三峡库区消落带土壤和人工污水处理系统中与氮转化相关的微生物研究
发布时间：2018-09-13 15:10
【摘要】：亚硝酸盐驱动型甲烷厌氧氧化是最近发现的一个耦合地球碳循环和氮循环的重要反应,该反应主要是由一类归属于NC10门的细菌M.oxyfera-like细菌来完成。该类菌能将甲烷在厌氧和亚硝酸盐存在的条件下转化为二氧化碳、水和氮气,因而能缓解甲烷造成的温室效应,但是,它们在自然界的分布和存在状态还远没有得到充分描述。除了亚硝酸盐驱动型甲烷厌氧氧化外,在厌氧环境中,氨和硝酸盐也可以通过厌氧氨氧化菌介导的厌氧氨氧化途径转化为氮气,该反应被认为是最经济有效的废水脱氮过程,并且被认为是自然界厌氧条件下氮转化的重要途径。虽然厌氧氨氧化菌在很多自然环境中的生态分布和作用得到了详细研究,但是,对于它与Methylomirabilis oxyfera-like菌在自然环境中的共存状况报道的还很少。三峡大坝建成后所形成的三峡库区消落带是一个半年淹没半年暴露的特殊水生生境,其中的甲烷排放一直是研究者关注的焦点。在本论文中,我们首先系统研究了三峡库区消落带土壤中反硝化甲烷厌氧氧化菌M.oxyfera及厌氧氨氧化菌的生态分布、多样性、及其与环境理化性质之间的相关性;其次,我们在实验室构建了序批式生物反应器(SBR)硝化反应器和以厌氧氨氧化为主的全自养脱氮反应器(CANON反应器),通过添加一定浓度的联氨(H2N4),探索其对脱氮过程中相关功能微生物的影响,并利用Q-PCR技术探究其丰度的变化。主要取得如下结果:(1)在三峡水库水位明显下降的3月份,沿着三峡库区消落带,在香溪河河(XXH)、长江巫山干流(WS)、涪陵王家沟(WJG)、小江(XJ)、苎溪河(ZXH)及涪陵附近的一稻田土进行取样,分析了土壤中的pH、总有机碳、干物质含量、氨氮、亚硝酸盐、硝酸盐和铁含量。结果表明,氨氮和亚硝酸盐的含量低于检出限。通过文献调研,在该香溪河、巫山、小江消落带取样点甲烷的年均排放通量为0.34mg/m2h,水稻田的排放量为4.86mg/m2h,其他取样点的甲烷排放量未见报道。(2)对上述土壤样品进行总DNA提取,利用专一于M.oxyfera-like细菌和Anammox细菌的16S rRNA基因的引物进行定量PCR测定,分析M.oxyfera-like和Anammox两类细菌在上述土壤样品中的存在和丰度。结果表明:在所有的样品中均有M.oxyfera-like细菌和Anammox细菌存在。消落带土壤中M.oxyfera-like的丰度为1.15±0.2×105-1.48±0.16×107 copies/g干重,这显著高于该区域水稻田土壤(p0.01);在消ii落带土壤中anammox的丰度明显低于m.oxyfera-like细菌,丰度为1.07±0.42×103-7.77±0.87×104copies/g干重;而在水稻田中该菌的丰度则明显高于m.oxyfera-like细菌,并且也明显高于所有消落带土壤中的anammox菌丰度(p0.01),达到9.78±0.78×103-3.65±0.76×106copies/g干重。皮尔逊相关性分析表明m.oxyfera-like细菌的丰度和no3--n的含量显著正相关。anammox丰度和环境因子之间的相关性也不显著。(3)在以上结果的基础上,我们分别构建了m.oxyfera-like细菌在样品ws、wjg和zxh(分别代表了两种菌的最高、中间和最低的丰富度)中的16srrna基因、甲烷氧化过程的关键酶—甲烷单加氧酶α亚基(pmoa)基因和厌氧氨氧化的关键酶—联氨合酶β亚基(has_b)基因文库。通过测序和进化树构建,我们对这两类菌在三峡库区消落带土壤中的多样性进行了系统研究。结果表明:m.oxyfera-like细菌的134条16srrna序列中,128条与m.oxyfera表现出了84.38-98.92%的相似性,这些序列分布在进化树的4个group中;141条pmoa基因序列中134条和m.oxyfera表现出了84.62-95.38%的一致性,在进化树中分布于6个cluster中。在获得的103条anammox的hzs_b序列中,分别有57.28%和24.27%与anammox的candidatus‘brocadiafulgida,candidatus‘brocadiaanammoxdianas聚在一起,相似性达到88.02-88.28%和84.11%-85.68%;13.59%的序列和candidatus‘kuenenia’聚在一起,相似性达到89.58%-98.44%;4.85%序列独立形成一支。(4)sbr硝化反应器以污水处理厂好氧池和厌氧池混合污泥作为种泥,开始进水nh4+-n浓度为51mg/l左右,逐渐增加至505mg/l左右,氮负荷率从起始阶段的102.9mg/l*d逐渐增加到1056.2mg/l*d。此时添加2mg/l的n2h4,反应器的运行效率出现了急剧的下降,氨氧化率从100%下降到最后的2.45%。氨氧化分为两个过程:即nh4+-n氧化为no2--n过程,关键微生物为氨氧化细菌(aob);no2--n氧化为no3--n的过程,关键微生物为亚硝酸盐氧化还原菌(nob)。分别采用这两类微生物的关键酶氨单加氧酶的α亚基(amoa)基因和亚硝酸盐氧化还原酶β亚基(nxrb)基因的特异性引物对种泥,富集培养后和添加n2h4后三个时期的活性污泥中两种微生物进行定量分析。结果表明,amoa基因拷贝数从2.01×106copies/g干污泥增加到1.0×109copies/g干污泥,nxrb基因的拷贝数为从4.20×105copies/g增加到1.28×107copies/g。添加n2h4后,这两种功能微生物的丰度分别下降为2.09×104copies/g和2.56×105copies/g。采用同样的引物构建克隆文库测序分析表明:获得的19条amoa基因序列中52.63%的序列属于Nitrosococcu;31.58%的的序列属于Nitrosomonas;15.79%序列属于亚硝化螺菌属Nitrosospira。获得的20条nxrB基因序列中,55%属于Nitrobacter winogradskyi;20%属于Nitrobacter vulgaris;15%属于Nitrobacter hamburgensis.(5)实验室稳定运行的CANON反应器,进水NH4+-N浓度维持在200mg/L左右时,NH4+-N和TN的去除率可以达到68%和65%,NLR为269mg/L*d左右。添加N2H4强化后,在进水NH4+-N浓度高达310mg/L时,NH4+-N和TN的去除率分别稳定在82.13%和72.65%左右,反应器的NLR率高达456.80mg/L*d。除AOB和NOB外,对该反应器中另一类关键微生物anammox进行了定量和多样性分析,采用的引物为该菌特异性的联氨合酶α亚基(hzs_A)基因。定量PCR分析表明:未添加N2H4前amoA的基因和nxrB基因拷贝数分别为1.03×107copies/g干污泥和1.30×106copies/g干污泥,而在添加N2H4后其拷贝数为6.26×104copies/g干污泥和1.66×105copies/g干污泥。Anammox的hzsA基因的拷贝数从3.14×109copies/g增加到5.84×1010opies/g。克隆文库测序分析表明:在获得的15条amoA基因中,66.67%的序列分布在Nitrosomonas属;33.33%的序列分布在Nitrosococcus属。获得的15条nxrB基因序列中,26.67%属于Nitrobacter vulgaris;53.33%属于Nitrobacter winogradskyi;20%属于Nitrobacter hamburgensis。anammox的hzsA基因的13条序列中38.46%属于待定的阶梯烷菌属(Candidatus Scalindua);15.38%属于待定的厌氧氨氧化丙酸球菌属(Candidatus Anammoxoglobus propionicus);46.15%的序列未能确定具体的种属。
