【作者】 李平；

【导师】 王焰新；

【作者基本信息】 中国地质大学 ， 环境工程， 2009， 博士

【摘要】 我国是世界养猪第一大国，生猪产量超过全世界总量的一半。近年来，我国养猪业飞速发展，给环境带来的污染问题已经成为社会关注的热点之一。养猪废水具有机物浓度高、悬浮物多、臭味大、污染成分复杂、排放量大，各项指标常超出国家排放标准几倍甚至几十倍的特点。据估算，我国养猪业废水COD的排放量超过工业废水和生活污水的排放总和。一个万头的猪场COD的排放量相当于4-6万人口的排放量。UASB厌氧生物技术是目前我国养猪废水处理的关键技术。而现有的畜禽厌氧生物处理技术研究往往仅停留在研究脱氮、除磷、COD的去除效率和产气率等的工艺参数方面，而对废水中主要有机污染物的生物降解机理，降解效率与微生物群落结构的关系，不同工艺参数条件下微生物群落结构的动态变化以及人工强化生境下，外源微生物对处理系统中微生物群落结构和功能的影响等方面研究还比较缺乏。本研究将传统微生物培养和现代分子生物学技术相结合，通过梯度变性凝胶电泳（DGGE）、16S rDNA限制性内切酶消化文库分析（ARDRA）等分子生物学技术和细菌筛选、分离鉴定以及代谢机理研究，解析了养猪废水厌氧生物处理的快速启动和稳定运行前后微生物群落结构的演变和微生物多样性；分析了典型难降解有机污染物（包括含氮杂环化合物和纤维素）的生物降解机理；解析了人工强化生境中，外源菌和本土菌共同作用的降解效率，以及相应的微生物群落的变化，初步明确了养猪废水厌氧处理环节的重要功能种群，为优化微生物群落结构、调节群落功能提供了科学依据，为实现养猪废水厌氧生物高效处理、可控运行提供理论和实践依据。研究的主要成果如下：1．全面系统地分析养猪废水厌氧处理环节微生物群落结构演变和多样性采用两种不同的接种源，反应器A接种鄂州市某猪场排污口沉积底泥，反应器B接种武汉市某啤酒厂活性污泥，实现了反应器的快速启动。对比了不同接种源条件下，反应器的启动速率；活性污泥的生长速度以及微观结构；COD去除效率；产气率和微生物的活性。研究结果表明：两个反应器均能在36天内实现快速启动，反应器B的启动速率略高于反应器A的。反应器B的颗粒污泥的生长速度快于反应器A的。当进水浓度为3000-6000 mg L^-1时，反应器A和B在稳定运行期COD的去除率分别在84-88％和90-95％，产气量分别为5．5-10．5和9．0-14．0 L day^-1，从VSS和VSS／TSS值来看，反应器B的微生物活性略高于反应器A。两个反应器的VFA值均适合微生物的生长。通过DGGE分析发现，反应器在启动前后的微生物群落结构发生了很大变化，而在稳定运行期变化并不大。反应器A和B在启动前后微生物群落结构的相似性仅为59．2％和60．0%。而在稳定期，反应器A和B在两个月稳定运行之间微生物群落结构的相似性为78．5％和74．0％。并且在启动前，两个反应器之间的微生物群落结构相差较大，而在稳定运行期差异较小。在启动前，反应器A和B的微生物群落结构相似性为62．4％，在稳定运行期，两个反应器的微生物群落结构相似性为79．9％。这种微生物群落的结构变化与其功能的转变相对应。通过ARDRA分析反应器B稳定运行期的微生物群落多样性，获得346个阳性克隆，经酶切和测序分析，划分为55个分类单元，微生物群落的组成为厚壁菌门33％，酸杆菌门23％，变形菌门19％，拟杆菌门11％，硝化螺旋菌门8％，绿弯菌门4％和其它2％。16S rDNA克隆文库分析的结果表明，养猪废水UASB厌氧处理过程中的微生物群落结构和多样性与以往的其它养猪废水处理技术，包括膜处理法和厌氧贮存式处理过程中的有较大差异。2．解析了养猪废水中典型难降解污染物的生物降解机理含氮杂环化合物是养猪废水中典型的难降解有机污染物，也是养猪废水中主要的恶臭成分之一。它们由色氨酸在动物体内分解而产生。未被完全处理的该类化合物排放于环境中，导致了土壤、地表水和地下水的污染，对人体和动物健康造成了严重的威胁。吲哚和粪臭素可以被人体和动物吸收进入血液，经生物活化后产生有毒的中间产物而影响血液中复合胺的产生，甚至产生溶血现象。有研究表明，动物体内雌激素酮浓度的升高与粪臭素含量直接相关。另外，粪臭素还是一种肺炎球菌毒素。暴露在吲哚环境下的植物组织由于蒽醌合成的影响而表现出低色素沉着。吲哚和粪臭素同样对许多微生物存在毒性效应。它们对细菌均有相当广泛的生物抑制作用，同时对瘤胃纤毛微生物也有毒性作用。本文采用传统的微生物培养技术，从USAB厌氧反应器活性污泥中，通过直接涂布法筛选到一株能有效降解吲哚和粪臭素的细菌LPC24，通过生理生化和分子水平鉴定细菌为恶臭假单胞菌，该细菌能以吲哚和粪臭素为唯一碳源生长，当吲哚和粪臭素浓度小于3．0 mM和2．5 mM时，该细菌能有效降解这两种化合物而没有滞后现象，且能分别在18天和30天之内完全降解3．0 mM吲哚和2．0mM粪臭素。而当吲哚和粪臭素浓度分别超过3．5 mM和3．0 mM时，该菌的生长速度以及降解速率明显受到抑制；当两者浓度分别大于5．0 mM和4．0 mM时，该菌几乎不能生长，也不能降解这两种化合物。另外，该菌能分别在24小时和30小时之内将10 mM的氨氧化和硝酸盐还原，并且没有亚硝酸盐的积累。GC-MS和HPLC研究结果表明，细菌LPC24降解吲哚的途径为羟吲哚-靛红-邻氨基苯甲酸，这与前人报道的相似。细菌LPC24降解粪臭素的途径为3-甲基羟吲哚-N甲酸基氨基苯乙酮-2-氨基苯乙酮，不同于以往研究中关于粪臭素生物降解途径的报道。纤维素是陆地环境中光合作用的初始产物，是生物圈最丰富的可再生生物资源。大量的难降解纤维素污染物存在于农业和林业废弃物以及造纸废水中。纤维素也是养猪废水中COD和SS的主要来源，是养猪废水中难降解的主要有机污染物之一。将天然纤维素水解成葡萄糖等小分子，必须依靠内切型葡聚糖酶（EG）、外切型葡聚糖酶（CBH）和纤维二糖酶（BG）三种酶协同作用才能完成。现有的研究通常着重于单个纤维素降解菌的筛选和相关酶活的分析，所筛选的细菌往往产酶类型单一，很难应用到实际废弃物及废水处理中。本文从猪场粪污中分离出26株能降解纤维素的细菌，通过初步筛选，从其中挑选出10株细菌，全面系统分析了各细菌的纤维素的三种酶活。酶活分析结果表明：十株细菌的EG酶活在7．0-16．0 IU mL^-1之间，其中菌株LCB12和LCD51的EG酶活最高，分别达到15．