发酵木糖产乙醇菌种筛选及应用

采用传统的菌种筛选方法,对样品进行了发酵木糖生产酒精的酵母菌的筛选,得到一株能够发酵木糖生产酒精的酵母菌,经初步鉴定为假丝酵母菌.其在木糖含量2%、30℃、100r/min的条件下发酵,酒精产量达到3.10g/L,基本达到了一般野生酵母的水平.再利用其发酵麦糟水解液,乙醇产量最高为4.24g/L.结果表明,Y-4T-1具有抵抗麦糟水解液中的乙酸、糠醛、羟甲基糠醛等抑制物生长的能力,为发酵麦糟生产酒精的进一步研究提供了菌种支持,为生物质燃料生产燃料乙醇的研究提供了新的思路.     

新型木糖发酵酵母Spathaspora passalidarum NRRL Y-27907与传统木糖发酵酵母Pichia stipitis NRRL Y-7124乙醇发酵性能比较

木糖乙醇转化率低是木质纤维素乙醇工业化生产尚未突破的技术瓶颈,选育高效发酵木糖产乙醇菌株一直是木质纤维素乙醇领域的研究热点。Spathaspora passalidarum是新近从自然界筛选到的一种新型发酵木糖产乙醇酵母,Pichia stipitis是已知木糖乙醇转化效率相对较高的酵母菌。从胁迫耐受性(乙醇、温度、渗透压),糖发酵能力(葡萄糖、木糖、葡萄糖和木糖混合糖),高温发酵能力方面对S.passalidarum NRRLY-27907和P.stipitis NRRLY-7124进行了比较。结果表明,NRRL Y-27907对高浓度乙醇、高温、高渗条件的耐受性比NRRL Y-7124更强。NRRL Y-27907葡萄糖发酵能力略低于NRRL Y-7124,但木糖发酵能力优于NRRL Y-7124。更为突出的是,NRRL Y-27907的葡萄糖阻遏效应较弱,葡萄糖和木糖混糖发酵能力明显优于NRRL Y-7124。在30~39℃下,NRRL Y-27907的木糖乙醇发酵能力皆优于NRRL Y-7124,具有更好的高温发酵潜力。因此,Spathaspora passalidarum在木质纤维素-生物乙醇转化方面具有很好的应用潜力。     

酵母菌性能优化关键技术研究进展

利用木质纤维素产乙醇能有效缓解传统能源危机与环境污染问题,而提升酵母菌发酵性能是提高木质纤维素乙醇产量的关键步骤之一.从酵母菌优化和改造等方面总结并分析了提高酵母菌厌氧乙醇发酵综合效率的技术发展、存在问题及发展趋势.首先介绍了酵母菌五碳糖代谢途径的构建,以进一步提高酵母菌对原料的综合利用率;其次阐述了通过筛选、驯化及分子生物工程等手段提升酵母菌对限制因素尤其是终产物乙醇的耐受性研究,保证菌体在高乙醇浓度环境下保持良好的发酵性能,提高最终产率及产量,推广木质纤维素乙醇的工业化生产.     

木质纤维素生产乳酸研究获突破

日前,中国科学院过程工程研究所万印华研究员的团队分离得到一株高温乳酸生产菌BacilluscoagulanslPE22。该菌株能够利用葡萄糖、木糖和阿拉伯糖同型发酵生产乳酸,并且对木质纤维素水解液中多种发酵抑制物具有耐受性。利用木质纤维素原料生产乳酸是目前的研究热点。然而,受限于乳酸生产菌种,以木质纤维素为底物进行乳酸发酵时,通常分为多个独立的操作单元导致发酵效率不高,影响了木质纤维素发酵生产乳酸的工业化进程。     

基因工程菌在木质纤维素生产燃料乙醇中的应用

利用基因工程技术对大肠杆菌(Escherichia coli)、运动发酵假单胞菌(Zymomonas mobilis)、啤酒酵母菌(Saccharomyces cerevisiae)进行改造,构建出一系列能够发酵木质纤维素水解产物(木糖、葡萄糖)的基因重组菌株,以提高发酵木糖和葡萄糖转化乙醇的能力,成为目前研究和开发的重点.文中对其研究进展及相关菌株载体的构建方法进行了综述.     