[Abstract]:Nitrite-driven anaerobic oxidation of methane is a recently discovered important reaction coupled with the Earth's carbon and nitrogen cycles. This reaction is mainly accomplished by a group of bacteria belonging to the NC10 family, M. oxyfera-like bacteria, which can convert methane to carbon dioxide, water and nitrogen in the presence of anaerobic and nitrite. In addition to nitrite-driven anaerobic oxidation of methane, ammonia and nitrate can also be converted to nitrogen through anaerobic ammonia oxidation mediated by anaerobic ammonia-oxidizing bacteria in an anaerobic environment. Anaerobic ammonia-oxidizing bacteria are considered to be the most economical and effective process for nitrogen removal from wastewater and an important way of nitrogen transformation under anaerobic conditions in nature. Although the ecological distribution and function of anaerobic ammonia-oxidizing bacteria in many natural environments have been studied in detail, the co-existence of anaerobic ammonia-oxidizing bacteria with Methylomirabilis oxyfera-like bacteria in natural environments has been reported. In this paper, we first studied the denitrifying methane anaerobic bacteria M. oxyfera and anaerobic ammonia in the soil of the Three Gorges reservoir area. Secondly, the sequencing batch bioreactor (SBR) nitrification reactor and the anaerobic ammonia oxidation (CANON) full autotrophic denitrification reactor (CANON reactor) were constructed in the laboratory to explore the phase of denitrification process by adding a certain concentration of hydrazine (H2N4). The main results are as follows: (1) In March, the water level of the Three Gorges Reservoir dropped markedly, along the Yangtze River (XXH), the Wushan River (WS), the Wangjiagou (WJG), the Xiaojiang (XJ), the Zhuxi River (ZXH) and a paddy field soil near Fuling along the Yangtze River (WS), the Xiangxi River (XXH), the Wushan River (WS), the Wangjiagou (WJG), the Xiaojiang (XJ), the Zhuxi River (ZXH) and the downstream of the Three Gorges Reservoir. Soil pH, total organic carbon, dry matter content, ammonia nitrogen, nitrite, nitrite and iron content were analyzed. The results showed that the contents of ammonia nitrogen and nitrite were below the detection limit. Methane emissions from other sampling sites were not reported at m2h. (2) Total DNA was extracted from the above soil samples, and 16S rRNA gene primers specific to M. oxyfera-like bacteria and Anammox bacteria were used for quantitative PCR analysis to analyze the presence and abundance of M. oxyfera-like and Anammox bacteria in the above soil samples. The abundance of M. oxyfera-like bacteria was 1.15 65 The abundance of m.oxyfera-like bacteria in paddy field was significantly higher than that of m.oxyfera-like bacteria, and the abundance of m.oxyfera-like bacteria was also significantly higher than that of anammox bacteria in soil of all the ebb zones (p0.01), reaching 9.78.78 (103-3.65) 0.76 (106) copies/g dry weight. There was no significant correlation between the abundance of anammox and environmental factors. (3) On the basis of the above results, we constructed the 16S rRNA gene of M. oxyfera-like bacteria in samples ws, WJG and zxh (representing the highest, middle and lowest abundance of the two bacteria), and the key enzyme of methane oxidation process, methane monooxygenase, respectively. Alpha subunit (pmoa) gene and