98 IU mL^-1和15．40 IU mL^-1，高于文献相关报道。各菌株的CBH酶活介于EG酶和BG酶之间，为2-9 IU mL^-1，其中菌株LCB03和LCD12酶活最高，分别达到8．53 IU mL^-1和10．60 IU mL^-1。各菌株的BG酶活与EG酶和CBH酶相比较小，在1-3 IU mL^-1之间，其中菌株LCB12和LCD51酶活最高，分别达到2．68 IU mL^-1和1．74 IU mL^-1。选取其中具较高各种酶活的五株细菌（LCB03，LCB12，LCB52，LCD12和LCD51）构建复合菌系协同降解天然纤维素。混合培养实验结果表明：这五株细菌两两混合培养后可以共存，不相互拮抗。降解实验表明，复合菌系能在一周之内降解0．36克稻草和0．1克滤纸，各种酶活分别在第三和第四天达到最高，EG为3．15 IU，CBH为1．12 IU，BG为0．55 IU，滤纸酶活为0．92 IU。经生理生化和分子水平鉴定，这五株细菌分别鉴定为香茅醇假单胞菌，嗜麦芽窄食单胞菌，铜绿假单胞菌，铜绿假单胞菌和水氏黄杆菌。在前人的研究中，至今没有关于嗜麦芽窄食单胞菌和水氏黄杆菌降解纤维素的报道。3．构建了人工强化生境中，并分析各污染物的降解效率以及微生物群落的结构变化人工强化生境的构建是通过投加外源微生物于处理系统中或扩大培养本土微生物，以加速和提高处理效率的一种生物处理方法。该方法将具有特殊功能效微生物个体或群落应用于实际处理系统中，以提高目标污染物的去除效率。由于处理系统的复杂性，微生物群落之间的竞争和相互抑制很难预见和调控，所以不是所有的人工强化生境都能构建成功，它在很大程度上取决于投加的微生物个体或群落在目标环境中的生存能力及其和本土微生物群落的协同能力。本文通过将所筛选的降解吲哚、粪臭素和纤维素的细菌投加到稳定运行的UASB的反应器中，构建人工强化生境，提高处理系统对典型难降解污染物，包括含氮杂环化合物和纤维素的去除效率，并通过DGGE技术解析强化生境中微生物群落的变化。结果表明：反应器的COD、吲哚、粪臭素和纤维素去除率均有不同程度的提高。在45天的运行过程中，COD去除率提高了2-7％，纤维素降解效率提高20％，吲哚和粪臭素的降解效率提高了15-20％。同时反应微生物活性的指标VSS和VSS／TSS值也有所提高。DGGE分析表明，人工强化生境的微生物群落结构变化明显。所接种的水氏黄杆菌和假单胞菌在整个微生物生态系统中形成了优势菌群，而嗜麦芽窄食单胞菌没有形成明显的优势群落。在投加细菌的影响下，出现了一些新的微生物群落，如梭菌纲，酸杆菌纲和硝化螺旋菌纲的菌群。另外，在低温培养下，微生物群落结构发生了很大程度地改变，一些优势群落优势度明显降低，三组拟杆菌纲，一组拟杆菌门和一组厚壁菌门的优势菌群消失，而新出现了另外两组拟杆菌门和厚壁菌门的菌群。这为选育低温厌氧细菌提供可供参考的依据。更多还原

【Abstract】 In recent years, pig breeding industry has been developing rapidly in China, the biggest pig breeding country. Half of the pigs in the world are bred in China. Available data show that the environmental due to pig farming is quite serious. Its discharged waste contains highly concentrated organic matter, excessive suspended solids, large content of ammonia nitrogen and phosphorus. All of its water quality indices exceed the national discharge standards several times or even dozens of times, becoming one of the major sources of water pollution. According to statistics, the annual COD discharge of the swine wastewater is far more than the total amount of national industrial wastewater and domestic sewage. The incomplete treated pollutants discharged into lakes, rivers or other water bodies, cause serious pollution of lakes and rivers. UASB is the typical anaerobic treatment technology of swine wastewater. Previous work on swine wastewater anaerobic treatment with UASB mainly focused on technical aspects such as start-up method, rector design, and processing parameters and few efforts have been made to understand mechanisms of organic pollutant degradation and the relationship between the structure and the function of the microbial community in the reactors.The objective of this study is to investigate the anaerobic biodegradation mechanism of organic pollutants in swine wastewater using microbial molecular ecology