发酵五碳糖和六碳糖产乙醇染色体整合大肠杆菌的构建

为了保证外源基因表达的稳定性,减少发酵副产物的生成,根据同源重组原理,将大肠杆菌丙酮酸甲酸裂解酶pfl基因两侧的基因片段作为同源片断,构建了带有运动发酵单胞菌丙酮酸脱羧酶基因pdc和乙醇脱氢酶基因adhB的整合重组质粒PA-pfl,用化学法转入大肠杆菌TOP10的感受态细胞,将经过氨苄青霉素筛选得到的重组子进行PCR扩增,证明pdc和adhB基因整合到了大肠杆菌的染色体基因组丙酮酸甲酸裂解酶基因pfl位点上.乙醇发酵结果表明,重组菌E.coli TOP10-pfl不但能稳定地利用葡萄糖产乙醇,也能稳定地利用木糖产乙醇,在大肠杆菌中建立了一条新的代谢糖生成乙醇的途径.     

嗜鞣管囊酵母(Pachysolen tannophilus 1622)和重组大肠杆菌(E.coli(pGM-PA))乙醇发酵特性研究

通过对嗜鞣管囊酵母和重组大肠杆菌代谢木糖及混合糖产乙醇研究,阐明两种菌的不同发酵能力.结果表明:重组大肠杆菌在5%木糖培养基中乙醇产率为理论值的100%,在10%木糖的培养基中乙醇产率达到理论值的96%.重组大肠杆菌木糖转化乙醇的能力明显高于嗜鞣管囊酵母,但嗜鞣管囊酵母与重组大肠杆菌相比乙醇耐受度更高(可达8%),且代谢过程中pH降低幅度不大.固定化技术对于提高P. tannophilus 1622乙醇发酵表现作用显著,而对重组大肠杆菌乙醇发酵的产率提高没有明显作用.     

固定化运动发酵单胞菌乙醇发酵研究

以运动发酵单胞菌(zymomonas mobilis 10225)为菌种。以葡萄糖为底物进行乙醇发酵。对不同底物浓度[5％，7．5％。10％(W／V)]、不同温度(25℃,30℃，35℃)条件下，游离细胞和固定化细胞乙醇发酵的特性进行研究。实验结果表明。葡萄糖浓度为5％。25℃时可达到最大的乙醇产率0．50g乙醇／g葡萄糖。以海藻酸钙为包埋介质，对Zymomonas mobilis进行固定化。在10％葡萄糖培养基中多批次半连续发酵，可在8h内使乙醇产率系数达到0．50。短的发酵周期和高的乙醇产率为后续的葡萄糖和木糖两步乙醇发酵提供理想的实验数据。     

酵母菌复合培养物对木质纤维素稀酸水解液原位脱毒乙醇发酵

为了解决木质纤维素稀酸水解产物中发酵抑制剂对微生物的抑制作用以及木糖的乙醇发酵问题,该研究用本实验室开发的能高效代谢葡萄糖产乙醇并代谢糠醛和5-羟甲基糠醛的2株酵母菌种Saccharomyces cerevisiae Y5和Ismtchenkia/orientalis Y4分别与Pichia.stipitis CBS6054组成2个复合菌种,用复合菌种对木质纤维素稀酸水解产物进行原位脱毒乙醇发酵.结果证明,复合菌种S.cerevisiae Y5,P.stipitis CBS6054显示出了很好的代谢稀酸水解液中的葡萄糖和木糖产乙醇并快速代谢糠醛和5-羟甲基糠醛的能力,乙醇产率为0.43g/g(达到理论值的85.1%).该复合培养物可作为木质纤维索稀酸水解产物不需任何脱毒处理直接进行乙醇发酵的复合菌种.     

优良酵母菌的筛选、鉴定及发酵生产乙醇的研究

从几种可能富含发酵木糖-葡萄糖酵母菌的材料中分离得到68株酵母菌,分别对其发酵、产乙醇、耐乙醇、木糖利用及木糖-葡萄糖发酵能力进行研究,筛选出发酵木糖-葡萄糖能力较强的酵母菌株。通过镜检观察、耐渗透压、耐受pH值、同化碳源和氮源试验,研究筛选菌株的形态和生理特性,并通过26S rRNA序列测定及系统进化分析对其进行分子鉴定,最后利用木糖-葡萄糖发酵培养基进行产乙醇试验。结果表明:分离菌株中S3和Y4菌株发酵木糖-葡萄糖能力较强,经分子鉴定分别为异常毕赤酵母(Pichia anomala)和热带假丝酵母(Candida tropicalis)。菌株Y4和对照菌株酿酒酵母(Saccharomyces cerevisiae)(N)产乙醇能力显著高于菌株S3(P0.05),且菌株Y4和N间无显著差异(P0.05);而S3与N组合其乙醇产量显著高于菌株Y4,N,S3单独及Y4与N组合,但显著低于S3与Y4组合及S3,N,Y4三者组合(P0.05),其中三者组合的乙醇产量最高,为39.74 g/L。     

