维生素在丙酮酸过量合成中的重要作用

研究了烟酸、硫胺素、吡哆醇、生物素和核黄素对一株光滑球拟酵母(\%Torulopsis glabrata\%) WSH\|IP303以葡萄糖为碳源、以氯化铵为唯一氮源生产丙酮酸的影响。利用正交试验方法,确证了硫胺素是影响WSH\|IP303生产丙酮酸的最重要因素。在硫胺素浓度一定(0.01～0.015mg/L)的前提下,提高烟酸浓度有助于加快耗糖速度。当烟酸、硫胺素、吡哆醇、生物素和核黄素的浓度分别为8、0.015、0.4、0.04和01mg/L时,摇瓶发酵48h,丙酮酸产量和产率可分别达到52.4g/L和0525g/g。采用优化的维生素组合方式,进行2.5L罐分批发酵,在初糖浓度120g/L的条件下发酵57.5h,丙酮酸产量和产率分别达到69.4g/L和0593g/g,分别比摇瓶培养的最好结果提高了32.%和13%。     

添加TCA循环中间产物加速光滑球拟酵母积累丙酮酸

在维生素限制的条件下,研究了添加TCA循环中间产物对光滑球拟酵母多重维生素营养缺陷型菌株CCTCC M202019生长和积累丙酮酸的影响。该菌株能以TCA循环中间产物为唯一碳源进行生长,且在以葡萄糖、乙酸和TCA循环中间产物为复合碳源的平板上菌落数高于分别以葡萄糖和乙酸或TCA循环中间产物为唯一碳源时的菌落数。与其它TCA循环中间产物相比,草酰乙酸更能促进细胞的生长、提高丙酮酸产量和对葡萄糖的得率。草酰乙酸能够促进细胞生长,是因为T. glabrata CCTCC M202019菌株能够利用乙酸作为乙酰辅酶A供体。在含有100 g/L葡萄糖和6 g/L乙酸钠的培养基中再添加10 g/L草酰乙酸进行分批发酵实验,可使菌体浓度从11.8 g/L提高到 13.6 g/L,增长幅度为15%;丙酮酸对葡萄糖的得率(0.66 g/g)以及生产强度(1.19 g·L^{-1<、sup>·h-1<、sup>)分别高出6%和24%,使发酵结束时间提前8～12h。}     

营养条件对光滑球拟酵母积累α-酮戊二酸的影响

以光滑拟球酵母为研究模型,研究α-酮戊二酸的浓度情况。通过单因素实验得到α-酮戊二酸积累最佳浓度的各单因素条件为：葡萄糖浓度140g／L,NH4Cl浓度5g／L。在碳源（30g/L葡萄糖初始浓度）匮乏条件下加入丙酮酸30g／L,在此条件下丙酮酸转化为α－酮戊二酸的转化率最高达53．7％。以30g／L丙酮酸为唯一碳源时在7L发酵罐中光滑拟球酵母可生成浓度为10．7g/Lα-酮戊二酸,外源丙酮酸的转化率可达66．9％。这一结果表明,T．glabrata具有将丙酮酸转化为α-KG的能力。     

过量表达NADH氧化酶加速光滑球拟酵母合成丙酮酸

[目的]进一步提高光滑球拟酵母(Torulopsis glabrata)发酵生产丙酮酸的生产强度.[方法]将来源于乳酸乳球菌(Lactococcus lactis)中编码形成水的NADH氧化酶noxE基因过量表达于丙酮酸工业生产菌株T. glabrata CCTCC M202019中,获得了一株NADH氧化酶活性为34.8 U/mg蛋白的重组菌T. glabrata-PDnoxE.[结果]与出发菌株T. glabrata CCTCC M202019相比,细胞浓度、葡萄糖消耗速率和丙酮酸生产强度分别提高了168%、44.9%和12%,发酵进行到36 h葡萄糖消耗完毕.补加50 g/L葡萄糖继续发酵20 h,则使丙酮酸浓度提高到67.2 g/L.葡萄糖消耗速度和丙酮酸生产强度增加的原因在于形成水的NADH氧化酶过量表达,导致NADH和ATP含量分别降低了18.1%和15.8%.而NAD<' 增加了11.1%.[结论]增加细胞内NAD<' 含量能有效地提高酵母细胞葡萄糖的代谢速度及目标代谢产物的生产强度.     

