,通过电转化导入兽疫链球菌中。通过一氧化碳(CO)差光谱检测到了VHb的表达。在摇瓶实验中,同时带有hasABC和vgb基因的重组菌比野生菌的透明质酸产量提高了30％。而在发酵罐中,带有这2个基因的重组菌的透明质酸产量达到了6．9g／L,高于重组菌5．5g／L的产量。实验结果表明,vgb基因的存在促进了细胞的生长,hasABC操纵子的过表达增强了透明质酸的合成。首次将VHb导入兽疫链球菌中,获得了表达,并证明其对菌体生长及透明质酸合成有促进作用。通过研究,VHb将可以在阳性菌中获得更广泛的应用。     

Poly(3-Hydroxybutyrate) Production with High Productivity and High Polymer Content by a Fed-Batch Culture of Alcaligenes latus under Nitrogen Limitation

Alcaligenes latus has been known to produce poly(3-hydroxybutyrate) (PHB) in a growth-associated manner even under nutrient-sufficient conditions. However, the PHB content obtained by fed-batch culture was always low, at ca. 50%, which makes the recovery process inefficient. In this study, the effect of applying nitrogen limitation on the production of PHB by A. latus was examined. In flask and batch cultures, the PHB synthesis rate could be increased considerably by applying nitrogen limitation. The PHB content could be increased to 87% by applying nitrogen limitation in batch culture, which was considerably higher than that typically obtainable (50%) under nitrogen-sufficient conditions. In fed-batch culture, cells were first cultured by the DO-stat feeding strategy without applying nitrogen limitation. Nitrogen limitation was applied at a cell concentration of 76 g (dry cell weight)/liter, and the sucrose concentration was maintained within 5 to 20 g/liter. After 8 h of nitrogen limitation, the cell concentration, PHB concentration, and PHB content reached 111.7 g (dry cell weight)/liter, 98.7 g/liter, and 88%, respectively, resulting in a productivity of 4.94 g of PHB/liter/h. The highest PHB productivity, 5.13 g/liter/h, was obtained after 16 h.     

Highly efficient strategy for enhancing taxoid production by repeated elicitation with a newly synthesized jasmonate in fed-batch cultivation of Taxus chinensis cells

A highly efficient bioprocessing strategy was developed for enhancing the production of plant secondary metabolites by repeatedly eliciting a fed-batch culture with a newly synthesized powerful jasmonate analog, 2,3-dihydroxypropyl jasmonate (DHPJA). In suspension cultures of a high taxuyunnanine C (Tc)-producing cell line of Taxus chinensis, 100 microM DHPJA was added on day 7 to fed-batch cultures with feeding of 20 g L(-1) sucrose on the same day. The synergistic effect of elicitation and substrate feeding on Tc biosynthesis was observed, which resulted in higher Tc accumulation than that by elicitation or sucrose feeding alone. More interestingly, both specific Tc yield (i.e., Tc content) and volumetric yield was further improved by a second addition of 100 microM DHPJA (on day 12) to the fed-batch cultures. In particular, with repeated elicitation and sucrose feeding the Tc volumetric yield was increased to 827 +/- 29 mg L(-1), which was 5.4-fold higher than that of the nonelicited batch culture. Furthermore, the above novel strategy was successfully applied from shake flask to a 1-L airlift bioreactor. A high Tc production and productivity of 738 +/- 41 mg L(-1) and 33.2 +/- 1.9 mg L(-1) d(-1), respectively, was achieved, which is higher than previous reports on Tc production in bioreactors. The results suggest that the aforementioned bioprocessing strategy may potentially be applied to other cell culture systems for efficient production of plant secondary metabolites.     

Production of ginsenoside and polysaccharide by two-stage cultivation of Panax quinquefolium L. cells

Based on the results of carbon source consumption in cell suspension culture of Panax quinquefolium L., 30 g L⁻¹ sucrose was fed into a 5-L stirred tank bioreactor on day 16 of culture to enhance cell density and metabolite production. Using a fed-batch cultivation strategy, polysaccharide production was enhanced to 1.608 g L⁻¹, which was 1.96-fold greater than with batch cultivation. The maximum saponin yield (7.828 mg L⁻¹) was obtained on day 24 and was about 36% higher than the yields obtained using batch cultivation. In a two-stage culture process, a combined treatment with sucrose, lactoalbumin hydrolysate, and methyl jasmonate caused a significant increase in total saponin yield (31.52 mg L⁻¹) in cell cultures after 27 d. This value represents an increase of 4.03-fold compared with the total saponin yield in fed-batch cultivation. The two-stage culture mode provided the best method for the in vitro production of secondary metabolites from P. quinquefolium.     

营养条件和流加发酵对放射型根瘤菌(Rhizobium radiobacter)产辅酶Q10的影响

利用放射型根瘤菌WSH2 6 0 1(RhizobiumradiobacterWSH2 6 0 1)重点考察了葡萄糖、蔗糖、玉米浆和蛋白胨、添加物以及流加发酵对细胞生长和产辅酶Q1 0 的影响 ,结果表明 ,葡萄糖和蔗糖适合于生产辅酶Q1 0 的最佳浓度分别为 30g L和 40g L ;辅酶Q1 0 发酵时玉米浆和蛋白胨的最适浓度分别为 11g L和 16g L ;添加蕃茄汁、玉米浆能提高发酵液的生物量 ,玉米浆、异戊醇、L 甲硫氨基酸等能促进辅酶Q1 0 的积累 ;与分批发酵相比 ,在 7L罐上流加蔗糖其细胞生物量 (DCW)和辅酶Q1 0 积累量增加 ,若在流加蔗糖的同时流加适当浓度的玉米浆能显著提高辅酶Q1 0 的产量 ,最大产量达到 5 2 .4mg L ;最大生物量 (DCW)和胞内辅酶Q1 0 含量 (C B值 )分别达到 2 6 .4g L和 2 .38mg g DCW ,比不流加的分批发酵分别提高 5 3 %和 33% ,比只流加蔗糖分别提高 2 4%和 2 6 %。