【作者】 高灵芳；

【导师】 王爱杰；

【作者基本信息】 哈尔滨工业大学 ， 环境科学与工程， 2013， 博士

【摘要】 为了减轻国内能源产业对国外进口燃油的依赖，木质纤维素能源化产业对传统的石油信赖型能源模式的转型提供了一种可持续发展的能源发展模式。木质纤维素能源产业的发展将对我国的经济增长，国家能源安全及环境质量产生深远的影响。当前木质纤维素生物能源产业的已经发展到瓶颈阶段，如何高效地降解木质纤维素释放出高质量及产能潜力大的糖类进行后续生物能源的生产是木质纤维素能源化的限速步骤。木质纤维素传统的化学糖化模式使得糖化成本居高不下难以投入实际生产应用，而生物糖化模式基于其低能耗、环境友好、工艺简单等特性成为当前木质纤维素糖化研究前沿。本研究针对高效降解木质纤维素产糖的微生物，从微生物降解木质纤维素产糖特性出发，结合微生物糖化水解液的产能潜力分析，选取降解木质纤维素产糖能力较高的菌株作为研究对象，利用基因组、转录组及蛋白质组分析手段对该微生物降解木质纤维素的糖化机制进行全面的解析，从系统生物学的角度解析降解木质纤维素产糖及高木质纤维素降解酶活的内在联系。优化了中温条件下降解木质纤维素产糖的细菌Shigella flexneri G3降解纤维素的产糖条件并对G3降解木质纤维素产糖能力进行全面的解析。以0.3%的AVICEL为底物，经过60h降解，G3对AVICEL的降解率达到～75%，产物中糖的含量为375mg g^-1AVICEL，产糖速率达6.25mg g^-1Avicel h^-1，糖的组成为纤维二糖（50%）和葡萄糖（30%）。G3是中温条件下降解纤维素产糖效率最高的菌株。这也是关于志贺氏菌株能够降解纤维素产糖的首次报道。通过对青霉菌Penicillium expansum YT02降解木质纤维素产糖能力的研究，证实了YT02能够高效地解聚木质纤维素原料产糖。以里氏木霉Trichdermareesei ATCC24449为对照菌株，YT02降解典型的木质纤维素原料的半纤维素酶活性及β-葡萄糖苷酶活性为对照菌株T.reesei的酶活的3倍。YT02的粗酶液对木质纤维素的平均糖化能力达到0.665g g^-1底物，除了较商业的纯化的混合酶产品的糖化能力0.7g g^-1底物略低外，YT02粗酶液的糖化能力较目前所报道的各类真菌及酶液的糖化能力高。YT02不能降解木质素，因此不能有效地降解木质纤维素高的木材原料。采用“低木质素生物质诱导”方法极大地促进了高木质含量木质纤维素原料的糖化。这一方法简要概括为在对高木质素原料采用白腐真菌生物预处理时，在木材原料中添加不同比例的苜蓿，利用其丰富的氮源来维持白腐真菌的生长与代谢并诱导氧物酶（LDPs）的产生，达到快速启动白腐真菌对木质素原料的降解的目的。最终确定橡木原料与苜蓿混合比为5：1，以及松木与苜蓿的混合比为3：1的方式为最佳的生物预处理方式。通过改进以后，木材原料的糖化率较采用酸化汽爆的预处理方法的木材原料的糖化提高了40%。通过菌种复配策略探讨了生物糖化液的产能潜力，细菌—细菌复配策略中，高效纤维素糖化菌Shigella flexneri str. G3与纤维素产氢菌Clostridiumacetobutylicum X9，降解AVICEL产氢量达到1.3mol H2（mol glucose）1，纤维素的降解率较C. acetobutylicum X9单独培养培养提高了50%。菌种复配体系还提高木质纤维素生物制氢的底物利用范围，以天然木质纤维素为原料也能够达到较高的产氢量。真菌—真菌复配策略中，酵母菌Candida shehatae CBS5813直接利用木质纤维素经P. expanusm YT02二步升温糖化后得到的水解液产乙醇能力为0.11～0.21g g^-1干物质，达到乙醇理论产值的88.2%。通过比较模拟水解性的乙醇产量，发现木质纤维素水解液中的抑制物对产乙醇产量的影响可忽略，发酵液中没有显著的木糖醇积累现象。选取降解木质纤维素产糖能力较高的真菌YT02为研究对象，以基因组、转录组及蛋白质组技术为研究手段全面解析了YT02降解木质纤维素产糖机制。基因组研究发现YT02中碳水化合物活性酶（CAZymes）的高丰度与广谱性是YT02较T. reesei更能有效地降解木质纤维素最主要的原因。通过对YT02的糖代谢途径进行解析，发现YT02中葡萄糖代谢途径中己糖激酶HK、磷酸果糖激酶PFK^-1和丙酮酸激酶PK基因的低丰度及木糖代谢途径中编码木酮糖激酶（XK）基因的缺失导致的YT02对葡萄糖、木糖代谢利用率低是其对降解纤维素后发生糖“累积”的主要原因。转录组分析揭示了YT02中编码纤维素酶、半纤维素酶及果胶质酶的基因底物诱导下的动态表达差异调空了YT02降解不同木质纤维素原料时酶活性差异。YT02中编码木质纤维素降解酶的基因在复杂底物条件下的高表达水平以及YT02中糖转运蛋白及cellobiose/cellodextrin转运蛋白的高丰度是YT02能够高效地降解木质纤维素为低聚糖及单糖的主要原因。更多还原

