本文關鍵詞：海洋石油降解菌的多樣性研究　出處：《山東大學》2016年碩士論文　論文類型：學位論文

  更多相關文章： 海洋 柴油 降解菌 降解率 高通量測序

【摘要】：目前,海洋污染日趨加劇,而其中的石油污染尤為嚴重,不僅威脅海洋生物的生存、生長和繁殖,降低海洋的使用價值,破壞海岸設施,還會影響該海區(qū)的水文氣象條件和降低海洋的自凈能力。治理海洋石油污染的主要方法有物理法、化學法和生物法。而其中生物治理憑借其費用低且不產生二次污染等優(yōu)勢表現出巨大的潛力,由此石油降解菌成為國內外學者的研究熱點。本文從2013年9月份開始,以石油降解菌為研究對象,選取威海小石島水樣進行預實驗,確定了切實可行的試驗方案。于2014年3月至2015年7月分批多次從不同站位取得海水水樣進行試驗。一方面水樣用于石油降解菌的分離篩選工作,同時對篩得的細菌進行16S rDNA序列測定,最終確定其種屬類別；另一方面對部分水樣進行高通量測序,對石油降解菌的多樣性做進一步深入的研究。主要工作如下：(1)選取全國13個不同油污染的地區(qū)水樣進行研究,地點包括：大連的大連灣和金石灘、威海小石島和金沙灘、青島團島和薛家島碼頭、煙臺、天津港、秦皇島、上海、廈門公務碼頭、深圳東角頭。試驗以柴油作為培養(yǎng)基的唯一碳源,篩選出具有柴油降解能力的菌株,已做序列測定的有19株,鑒定結果為：芽孢桿菌屬(Bacillus)6株,假單胞菌屬(Pseudomonas)3株,鞘脂菌屬(Sphingobium) 2株,弧菌屬(Vibrio)2株,土芽孢桿菌屬(Geobacillus)1株,短芽孢桿菌屬(Brevibacillus)1株,短桿菌屬(Brevibacterium)1株,亞硫酸鹽桿菌屬(Sulfitobacter)1株,副球菌屬(Paracoccus)1株,不動桿菌屬(Acinetobacter)1株；其中4株為新種,有待于進一步的研究。并測得18℃、28℃和30℃下細菌的生長曲線和對柴油的降解率,可以看出：所有菌株生長趨勢均為“細菌型曲線生長,分為調整期、對數生長期、穩(wěn)定期和衰退期。在28℃和30℃下細菌的調整期、對數生長期均比18℃下的時間短,細菌數量多。三種溫度下細菌對柴油降解率均在30%以上,不同菌株在不同溫度下對柴油的降解率略有差異。(2)同時本試驗對大連、石島、深圳地區(qū)添加1%(V/V)柴油前后的水樣進行高通量測序,可以看出：添加柴油前后的水樣中共有OTU含量僅為10%左右,且細菌的總量、種類都發(fā)生了顯著差異。大連水樣添加柴油前約有221屬,添加柴油后減少為173個屬；石島水樣添加柴油前約有321屬,添加柴油之后減少為196屬,深圳水樣在添加柴油前有241屬,添加柴油后變?yōu)?03屬。其中深圳水樣中菌落結構變化最少,大連次之,石島水樣中菌群差異最為顯著,這和采樣水域石油污染程度不同一致。假螺菌屬(Pseudospirillum)在三個地區(qū)均相對豐富,添加柴油之后出現不同程度的減少；科爾韋爾氏菌屬(Colwellia)為石島和深圳水樣的優(yōu)勢種,在大連地區(qū)卻未發(fā)現,添加柴油之后明顯減少,幾乎不能適應新環(huán)境；食烷菌屬(Alcanivorax)和單胞菌屬(Alteromonas)在添加柴油之后的三組水樣中顯著增加,體現了這兩種菌對柴油的偏好性�？傮w來說,柴油對海洋生態(tài)環(huán)境菌群的結構和數量均有較大影響。
[Abstract]:At present, the marine pollution is becoming more and more serious, and the oil pollution is particularly serious. It not only threatens the survival, growth and reproduction of marine organisms, reduces the use value of the ocean, destroys the coastal facilities, but also affects the hydrometeorological conditions in the sea area and reduces the self purification capacity of the ocean. The main methods for controlling marine oil pollution include physical, chemical and biological methods. Biological treatment has great potential because of its low cost and no two pollution. Therefore, petroleum degrading bacteria have become the research focus of scholars at home and abroad. Starting from September 2013, this paper takes the petroleum degrading bacteria as the research object, selects the Weihai small Shidao water sample to carry on the pre experiment, and confirms the practical test plan. Water samples from different stations were tested in different stations from March 2014 to July 2015. On the one hand, water samples are used for isolation and screening of petroleum degrading bacteria. Meanwhile, the 16S rDNA sequence of the screened bacteria is determined, and finally the species categories are determined. On the other hand, some water samples are sequenced with high flux, and further research is done on the diversity of petroleum degrading bacteria. The main work is as follows: (1) selected the 13 different oil polluted areas of water, place include: Dalian and Dalian Bay, Weihai Golden Pebble Beach small Shidao and Jinshatan, Qingdao, Yantai