休哈塔假丝酵母乙醇发酵性能的研究

木糖的高效发酵是制约纤维素燃料乙醇生产的技术瓶颈之一,高性能发酵菌种的开发是本领域研究的重点。以木糖发酵的典型菌株休哈塔假丝酵母为材料,研究氮源配比、葡萄糖和木糖初始浓度、葡萄糖添加及典型抑制物等因素对其木糖利用和乙醇发酵性能的影响规律。结果表明,硫酸铵更适宜于木糖和葡萄糖发酵产乙醇。在摇瓶振荡发酵条件下,该酵母可发酵164.0 g/L葡萄糖生成61.9 g/L乙醇,糖利用率和乙醇得率分别为99.8%和74.0%;受酵母细胞膜上转运体系的限制,对木糖的最高发酵浓度为120.0 g/L,可生成45.7 g/L乙醇,糖利用率和乙醇得率分别达到94.8%和87.0%。休哈塔假丝酵母发酵木糖的主要产物为乙醇,仅生成微量的木糖醇;添加葡萄糖可促进木糖的利用;休哈塔假丝酵母在葡萄糖发酵时的乙酸和甲酸的耐受浓度分别为8.32和2.55 g/L,木糖发酵时的乙酸和甲酸的耐受浓度分别为6.28和1.15 g/L。     

休哈塔假丝酵母发酵木糖制乙醇过程研究

木质纤维素原料水解产物的主要成分是葡萄糖和木糖,其中葡萄糖很容易发酵,致使木糖成为木质纤维素发酵的关键,休哈塔假丝酵母(Candida shehatae)1766是自然界木糖发酵性能较好的天然酵母之一。研究了发酵温度、发酵时间、接种量、初始pH值、摇床转速等因素对休哈塔假丝酵母1766发酵木糖生产乙醇的影响,由正交试验初步确定了休哈塔假丝酵母发酵木糖制乙醇工艺的适宜条件为好氧条件,发酵时间为2d,发酵温度为28℃,摇床转速为150r/min,初始pH值为5,此时乙醇收率最高可达68.62%。     

休哈塔假丝酵母HDYXHT-01利用木糖生产乙醇的发酵工艺优化

采用Plackett-Burman (PB) 方法和中心组合设计 (Ccentral composit design,CCD) 对休哈塔假丝酵母Candida shehataeHDYXHT-01利用木糖发酵生产乙醇的工艺进行优化。PB试验设计与分析结果表明：硫酸铵、磷酸二氢钾、酵母粉和接种量是影响木糖乙醇发酵的4个关键因素,以乙醇产量为响应目标,采用CCD和响应面分析法 (Response surface methodology,RSM),确定了木糖乙醇发酵的最佳工艺为：硫酸铵1.73 g/L、磷酸二氢钾3.56 g/L、酵母粉2.62 g/L和接种量5.66％,其他发酵条件为：木糖80 g/L,MgSO4·7H2O 0.1 g/L,pH 5.0,培养温度30 ℃,装液量100 mL/250 mL,摇床转速140 r/min,发酵时间48 h,在该条件下发酵液中乙醇产量可以达到26.18 g/L,比未优化前提高了1.15倍。     

休哈塔假丝酵母转化木糖和葡萄糖为乙醇的发酵转录谱

[目的]探究木糖发酵典型菌株休哈塔假丝酵母在己糖和戊糖发酵中的转录谱及差异,筛选出与木糖利用和乙醇发酵代谢途径及调控相关的关键性酶和功能蛋白质基因.[方法]应用新一代高通量测序技术454 GS FLX Titanium分别构建了休哈塔假丝酵母木糖、葡萄糖发酵的cDNA文库,并进行De novo转录组的表达序列标签大规模测序和序列比较分析,进而挖掘出该酵母中参与木糖代谢和乙醇发酵的相关基因.[结果]分别对木糖和葡萄糖发酵样本进行二分之一RUN测序并各自得到60万条reads,序列平均长度400 bp.共拼接得到7250条(木糖)和7168条(葡萄糖)contigs,并利用BLAST对木糖样品和葡萄糖样品中的2421个基因(contig)和2456个基因(contig)进行了功能注释和GO分类.通过两个文库间的序列对比分析,共发现158个基因属于差异表达状态(P＜0.05).基于经典的糖酵解及乙醇发酵途径筛选出与木糖乙醇发酵相关的候选基因,并且比较分析其转录水平的差异.[结论]基于大规模转录谱测序和比较分析首次筛选出休哈塔假丝酵母中参与木糖代谢和乙醇发酵的基因群,可为后续的分子生物学及代谢调控研究提供基础数据.     

