【作者】 孙宇；

【导师】 王彦杰； 王伟东；

【作者基本信息】 黑龙江八一农垦大学 ， 生物化学与分子生物学， 2013， 硕士

【摘要】 为提高牛粪厌氧发酵的甲烷产量，利用两相厌氧发酵技术研究牛粪酸化处理中的酸化时间、搅拌频率、料液浓度和氮素添加量等条件对产甲烷量的影响。在单因素试验基础上选取酸化时间、料液浓度、氮素添加量为自变量，以甲烷总产量为响应值，采用三因素五水平的响应面分析法建立酸化处理条件对甲烷产量影响的回归模型，并进行显著性和交互作用分析。利用16S rDNA克隆文库技术分析了酸化处理前后细菌的多样性。利用全混式固定床作为厌氧发酵反应器，以酸化处理后的牛粪为原料进行厌氧发酵，研究不同载体材料对厌氧发酵甲烷产量和化学需氧量(chemical oxygen demand,COD)去除率的影响。同时以筛选所得材料为固定床载体，研究了固定床运行参数对厌氧发酵的影响。酸化处理单因素试验结果表明最佳的酸化时间、搅拌频率、料液浓度、氮素添加量分别为96.0h、3times/24h (60r/min，每次1min)、8.0%、0.58g/L。响应面分析结果表明在35℃、搅拌频率为3times/24h(60r/min，每次1min)酸化条件下，对甲烷产量影响效应顺序依次为：酸化料液浓度>氮素添加量>酸化时间；优化所得酸化处理条件为：酸化时间93.7h、料液浓度8.3%、氮素添加量0.58g/L。在优化酸化处理条件下的沼气产量、甲烷含量、VS去除率和COD去除率分别提高了44.6%、14.3%、41.7%和33.9%；挥发性有机酸(volatile fatty acid, VFA)含量提高了1.1倍，其中乙酸含量提高了1倍；同时对纤维素、半纤维素和木质素的降解率均有不同程度的提高。酸化处理前后料液微生物多样性的分析结果表明，酸化处理前的细菌主要包括专性厌氧的拟杆菌属(Bacteroidetes)、兼性厌氧的肠杆菌属(Enterobacter)、好氧的分散泛菌(Pantoea dispersa)、寡养单胞菌属(Stenotrophomonas)以及一部分未培养菌(Uncultured bacterium)。酸化处理后的细菌主要包括拟杆菌属(Bacteroides)、毛螺菌属(Lachnospiraceae)、梭菌属(Clostridiales)、理研菌属(Rikenellaceae)和瘤胃细菌(Uncultured rumen bacterium)以及一部分未培养菌(Uncultured bacterium)。固定床载体材料的筛选试验结果表明，在竹纤维、碳纤维以及塑胶网等材料中，使用碳纤维薄膜作为固定床载体具有高的甲烷产量和COD去除率，与对照相比，沼气产量、甲烷产量和COD去除率分别提高了12.1%、15.6%和17.5%。以碳纤维薄膜为固定床载体材料对酸化处理的牛粪进行厌氧发酵，通过对固定床搅拌间隔、进料浓度、运行温度以及水力停留时间等参数的单因素试验，根据对甲烷产量和COD去除率显著性的影响，分析得出在最佳的机械搅拌间隔为24h、最佳的料液浓度为8%、最佳运行温度35℃、最佳HRT12d。本试验通过对酸化处理条件的优化、固定床载体材料筛选以及固定床运行参数的研究得出一套以牛粪为发酵原料全混式固定床两相厌氧发酵工艺。该工艺有利于甲烷的产生，可提高甲烷含量及去除率。更多还原

【Abstract】 In order to improve the production of methane of anaerobic fermentation of the cow dung, the effect ofacidizing time, stirring frequency, materials density and nitrogen content of acidizing of cow dung on theproduction of methane was studied by the two phase anaerobic fermentation technology. Base on single factorexperiment, using acidizing time, materials density and nitrogen content as independent variable, andproduction of methane as response values, the regression model of the effect of acidulation on production ofmethane was built by response surface analysis of three factors and five levels, then the significant differenceand interaction were analyzed. This method could be used on experimental analysis according to theverification test, and the production of methane and removal rate of COD could be improved under theoptimization condition. The microbial diversity was investigated using the methods of plating and16S rDNAclone technology. Using the completly mixed fixed-bed as the reactor of anaerobic fermentation, cow dungthrough acidulation as raw material, and By the production of methane t as index,carrier material of fixed-bedwas screened,and the operating parameter of fixed bed reactor was optimized. The main results were asfollows:According to the result of single factor of acidulation, the optimum acidizing time, stirring frequency,materials density and nitrogen content was96h,3times/24h,8.0%and1.28g/L respectively. Under thecondition of acidulation that35℃of temperature,3times/24h of stirring frequency, acidificationconcentration had the greatest impact on the production of methane,the following was addition of nitrogen, andthe minima was acidification time, through the analysis by response surface analysis of three factors and fivelevels. The optimum conditions of acidulation were93.7h of acidizing time,8.3%of materials density,0.58g/L of nitrogen content,under this condintion, the methane production, biogas production, removal rate of VSand COD were increased by14.3%、44.6%、41.7%and33.9%respectively, however there was non-significantimpact on the change of content of cellulose, hemicellulose and lignose under acidulation.The microbial diversity of cow dung-liquor of control and acidulation was analyzed by16S rDNA clonetechnology. The acid-forming bacteria of control was composed with Bacteroidetes, Enterobacter, Pantoeadispersa and Uncultured bacterium. The acid-forming bacteria of acidulation had a high microbial diversity.The acid-forming bacteria of acidulation was composed with Uncultured rumen bacterium, which coulddecompose saccharides into VFA,and involved in body metabolism by gastric absorption, and Unculturedbacterium.49,which could improve the content of VFA in activated sludge.Using the completly mixed fixed-bed as the reactor of anaerobic fermentation, cow dung throughacidulation as raw material, The influence of carbon fiber, bamboo carbon fiber and plastic as fixed-bed carrieron the production of methane were researched. The optimal material was carbon fiber. Compared with thecontrol group,the biogas production, methane production and removal rate of COD were increased by12.1%、15.6%and17.5%respectively.Using the carbon fiber as fixed-bed carrier, and cow dung through acidulation, the anaerobic fermentationwas carried out.Through the single factor experiment of stirring frequency, materials density, runningtemperature and hydraulic retention time of static bed, the optimum stirring frequency, materials density,operating temperature and HRT were24h,8%,35℃and12d respectively,which were analyzed base on the influence of significance on the methane production and removal rate of COD.The results of the above research indicated that after acidulation,the microbial diversity of acid-formingbacteria was improved, which leaded to generation of organic acid produced through metabolism with differenttypes of saccharide,and the content of VFA utilized by methane bacteria was also improved,and it wasconducive to the generation of methane, thereby could increase the content of methane, and the removal rate ofCOD had also been improved.更多还原