【作者】 刘艳；

【导师】 曲音波；

【作者基本信息】 山东大学 ， 微生物学， 2008， 博士

【摘要】 木质纤维素是最丰富的可再生资源和能够转化成燃料的底物,将纤维质的生物量转化为可再生能源具有重大的意义。由于纤维素具有高度有序、紧密结合的晶体结构,又与半纤维素相联结,外周还由木质素包围,要把它直接水解成可利用的葡萄糖相当困难。目前对木质纤维素酶解多使用来源于丝状真菌的纤维素酶,但由于酶降解效率低下,酶解过程受产物抑制,酶蛋白不耐高温等,制约了其大规模应用。某些纤维素降解菌,如热纤梭菌具有高效的纤维素降解复合体——纤维小体,能有效降解纤维素,具有直接把生物质转化为乙醇和H₂的能力,为利用一种微生物直接转化生物质的设想提供了可能。但在利用热纤梭菌进行乙醇发酵过程中往往伴有乙酸和丁酸等副产物的产生,而且最终产物的最高积累浓度仅为3%左右,只及酿酒酵母与发酵单胞菌属的25～30%。利用DNA重组技术重新设计糖酵解途径,阻断副产物的形成路线,解除乙醇生物合成的代谢阻遏作用,同时提高细胞对高浓度乙醇的耐受性,是高产乙醇的梭菌属工程菌构建的主要内容。高温厌氧菌还具有产H₂的能力。氢气具有热值高,无污染的特点。生物产氢包括光合产氢和发酵产氢。发酵产氢需要设备简单,不需光照,但H₂的摩尔产量低和原材料价格高仍是未解决的问题。发酵产氢只能利用有机物降解的15%能量。因此提高H₂的产量将是今后研究的重点。另外,也可利用两步法,将发酵产氢与光合产氢或与产甲烷结合,提高对生物质的利用效率。使用纤维素或废水产氢,可降低原料的成本。本论文通过研究嗜热厌氧纤维素降解菌的降解特性,对其纤维素酶系和调控机制有更深入的了解,同时可利用其能够转化纤维素生成氢气的特性,研究纤维素发酵产氢的可行性。本论文的主要研究内容如下:1.嗜热厌氧纤维素降解菌的筛选与鉴定以纤维素为唯一碳源,对嗜热厌氧纤维素降解菌进行富集。采用纤维素粉和纤维二糖滚管法对富集后的菌群进一步纯化,得到纤维素降解菌JN4和其伴生菌GD17。JN4能有效降解微晶纤维素和滤纸,也可利用玉米芯粉、玉米秸粉、木糖渣等天然底物作为碳源生长,GD17在富集液中数量较多,能利用纤维二糖、葡萄糖和木糖生长。通过形态观察,生理生化特性和16S rDNA分析进行鉴定,确定JN4为热纤梭菌（Clostridium thermocellum）,GD17为热解糖高温厌氧杆菌（Thermoanaerobacterium thermosaccharolyticum）。2.C.thermocellum JN4和伴生菌T.thermosaccharolyticum GD17的生长和产氢特性以纤维二糖为碳源,确定了C.thermocellum JN4和T.thermosaccharolyticumGD17的最适生长温度为55～60℃,并进行了生长曲线的测定。利用GC和HPLC对发酵产物的分析表明,C.thermocellum JN4分解纤维素生成H₂、乙酸、乙醇和乳酸,T.thermosaccharolyticum GD17可利用各种还原糖生成乙酸、乙醇、乳酸和丁酸。C.thermocellum JN4可降解微晶纤维素、滤纸,未经预处理的玉米秸粉和玉米芯粉。对C.thermocellum JN4的胞外蛋白酶活测定表明,C.thermocellum JN4以微晶纤维素为碳源生长时,发酵液具有CMC酶活、磷酸膨胀纤维素酶活和微晶纤维素酶活。以纤维二糖为碳源时,C.thermocellum JN4的H₂产率(Y_H2)约为0.7mol H₂/mol二糖;以纤维二糖、葡萄糖和木糖为碳源时,T.thermosaccharolyticum GD17的H₂产率(Y_H2)分别为4mol H₂/mol纤维二糖,2.2mol H2/mol葡萄糖和1.7mol H₂/mol木糖。3.C.thermocellum JN4单独培养和与T.thermosaccharolyticum GD17混合培养利用纤维素生长和产氢的动力学研究以微晶纤维素为碳源,研究了厌氧管内C.thermocellum JN4单独培养和与T.thermosaccharolyticum GD17混合培养的生长和产氢动力学。结果表明单独培养C.thermocellum JN4可有效降解0.5%微晶纤维素,生长后期有纤维二糖、葡萄糖的积累,乳酸、乙酸和乙醇的量逐渐增加,到60 h达到稳定,产H₂能力为0.8 mol H₂/mol葡萄糖。加入伴生菌T.thermosaccharolyticum GD17,与Cthermocellum JN4共同培养,可将C.thermocellum JN4产生的纤维二糖和葡萄糖完全利用,乙酸和乙醇产量与C.thermocellum JN4单独接种相比变化不大,有端岵?并在60 h后仍有增长,乳酸产生在60 h之后开始下降,最终完全检测不到,而混合菌的产H₂能力提高到1.8 mol H₂/mol葡萄糖。4.2L反应器中C.thermocellum JN4单独培养和与T.thermosaccharolyticumGD17混合培养利用纤维素的生长和产氢情况用自制的2L厌氧反应器在不控制pH的情况下,以滤纸为碳源单独培养C.thermocellum JN4,产氢量约为18mmol/1培养基,其它发酵产物有乙酸、乳酸和乙醇,其中乙醇的含量较高,达到24 mmol/1培养基以上,显示该菌有改造成为纤维素乙醇生产菌的可能。与用厌氧管培养不同,发酵液中没检测到纤维二糖和葡萄糖,表明扩大培养较有利于JN4的生长和代谢。C.thermocellum JN4与T.thermosaccharolyticum GD17混合培养,在不控制pH的情况下,产氢量约为35mmol/1培养基,其它发酵产物有乙酸、乳酸、丁酸和乙醇。乳酸含量在50h到达最高,然后逐渐减少,同时丁酸含量逐渐增加,与厌氧管培养结果相似。控制pH在pH6.0-6.5,产氢量约为25mmol/1培养基,比不控制pH要少。其它发酵产物中,乳酸含量最高,这表明pH在6.0-6.5的情况下,更有利于乳酸的生成,造成产氢量的减少。如果延长培养时间,T.thermosaccharolyticum GD17可利用乳酸,会有更多的H₂产生。5.C.thermocellum JN4外切葡聚糖酶基因celS的原核表达以C.thermocellum JN4的基因组为模板,以C.thermocellum ATCC 27405的celS基因为参考设计引物,PCR扩增得到的序列与Genbank的已知序列进行比对,结果表明扩增获得的片段为celS基因,且与Clostridium thermocellum ATCC27405的celS基因仅有3个碱基的差异。将C.thermocellum JN4的celS基因和质粒pET-22b连接,构建表达载体,并利用大肠杆菌BL21（DE3）进行CelS的异源表达,得到的重组蛋白rCelS具有水解磷酸膨胀纤维素的能力。6.C.thermocellum JN4外切葡聚糖酶基因CelS的定点突变结合蛋白质结构生物学信息,对C.thermocellum JN4的rCelS蛋白催化中心的相关位点进行D255N和Y351F定点突变和原核表达,结果表明定点突变后的两种rCelS蛋白均没有水解磷酸膨胀纤维素的能力。表明Asp255有可能作为广义碱基团在外切葡聚糖酶CelS的催化反应中与广义酸基团Glu87进行翻转型催化反应。Tyr351是CelS的保守位点,将其变成Phe可能对CelS的结构与催化功能造成影响。更多还原