methods in combination with traditional microbial methods. The results can be summarized as follows:Microbial community structure, dynamics and diversity analysis Two UASB reactors inoculated with different inocula sources were started up in 36 days. These two reactors operated steadily for another two months with the COD removal rate of 84-88 % and 90-95 %. The methane production of 5.5-10.5 and 9.0-14.0 L day^-1 respectively as the influent COD was between 3000-6000 mg L^-1. The start-up rate, COD removal rates and methane production, microbial activity characterized by VSS and VSS/TSS of reactor A were slightly lower than those of the reactor B in the same operation time. The VFAs level of the both reactors was appropriate for microorganism activities. According to the results of DGGE and ARDRA, the bacterial communities were diversified after a long time of acclimation and were similar to each other in the two reactors inoculated with different sources but changed insignificantly during the steadily operation time. This community structure shift of the reactors was in accordance with their functions. 55 operational taxonomic units （OTUs） were identified from 346 positive clones. The microbial population percentages distributions were 33 % （Firmicutes）, 23 % （Acidobacteria）, 19 % （Proteobacteria）, 11 % （Bacteroidetes）, 8 % （Nitrospira）, 4 % （Chloroflexi） and 2 % （others）. The analysis of 16S rRNA clone library showed that the bacterial community was quite different from those related with swine waste treatment by other technologies such as biofilter and pig slurry anaerobic storage in previous studies.Typical reluctant organic compounds biodegradation mechanism analysis One bacterial strain named LPC24 was isolated from the activated sludge from the USAB reactor for swine wastewater treatment by direct spreading plate method. This bacterial strain was finally identified as Psedomonas putida based on its phenotype and genotype characteristics. This isolate could utilize indole or 3-MI as the sole source of carbon source. 3.0 mM indole and 2.0 mM 3-MI could be degraded in 18 days and 30 days respectively by this bacterium. Additionally, this strain could oxidize ammonia and deoxidize nitrate effectively without nitrite accumulation. According to the results of HPLC and GC-MS, the metabolic pathways of indole by strain LPC24 was oxindole /isatin /anthranilic acid which was similar to that proposed previously. The degrading pathway of 3-MI by strain LPC24 was identified as 3-methyloxindole /formylaminoacetophenone /2-aminoacetophenone which was different from the pathways proposed so far in other studies. 