细菌Aquabacterium parvum B6的硝酸盐依赖型铁氧化代谢特性研究

发布时间：2019-06-11 01:47

【摘要】：针对我国水资源污染现状,本文基于铁氧化细菌(Fe-oxidizing Bacteria,FOB)的研究理论,开展对其硝酸盐依赖型铁氧化(nitrate-dependent Fe(II)oxidation,NDFO)代谢特性研究。通过菌株筛选、效能考察、代谢机理解析、反应器建立与运行等手段及方法,首次明确Aquabacteria种属菌株的NDFO代谢特性及机理。本研究在实验室现保存的不同种属的铁氧化细菌中,通过对照试验,筛选出一株水小杆菌属菌株Aquabacteriaum parvum B6。该菌株在厌氧培养过程中对Fe(II)和硝酸盐氮的代谢特性十分显著,细胞数目也有明显增加。厌氧连续培养试验发现,菌株B6在厌氧培养条件下,可以达到理想的NDFO效果,培养瓶中微生物数量也可达到最高值,约为2.73×105 cell/m L。考察不同环境因子对菌株B6的NDFO效能影响。经过综合比较发现:以酵母提取物或乙酸钠为碳源的培养条件下菌株B6对NO-3-N的去除和对Fe(II)的氧化效能较高;葡萄糖是最有利于微生物生长的碳源类型;柠檬酸钠作为单一碳源时,菌株B6的NDFO效能较低。通过响应面BBD法考察初始p H值、初始温度和C/N比对菌株B6的NDFO效能影响,发现初始p H值对于菌株B6的硝酸盐氮去除效能影响较大。对菌株B6的Fe(II)氧化效能而言,初始p H值和C/N比的影响大于初始温度,通过Optimization Numedcal Solution对其NDFO效能进行优化。利用Illumina Hi Seq 2000测序技术,对菌株B6进行了全基因组测序。由RAST annotation server基因注释结果可知:菌株B6的基因组大小为4 592 999 bp,GC含量约65.3%。基因按功能可分为27类:与蛋白质代谢相关的基因252个;与碳水化合物代谢相关的基因236个;与氮代谢相关的基因59个;与铁代谢相关的基因22个。由菌株B6的基因序列注释结果得知:菌株B6在厌氧条件下存在硝酸盐→亚硝酸盐→一氧化氮→一氧化二氮的代谢途径,这一过程与菌株所含有的Nar G、Nar H、NarJ、Nar I、q Nor、NnrS、DNR和Nnru编码基因密切相关;硝酸盐对Fe(II)的氧化过程与Nar亚基的铁硫簇有关,亚硝酸盐对Fe(II)的氧化过程可能与Nir的heme c相关,而一氧化二氮则通过q Nor实现对Fe(II)的氧化。构建以菌株B6为主体的生物脱氮除铁反应器,并考察反应器在启动及稳定运行期间对于氨氮、硝酸盐氮、亚硝酸盐氮、Fe(II)和总铁的去除效能,发现生物脱氮除铁反应器的总氮和总铁的去除率可达到79.70%和75.16%。对于投加了附着菌株B6的载体,反应器缺氧区其硝酸盐氮及Fe(II)去除率则可分别达到46.9%和60.33%,其硝酸盐氮还原速率可达到0.07mg NO-3-N/(L·h)。生物脱氮除铁反应器的微生物群落结构较为稳定多样。缺氧区中DO含量和进水C/N值有利于兼性厌氧微生物生长,菌株B6所属水小杆菌属(Aquabacterium)相对丰度为8.06%,其NDFO代谢效能得以保证。此外与菌株B6功能较为相近的芽孢杆菌属(Bacillus)在缺氧区中也具有优势地位,相对丰度为22.58%,菌株B6不仅可以保持较为优势的地位,也可以强化系统中与其功能相近的菌属的优势地位,因而得以保证反应系统具有较高的氨氮和总氮处理效能。而好氧区中的DO含量和C/N比值则有利于鞘氨醇杆菌属(Sphingobacterium),金黄杆菌属(Chryseobacterium)和芽孢杆菌属(Bacillus)的具有异养硝化、好氧反硝化代谢特性的微生物种群逐步处于优势地位。
[Abstract]:In view of the present situation of water pollution in China, this paper studies the metabolic characteristics of nitrate-dependent iron (NFFO) based on the theory of iron-oxide-bacteria (FOB). The NDFO metabolic characteristics and mechanism of the Aquabacteria species were identified for the first time by means of strain screening, efficiency investigation, analysis of metabolic mechanism, establishment and operation of the reactor and the like. In this study, a small genus of the genus Aquabacteriaum parvum B6 was screened by control test in different species of iron-oxidizing bacteria that were currently stored in the laboratory. The metabolic characteristics of Fe (II) and nitrate nitrogen were very significant in the process of anaerobic culture, and the number of cells increased significantly. The results of the anaerobic continuous culture show that under the condition of anaerobic culture, the strain B6 can achieve the ideal NFFO effect, and the number of the microorganisms in the culture bottle can reach the highest value, which is about 2.73 to 105 cell/ mL. The effect of different environmental factors on the NDF of the strain B6 is investigated. The results of comprehensive comparison show that the removal of NO-3-N by the strain B6 and the high oxidation efficiency of the strain B6 to the Fe (II) under the condition of the culture of the yeast extract or the sodium acetate as the carbon source, the glucose is the carbon source type which is the most favorable for the growth of the microorganism, and the NDF of the strain B6 is lower when the sodium citrate is used as a single carbon source. The effect of initial p H value, initial temperature