【摘要】：自然界中存在廣泛的微生物資源,其生存環(huán)境各異,且有豐富的基因多樣性。從自然界中廣泛分離污染物分解菌,研究其污染物降解活性和基因特征之間的關(guān)系是從分子水平揭示微生物的污染物降解活性及其影響因素,從而研制出高效污染物降解菌的最有效方法。本文旨在揭示自然高鹽環(huán)境中耐鹽苯酚降解菌的生物多樣性、代謝多樣性以及對苯酚的降解活性,為今后基因序列和苯酚降解活性之間的關(guān)系研究提供參考和依據(jù)。本文以內(nèi)蒙古地區(qū)額濟(jì)淖爾鹽湖、吉蘭泰鹽湖以及查干淖爾鹽湖為研究對象,馴化分離了耐鹽苯酚降解菌。研究發(fā)現(xiàn),從額濟(jì)淖爾湖馴化培養(yǎng)出的菌群能夠顯著降低培養(yǎng)液中的苯酚濃度。應(yīng)用以苯酚為唯一碳源的固體培養(yǎng)基分離出了該菌群中的微生物,并挑選其中兩株進(jìn)行了16SrDNA法鑒定。結(jié)果表明挑選出的兩株苯酚降解菌分別屬于丙酸桿菌屬和鹽單胞菌屬。利用苯酚單加氧酶基因、鄰苯二酚1,2雙加氧酶基因以及鄰苯二酚2,3雙加氧酶基因的簡并引物檢測了苯酚降解過程中的關(guān)鍵酶的編碼基因。在屬于丙酸桿菌屬的苯酚降解菌中檢測到了苯酚單加氧酶基因和鄰苯二酚1,2雙加氧酶基因,說明該菌可能利用苯酚單加氧酶將苯酚轉(zhuǎn)化為鄰苯二酚,并將鄰苯二酚進(jìn)一步分解為粘康酸。雖然對PCR條件進(jìn)行了多次優(yōu)化,在屬于鹽單胞菌的苯酚降解菌中未能檢測到任何功能基因,說明該菌降解基因序列與目前發(fā)現(xiàn)的降解基因序列有較大的差異。應(yīng)用含200 mg／L苯酚,10%鹽度的培養(yǎng)液初步試驗了分離出的菌株對苯酚的降解活性。經(jīng)一輪的馴化培養(yǎng)后,丙酸桿菌能夠在12天之內(nèi)降解99.5%的苯酚,鹽單胞菌在12天之內(nèi)降解99.0%的苯酚。研究發(fā)現(xiàn),50mg／L的酵母提取物對丙酸桿菌的苯酚降解活性沒有影響,而不存在酵母提取物時鹽單胞菌對苯酚的降解活性顯著降低。本文對于自然環(huán)境中耐鹽苯酚降解菌多樣性和降解基因多樣性了解以及污染物處理工程菌的開發(fā)利用具有重要的科研價值。
[Abstract]:There are a wide range of microbial resources in nature, their living environment is different, and there is abundant genetic diversity. Pollutant decomposing bacteria were widely isolated from nature. The relationship between their pollutant degradation activity and genetic characteristics was revealed at molecular level. Therefore, the most effective method for the degradation of pollutants was developed. The purpose of this paper is to reveal the biological diversity, metabolic diversity and degradation activity of phenol-tolerant bacteria in natural high-salt environment, and to provide a reference and basis for the study of the relationship between gene sequence and phenol degradation activity in the future. In this paper, salt-tolerant phenol degrading bacteria were domesticated and isolated from Ejinaoer Salt Lake, Jilantai Salt Lake and Chagannur Salt Lake in Inner Mongolia. It was found that the microbial communities domesticated from Eziannur Lake could significantly reduce the concentration of phenol in the culture medium. The microbes in the microflora were isolated in solid medium with phenol as the sole carbon source and two of them were selected for identification by 16SrDNA. The results showed that the two phenol-degrading bacteria belonged to Propionibacterium and Salmonella respectively. The degenerate primers of phenol monooxygenase gene, catechol 1 + 2 dioxygenase gene and catechol 2 + 3 double oxygenase gene were used to detect the key enzyme coding genes in the process of phenol degradation. Phenol monooxygenase gene and catechol 1 / 2 dioxygenase gene were detected in phenol-degrading bacteria belonging to Propionibacterium, indicating that phenol could be transformed into catechol by phenol monooxygenase. The catechol was further decomposed into mucoronic acid. Although the PCR conditions were optimized many times, no functional gene was detected in phenol degrading bacteria belonging to Salmonella, which indicated that the degradation gene sequence of this strain was different from that of the current one. The degradation activity of phenol was preliminarily tested in the culture medium containing 10% salt of 200 mg/L phenol. After one round of acclimation, propionic acid bacillus could degrade 99.5% phenol within 12 days, and Salmonella could degrade 99.0% phenol within 12 days. It was found that the yeast extract of 50 mg / L had no effect on phenol degradation activity of Propionibacterium propioniae, but the phenol degradation activity of Salmonella Salmonella was significantly decreased when there was no yeast extract. This paper has important scientific research value for understanding the diversity of phenol-tolerant bacteria and degrading gene diversity in natural environment and for the development and utilization of pollutant treatment engineering bacteria.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：X172;X703

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