diammonia synthase beta subunit (has_b) gene library, the key enzyme of anaerobic ammonia oxidation, were used to study the diversity of the two bacteria in the soil of the Three Gorges Reservoir Area by sequencing and phylogenetic tree construction. The results showed that 128 of 134 16S rRNA sequences of M. 84.38-98.92% similarity was found in the four groups of the evolutionary tree; 134 of the 141 pmoA gene sequences and m.oxyfera showed 84.62-95.38% consistency and were distributed in six clusters in the evolutionary tree. Among the 103 anammox hzs_b sequences, 57.28% and 24.27% were candidatus'brocadiafulgi'brocadiagi' respectively. Da, candidatus'brocadia anammox Dianas aggregated together, similarity reached 88.02-88.28% and 84.11% - 85.68%; 13.59% sequence and candidatus'kuenenia'aggregated together, similarity reached 89.58% - 98.44%; 4.85% sequence formed an independent. (4) SBR nitrification reactor with sewage treatment plant aerobic tank and anaerobic tank mixed sludge as a kind of sludge, began. The influent NH4 + - N concentration was about 51 mg / l, and gradually increased to about 505 mg / L. The nitrogen loading rate gradually increased from 102.9 mg / L * D in the initial stage to 1056.2 mg / L * D. When 2 mg / L N2H4 was added, the operating efficiency of the reactor decreased sharply, and the ammonia oxidation rate decreased from 100% to 2.45%. Ammonia-oxidizing bacteria (aob) and nitrite-oxidizing-reducing bacteria (nob) are the key microorganisms in the process of ammonia-oxidizing bacteria (aob) and nitrite-oxidizing-reducing bacteria (no2-n) oxidation to no3-n. The results showed that the copy number of amoA gene increased from 2.01 *106 copies/g dry sludge to 1.0 *109 copies/g dry sludge, and the copy number of nxrb gene increased from 4.20 *105 copies/g to 1.28 *107 copies/g. Sequencing analysis showed that 52.63% of the 19 amoA gene sequences belonged to Nitrosococcu, 31.58% belonged to Nitrosomonas and 15.79% belonged to Nitrosospira. Among them, 55% belonged to Nitrobacter winogradskyi, 20% belonged to Nitrobacter vulgaris, 15% belonged to Nitrobacter hamburgensis. (5) The removal rate of NH4 + - N and TN could reach 68% and 65% when the influent NH4 + - N concentration was maintained at about 200 mg / L, and NLR was about 269 mg / L * D. The removal rates of NH4 + - N and TN were 82.13% and 72.65% respectively at 310 mg/L. The N LR rate of the reactor was 456.80 mg/L * D. Besides AOB and NOB, the key microorganism anammox in the reactor was analyzed quantitatively and diversity. The primers were the hzs_A gene. Quantitative PCR analysis table was used. Ming: The copies of amoA gene and nxrB gene were 1.03 x 107 copies/g dry sludge and 1.30 x 106 copies/g dry sludge without N2H4, respectively. The copies of hzsA gene were 6.26 x 104copies/g dry sludge and 1.66 x 105copies/g dry sludge when N2H4 was added. The copies of hzsA gene in Anammox increased from 3.14 x 109 copies/g to 5.84 x 1010 opies/g. Sequencing analysis showed that 66.67% of the 15 amoA genes were located in Nitrosomonas, 33.33% in Nitrosococcus, 26.67% in Nitrobacter vulgaris, 53.33% in Nitrobacter winogradskyi, 20% in Nitrobacter hamburgensis. Among the sequences, 38.46% belonged to the undetermined genus Candidatus Scalindua, 15.38% belonged to the undetermined genus Candidatus Anammoxoglobus propionicus, and 46.15% could not identify the specific species.