【Abstract】 Cellulose is the most abundant renewable resource that can be turned into fuel. Effective utilization of cellulose effectively is of great significance.However. cellulose is very difficult to hydrolyze because of lack of cellulases with high activities,which has limited its uses.At present,cellulases are mainly from fungi. Some anaerobic bacteria such as Clostridium thermocellum produce cellulases which can hydrolyze cellulose effectively.C.thermocellum can produce ethanol and H₂. However,the use of C.thermocellum for ethanol fermentation often has organic acids as by-products.The highest concentration of ethanol is only about 3%. Recombinant DNA technology was used to re-design the glycolysis pathway to block the synthetic pathway of by-products and to improve the cell tolerance to ethanol.Hydrogen is a clean and sustainable energy carrier for the future because of its high conversion efficiency and nonpolluting nature when used in fuel cells.Anaerobic fermentation has been demonstrated as a technically feasible way to produce hydrogen.Some anaerobic bacteria can use for H₂ production.However.the cost of hydrogen production and low hydrogen yield could limit its widespread application as an energy source in the future.Cellulose is the promising economical source for hydrogen production.Hydrogen production by fermentation can use only 15% energy of the organic matter.Therefore,to improve the H₂ production is becoming the focus of energy research.Combination of hydrogen production by fermentation with photosynthetic hydrogen production or methane production can improve the efficiency of the use of biomass.Thermophilic anaerobic bacteria can effectively utilize cellulose and have great potential for H₂ production.The study of cellulose degradation characteristics of thermophilic anaerobic bacteria can help us have a better understanding of their cellulases and regulation mechanisms.Furthermore,we can make use of cellulose fermentation to product hydrogen.The main results of the thesis are as follows: 1.Screening and identification of thermophilic anaerobic bacteriaUsing cellulose as the sole carbon source,the thermophilic anaerobic bacteria that can utilize cellulose were enriched from rotten wheat straw.Single colonies were isolated by using agar roll tubes containing cellobiose.A cellulose-degrading bacterium JN4 was obtained and identified as C.thermocellum by 16S rDNA analysis and morphological observation.Another bacterium that was the most abundant in the sample was isolated and identified as Thermoanaerobacterium thermosaccharolyticum.2.Growth and H₂ production properties of C.thermocellum and T. thermosaccharolyticumThe optimal growth temperature of C.thermocellum JN4 and T. thermosaccharolyticum GD 17 was 60℃by using cellobiose as carbon source.The growth curves of the two strains were determined.GC and HPLC analysis showed that the fermentation products of C.thermocellum JN4 were H₂,acetate,lactate and ethanol using cellulose as carbon source.C.thermocellum JN4 could utilize microcrystalline cellulose,filter paper and several kinds of natural substrates such as corn cob powder and corn stalk powder.The extracellular proteins of C. thermocellum JN4 showed CMCase,PASCase and cellobiohydrolase activity when grown on microcrystalline cellulose.The fermentation products of T. thermosaccharolyticum GD17 were H₂,acetate,butyrate and ethanol using reducing sugar as carbon source.The production of hydrogen by both strains was investigated. The hydrogen yield of C.thermocellum was 0.7mol H₂（mol cellobiose）^-1.The hydrogen yield of T.thermosaccharolyticum GD17 was 4mol H₂（mol cellobiose）_-1, 2.2 tool H₂（mol glucose）^-1 and 1.7mol H₂（mol xylose）^-1.3.The growth kinetics and hydrogen production of C.thermocellum JN4 and co-cultures of C.thermocellum