提高木糖代谢能力的酿酒酵母Y5-X3的初步构建

为获得能够发酵木糖和葡萄糖的新菌种,文中采用实验室专利菌株Saccharomyces cerevisiae Y5为宿主菌,分别采用热带假丝酵母(Candida tropicalis)和休哈塔假丝酵母(Candida shehatae)作为木糖代谢关键酶XYL1,XYL2的来源,利用酵母整合载体将XYL1,XYL2基因导入S.cerevisiae Y5基因组,同时超表达S.cerevisiae Y5内源木酮糖激酶基因XKS1,获得了酿酒酵母新菌种S.cerevisiae Y5-X3。结果表明,S.cere-visiae Y5-X3在摇瓶共发酵3%葡萄糖和2%木糖时能够利用木糖产乙醇,木糖消耗量和乙醇产量分别比宿主菌提高102.93%和12.54%,发酵2%木糖时木糖的消耗量是宿主菌的2.7倍。XKS1基因的超表达促进了木糖代谢,大大减少了木糖醇积累,提高了乙醇产量。     

生产纤维素乙醇的原生质体融合菌株的构建

为了获得耐发酵抑制剂、耐乙醇并利用木糖的纤维素乙醇生产菌种,以实验室保藏菌种Saccharomyces cerevisiae Y5和Pichia stipitis CBS6054为亲本,采用双亲灭活原生质体融合技术,选育出了共代谢葡萄糖和木糖,且耐受发酵抑制剂的酵母菌株Y10-F.该菌在以木糖为唯一碳源的培养基中培养96h,木糖的利用率达到58.8％,乙醇浓度为5.2g/L.在外加6.0、8.0g/L乙醇的培养液中,Y10-F的生长优于亲株.外加3.0g/L的糠醛时,Y10-F的延滞期较亲本Y5和CBS6054分别缩短了6h和18h.对融合菌株Y10-F进行了汽爆法预处理玉米秸秆的酶解液发酵实验,具有较好的耐毒和产乙醇能力.     

木质纤维素酒精发酵菌种的筛选

为对本质纤维素水解糖液进行酒精发酵，同时代谢葡萄糖和木糖成酒精的9个菌种以木糖为唯一碳源进行驯化，从中选出3个菌株。并对3个菌株进行固定化，进行酒精发酵研究，以进一步阐明该菌株代谢葡萄糖和木糖成酒精的能力。以总糖利用率、葡萄糖和木糖利用率、酒精产率为指标，3株菌具有较好的同时发酵葡萄糖和木糖成酒精的能力。Pachysolen tannophilis ATCC 32728的总糖(5％葡萄糖和2％木糖)利用率达到了85．08％(36h)，葡萄糖的利用率为99．21％(36h)，木糖利用率80．38％(36h)，菌株Cadida shehatae ATCC 34887的酒精产率达到理论值的82．91％。     

重组酿酒酵母发酵木糖产乙醇的研究进展

木糖发酵是利用木质纤维素原料制取乙醇商业化生产的基础和关键,但传统的乙醇生产菌株酿酒酵母(Saccharomyces cerevisiae)不能利用木糖,因而无法满足商业生产的需求.人们利用各种基因工程手段对S. cerevisiae实施基因改造,包括对木糖运输途径的改造,木糖代谢途径的引入,增强其对发酵抑制剂的耐受性等方面都得到广泛研究,各重组菌的木糖发酵能力有了不同程度的改善,但是仍然未能用于商业化生产.酿酒酵母的木糖代谢工程有待于进一步深入研究.     