氦氖激光和亚硝基胍复合诱变选育高产乙醇的休哈塔假丝酵母

木糖的乙醇发酵一直被视为木质纤维原料生物转化产生乙醇的关键因素,休哈塔假丝酵母(Candidashehatae)是木糖发酵性能较好的天然酵母之一。对Candida shehatae HDYXHT-01进行了氦氖激光诱变和NTG诱变,力求选育出发酵木糖产乙醇能力强的菌株。氦氖激光诱变得到的突变株HN-3乙醇产量为17.03g/L,乙醇得率达到0.3393g/g,相比原始菌株提高20.36%。再对HN-3进行NTG诱变,得到的突变株NTG-2乙醇产量为24.20g/L,相比HN-3提高42.10%。进而对NTG-2菌株进行了摇瓶48h连续发酵试验,测得其乙醇产量、木糖利用率、乙醇得率和乙醇产率分别达到24.16g/L,69.26%,0.4360g/g和0.7075g/(L·h)。     

休哈塔假丝酵母（Candida shehatae）木糖醇脱氢酶基因（xyl2）克隆与序列分析

根据木糖醇脱氢酶基因序列相似的特点,根据树干毕赤酵母（Pichia．sfipitis）,热带假丝酵母（Candidatropicalis）设计1对简并引物获得C．shehatae HDYXHT－01木糖醇脱氢酶基因的序列,此片段长为1095bp。利用生物信息学软件对此序列进行了同源性分析、氨基酸组成分析、疏水性分析、磷酸化位点预测、CDS分析、二级结构预测。结果表明,该克隆片段为C．shehataeHDYXHT－01木糖醇脱氢酶基因的序列。     

酿酒酵母W5及休哈塔假丝酵母20335原生质体制备条件的确定

确定了酿酒酵母W5及休哈塔假丝酵母20335原生质体制备的最佳条件。选取不同脱壁预处理时间及不同酶解时间,对酿酒酵母W5、休哈塔假丝酵母20335进行原生质体制备和再生,比较制备率和再生率。确定脱壁预处理30 min后,以终浓度2%的蜗牛酶,30℃、100 r/min酶解处理15 min为双亲株原生质体制备的最佳条件。利用原生质体融合的方法,以酿酒酵母W5和休哈塔假丝酵母20335为亲本株,构建可以利用木糖生产生物乙醇的新型酿酒酵母融合株,该前期工作为W5、20335原生质体融合工作奠定了重要的基础,对于将木质纤维素原料转化为生物乙醇的研究具有极其重要的意义。     

一株中型假丝酵母发酵木糖产乙醇的特性研究

本研究对自然界中筛选得到的1株可以发酵木糖产乙醇的中型假丝酵母(Candida intermedia)的特性进行了研究.该菌株在28℃、120 r/min、72 h条件下,发酵3%木糖的乙醇产率最高,达到理论值的43.70%,发酵7%木糖得到的乙醇产量最高,为6.480 g/L.发酵时间延长到156 h,可以利用8%木糖产乙醇21.225 g/L,产率为理论值的72.87%.该菌株还可以在同样条件下,发酵13%葡萄糖得到乙醇50.965 g/L,达到理论值的76.90%.以3% 2% 3%分批补加糖,比一次性发酵8%木糖的乙醇产量提高9.91%.在葡萄糖和木糖的混合培养基中,优先利用葡萄糖,同时还表现出葡萄糖对木糖发酵的促进作用,当2%的木糖与6%葡萄糖混合时,乙醇产量比两者单独发酵的加和提高了25%.     

C．shehatae TZ8耐乙醇菌株的筛选及发酵性能研究

以C.shehataeTZ8为出发茵株,利用1％溶壁酶和1％蜗牛酶酶解1．5h,制备成C.shehataeTZ8原生质体,并对原生质体进行紫外诱变,以含不同浓度乙醇的木糖液体培养基培养进行初筛和复筛,获得一株遗传性能稳定、耐乙醇能力达5．5％（v／v）的蕾株C．shehataeTZ8-4,比初始菌株耐乙醇能力提高了2％。对突变株C．shehataeTZ8-4发酵性能的研究结果表明：C．shehataeTZ8-4发酵糖能力从80g/L（葡糖糖和木糖比为2：1）提高到120g／L,最大乙醇产量从27．41g／L提高到43．12g／L。     

Xylose metabolism by Candida shehatae in continuous culture

Summary Xylose metabolism by Candida shehatae in continuous culture was examined under both fully-aerobic and semi-aerobic conditions. Growth did not occur in the absence of respiration. Under fully-aerobic conditions, the cell yield was constant at 0.51 g/g and the specific respiration rate Q _{o 2} was linearly related to the specific growth rate with a slope of 15 mmol g^-1 and an intercept of 1.2 mmol g^-1 h^-1. Under semi-aerobic conditions, Q _{O 2} was proportional to with a slope consistent with a cell yield from oxygen Y _{O 2} of 35±7 g mol^-1. The specific xylose uptake rate was a constant independent of and equal to the maximum rate of xylose transport. Ethanol production was observed under semi-aerobic conditions only, and the specific fermentation rate was inversely related to .The use of trade, firm, or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the U.S. Department of Agriculture of any product or service to the exclusion of others which may be suitableMaintained in cooperation with the University of Wisconsin-Madison     

