【作者】 何江；

【导师】 毛忠贵；

【作者基本信息】 江南大学 ， 发酵工程， 2012， 硕士

【摘要】 含有大量木质纤维素的木薯渣是木薯酒糟厌氧消化过程中的主要底物,它的不溶性和复杂性使其难于降解,因此木薯渣的高效水解是提高木薯酒糟厌氧发酵性能和产甲烷效率的关键。近年来,随着纤维质底物协同降解机理的研究,开发高效木质纤维素分解复合菌群对于木薯渣、甘蔗渣等工农业废弃物残渣向甲烷、氢气、酒精等高附加值产品转化具有重要的价值。本文通过底物限制性培养技术得到一组能够高效降解纤维质的功能菌群RXS,该菌群能够在40h内将滤纸和木薯渣降解明显,CMC酶和木聚糖酶活力分别为38.5U和90.2U。连续传代、-80℃冷冻保存半年和90℃高温处理20min后,该菌群对底物的降解效果和酶活力表达基本不变,表明该菌群稳定性良好;利用PCR-DGGE技术分析不同代时的微生物结构并进行初步鉴定。结果发现条带基本不变,一些兼性微生物（Beta proteobacterium HMD444）、厌氧微生物（Thermoanaerobacterium thermosaccharolyticum strain M18, Therm -anaerovibrio acidaminovorans DSM 6589, and Clostridium sp.strains LDC-8-c12, 5-8, CO6- 72, etc.）和不可培养细菌良好的共存于此体系中。对该菌群的酶活表达及其关键水解酶的酶学特性分析表明,该菌群分泌的胞外水解酶是一组包含木聚糖酶、CMC酶、β-葡萄糖苷酶、微晶纤维素酶、滤纸酶和果胶酶等的多酶复合体系;CMC酶和木聚糖酶的最适反应温度、pH和酶促反应时间分别为60℃、6.0和10min;温度和pH稳定性较好;同时发现Mn²⁺对两种酶具有激活作用,Fe²⁺、Cu²⁺和SDS具有抑制作用。而Mg²⁺对CMC酶有抑制作用,对木聚糖酶有促进作用。吐温-80、曲拉通-100、EDTA、K⁺、Ca²⁺、Li+、Ba²⁺等对CMC酶和木聚糖酶活力影响不明显。在该菌群应用于木薯渣的水解过程中,监测发现CMC酶、木聚糖酶、果胶酶等关键酶的酶活力分别在第2～3d达到最大值34.4、80.5和15.8U;经过10d的发酵后,木薯渣中的纤维素、半纤维素及木质素分别降解了79.8%、85.9%和19.4%,且木薯渣的失重高达61.5%;此外,代谢产物主要是乙酸、丁酸、己酸和甘油;而溶解性COD、总糖和总挥发酸的变化表明第2d时木薯渣的水解率最高。培养基及其培养条件对复合菌群发酵性能的影响分析表明,复合菌群能够更好的分解滤纸、棉花等纤维素;而以木薯渣为唯一碳源时,20g/L的添加量分解效率最佳;利用蛋白胨和酵母粉做氮源时的纤维素分解活性远高于硝酸铵、尿素等无机氮源;在发酵温度、接种体积分数、初始pH、转速分别为50⁶0℃、5¹0%、5.0⁹.0和75¹35r/min的范围内,菌群RXS对纤维质底物的分解能力较强。上述结果表明,该菌群能够有效地水解木薯燃料酒精生产过程中的废弃物—木薯渣,并有望用于木薯渣高效沼气发酵的前处理中。在有效体积为3L的水解反应器（CSTR）和13L的高温厌氧反应器（ASBR）中,考察了菌群RXS和高温厌氧出水循环耦合对木薯酒糟水解、产甲烷的影响。结果表明在木薯酒糟废液与高温消化液的比例为1:2、通气量为0.25vvm的水解条件下,经过24h的水解,出水TVFA（乙酸）含量由最初的0.89 g/L累积到3.57 g/L,水解液的累计甲烷产量相比处理0h时提高了17.7%,产气速率也明显升高。分析了不同OLR的木薯酒糟单相和两相甲烷发酵的产甲烷阶段的运行状况,结果表明单相厌氧发酵时,OLR在12.0gCOD.L-1.d-1的范围内,COD去除率在70%左右,TVFA/Talk在0.37附近波动,比产甲烷速率为0.115L CH4/g COD。两相发酵时,OLR在20.0gCOD.L-1.d-1范围内,COD去除率在75%左右,TVFA/Talk在0.20以下,比产甲烷速率为0.128L CH4/g COD。而高于这些OLR时,出水出现酸化现象,反应器运行不正常。这些结果表明通过纤维质水解功能菌群强化木薯渣水解过程的两相沼气发酵工艺,可以提高甲烷产量,并且使产甲烷阶段高效稳定运行,出水中可能含有更少的酒精发酵抑制物质。更多还原