温度对丙酮酸生物合成动力学、能荷和氧化-还原度的影响

在光滑球拟酵母(Torulopsis glabrata620)生产丙酮酸的过程中,温度对丙酮酸生物合成有着重要的影响。考察了不同发酵温度下基质消耗、细胞生长、丙酮酸合成及能荷水平和氧化-还原度等方面的差异。在恒温发酵中,维持较高的发酵温度可以增强糖耗,促进菌体生长,加速丙酮酸积累,但前期胞内能荷水平较高,菌体消耗较多葡萄糖合成菌体,后续产酸能力不足,导致丙酮酸得率降低;维持较低的发酵温度可以在发酵后期提供稳定的产酸能力,但菌体代谢缓慢,后期胞内NADH/NAD 水平较高,丙酮酸生产强度降低。因此仅仅采取单一的温度控制策略很难达到丙酮酸高产量、高产率和高生产强度的统一。     

pH及流加葡萄糖对酵母分批发酵生产谷胱甘肽的影响

在5 L的发酵罐中研究了pH及流加葡萄糖对酵母分批发酵生产谷胱甘肽(GSH)的影响。实验考察了不同浓度的流加葡萄糖和不同的恒pH值的分批发酵对于酵母生产GSH产量的变化。实验结果表明,当pH值控制为5.0,流加葡萄糖流速为5g.L-1.h-1,连续流加30 h,可使GSH产量最高,与之前未流加葡萄糖和控制pH相比,其产量提高了6倍。     

汉逊德巴利酵母发酵葡萄糖生产D-阿拉伯糖醇

从378株耐高渗酵母中,筛选到1株由葡萄糖发酵高产D-阿拉伯糖醇的酵母。通过生理生化和分子生物学的鉴定,证实该菌株为Debaryomyces hansenii,保藏编号CICIM Y 0504。研究该酵母摇瓶发酵的主要影响因素,确定其摇瓶发酵条件为:葡萄糖200 g/L,酵母膏10 g/L,初始pH值3,装液量20 mL/250 mL,温度30℃。在此条件下发酵120 h,D-阿拉伯糖醇浓度达90.37 g/L,转化率45.18%。在15 L发酵罐对该酵母进行扩大培养,结果表明,初始葡萄糖浓度200 g/L的分批发酵产D-阿拉伯糖醇64.07 g/L,转化率33.94%;葡萄糖浓度控制在30～50 g/L的分批补料发酵产D-阿拉伯糖醇125 g/L,转化率37.5%。研究结果对葡萄糖发酵生产D-阿拉伯糖醇工业化的实现具有重要启示。     

温度对丙酮酸生物合成动力学、能荷和氧化 还原度的影响

在光滑球拟酵母（Torulopsis glabrata 620）生产丙酮酸的过程中,温度对丙酮酸生物合成有着重要的影响。考察了不同发酵温度下基质消耗、细胞生长、丙酮酸合成及能荷水平和氧化还原度等方面的差异。在恒温发酵中,维持较高的发酵温度可以增强糖耗,促进菌体生长,加速丙酮酸积累,但前期胞内能荷水平较高,菌体消耗较多葡萄糖合成菌体,后续产酸能力不足,导致丙酮酸得率降低;维持较低的发酵温度可以在发酵后期提供稳定的产酸能力,但菌体代谢缓慢,后期胞内NADH/NAD⁺水平较高,丙酮酸生产强度降低。因此仅仅采取单一的温度控制策略很难达到丙酮酸高产量、高产率和高生产强度的统一。     