26 strains were isolated from piggery sludge and manure from one big pig farm, and ten of them with larger halos were selected to further study. A composite microbial system was constructed by five bacteria named as LCB03, LCB12, LCB52, LCD12 and LCD51 with higher cellulases activities from the ten isolates. About 0.36 gram of rice straw and 0.10 gram filter paper were degraded by the composite microbial system within 1 -week individually. These five bacteria were identified as Pseudomonas citronellolis. Stenotrophomonas maltophilia, Pseudomonas aeruginosa, Pseudomonas aeruginosa and Flavobacterium mizutaii respectively according to the results of phenotype and genotype. To our best knowledge, little work has been done before on cellulose degradation using bacteria S. maltophilia and F. mizutaii.Bio-augmentation of recalcitrant pollutants degradation and microbial community dynamics analysis Six isolates with strong capability of degrading indole and skatole or cellulose were inoculated into an UASB reactor to strengthen the degradation capability of these recalcitrant pollutants. The results showed that the capability of degrading the recalcitrant pollutants including indole, skatole and cellulose was enhanced to different degrees in the whole process of bio-augmentation. COD removal performance was improved 2-7 %, the cellulose degrading capability of was increased more than 20 %, and the removal rates of indole and skatole increased 15-20 %. The bacteria activity characterized as the VSS and VSS/TSS was also improved; the microbial community structure analyzed by means of molecular tools PCR-DGGE was changed apparently. The inoculated bacteria Flavobacterium mizutaii and Pseudomonas dominated, and the strain Stenotrophomonas maltophilia failed to dominate in the microbial community in the bio-augmentation system. With the effect of bio-augmentation, some new bacterial community identified as Clostridia, Acidobacteria and Nitrospira appeared in the bioaugmented system. Additionally, the bacterial structure and diversity under low temperature incubation were detected. The bacterial community structure was changed dramatically. The density of all of the dominant groups of Chloroflexi and Acidobacteria, and three Bacteroidetes, one Proteobacteria and one Firmicutes group substantially decreased or even disappeared from the bacterial system incubated under the temperature of 30-35℃. Some bacterial groups which could dominant in the community under low temperature were found. These bacterial groups are of ecological significance to lower energy requirement in anaerobic digestion in cold climates which might serve as an alternative strategy for practical swine wastewater treatment.更多还原