【学位授予单位】：河南师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X703;X172;X53
本文编号：2241536
[Abstract]:Nitrite-driven anaerobic oxidation of methane is a recently discovered important reaction coupled with the Earth's carbon and nitrogen cycles. This reaction is mainly accomplished by a group of bacteria belonging to the NC10 family, M. oxyfera-like bacteria, which can convert methane to carbon dioxide, water and nitrogen in the presence of anaerobic and nitrite. In addition to nitrite-driven anaerobic oxidation of methane, ammonia and nitrate can also be converted to nitrogen through anaerobic ammonia oxidation mediated by anaerobic ammonia-oxidizing bacteria in an anaerobic environment. Anaerobic ammonia-oxidizing bacteria are considered to be the most economical and effective process for nitrogen removal from wastewater and an important way of nitrogen transformation under anaerobic conditions in nature. Although the ecological distribution and function of anaerobic ammonia-oxidizing bacteria in many natural environments have been studied in detail, the co-existence of anaerobic ammonia-oxidizing bacteria with Methylomirabilis oxyfera-like bacteria in natural environments has been reported. In this paper, we first studied the denitrifying methane anaerobic bacteria M. oxyfera and anaerobic ammonia in the soil of the Three Gorges reservoir area. Secondly, the sequencing batch bioreactor (SBR) nitrification reactor and the anaerobic ammonia oxidation (CANON) full autotrophic denitrification reactor (CANON reactor) were constructed in the laboratory to explore the phase of denitrification process by adding a certain concentration of hydrazine (H2N4). The main results are as follows: (1) In March, the water level of the Three Gorges Reservoir dropped markedly, along the Yangtze River (XXH), the Wushan River (WS), the Wangjiagou (WJG), the Xiaojiang (XJ), the Zhuxi River (ZXH) and a paddy field soil near Fuling along the Yangtze River (WS), the Xiangxi River (XXH), the Wushan River (WS), the Wangjiagou (WJG), the Xiaojiang (XJ), the Zhuxi River (ZXH) and the downstream of the Three Gorges Reservoir. Soil pH, total organic carbon, dry matter content, ammonia nitrogen, nitrite, nitrite and iron content were analyzed. The results showed that the contents of ammonia nitrogen and nitrite were below the detection limit. Methane emissions from other sampling sites were not reported at m2h. (2) Total DNA was extracted from the above soil samples, and 16S rRNA gene primers specific to M. oxyfera-like bacteria and Anammox bacteria were used for quantitative PCR analysis to analyze the presence and abundance of M. oxyfera-like and Anammox bacteria in the above soil samples. The abundance of M. oxyfera-like bacteria was 1.15 65 The abundance of m.oxyfera-like bacteria in paddy field was significantly higher than that of m.oxyfera-like bacteria, and the abundance of m.oxyfera-like bacteria was also significantly higher than that of anammox bacteria in soil of all the ebb zones (p0.01), reaching 9.78.78 (103-3.65) 0.76 (106) copies/g dry weight. There was no significant correlation between the abundance of anammox and environmental factors. (3) On the basis of the above results, we constructed the 16S rRNA gene of M. oxyfera-like bacteria in samples ws, WJG and zxh (representing the highest, middle and lowest abundance of the two bacteria), and the key enzyme of methane oxidation process, methane monooxygenase, respectively. Alpha subunit (pmoa) gene and diammonia synthase beta subunit (has_b) gene library, the key enzyme of anaerobic ammonia oxidation, were used to study the diversity of the two bacteria