五碳糖发酵生产乙醇的菌种研究进展

当前以木质纤维素为原料制备燃料乙醇已成为全球研究热点,发酵五碳糖产乙醇的菌种选育是其关键技术之一。文中综述了微生物发酵五碳糖产乙醇的机理,五碳糖发酵自然微生物的种类及菌种遗传改良的研究进展,并对今后菌种的研究方向进行了简要论述。     

玉米秸秆生产燃料乙醇技术

玉米秸秆经预处理后可得到纤维素和半纤维素,用酸或酶将其水解成单糖,再进行发酵就可以生产燃料乙醇。对玉米秸秆生产燃料乙醇的原料预处理、水解产生可发酵单糖和乙醇发酵等技术方法进行了综述。     

丝状真菌发酵木糖产乙醇的研究

以木质纤维素为原料生产燃料乙醇是目前的一个研究重点,其中利用木糖产乙醇微生物的研究尤为重要。本文简要概述了自然界中能利用木糖产乙醇的几种主要丝状真菌及基因工程技术在之研究中的应用,并展望了今后的研究方向。     

葡萄糖、木糖共发酵菌株扩培原料优选

燃料乙醇将成为未来最重要的可再生清洁能源之一。利用来源广泛的农、林废弃物等各类木质纤维质原料生产燃料乙醇具有极为广阔的前景。纤维质原料含有大量的纤维素、半纤维素,XWS-Y菌株能够同时代谢葡萄糖和木糖,共同发酵生成乙醇,从而提高发酵乙醇浓度。最终发酵效果与XWS-Y菌株的扩大培养有着直接关系,本文详细评价几种不同原料在相同的条件下扩培XWS-Y菌株,在酶解液中的发酵性能。     

生物质发酵生产乙醇的研究进展

生物质是一种广泛存在的可再生资源,经发酵生产乙醇所用的天然生物质资源原料主要分为3类:糖、淀粉和纤维素物质.木质纤维原料发酵生产乙醇,要先对原料进行热机械法、自动水解法、酸处理法、碱处理法、有机溶剂处理法、生物法等预处理;发酵工艺方式有直接发酵法、间接发酵法、混合菌种发酵、同步糖化发酵法(SSF法)、非等温同步糖化发酵法和固定化细胞发酵法(NSSF法).用木糖发酵生产乙醇的微生物有管囊酵母(Pachysolen tannophilus)、树干毕赤酵母(Pichia stipits)和休哈塔假丝酵母(Candida shechatae)等.对利用生物质资源生产乙醇还应在纤维素酶、混合糖的发酵及生产工艺上进行深度的研究.     

Ethanol production by a new pentose-fermenting yeast strain, Scheffersomyces stipitis UFMG-IMH 43.2, isolated from the Brazilian forest

The ability of a recently isolated Scheffersomyces stipitis strain (UFMG-IMH 43.2) to produce ethanol from xylose was evaluated. For the assays, a hemicellulosic hydrolysate produced by dilute acid hydrolysis of sugarcane bagasse was used as the fermentation medium. Initially, the necessity of adding nutrients (MgSO(4)·7H(2)O, yeast extract and/or urea) to this medium was verified, and the yeast extract supplementation favoured ethanol production by the yeast. Then, in a second stage, assays under different initial xylose and cell concentrations, supplemented or not with yeast extract, were performed. All these three variables showed significant (p < 0.05) influence on ethanol production. The best results (ethanol yield and productivity of 0.19 g/g and 0.13 g/l/h, respectively) were obtained using the hydrolysate containing an initial xylose concentration of 30 g/l, supplemented with 5.0 g/l yeast extract and inoculated with an initial cell concentration of 2.0 g/l. S. stipitis UFMG-IMH 43.2 was demonstrated to be a yeast strain with potential for use in xylose conversion to ethanol. The establishment of the best fermentation conditions was also proved to be of great importance to increasing the product formation by this yeast strain. These findings open up new perspectives for the establishment of a feasible technology for ethanol production from hemicellulosic hydrolysates.     

Bioethanol production from rice straw by a sequential use of Saccharomyces cerevisiae and Pichia stipitis with heat inactivation of Saccharomyces cerevisiae cells prior to xylose fermentation

In order to establish an efficient bioethanol production system from rice straw, a new strategy to ferment the mixture of glucose and xylose by a sequential application of Saccharomyces cerevisiae and Pichia stipitis was developed, in which heat inactivation of S. cerevisiae cells before addition of P. stipitis was employed. The results showed that heating at 50°C for 6h was sufficient to give high xylose fermentation efficiency. By application of the inactivation process, 85% of the theoretical yield was achieved in the fermentation of the synthetic medium. At the same time, the xylitol production was reduced by 42.4% of the control process. In the simultaneous saccharification and fermentation of the lime-pretreated and CO(2)-neutralized rice straw, the inactivation of S. cerevisiae cells enabled the full conversion of glucose and xylose within 80 h. Finally, 21.1g/l of ethanol was produced from 10% (w/w) of pretreated rice straw and the ethanol yield of rice straw reached 72.5% of the theoretical yield. This process is expected to be useful for the ethanol production from lignocellulosic materials in the regions where large-scale application of recombinant microorganisms was restricted.     

