【摘要】：針對我國水資源污染現(xiàn)狀,本文基于鐵氧化細菌(Fe-oxidizing Bacteria,FOB)的研究理論,開展對其硝酸鹽依賴型鐵氧化(nitrate-dependent Fe(II)oxidation,NDFO)代謝特性研究。通過菌株篩選、效能考察、代謝機理解析、反應器建立與運行等手段及方法,首次明確Aquabacteria種屬菌株的NDFO代謝特性及機理。本研究在實驗室現(xiàn)保存的不同種屬的鐵氧化細菌中,通過對照試驗,篩選出一株水小桿菌屬菌株Aquabacteriaum parvum B6。該菌株在厭氧培養(yǎng)過程中對Fe(II)和硝酸鹽氮的代謝特性十分顯著,細胞數(shù)目也有明顯增加。厭氧連續(xù)培養(yǎng)試驗發(fā)現(xiàn),菌株B6在厭氧培養(yǎng)條件下,可以達到理想的NDFO效果,培養(yǎng)瓶中微生物數(shù)量也可達到最高值,約為2.73×105 cell/m L。考察不同環(huán)境因子對菌株B6的NDFO效能影響。經(jīng)過綜合比較發(fā)現(xiàn):以酵母提取物或乙酸鈉為碳源的培養(yǎng)條件下菌株B6對NO-3-N的去除和對Fe(II)的氧化效能較高;葡萄糖是最有利于微生物生長的碳源類型;檸檬酸鈉作為單一碳源時,菌株B6的NDFO效能較低。通過響應面BBD法考察初始p H值、初始溫度和C/N比對菌株B6的NDFO效能影響,發(fā)現(xiàn)初始p H值對于菌株B6的硝酸鹽氮去除效能影響較大。對菌株B6的Fe(II)氧化效能而言,初始p H值和C/N比的影響大于初始溫度,通過Optimization Numedcal Solution對其NDFO效能進行優(yōu)化。利用Illumina Hi Seq 2000測序技術(shù),對菌株B6進行了全基因組測序。由RAST annotation server基因注釋結(jié)果可知:菌株B6的基因組大小為4 592 999 bp,GC含量約65.3%�；虬垂δ芸煞譃�27類:與蛋白質(zhì)代謝相關的基因252個;與碳水化合物代謝相關的基因236個;與氮代謝相關的基因59個;與鐵代謝相關的基因22個。由菌株B6的基因序列注釋結(jié)果得知:菌株B6在厭氧條件下存在硝酸鹽→亞硝酸鹽→一氧化氮→一氧化二氮的代謝途徑,這一過程與菌株所含有的Nar G、Nar H、NarJ、Nar I、q Nor、NnrS、DNR和Nnru編碼基因密切相關;硝酸鹽對Fe(II)的氧化過程與Nar亞基的鐵硫簇有關,亞硝酸鹽對Fe(II)的氧化過程可能與Nir的heme c相關,而一氧化二氮則通過q Nor實現(xiàn)對Fe(II)的氧化。構(gòu)建以菌株B6為主體的生物脫氮除鐵反應器,并考察反應器在啟動及穩(wěn)定運行期間對于氨氮、硝酸鹽氮、亞硝酸鹽氮、Fe(II)和總鐵的去除效能,發(fā)現(xiàn)生物脫氮除鐵反應器的總氮和總鐵的去除率可達到79.70%和75.16%。對于投加了附著菌株B6的載體,反應器缺氧區(qū)其硝酸鹽氮及Fe(II)去除率則可分別達到46.9%和60.33%,其硝酸鹽氮還原速率可達到0.07mg NO-3-N/(L·h)。生物脫氮除鐵反應器的微生物群落結(jié)構(gòu)較為穩(wěn)定多樣。缺氧區(qū)中DO含量和進水C/N值有利于兼性厭氧微生物生長,菌株B6所屬水小桿菌屬(Aquabacterium)相對豐度為8.06%,其NDFO代謝效能得以保證。此外與菌株B6功能較為相近的芽孢桿菌屬(Bacillus)在缺氧區(qū)中也具有優(yōu)勢地位,相對豐度為22.58%,菌株B6不僅可以保持較為優(yōu)勢的地位,也可以強化系統(tǒng)中與其功能相近的菌屬的優(yōu)勢地位,因而得以保證反應系統(tǒng)具有較高的氨氮和總氮處理效能。而好氧區(qū)中的DO含量和C/N比值則有利于鞘氨醇桿菌屬(Sphingobacterium),金黃桿菌屬(Chryseobacterium)和芽孢桿菌屬(Bacillus)的具有異養(yǎng)硝化、好氧反硝化代謝特性的微生物種群逐步處于優(yōu)勢地位。
[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.
【學位授予單位】：哈爾濱工業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：X172

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