JN4 and T.thermosaccharolyticum GD17 using microcrystalline cellulose as carbon source Using microcrystalline cellulose as carbon source and Hungate tubes,batch fermentation was performed to investigate the growth characteristics and H₂ production of C.thermocellum JN4 cultures and co-cultures of C.thermocellum JN4 and T.thermosaccharolyticum GD17.The results showed that C.thermocellum JN4 can degrade microcrystalline cellulose to produce hydrogen,ethanol,acetate and lactate,but cannot completely utilize cellobiose and glucose produced by the cellulose degradation.Its hydrogen yield was about 0.8 mol H₂（mol glucose）^-1,with lactate as the main product.When C.thermocellum JN4 was co-cultured with T. thermosaccharolyticum GD17,hydrogen production increased about 2-fold and H₂ yield reached a high level of 1.8 mol H₂（mol glucose）^-1.Butyrate was the most abundant byproduct and lactate was not detected at the end of the co-culture process.4.The growth and H₂ production of C.thermocellum JN4 and co-cultures of C. thermocellum JN4 and T.thermosaccharolyticum GD17 in 2L anaerobic reactorUsing filter paper as carbon source in 2L anaerobic reactor.C.thermocellum JN4 can produce 18mmol H₂/1 culture.Other fermentation products are ethanol,acetic acetate and lactate.The higher ethanol content of 24 mmol/1 culture indicated that the strain had the potential of cellulosic ethanol production.Differences from cultures in Hungate tubes,cellobiose and glucose were not detected.That means that the scale-up of cultivation may benefit the growth and metabolism of C thermocellum JN4.When C thermocellum JN4 was co-cultured with T. thermosaccharolyticum GD17 without pH control,the H₂ production increased to 35mmol/1 culture.Other fermentation products are ethanol,acetate,lactate and butyrate.Lactate increased until 50h and then decreased.At the same time, butyrate increased,which was similar to the cultures in Hungate tubes.When C. thermocellum JN4 was co-cultured with T.thermosaccharolyticum GD17 with pH control at pH 6.0～6.5,the H₂ production was 25mmol/l culture.Lactate was the predominant fermentation product.Because no H₂ was produced in lactate fermentation,the H₂ production decreased. 5.Cloning and expression of Clostridium thermocellum JN4 celS gene in Escherichia coliThe gene was cloned by using the genome of Clostridium thermocellum JN4 as template.The primers were designed according to C.thermocellum ATCC 27405 celS gene.The nucleotide sequence data were submitted to GenBank.There were only 3 bases’ difference between the submitted sequence and C.thermocellum ATCC 27405 celS gene.So the fragment we cloned should be celS gene.The PCR-amplified gene fragment,following restriction-enzyme digestions,was ligated with the similarly digested plasmid pET-22b.The ligated plasmid was introduced into E.coli BL21（DE3）.The recombinant protein rCelS had PASCase activity. But the activity was low.6.Site-directed mutagenesis of Clostridium thermocellum JN4 celS gene and expression in Escherichia coliAccording to protein structural information,two sites were site-directed mutagenesis.The one was D255N and the other was Y351F.The sites were chosed because they were key sites of catalytic function.The mutations were expressed in Escherichia coli but neither of them had PASCase activity.So we speculated that Asp255 may be the base group of the catalytic center.Tyr351 is a conservative site and the change to Phe may impact the structure of CelS catalytic center.更多还原