木糖浓度及补料发酵对树干毕赤酵母乙醇发酵的影响

探究不同浓度木糖及补料对树干毕赤酵母（Pichia stipitis）菌株1K-9发酵木糖产乙醇的影响,提高木糖产乙醇的发酵水平,为扩大规模发酵木糖产乙醇打下基础。结果表明,菌株1K-9先采用10%木糖进行乙醇发酵,36 h补加与10%木糖培养基等体积的20%木糖培养基继续发酵,发酵至108 h时菌数也达到了（12.16±0.07）×108个/mL,较未补料发酵时有所提高;发酵108 h时醪液中残留的木糖含量为（1.03±0.02）g/L,较未补料发酵时有所降低;乙醇含量达到了6.56%vol,较未补料时提高了1.85%vol。因此补料发酵是有效的。     

热带假丝酵母木糖还原酶的酶学性质研究

研究了从热带假丝酵母(Candida tropicalis)菌体中获得的木糖还原酶(XR)的酶学性质。实验结果证实,C.tropicalis的细胞浆粗提液经盐析、透析及阴离子交换柱层析后得到的酶液中木糖还原酶比酶活为9·3U/mg、最适酶反应pH为6·0、最适反应温度为35℃;以木糖为底物时,Km·Xyl为64·8mmol/L、Km·N·X为0·0622mmol/L;以阿拉伯糖为底物时,Km·Ara为172mmol/L、Km·N·A为0·0375mmol/L。Zn2+是木糖还原酶的激活剂,Fe3+为抑制剂。固定木糖为反应底物,分别以NADPH及NADH为辅酶测定酶活,实验结果显示该菌体中木糖还原酶的活性主要依赖于辅酶NADPH。     

稀酸预处理玉米芯酶解工艺响应面优化研究

木质纤维原料还原糖（葡萄糖、木糖）转化是燃料乙醇生产的关键步骤之一,该文以玉米芯为原料,采用稀硫酸处理、酶水解以提高还原糖转化量。以还原糖转化量为考核指标,采用单因素试验及响应面试验设计优化稀酸处理玉米芯酶解条件,拟合硫酸体积分数、加酶量、酶解时间3个因素对还原糖转化量的回归模型。结果表明,最佳酶解工艺为121 ℃条件下预处理60 min,硫酸体积分数0.8%,料液比1∶15（g∶mL）,加酶量7%（纤维素酶∶半纤维素酶1∶1）,酶解时间70.9 h。在此最佳条件下,采用高效液相色谱（HPLC）法测定酶解液中还原糖转化量为462.62 mg/g,其中木糖、葡萄糖转化量分别为330.02 mg/g、132.60 mg/g,还原糖转化率可达46.3%。     

在酿酒酵母中共表达sXYLA和XKS1及木糖发酵的初步研究

为改善重组酵母发酵木糖生产乙醇的能力,将定点突变改造后的Thermus thermophilus木糖异构酶基因sXYLA克隆到酵母表达载体pYX212并用于转化酸酒酵母Saccharomyces cerevisiae YPH499进行表达研究。酶活检测表明,改造后的木糖异构酶活性是未改造的1.91倍。在此基础上将改造后具有良好特性的木糖异构酶基因sXYLA和来自酸酒酵母的木酮糖激酶基因XKS1耦联,构楚得到重组表达质粒pYX-sXYLA- XKS1,在酿酒酵母YPH499中实现组成型共表达。结果表明,在84 h时重组菌发酵液酶活达到最高,木糖异构酶为0.624 U/mg蛋白,木酮糖激酶为0.688 U/mg蛋白。以葡萄糖和木糖为混合碳源初步进行半通氧发酵,代谢产物分析表明酸酒酵母重组菌木糖的消耗为4.75 g/L,乙醇的产量为0.839 g/L,分别比出发菌提高20.9%和14.8%,为酿酒酵母利用木糖发酵乙醇奠定基础。     

Biological detoxification of waste house wood hydrolysate using Ureibacillus thermosphaericus for bioethanol production

Hydrolysates of lignocelluloses hydrolyzed by diluted sulfuric acid contain toxic compounds that inhibit ethanol production by Saccharomyces cerevisiae and the ethanologenic recombinant Escherichia coli KO11. We investigated the biological detoxification of a hydrolysate of waste house wood (WHW) by a thermophilic bacterium, Ureibacillus thermosphaericus. When the hydrolysate was treated with this bacterium at 50 degrees C for 24 h, the ethanol production rate by S. cerevisiae increased markedly and was comparable to that for the hydrolysate treated with an excess amount of calcium hydroxide (overliming). Chromatographic analysis of synthetic hydrolysates containing furfural or 5-hydroxymethyl furfural that are considered to be major toxic compounds in hydrolysates revealed that U. thermosphaericus degrades these compounds. In the WHW hydrolysates, however, the concentrations of these compounds were not decreased markedly by the bacterium. These results suggest that the bacterium degrades minor but more toxic compounds or phenolic compounds in the WHW hydrolysates. The combination of bacterial and overliming treatments of hydrolysates minimized significantly the decrease in ethanol production rate by E. coli KO11 as fermentation proceeded. Because the bacterium grows rapidly and does not consume sugars, our biological detoxification should be useful for bioethanol production from acid hydrolysates of lignocelluloses.     