【Abstract】 Cassava residues composed by vast lignocellulose are the main substrates during the anaerobic digestion of cassava alcohol distillation wastewater. Because of their insolubility and complexity, they are extremely difficult to be degraded, so hydrolysis of cassava residues efficiently is the crucial step to improve the performance of anaerobic digestion and methane production rates. With the research of mechanism about cellulose degradation by synergistic relationships recently, study on complex microbial community with capacity of degrading lignocellulosic agro-industrial residues such as cassava residues and sugarcane bagasse is a promising approach which can provide efficient biomass conversion to value-added products,such as methane, hydrogen, alcohol.In this study, a microbial consortium with high effective and stable cellulosic degradation ability was constructed by successive enrichment and incubation using cassava residues and filter paper as carbon sources, where the substrates in the culture could be evidently broken down, otherwise, CMCase and xylanase activity was 38.5U and 90.2U after incubated 40 hours. This consortium could remain a stable degrading ability and high enzymatic activity after subcultured 60 generations, preserved in -80℃for half a year and treated 20min under conditions of high temperature below 90℃. what’s more, PCR-DGGE technique analysis showed that the composition of this microbial complex remained stable after subcultured for several times. Moreover, some aerobic/facultative anaerobic （Beta-proteobacterium HMD444） , strictly anaerobic bacteria （Thermoanaerobacterium thermosaccharolyticum strain M18, Thermanaerovibrio acidaminovorans DSM 6589, and Clostridium sp. strains LDC-8-c12, 5-8, CO6-72, etc.） coexisted in this constructed microbial consortium. In addition, a few unidentified uncultured bacteria also stably coexisted in this consortium.The excreted extracellular enzymes of this microbial community are a group of enzyme complex community including Xylanase, CMCase,β-glucosidase, Avicelase, FPA and Pectinase. Besides, the basic enzymatic activity characteristics of CMCase and Xylanase were analysed. Both were optimally active at 60℃and pH 6.0; Both was remained 80% original activity at a temperature between 20℃and 70℃,while retained at least 70% original activity for 60min in the pH range from 5.0⁹.0; the presence of Mn²⁺ positively influenced both of activity, but the activity was greatly inhibited in the presence of Cu²⁺, Fe²⁺ and SDS, while Mg²⁺ have inhibition effect to CMCase but positively influence to xylanase; Meanwhile, tween-80, triton-100, EDTA, K+, Ca²⁺ have no difference to enzyme activity.During the degradation process of cassava residues, the key hydrolytic enzymes including CMCase, xylanase and pectinase showed a maximum enzyme activity of 34.4, 90.5 and 15.8U on the second or third day, respectively. After 10 days’ fermentation, the degradation ratio of cellulose, hemicellulose and lignin of cassava residues was 79.8%, 85.9% and 19.4% respectively, meanwhile the loss ratio of cassava residues reached 61.5%. Otherwise,it was found that the dominant metabolites are acetic acid, butyric acid, caproic acid and glycerol, and the highest hydrolysis ratio was obtained on the second day by monitoring sCOD, total volatile fatty acids and total sugars. In addition, effect of culture mediums and cultural conditions on the fermentation capacity of complex microbial community bred by laboratory was investigated. The results showed that composite microbial system could directly degrade carbon source with high natural cellulose content （such as filter paper and cotton） effectively. When the addition amount of cassava residues as the sole carbon source was 20g/L, cellulase and hemicellulase showed a maxmium activity. what’s more, state of hydrolysis using peptone and yeast powder as nitrogen source was higher than that of inorganic nitrogen source such as urea and ammonium nitrate. The optimum fermentation temperature, inoculum concentration, pH and rotate speed were 50⁶0℃, 5¹0%, 5.0⁹.0 and 75¹35r/min. The above results revealed that this consortium can efficitvely hydrolyze cassava residues and has great potential to be utilized for the pretreatment of cassava residues for biogas fermentation.Effect of coupled microbial consortium and anaerobic effluent circulation on hydrolysis and methanogenic of cassava distillage were studied in hydrolytic-acidogenic reactor with a valid volume 3L and methanogenic reactor with a effective volume of 13L. The results demonstrated that the maximum total volatile fatty acid concentration in the hydrohynates was accumulated from 0.89g/L to 3.57g/L after treated 24h under conditions of 1:2（the ratio of cassava residues and effluent） and 0.25vvm（The volume of aeration）. The total methane production volume was improved by 17.7% compared to untreated. What’s more, the biogas production rate was rose greatly. Methagenic of cassava distillations untreated and treated in ASBR, the results showed that the methanogenic reactor operated normally for OLRs lower than 12.0gCOD.L-1 during single fermentation. This behaviour was shown by the total volatile fatty acids/total alkalinity ratio, whose values were always kept lower than 0.37 and the total COD removal rate was kept around 70%. A methane yield of 0.119L CH4 g-1COD eliminated was achieved. But while operated as two phase biogas digestion, The methanogenic reactor operated with high stability for OLRs lower than 20.0 gCOD.L-1, this behaviour was shown by the volatile fatty acids/total alkalinity ratio, whose values were always kept lower than 0.12. The total COD removal rate was kept around 75%. A methane yield of 0.128L CH4 g-1COD eliminated was achieved. Once higher this OLR, the effluent acided soon and the reactor operated failure. All these results demonstrated that the integrated digestion process promoted the biotransformation of cassava residues to biogas from the high-rate hydrolytic-acidification phase to the methanogenic phase and ultimately make the methanogenic digestion process operating much more stable. More importantly, less inhibitor of alcoholic fermentation produced in this system.更多还原