城市黑臭河道氮转化途径分型表征及微生物作用机理研究

发布时间：2018-04-23 21:00

本文选题：黑臭河道 + ATU　；参考：《华东师范大学》2017年硕士论文

【摘要】：为了尽快遏制我国城市水污染日益加重的趋势、改善城市的水环境质量,近年来许多城市根据其自身特点,开展了相应的黑臭河道治理工程。然而,随着相应的黑臭河道治理工程的实施,污染负荷得到了大幅度削减,氮去除难以达标的问题却逐渐突显出来。其主要原因是目前黑臭河道治理过程中侧重于对氨氮的硝化转化,而忽视氮的脱除过程,导致总氮去除率偏低的现象普遍存在。为了解决黑臭河道"氮超标"难题,本研究基于曝气、调控C/N比和底泥投加硝酸钙三种治理措施,借助同位素示踪技术和物料平衡分析,从氨氧化、反硝化、厌氧氨氧化和硝酸盐异化还原成铵4个方面对相应措施下的氮转化途径进行分型表征,探讨在黑臭河道氮转化途径中的地位和作用,并通过高通量测序和荧光定量PCR技术解析对应的微生物作用机制,以期为底泥内源氮的有效控释和黑臭河道治理工程提供基础的理论指导和借鉴。主要研究结果如下:(1)在对氨苄青霉素、双氰胺(DCD)、烯丙基硫脲(ATU)、辛炔、氯仿和乙炔抑制氨氧化细菌(AOB)性能评定的基础上,筛选出适用于黑臭河道相对较优的硝化抑制剂为ATU,适宜浓度为25μmol·L-1,其主要通过降低AOB(主要是亚硝化螺菌属)种群丰度且抑制AOB菌群活性的方式实现其良好的硝化抑制效果。(2)基于曝气、调控C/N比和底泥投加硝酸钙三种治理措施的研究发现,曝气(工况3)对于氨氮的削减最为有效,在第18 d浓度便降低为2.07±0.24 mg·L-1,曝气条件下增加C/N比(工况5和工况7)不利于氨氮的削减,使得氨氮降为2 mg·L-1的时间推迟10～15d;但对于TN来说,增大C/N比有利于TN的快速削减,TN均在34～38d后降为2mg·L-1以下,相比曝气组达标的时间平均缩短22～26 d,C/N比为10(工况5)或者20(工况7)无显著影响差别,表明当C/N比为10条件下,碳源不是影响TN去除的限制因素;投加硝酸钙(工况9)不利于氨氮和TN的去除;所有添加抑制剂组均使氨氮和TN达标时间显著延迟。(3)曝气调控条件下,AOB在黑臭河道氨氧化过程中起主导作用,其贡献率为62.63～78.28%,而AOA占21.72～37.37%;同时发现稳定时氮素的削减以反硝化作用为主导,其速率高达3.61μmol N·kg-1·h-1,对氮素的削减贡献率约为83.26～92.46%;各工况厌氧氨氧化速率均在0.27～0.48μmol N·kg-1·h-1,贡献率为7.54～16.74%;曝气过程中DNRA速率低于检测限。(4)不同C/N比调控条件下,AOB均在氨氧化过程中起主导作用,其贡献率约为72.74～83.74%,AOA占16.26～27.26%,且受C/N比影响不大;除C/N比为20(工况7)条件下,厌氧氨氧化速率与反硝化速率相近外,各工况氮素削减均以反硝化过程为主,贡献率分别为64.58～89.17%,厌氧氨氧化贡献约为10.83～35.42%。特别地,在C/N比为10(工况5)中检测到DNRA过程,但其对氮素削减的贡献率较低,约为5.18～7.45%。(5)投加硝酸钙能够增大AOA对氨氧化过程的贡献(24.37～50.26%),且能够促进反硝化速率和厌氧氨氧化速率,但仍然以反硝化过程为主,其贡献率约为79.56～85.52%,厌氧氨氧化作用的贡献率为14.48～20.44%;且整个过程中发现DNRA过程速率低于检测限。(6)黑臭河道中细菌以变形菌门、厚壁菌门和绿弯菌门为优势菌群,不同调控措施均能显著降低厚壁菌门丰度,而对变形菌门来说,投加硝酸钙调控对其丰度的影响最大;古菌中优势菌群主要为深古菌门、广古菌门、泉古菌门和奇古菌门,与起始工况相比,各工况运行稳定时优势菌群丰度均显著增加,但受不同调控措施影响较小。(7)基于amoA基因对氨氧化微生物的分子生物学分析表明,各工况AOB数量明显高于AOA数量(4～9倍),约为3.71×105～1.13×106拷贝数/(克·干重),添加抑制剂主要抑制AOB中的Nitrosomonas和Nitsomospira菌属,而对AOA无明显影响,同时发现溶解氧是影响AOA菌群结构差异性的主要环境因子。(8)对反硝化微生物来说,各工况nirS型反硝化菌的数量明显高于narG、norB和nosZ型反硝化菌数量,约为5.39×107～5.47×108拷贝数/(克·干重),其优势菌属为Pseudowonas、Sulfuritalea和Dechloromonas;在不同调控因子中,硝态氮是影响反硝化菌菌群结构的主要因素,投加硝酸钙后Thiobacillus菌属的丰度剧烈增加,成为优势菌属,推测可能促进了硫自养反硝化过程的发生,其中抑制剂的添加影响了部分narG型反硝化菌,对其他反硝化菌无显著影响。(9)对于厌氧氨氧化微生物来说,各工况优势菌属为Candidatus_Jettenia和Candidatus_Kuenenia,在不同调控措施中,底泥投加硝酸钙能够显著改变厌氧氨氧化菌的丰度和菌群组成,其数量由2.06×104～8.79×104拷贝数/(克·干重)增加到2.66×105～1.95×106拷贝数/(克·干重),其中Candidatus_Jettenia菌属的相对丰度显著增加,由19.53%增加为73.30～73.83%,而其他调控措施相对来说影响不大。(10)对于DNRA微生物来说,发现增加C/N比能够促进其生长,各工况数量为3.45×106～9.19×106拷贝数/(克·干重),其优势菌属均为Desulfovibrio属,丰度约为 0.7%～1.4%,主要以 Desulfovibrio_vulgaris 和 Desulfovibrio_termitidis种为主,但可能其活性较低或者相关功能酶未表达。
[Abstract]:In order to restrain the increasing trend of urban water pollution and improve the quality of urban water environment as soon as possible, in recent years, many cities have carried out corresponding control projects of black and smelly river channels according to their own characteristics. However, with the implementation of the corresponding control projects of the black and smelly river, the negative pollution is greatly reduced and the nitrogen removal is difficult to reach the standard. The main reason is that the main reason is that at present, the process of nitrification and conversion of ammonia nitrogen is emphasized in the process of controlling the black and smelly river, but the removal process of nitrogen is ignored and the removal rate of total nitrogen is low. In order to solve the problem of "nitrogen exceeding standard" in the black and smelly channel, this study is based on aeration, regulating the C/N ratio and adding calcium nitrate to the sediment. With the help of isotopic tracer and