利用木糖产乙醇的真菌筛选及发酵条件优化

以木糖为唯一碳源,从高、中温酒曲中分离到16株能利用木糖的丝状真菌;通过发酵试验复筛,获得一株能产乙醇的丝状真菌Z7;综合形态学和ITS序列分析,初步鉴定为Aspergillus flavus。通过单因素试验确定最佳氮源和发酵温度;通过正交试验和SPSS软件分析得到了不同N、P、K成分对乙醇、残糖和菌体干重的影响,获得最佳的发酵条件为:尿素1g/L,NH_4NO_3 1g/L,K_2 HPO_4 2g/L,KCl 0.5g/L,MgSO_4·7H_2O 0.5g/L,NaNO_3 1g/L,pH自然,培养温度33℃。以玉米芯半纤维素水解液为底物进行乙醇发酵,根据稀酸水解的单糖释放量和乙醇产量,确定115℃,1h为最佳玉米芯预处理条件;添加1 g/L的吐温20能获得最大的乙醇浓度8.31 g/L。因此,Aspergillus flavusZ7能利用半纤维素水解产物产乙醇,其中木糖的利用率在80%以上。     

Simultaneous hydrogen and ethanol production from a mixture of glucose and xylose using extreme thermophiles I: Effect of substrate and pH

The simultaneous hydrogen and ethanol production from glucose and xylose was investigated. The effect of carbon sources on hydrogen and ethanol production was examined in batches. When the substrate concentration was increased from 1 g/L to 7 g/L, the hydrogen yield decreased from 0.74 mol/mol to 0.15 mol/mol and from 0.67 mol/mol to 0.07 mol/mol for glucose and xylose. The highest ethanol yield of 1.19 mol/(mol·glucose) was obtained at 5 g/L glucose and 6 g/L xylose concentrations. For the co-fermentation of glucose and xylose, the highest ethanol yield 1.54 mol/(mol·hexose) was obtained at 2.5 g/L glucose to 2.5 g/L xylose (1:1). However, the hydrogen yield was not significantly affected by the glucose to xylose ratio. Continuous co-fermentation of glucose and xylose by extreme thermophiles was successfully demonstrated using an upflow anaerobic reactor. The hydrogen production rate, the ethanol concentration, and the substrate degradation efficiency increased along with pH. The optimal pH for the continuous mode was determined to be in the range of 5.8–6.6.     

Simultaneous glucose and xylose utilization for improved ethanol production from lignocellulosic biomass through SSFF with encapsulated yeast

Simultaneous glucose and xylose uptake was investigated for ethanol production using the simultaneous saccharification, filtration and fermentation (SSFF) process with pretreated wheat straw as a xylose-rich lignocellulosic biomass. A genetically engineered strain of Saccharomyces cerevisiae (T0936) with the ability to ferment xylose was used for the fermentations. SSFF was compared with a conventional method of simultaneous saccharification and fermentation (SSF) for glucose and xylose uptake, ethanol production, and cell viability on 10% and 12% suspended solids (SS) basis. With 10% SS, an ethanol yield of 90% of the theoretical level was obtained during SSFF with 80% xylose uptake while only 53% ethanol yield was observed during the SSF process. Increasing the solid load to 12% resulted in an ethanol yield of 77% of the theoretical value and 36% xylose uptake during SSFF while only 27% ethanol yield and no xylose uptake was observed during the corresponding SSF process. The SSFF process preserved the viability of the genetically engineered yeast throughout the fermentation, even when reused for 2 consecutive cultivations. The results show that the SSFF process does not only enhance effective cell performance but also facilitates simultaneous glucose and xylose utilization, which is important for broad range of biomass utilization for lignocellulosic ethanol production.     

稻壳粉水解液发酵生产燃料酒精的研究

在试验室内分离、筛选、驯化培养出一株高效木糖酒精发酵菌株g-13,采用该菌株发酵稻壳粉水解液,可以同时将水解液中的葡萄糖和木糖转化为酒精,酒精转化率为0.38g/g（酒精/消耗的糖）.该试验技术可操作性强,发酵条件易于控制.文章最后还对稻壳粉的水解条件进行了优化探索.     

Simultaneous saccharification and co-fermentation of whole wheat in integrated ethanol production

Two of the most important ways of reducing the production cost of lignocellulosic ethanol are to increase the ethanol yield and the concentration in the fermentation broth. This can be facilitated by co-fermentation of glucose and xylose from agricultural residues such as wheat straw, due to the high amount of xylose in the hemicelluloses in these materials.Simultaneous saccharification and co-fermentation (SSCF) of steam-pretreated wheat straw (SPWS) with and without the addition of liquefied wheat meal (LWM) was performed using the pentose-fermenting yeast, TMB3400. The highest overall ethanol yield in batch operation, of around 70%, equivalent to an ethanol concentration of 43.7 g L⁻¹, was achieved using SPWS with 7.5% water-insoluble solids (WIS) and addition of LWM with 1% WIS. Using SPWS with a higher WIS (10%) resulted in a decreased yield, 60%, although the concentration of ethanol increased to 53.0 g L⁻¹. SSCF of 7.5% straw was also performed with a single (after 20 h) or fed-batch addition of 1% WIS LWM (after 20, 24 and 28 h) resulting in an increase in both ethanol yield and concentration compared to the reference, without wheat meal addition, but no significant difference compared to the batch experiments.The addition of wheat meal to SSCF did not improve xylose utilization significantly, probably due to the instant release of glucose from the liquefied meal, which hampers the uptake of xylose. The instant release of glucose was shown to be caused by the high amylase activity of the β-glucosidase enzyme preparation.     