溶氧对Candida glycerinogenes产甘油发酵过程的影响

研究了溶氧浓度对产甘油假丝酵母分批发酵生产甘油过程的影响。实验结果表明：当溶氧浓度控制在30%时,C. glycerinogenes的甘油产量、得率和产率达到最高,分别为120.7 g/L、0.575 g/g和1.69 g/(L•h),而糖酵解代谢副产物形成最少。当溶氧浓度为10%时,发酵过程呈现出“巴斯德效应”的特征,生成的酵解代谢副产物维持在较高水平。在快速生长阶段,随着溶氧从10%增加到60%,细胞呼吸类型表现为从厌氧呼吸向好氧呼吸转变,酵解代谢副产物依次减少。在生长稳定期,控制的溶氧浓度越高,酵解代谢副产物乙醇、乙酸等的生成减少。分别选用Logistic方程、Luedeking-Piret方程和Luedeking-Piret-like方程,能较好地模拟细胞生长、甘油合成和葡萄糖消耗的动力学过程。     

皮状丝孢酵母B3利用木薯淀粉发酵生产微生物油脂

对皮状丝孢酵母B3以木薯淀粉水解液为碳源发酵生产微生物油脂培养条件进行了优化,并在2 L发酵罐中对菌体生长和油脂积累进行了考察。摇瓶实验表明,木薯淀粉水解液的浓度高于90 g/L时不利于菌体的生长和油脂积累,皮状丝孢酵母B3发酵生产微生物油脂的最适氮源及其浓度、最适C/N比和pH分别为酵母提取物3.0 g/L、116、6.0,在此条件下培养144 h菌体生物量、油脂产量和油脂含量分别达到15.2 g/L、6.22 g/L和40.9％;在2 L发酵罐中分批发酵44 h后菌体生物量、油脂产量和油脂含量分别达28.7 g/L、12.27 g/L和42.8％。以皮状丝孢酵母B3所产油脂制备生物柴油,其主要组成包括棕榈酸甲酯、硬脂酸甲酯、油酸甲酯、亚油酸甲酯等,且理化特性符合相关国家标准,可作为一种有潜力的化石燃料替代品。     

Fed‐Batch Process for Pyruvate Production by Recombinant Escherichia coli YYC202 Strain

Fed‐batch fermentation was applied to the production of pyruvate by using a recombinant Escherichia coli YYC202 strain. This strain is completely blocked in its ability to convert pyruvate into acetyl‐CoA or acetate, resulting in acetate auxotrophy during growth in glucose minimal medium. By controlling acetate and glucose feed rate, a series of lab‐scale fed‐batch experiments were performed at pH 7 and 37 °C. CO₂ production rate (CTR) was used for on‐line regulation of the acetate feed rate. The correlation between CTR and acetate consumption rate (ACR) was determined experimentally. At optimal process conditions a final pyruvate concentration higher than 62 g/L, a space‐time yield of up to 42 g/L/d and pyruvate/glucose molar yield of 1.11 mol/mol were achieved. Experimental evidence was gathered that pyruvate export is active.     

Ethanol fermentation in an immobilized cell reactor using Saccharomyces cerevisiae