【Abstract】 Bioenergy from lignocellulose offers a great clean sustainable alternative toconventional petroleum-based energy sources which can dramatically impactnational economic growth, national energy security and environmental quality.However, the development of lignocellulose-derived bioenergy is still in its infancy.One of the great challenges is to depolymerize plant materials and release highquality, high quantity sugars for biofuel production. New microbial catalysts foreffectively disrupting plant polymers and efficiently releasing sugars are urgentlyneeded. Therefore, the objective of this study is to obtain and characterize novelfungal strains capable of effectively degrading plant polymers and efficientlyproducing sugars for biofuel production.A novel Shigella strain （Shigella flexneri str. G3） showing high cellulolyticactivity under mesophilic, anaerobic conditions was characterized. The bacteriumdisplays effective production of glucose, cellobiose and other oligosaccharides fromcellulose (Avicel PH^-101) at the optimal conditions of40oC and pH6.5.Approximately75%of cellulose was hydrolyzed in the modified ATCC1191medium containing0.3%cellulose, and the oligosaccharide production yield andspecific production rate reached375mg g^-1Avicel and6.25mg g^-1Avicel h^-1after a60-hour incubation, respectively. To our knowledge, this represents the highestoligosaccharide yield and specific rate from cellulose for mesophilic bacterialmonocultures reported so far. The results demonstrate that S. flexneri G3iscapable of rapid conversion of cellulose to oligosaccharides, with potential biofuelapplications under mesophilic conditions.We have obtained a novel fungal strain, Penicillium sp. YT02, which is capableof effectively depolymerizing plant materials such as alfalfa，switchgrass, cornstover and wheat straw. The isolated strain can efficiently produce sugars from plantmaterials with the highest realized theoretical sugar yields reported, and is able toproduce30%more sugars than the control fungus, Trichoderma reesei. The averageefficient of saccharificaiton from varied lignocellulosic substrates was amount to0.665g g^-1substrates, the highest oligosaccharides producity from raw fungalenzyme. An novel bio-delignin approach was proposed that in the presence of low C: Nbiomass, such as alfalfa, an essential nutrient source for high-lignin biomass （high C:N）, may improve the decomposition of the high-lignin materials for furtherbioenergy process. In this study, microbial pretreatment with alfalfa woodybiomass mixture system by white-rot P. chrysosprium was able to degrade lignin inhard and soft wood materials and has the potential to be an energy-saving, low cost,simple, two lignocellulosic biomass process simultaneously, and environmentfriendly approach which can reduce the severity of chemical pretreatments. Abalance between lignin degradation and availability of carbohydrates indicates that50g L^-1of raw oak biomass and10g L^-1of alfalfa and50g L^-1of raw pine and15gL^-1of alfalfa were the most promising pretreatment, which improve thesaccharification ratio to30%compared with traditional pretreatment methods.Bioaugmented fermentation of cellulosic substrates to produce biohydrogenvia co-culture of isolated strains Shigella flexneri str. G3&Clostridiumacetobutylicum X9was investigated. The ability of the selected strains to effectivelyconvert different cellulosic substrates to hydrogen was tested on carboxymethylcellulose （AVICEL）, as well as pretreated lignocellulosic material such as Bermudagrass, corn stover, rice straw, and corn cob. Results showed that co-culture ofShigella flexneri str G3and Clostridium acetobutylicum X9efficiently improvedcellulose hydrolysis and subsequent hydrogen production from carboxymethylcellulose. Hydrogen production yield approximately reached1.3mol H2（mol glucose）1, compared to0.32mol H2（mol glucose）1of the X9single culture,while the cellulose degradation efficiency increased by50%. Co-culture alsoefficiently improved hydrogen production from natural lignocellulosic materials（which was even70%-80%higher than mono-culture with X9）, and the highestperformance of24.8mmol L^-1was obtained on Bermuda grass. The resultsdemonstrate that co-culture of S. flexneri&G3&C. acetobutylicum X9was capableto efficiently enhance cellulose conversion to hydrogen, thus fostering potentialbiofuel applications under mesophilic conditions.In addition, the produced sugars were further examined for producing ethanolcoupled with a yeast strain. In this study, with alfalfa and other lignocellulosicsubstrates （except oak tree）, the ethanol yields ranged from0.11to0.21g ethanol gbiomass^-1after24hrs of fermentation, which corresponds to88.2%of the maximal theoretical value. To the best of our knowledge, this could be the highest efficiencyof ethanol production reported for monocultures of yeast with lignocellulosichydrolysate. These results suggested that the novel fungal isolate, YT02, can serveas an effective microbial catalyst for cellulosic ethanol production, and could be agood source of new enzymes for various industrial applications.In order to understand the mechanism that why YT02could produce highactivity enzymes, we sequenced its whole genome sequence and researched itsgenes expression profile of YT02. Consistent with the high saccharificationefficiency, the genome of YT02encodes more carbohydrate-active enzymes（CAZymes） than T. reesei. Less gene numbers encoding HK, PFK-1and PK in EMPpathway and The absence of endocing genes XK in predicted xylose utilizationpathway of YT02was the main reaon why oligosaccharides were “accumulated”during lignocellulosic saccharificaiton. RNA-seq data revealed the abundance ofxylanase and their active expression might attribute to the significantly higherxylanase activity of YT02than T. reesei. The expression of CAZymes genes wassignificantly induced by lingocellulose. Our analysis, coupled with the genomesequence data and RNA-seq data, provides a roadmap for further developingenhanced P. expansum strains for industrial applications such as biofuel production.更多还原