Xylitol production by recombinant Saccharomyces cerevisiae expressing the Pichia stipitis and Candida shehatae XYL1 genes

The xylose reductase gene (XYL1) was isolated from Pichia stipitis and Candida shehatae, cloned into YEp-based vectors under the control of ADH2 and PGK1 promoter/terminator cassettes and introduced into Saccharomyces cerevisiae Y294 by electroporation. Shake-flask fermentations were carried out with 5% xylose and 1% galactose, glucose or maltose as co-substrates. Xylose uptake was similar in both the recombinant strains when different co-substrates were used and slowed once the co-substrate was depleted. The recombinant strains converted xylose to xylitol with yields approaching the theoretical maxima. Xylitol production was most rapid when the co-substrate was still present. Approximately 50% of the xylose was not metabolized due to the depletion of the co-substrate. Received: 23 December 1999 / Received revision: 30 June 2000 / Accepted: 1 July 2000     

Enzymatic and physiological study of d-xylose metabolism by Candida shehatae

Summary Candida shehatae carbon metabolic pathways were correlated with fermentative activity under different growth conditions. Reduced nicotine adenine dinucleotide (NADPH) is the coenzyme preferred for xylose reductase by C. shehatae under in vitro anoxic cell culture conditions. To prevent a redox imbalance derived from intracellular accumulation of NADH in the second enzymatic step of xylose metabolism, the operation of phosphoketolase via in addition the classic pentose phosphate pathway essential for NADH dissimilation is suggested. Variation in cultivation conditions showed a different NADH/NADPH ratio coupled to xylose reductase activity. The existence of two xylose reductases is discussed. Like ethanol, xylitol accumulates only under oxygen-limited or anaerobic conditions. Xylitol accumulaiton under unaerobic conditions was higher when using respiring cells than respirofermentative cells. This fact suggests that cells pregrown under oxygen limitation are better adapted to starting alcoholic fermentation than cells previously grown under aerobic conditions.Offprint requests to: M. T. Amaral-Collaço     

Ethanol and sugar tolerance of Candida shehatae

Summary Candida shehatae ATCC 22984 fermented solutions of up to 260 g/L sugars derived by hydrolysis of whole barley. These solutions contained hexose: pentose 7030, the hexose being mainly glucose from the barley starch and the pentose being mainly xylose. At sugar concentrations of 180 g/L, fermentation was complete in 72 h, yielding 84 g/L ethanol, 0.47 g ethanol/g sugar. At 260 g/L, fermentation ceased when ethanol concentration reached 100 g/L, but resumed when the ethanol was removed by vacuum distillation, to yield finally 0.50 g ethanol/g sugar.     

Simultaneous utilization of glucose and D-xylose by Candida shehatae in a chemostat

The kinetics of biomass formation, D-xylose utilization, and mixed substrate utilization were determined in a chemostat using the yeast Candida shehatae. The maximum growth rate of C. shehatae grown aerobically on D-xylose was 0.42 h⁻¹ and the Monod constant, K _s, was 0.06 g L⁻¹. The biomass yield, Y _{X/S}, ranged from 0.40 to 0.50 g g⁻¹ over a dilution rate range of 0.2–0.3 h⁻¹, when C. shehatae was grown on pure D-xylose. Mixtures of D-xylose and glucose (∼1 : 1) were simultaneously utilized over a dilution rate from 0.15 to 0.35 h⁻¹ at pH 3.5 and 4.5, but pH 3.5 reduced μ_max and reduced the dilution rate range over which D-xylose was utilized in the presence of glucose. At pH 4.5, μ_max was not reduced with the mixed sugar feed and the overall or lumped K _s value was not significantly increased (0.058 g L⁻¹ vs 0.06 g L⁻¹), when compared to a pure D-xylose feed. Kinetic data indicate that C. shehatae is an excellent candidate for chemostat production of value added products from renewable carbon sources, since simultaneous mixed substrate utilization was observed over a wide range of growth rates on a 1 : 1 mixture of glucose and D-xylose. Received 21 August 1997/ Accepted in revised form 28 May 1998     

Unstable petite and grande variants of Candida shehatae

Summary Two strains of Candida shehatae (ATCC 22984 and CSIR Y492) exhibit marked variability in colony size (petite, grande) and respiratory activity (tetrazolium reaction) when grown on glucose, xylose, and--especially--xylitol agar. The transitions occur in both directions at high frequency. Strains showing a negative or weak tetrazolium reaction on xylitol ferment xylose better than those showing a strong tetrazolium reaction. The type strain (ATCC 34887) shows stable colonial morphology with moderate respiratory and fermentative activities. The objective of this report is to demonstrate these variations.