Fermentation of sugar by Saccharomyces cerevisiae, for production of ethanol in an immobilized cell reactor (ICR) was successfully carried out to improve the performance of the fermentation process. The fermentation set-up was comprised of a column packed with beads of immobilized cells. The immobilization of S. cerevisiae was simply performed by the enriched cells cultured media harvested at exponential growth phase. The fixed cell loaded ICR was carried out at initial stage of operation and the cell was entrapped by calcium alginate. The production of ethanol was steady after 24 h of operation. The concentration of ethanol was affected by the media flow rates and residence time distribution from 2 to 7 h. In addition, batch fermentation was carried out with 50 g/l glucose concentration. Subsequently, the ethanol productions and the reactor productivities of batch fermentation and immobilized cells were compared. In batch fermentation, sugar consumption and ethanol production obtained were 99.6% and 12.5% v/v after 27 h while in the ICR, 88.2% and 16.7% v/v were obtained with 6 h retention time. Nearly 5% ethanol production was achieved with high glucose concentration (150 g/l) at 6 h retention time. A yield of 38% was obtained with 150 g/l glucose. The yield was improved approximately 27% on ICR and a 24 h fermentation time was reduced to 7 h. The cell growth rate was based on the Monod rate equation. The kinetic constants (K(s) and mu(m)) of batch fermentation were 2.3 g/l and 0.35 g/lh, respectively. The maximum yield of biomass on substrate (Y(X-S)) and the maximum yield of product on substrate (Y(P-S)) in batch fermentations were 50.8% and 31.2% respectively. Productivity of the ICR were 1.3, 2.3, and 2.8 g/lh for 25, 35, 50 g/l of glucose concentration, respectively. The productivity of ethanol in batch fermentation with 50 g/l glucose was calculated as 0.29 g/lh. Maximum production of ethanol in ICR when compared to batch reactor has shown to increase approximately 10-fold. The performance of the two reactors was compared and a respective rate model was proposed. The present research has shown that high sugar concentration (150 g/l) in the ICR column was successfully converted to ethanol. The achieved results in ICR with high substrate concentration are promising for scale up operation. The proposed model can be used to design a lager scale ICR column for production of high ethanol concentration.     

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℃。     

A comparative study of glucose conversion to ethanol by Zymomonas mobilis and a recombinant Escherichia coli

Summary Zymomonas mobilis and recombinant Escherichia coli B (pLOI297) were compared in side-by-side batch fermentations using a synthetic cellulose hydrolysate (glucose/salts) medium with pH control at 6.0 and an inoculation cell density of 35–50 mg dry wt. cells/L. At a nominal glucose concentration of 6%, both cultures achieved near maximal theoretical ethanol yields; however, the Z. mobilis fermentation was complete at 13h compared to 33h for the E.coli fermentation. With approx.12% glucose, the Z. mobilis fermentation was complete in 20h with a process yield of 0.49 g ethanol/g added glucose compared to the E. coli fermentation which remained 20% incomplete after 6 days resulting in a process yield of only 0.32 g/g. Nutrient supplementation (10g tryptone/L) resulted in complete fermentation of 12% glucose (pH 6.3) by the recombinant E. coli in 4 days, with a yield of 0.48 g/g.     

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.     

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.     

Improved production of the anticancer compound 1403C by glucose pulse feeding of marine Halorosellinia sp. (No. 1403) in submerged culture

Effects of different pulse fed-batch methods on production of the anti-cancer compound 1403C by marine mangrove endophytic fungus Halorosellinia sp. (No. 1403) in a 5-L bioreactor were investigated. Since high glucose concentrations improved mycelial growth but inhibited 1403C production, the cultures were pulse fed with glucose solutions to keep the residual glucose lower than 4 g/L but higher than 0.5 g/L during rapid growth phase (0-50 h). In this way, a maximum dry biomass, 1403C production and yield coefficient (Y1403C/X) of up to 4.5 g/L, 2.64 g/L and 0.59 g/g dry cell weight, respectively were achieved. These values are 22.7%, 98.0% and 61.4%, respectively higher than those obtained with batch cultures. This strategy is valuable for fermentation scale-up of Halorosellinia sp. (No. 1403) for 1403C production, and might also be applicable to other marine fungi cultures.     

Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by Ralstonia eutropha in high cell density palm oil fermentations

Improved production costs will accelerate commercialization of polyhydroxyalkanoate (PHA) polymer and PHA-based products. Plant oils are considered favorable feedstocks, due to their high carbon content and relatively low price compared to sugars and other refined carbon feedstocks. Different PHA production strategies were compared using a recombinant strain of Ralstonia eutropha that produces high amounts of P(HB-co-HHx) when grown on plant oils. This R. eutropha strain was grown to high cell densities using batch, extended batch, and fed batch fermentation strategies, in which PHA accumulation was triggered by nitrogen limitation. While extended batch culture produced more biomass and PHA than batch culture, fed batch cultivation was shown to produce the highest levels of biomass and PHA. The highest titer achieved was over 139 g/L cell dry weight (CDW) of biomass with 74% of CDW as PHA containing 19 mol% HHx. Our data suggest that the fermentation process is scalable with a space time yield (STY) better than 1 g PHA/L/h. The achieved biomass concentration and PHA yield are among the highest reported for the fermentation of recombinant R. eutropha strains producing P(HB-co-HHx).     

Regulation of NADH Oxidase Expression <Emphasis Type="Italic">via</Emphasis> a Thermo-regulated Genetic Switch for Pyruvate Production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

As a chemical, pyruvate can be used as a raw material for drug, agrochemical, chemical, and food industries. In the microbial production of pyruvate, although continuous expression of exogenous NADH oxidase (noxE) can improve glucose consumption, it can lead to a decrease of pyruvate yield. For efficient pyruvate production, a thermo-regulated genetic switch was designed to dynamically control the expression of noxE from Lactococcus lactis on the Escherichia coli MP-XB010CN chromosome. At the initial stage of fermentation, switching on the genetic switch for efficient noxE expression can promote growth rate and biomass accumulation, then switching off noxE expression can weaken the TCA pathway and improve the pyruvate yield. High pyruvate concentration of 93.0 g/L and yield of 0.71 g/g glucose were achieved with the thermo-regulated two-phase fermentation. Efficient cell growth and pyruvate production were reached separately by switching cultivation temperature. The results indicated that the genetic switch for controlling the noxE gene accurate expression was an effective strategy for improving pyruvate production.     

High-titer n-butanol production by clostridium acetobutylicum JB200 in fed-batch fermentation with intermittent gas stripping

Acetone–butanol–ethanol (ABE) fermentation with a hyper‐butanol producing Clostridium acetobutylicum JB200 was studied for its potential to produce a high titer of butanol that can be readily recovered with gas stripping. In batch fermentation without gas stripping, a final butanol concentration of 19.1 g/L was produced from 86.4 g/L glucose consumed in 78 h, and butanol productivity and yield were 0.24 g/L h and 0.21 g/g, respectively. In contrast, when gas stripping was applied intermittently in fed‐batch fermentation, 172 g/L ABE (113.3 g/L butanol, 49.2 g/L acetone, 9.7 g/L ethanol) were produced from 474.9 g/L glucose in six feeding cycles over 326 h. The overall productivity and yield were 0.53 g/L h and 0.36 g/g for ABE and 0.35 g/L h and 0.24 g/g for butanol, respectively. The higher productivity was attributed to the reduced butanol concentration in the fermentation broth by gas stripping that alleviated butanol inhibition, whereas the increased butanol yield could be attributed to the reduced acids accumulation as most acids produced in acidogenesis were reassimilated by cells for ABE production. The intermittent gas stripping produced a highly concentrated condensate containing 195.9 g/L ABE or 150.5 g/L butanol that far exceeded butanol solubility in water. After liquid–liquid demixing or phase separation, a final product containing ～610 g/L butanol, ～40 g/L acetone, ～10 g/L ethanol, and no acids was obtained. Compared to conventional ABE fermentation, the fed‐batch fermentation with intermittent gas stripping has the potential to reduce at least 90% of energy consumption and water usage in n‐butanol production from glucose. Biotechnol. Bioeng. 2012; 109: 2746–2756. © 2012 Wiley Periodicals, Inc.