in the soil of the Three Gorges Reservoir Area by sequencing and phylogenetic tree construction. The results showed that 128 of 134 16S rRNA sequences of M. 84.38-98.92% similarity was found in the four groups of the evolutionary tree; 134 of the 141 pmoA gene sequences and m.oxyfera showed 84.62-95.38% consistency and were distributed in six clusters in the evolutionary tree. Among the 103 anammox hzs_b sequences, 57.28% and 24.27% were candidatus'brocadiafulgi'brocadiagi' respectively. Da, candidatus'brocadia anammox Dianas aggregated together, similarity reached 88.02-88.28% and 84.11% - 85.68%; 13.59% sequence and candidatus'kuenenia'aggregated together, similarity reached 89.58% - 98.44%; 4.85% sequence formed an independent. (4) SBR nitrification reactor with sewage treatment plant aerobic tank and anaerobic tank mixed sludge as a kind of sludge, began. The influent NH4 + - N concentration was about 51 mg / l, and gradually increased to about 505 mg / L. The nitrogen loading rate gradually increased from 102.9 mg / L * D in the initial stage to 1056.2 mg / L * D. When 2 mg / L N2H4 was added, the operating efficiency of the reactor decreased sharply, and the ammonia oxidation rate decreased from 100% to 2.45%. Ammonia-oxidizing bacteria (aob) and nitrite-oxidizing-reducing bacteria (nob) are the key microorganisms in the process of ammonia-oxidizing bacteria (aob) and nitrite-oxidizing-reducing bacteria (no2-n) oxidation to no3-n. The results showed that the copy number of amoA gene increased from 2.01 *106 copies/g dry sludge to 1.0 *109 copies/g dry sludge, and the copy number of nxrb gene increased from 4.20 *105 copies/g to 1.28 *107 copies/g. Sequencing analysis showed that 52.63% of the 19 amoA gene sequences belonged to Nitrosococcu, 31.58% belonged to Nitrosomonas and 15.79% belonged to Nitrosospira. Among them, 55% belonged to Nitrobacter winogradskyi, 20% belonged to Nitrobacter vulgaris, 15% belonged to Nitrobacter hamburgensis. (5) The removal rate of NH4 + - N and TN could reach 68% and 65% when the influent NH4 + - N concentration was maintained at about 200 mg / L, and NLR was about 269 mg / L * D. The removal rates of NH4 + - N and TN were 82.13% and 72.65% respectively at 310 mg/L. The N LR rate of the reactor was 456.80 mg/L * D. Besides AOB and NOB, the key microorganism anammox in the reactor was analyzed quantitatively and diversity. The primers were the hzs_A gene. Quantitative PCR analysis table was used. Ming: The copies of amoA gene and nxrB gene were 1.03 x 107 copies/g dry sludge and 1.30 x 106 copies/g dry sludge without N2H4, respectively. The copies of hzsA gene were 6.26 x 104copies/g dry sludge and 1.66 x 105copies/g dry sludge when N2H4 was added. The copies of hzsA gene in Anammox increased from 3.14 x 109 copies/g to 5.84 x 1010 opies/g. Sequencing analysis showed that 66.67% of the 15 amoA genes were located in Nitrosomonas, 33.33% in Nitrosococcus, 26.67% in Nitrobacter vulgaris, 53.33% in Nitrobacter winogradskyi, 20% in Nitrobacter hamburgensis. Among the sequences, 38.46% belonged to the undetermined genus Candidatus Scalindua, 15.38% belonged to the undetermined genus Candidatus Anammoxoglobus propionicus, and 46.15% could not identify the specific species.
【学位授予单位】：河南师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X703;X172;X53
【参考文献】
相关期刊论文 前4条
1 孙海兵;;三峡库区消落区农业利用初探[J];安徽农业科学;2007年34期
2 苏维词;三峡库区消落带的生态环境问题及其调控[J];长江科学院院报;2004年02期
3 肖文涛;;污水生物脱氮除磷工艺的现状与发展[J];环境保护与循环经济;2010年11期
4 朱群;沈李东;胡宝兰;楼莉萍;程东庆;;西湖底泥中的反硝化型甲烷厌氧氧化菌的分子生物学检测[J];环境科学学报;2013年05期
，本文编号：2241536
本文链接：https://www.wllwen.com/kejilunwen/huanjinggongchenglunwen/2241536.html
最近更新
教材专著