Cell growth and hydrogen production on the mixture of xylose and glucose using a novel strain of Clostridium sp. HR-1 isolated from cow dung compost

A novel mesophilic hydrogen-producing bacterium was isolated from cow dung compost and designated as Clostridium sp. HR-1 by 16S rRNA gene sequence. The optimum condition for hydrogen production by strain HR-1 was pH of 6.5, temperature of 37 °C and yeast extract as nitrogen sources. The strain HR-1 has the ability to utilize kinds of hexose and pentose as carbon sources for growth and H₂ production. Cell growth and hydrogen productivity were investigated for batch fermentation on media containing different ratios of xylose and glucose. Glucose was the preferred substrate in the glucose and xylose mixtures. The high glucose fraction had higher cell biomass production rate. The rate of glucose consumption was higher than xylose consumption, and remained essentially constant independent of xylose content of the mixture. The rate of xylose utilization was decreased with increasing of the glucose fraction. The average H₂ yield and specific H₂ production rates with xylose and glucose are 1.63 mol-H₂/mol xylose and 11.14-H₂ mmol/h g-cdw, and 2.02 mol-H₂/mol-glucose and 9.37 mmol-H₂/h g-cdw, respectively. Using the same initial substrate concentration, the maximum average H₂ yield and specific H₂ production rates with the mixtures of 9 g/l xylose and 3 g/l glucose was 2.01 mol-H₂/mol-mixed sugar and 12.56 mmol-H₂/h g-cdw, respectively. During the fermentation, the main soluble microbial products were ethanol and acetate which showed trends with the different ratios of xylose and glucose.     

Steam pretreatment of dilute H2SO4-impregnated wheat straw and SSF with low yeast and enzyme loadings for bioethanol production

Conversion of lignocellulosic material to monomeric sugars and finally ethanol must be performed at low cost, i.e. with limited consumption of chemicals, yeast and enzymes while still reaching high yields, if it is to compete with other fuel conversion processes. The objective of this study was thus to investigate ethanol production from steam-pretreated wheat straw by simultaneous saccharification and fermentation (SSF). The concentration of sulphuric acid in the impregnation liquid prior to pretreatment was kept low, 0.2%, and SSF was performed at low enzyme loadings, 3–14 FPU g⁻¹ water-insoluble solids (WIS), and a low yeast concentration, 2 g L⁻¹. The pretreatment conditions were optimised to give the highest overall glucose and xylose recovery after enzymatic hydrolysis of the residual WIS. The highest recovery of glucose (102%) and xylose (96%) was obtained after pretreatment at 190 °C for 10 min. Achieving high yields of glucose and xylose with the same pretreatment conditions is unusual and makes wheat straw a highly suitable raw material for bioethanol production. SSF was performed on whole slurry from straw pretreated under the optimal conditions. A high overall ethanol yield, 67% of the theoretical based on glucose in the raw material, was obtained.     

Improvement of hydrogen production by metabolic engineering of Escherichia coli: Modification on both the PTS system and central carbon metabolism

Lignocellulosic-based production of bio-hydrogen (H₂) by Escherichia coli requires efficient consumption of pentoses and hexoses. However, carbon catabolite repression (CCR) causes sequential utilization of carbohydrates and in some cases null consumption of less preferred carbohydrates, such as xylose. In this work, we evaluated the effect of elimination of the phosphotransferase system (PTS), responsible for CCR in strain E. coli WDH (ΔhycA) on H₂ production using mixtures of glucose-xylose as carbon source. Elimination of ptsG gene (glucose permease-enzyme IIB), allowed simultaneous consumption of glucose and xylose, and improved H₂ production 1.2-times with respect to the parenteral strain. Whereas, elimination of ptsG gene in combination with deletion of ldhA (d-lactate dehydrogenase) and/or frdD (fumarate reductase) genes, improved H₂ production 2.5-times with a H₂ yield of 0.27 mol·C-mol⁻¹, using mixtures of glucose/xylose or wheat straw hydrolysate. Interestingly, besides the improvement on H₂ production, E. coli WDH-GFA (ΔhycA, ΔptsG, ΔfrdD, ΔldhA) strain also produced ethanol as the main carbon by-product. These results show that elimination of ptsG, in combination with a modified central carbon metabolism improves the production of H₂.