Tuandao and Xuejiadao pier, Tianjin port, Qinhuangdao, Shanghai, Xiamen, Shenzhen official pier dongjiaotou. Experiment with diesel oil as sole carbon source in culture medium, and screened with diesel degrading strains, have been sequenced 19 strains, identification results: Bacillus (Bacillus) and 6 strains of Pseudomonas aeruginosa (Pseudomonas) of 3 strains of bacteria, sphingolipids (Sphingobium) and 2 strains of Vibrio (Vibrio) 2 strains of soil Bacillus (Geobacillus) of 1 strains, short bacillus (Brevibacillus) of 1 strains, Brevibacterium (Brevibacterium) of 1 strains of Bacillus, sulfite (Sulfitobacter) and 1 strains of Paracoccus sp. (Paracoccus) and 1 strains of Acinetobacter 1 strains (Acinetobacter); 4 plant species need to be further studied. The growth curve of bacteria and the degradation rate of diesel oil at 18, 28 and 30 degrees were measured. It can be seen that the growth trend of all strains is "bacterial curve growth", which is divided into adjustment period, logarithmic growth stage, stable stage and recession stage. At 28 and 30, the period of bacterial adjustment and the logarithmic growth period are shorter than those under 18, and the number of bacteria is more. The degradation rate of diesel oil was above 30% at three temperatures, and the degradation rate of diesel was slightly different at different temperatures. (2) at the same time, this experiment carried out high flux sequencing of water samples before and after adding 1% (V/V) diesel in Dalian, Shidao and Shenzhen regions. It can be seen that the OTU content of water samples before and after adding diesel oil is only about 10%, and the total amount and species of bacteria are significantly different. About 221 genera were added to the water samples of Dalian, and 173 genera were reduced after adding diesel oil. About 321 genera were added to Shidao water samples, and 196 genera after adding diesel oil. Shenzhen water samples had 241 genera before adding diesel oil, and then 203 after adding diesel fuel. Among them, the change of bacterial structure in Shenzhen water samples is the least, followed by Dalian. The difference of flora in Shidao water samples is the most significant, which is consistent with the degree of oil pollution in sampling waters. False Spirillum (Pseudospirillum) in the three regions are relatively abundant, add diesel after different degrees of reduction; Kerr Weir S sp. (Colwellia) is the dominant species in Shidao and Shenzhen samples, but not found in the Dalian area, after adding diesel was significantly reduced, almost can not adapt to the new environment; food bacteria (alkane Alcanivorax) and Aeromonas (Alteromonas) in three groups after adding water samples in diesel increased significantly, which embodies the two kinds of bacteria on diesel preference. In general, diesel has a great influence on the structure and quantity of the marine ecological environment bacteria.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：X172;X55

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