and C/ N ratio on the NDF of the strain B6 was investigated by the response surface BBD method, and the initial p H value was found to have a greater effect on the nitrate nitrogen removal efficiency of the strain B6. The effect of the initial p H value and the C/ N ratio is greater than the initial temperature for the Fe (II) oxidation performance of the strain B6, and the NDFO performance of the strain B6 is optimized. The full-genome sequencing of strain B6 was performed using the Illumina Hi Seq 2000 sequencing technique. The results showed that the genomic size of the strain B6 was 4 592 999 bp and the GC content was about 65.3%. The gene can be divided into 27 groups according to the function:252 genes related to protein metabolism,236 genes related to carbohydrate metabolism,59 genes related to nitrogen metabolism, and 22 genes related to iron metabolism. It is known from the results of the gene sequence of the strain B6 that the strain B6 has the metabolic pathway of nitrate, nitrite and nitrous oxide and nitrous oxide in the anaerobic condition, the process and the Nar G, Nar H, NarJ, Nar I, q Nor, NnrS contained in the strain, The oxidation process of the iron (II) is related to the iron-sulfur cluster of the Nar subunit, and the oxidation process of the nitrite to the Fe (II) may be related to the scheme c of the Nir, while the dinitrogen monoxide is oxidized by the q-Nor to the Fe (II). the biological nitrogen removal and deironing reactor with the strain B6 as the main body is constructed, and the removal efficiency of the ammonia nitrogen, the nitrate nitrogen, the nitrite nitrogen, the Fe (II) and the total iron during the startup and the stable operation of the reactor is investigated, It is found that the removal rate of total nitrogen and total iron of the biological nitrogen removal and deironing reactor can reach 79.70% and 75.16%. The removal rate of nitrate nitrogen and Fe (II) in the anoxic zone of the reactor can reach 46.9% and 60.33%, respectively, and the nitrate nitrogen reduction rate can reach 0.07 mg of NO-3-N/ (L 路 h). The microbial community structure of the biological nitrogen removal and deironing reactor is relatively stable and diverse. The DO and C/ N values in the anoxic zone are beneficial to the growth of facultative anaerobic microorganisms, and the relative abundance of the genus Aquabacterium belonging to the strain B6 is 8.06%, and the NFO metabolism performance of the strain B6 is ensured. In addition, the bacillus (Bacillus), which is similar to the function of the strain B6, also has the dominant position in the anoxic zone, the relative abundance is 22.58%, and the strain B6 can not only maintain the advantages of the advantages, but also can strengthen the dominant position of the bacteria with the similar function in the system, So that the reaction system has higher ammonia nitrogen and total nitrogen treatment efficiency. The DO content and the C/ N ratio in the aerobic zone are beneficial to the step-by-step advantage of the microbial population with heterotrophic nitrification, aerobic denitrification and metabolic characteristics of the genus Corynebacterium, Chryseobacterium and Bacillus.
【学位授予单位】：哈尔滨工业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：X172

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