material balance analysis, the nitrogen conversion pathway under the corresponding measures was characterized by 4 aspects of ammonia oxidation, denitrification, anaerobic ammonia oxidation and nitrate dissimilation reduction into ammonium. The status and application of the nitrogen conversion in the black and smelly channel were discussed, and the high throughput sequencing and fluorescence quantitative PCR technique were used. The corresponding mechanism of microbial action is analyzed in order to provide theoretical guidance and reference for effective controlled release of endogenous nitrogen in sediment and treatment of black odor river. The main results are as follows: (1) on the basis of the performance evaluation of ampicillin, Dicyandiamide (DCD), allyl thiourea (ATU), octyyne, chloroform and acetylene inhibition ammonia oxidizing bacteria (AOB) A relatively excellent nitrification inhibitor suitable for black and smelly rivers was selected as ATU, with a suitable concentration of 25 mu mol. L-1, which achieved good nitrification inhibition effect mainly by reducing the abundance of AOB (mainly the genus snnail) and inhibiting the activity of AOB bacteria. (2) three control measures based on aeration, control C/N ratio and sediment cast calcium nitrate. The study found that aeration (working condition 3) is the most effective for ammonia nitrogen reduction, and the concentration of eighteenth D is reduced to 2.07 + 0.24 mg L-1. The increase of C/N ratio under the aeration condition (working condition 5 and working condition 7) is not conducive to the reduction of ammonia nitrogen, and the time of ammonia nitrogen reduction to 2 mg. L-1 is delayed 10 to 15d; but for TN, the increase of C/N ratio is beneficial to the rapid reduction of TN, TN is both in After 34 ~ 38d to below 2mg L-1, the time of the aeration group was reduced by 22~26 D, the C/N ratio was 10 (working condition 5) or 20 (working condition 7). It showed that when the C/N ratio was 10, the carbon source was not the limiting factor for the removal of TN; the addition of calcium nitrate (working condition 9) was not conducive to the removal of ammonia nitrogen and TN; all addition inhibitor groups were all made The time of ammonia nitrogen and TN standard was delayed significantly. (3) under the condition of aeration, AOB played a leading role in the process of ammoxidation of black and smelly rivers, and its contribution rate was 62.63 to 78.28%, while AOA accounted for 21.72 ~ 37.37%. At the same time, the reduction of nitrogen was dominated by denitrification, and its rate was up to 3.61 mol N. Kg-1. H-1, and the contribution rate of nitrogen reduction was about The rate of anammox is 83.26 to 92.46%; the rate of anammox is 0.27 to 0.48 Mu mol N kg-1. H-1, and the contribution rate is 7.54 to 16.74%, and the DNRA rate is lower than the detection limit during aeration. (4) under the condition of different C/N ratio, AOB plays the leading role in the process of ammonia oxidation, and its contribution rate is about 72.74 to 83.74%, AOA is 16.26 ~ 27.26%, and influenced by C/N ratio. In addition to the C/N ratio of 20 (working condition 7), the anaerobic ammonia oxidation rate is similar to the denitrification rate, and the nitrogen reduction is mainly denitrification, the contribution rate is 64.58 to 89.17%, the anammox contributes about 10.83 to 35.42%., and the DNRA process is detected in the C/N ratio 10 (working condition 5), but its contribution to nitrogen reduction