Bioethanol production from ball milled bagasse using an on-site produced fungal enzyme cocktail and xylose-fermenting Pichia stipitis

Sugarcane bagasse is one of the most promising agricultural by-products for conversion to biofuels. Here, ethanol fermentation from bagasse has been achieved using an integrated process combining mechanical pretreatment by ball milling, with enzymatic hydrolysis and fermentation. Ball milling for 2 h was sufficient for nearly complete cellulose structural transformation to an accessible amorphous form. The pretreated cellulosic residues were hydrolyzed by a crude enzyme preparation from Penicillium chrysogenum BCC4504 containing cellulase activity combined with Aspergillus flavus BCC7179 preparation containing complementary β-glucosidase activity. Saccharification yields of 84.0% and 70.4% for glucose and xylose, respectively, were obtained after hydrolysis at 45 °C, pH 5 for 72 h, which were slightly higher than those obtained with a commercial enzyme mixture containing Acremonium cellulase and Optimash BG. A high conversion yield of undetoxified pretreated bagasse (5%, w/v) hydrolysate to ethanol was attained by separate hydrolysis and fermentation processes using Pichia stipitis BCC15191, at pH 5.5, 30 °C for 24 h resulting in an ethanol concentration of 8.4 g/l, corresponding to a conversion yield of 0.29 g ethanol/g available fermentable sugars. Comparable ethanol conversion efficiency was obtained by a simultaneous saccharification and fermentation process which led to production of 8.0 g/l ethanol after 72 h fermentation under the same conditions. This study thus demonstrated the potential use of a simple integrated process with minimal environmental impact with the use of promising alternative on-site enzymes and yeast for the production of ethanol from this potent lignocellulosic biomass.     

d-Xylose consumption by nonrecombinant Saccharomyces cerevisiae: A review

Xylose is the second most abundant sugar in nature. Its efficient fermentation has been considered as a critical factor for a feasible conversion of renewable biomass resources into biofuels and other chemicals. The yeast Saccharomyces cerevisiae is of exceptional industrial importance due to its excellent capability to ferment sugars. However, although S. cerevisiae is able to ferment xylulose, it is considered unable to metabolize xylose, and thus, a lot of research has been directed to engineer this yeast with heterologous genes to allow xylose consumption and fermentation. The analysis of the natural genetic diversity of this yeast has also revealed some nonrecombinant S. cerevisiae strains that consume or even grow (modestly) on xylose. The genome of this yeast has all the genes required for xylose transport and metabolism through the xylose reductase, xylitol dehydrogenase, and xylulokinase pathway, but there seems to be problems in their kinetic properties and/or required expression. Self-cloning industrial S. cerevisiae strains overexpressing some of the endogenous genes have shown interesting results, and new strategies and approaches designed to improve these S. cerevisiae strains for ethanol production from xylose will also be presented in this review.     

Investigation of limiting metabolic steps in the utilization of xylose by recombinant Saccharomyces cerevisiae using metabolic engineering

A Saccharomyces cerevisiae screening strain was designed by combining multiple genetic modifications known to improve xylose utilization with the primary objective of enhancing xylose growth and fermentation in xylose isomerase (XI)-expressing strains. Strain TMB 3045 was obtained by expressing the XI gene from Thermus thermophilus in a strain in which the GRE3 gene coding for aldose reductase was deleted, and the genes encoding xylulokinase (XK) and the enzymes of the non-oxidative pentose phosphate pathway (PPP) [transaldolase (TAL), transketolase (TKL), ribose 5-phosphate ketol-isomerase (RKI) and ribulose 5-phosphate epimerase (RPE)] were overexpressed. A xylose-growing and fermenting strain (TMB 3050) was derived from TMB 3045 by repeated cultivation on xylose medium. Despite its low XI activity, TMB 3050 was capable of aerobic xylose growth and anaerobic ethanol production at 30 degrees C. The aerobic xylose growth rate reached 0.17 l/h when XI was replaced with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes expressed from a multicopy plasmid, demonstrating that the screening system was functional. Xylose growth had not previously been detected in strains in which the PPP genes were not overexpressed or when overexpressing the PPP genes but having XR and XDH genes chromosomally integrated. This demonstrates the necessity to simultaneously increase the conversion of xylose to xylulose and the metabolic steps downstream of xylulose for efficient xylose utilization in S. cerevisiae.