The rate is low. The addition of 5.18 ~ 7.45%. (5) calcium nitrate can increase the contribution of AOA to the process of ammonia oxidation (24.37 ~ 50.26%), and can promote the denitrification rate and the anaerobic ammonia oxidation rate, but still mainly denitrification process, the contribution rate is about 79.56 to 85.52%, the contribution rate of anaerobic ammonia oxidation is 14.48 to 20.44%; and the whole process is in the whole process. It was found that the DNRA process rate was lower than the detection limit. (6) the bacteria in the black and smelly rivers were dominated by the deformable bacteria gate, the thick wall bacteria gate and the green flexural bacteria gate, and the different control measures could significantly reduce the abundances of the thick wall bacteria, while the control of calcium nitrate on the abundance of the bacteria was the most important for the deformable bacteria. Bacteria gate, spring Archaea and archaea door, compared with the starting condition, the abundance of dominant flora increased significantly when the operating conditions were stable, but affected by different control measures. (7) the molecular biological analysis of ammoxidation microorganism based on amoA gene showed that the number of AOB in each working condition was obviously higher than that of AOA (4~9 times), about 3.71 * 105 ~ 1.13 x 10 6 copy number / (g dry weight), adding inhibitor mainly inhibited Nitrosomonas and Nitsomospira in AOB, but had no obvious effect on AOA. At the same time, it was found that dissolved oxygen was the main environmental factor affecting the structural difference of AOA bacteria. (8) for denitrifying microorganisms, the number of nirS denitrifying bacteria in each condition was significantly higher than that of narG, norB and nosZ type. The number of nitrifying bacteria is about 5.39 x 107 ~ 5.47 * 108 copies / (k dry weight). The dominant bacteria are Pseudowonas, Sulfuritalea and Dechloromonas. In different regulatory factors, nitrate nitrogen is the main factor affecting the structure of denitrifying bacteria. After adding calcium nitrate, the abundance of Thiobacillus genera is greatly increased, and it is presumed possible. The process of sulfur autotrophic denitrification was promoted, in which the addition of inhibitors affected partial narG denitrifying bacteria and had no significant effect on other denitrifying bacteria. (9) for anaerobic ammonia oxidizing microorganisms, the dominant bacteria were Candidatus_Jettenia and Candidatus_Kuenenia in various operating conditions. In different control measures, calcium nitrate could be added to the sediment. The abundances and flora composition of anammox were significantly changed from 2.06 x 104 to 8.79 x 104 copies / (k dry weight) to 2.66 x 105 to 1.95 * 106 copies / (k dry weight). The relative abundance of Candidatus_Jettenia bacteria increased significantly from 19.53% to 73.30 ~ 73.83%, and the other regulation measures were relatively not affected. (10) for DNRA microbes, it was found that the increase of C/N ratio could promote its growth. The number of working conditions was 3.45 x 106 ~ 9.19 x 106 copies / (k dry weight). The dominant genus were all Desulfovibrio genera, and the abundance was about 0.7% to 1.4%, mainly with Desulfovibrio_vulgaris and Desulfovibrio_termitidis species, but their activity was lower or lower. The related functional enzymes were not expressed.

【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X522

【参考文献】