海洋石油降解菌的多样性研究

发布时间：2017-12-28 09:02

本文关键词：海洋石油降解菌的多样性研究　出处：《山东大学》2016年硕士论文　论文类型：学位论文

【摘要】：目前,海洋污染日趋加剧,而其中的石油污染尤为严重,不仅威胁海洋生物的生存、生长和繁殖,降低海洋的使用价值,破坏海岸设施,还会影响该海区的水文气象条件和降低海洋的自净能力。治理海洋石油污染的主要方法有物理法、化学法和生物法。而其中生物治理凭借其费用低且不产生二次污染等优势表现出巨大的潜力,由此石油降解菌成为国内外学者的研究热点。本文从2013年9月份开始,以石油降解菌为研究对象,选取威海小石岛水样进行预实验,确定了切实可行的试验方案。于2014年3月至2015年7月分批多次从不同站位取得海水水样进行试验。一方面水样用于石油降解菌的分离筛选工作,同时对筛得的细菌进行16S rDNA序列测定,最终确定其种属类别；另一方面对部分水样进行高通量测序,对石油降解菌的多样性做进一步深入的研究。主要工作如下：(1)选取全国13个不同油污染的地区水样进行研究,地点包括：大连的大连湾和金石滩、威海小石岛和金沙滩、青岛团岛和薛家岛码头、烟台、天津港、秦皇岛、上海、厦门公务码头、深圳东角头。试验以柴油作为培养基的唯一碳源,筛选出具有柴油降解能力的菌株,已做序列测定的有19株,鉴定结果为：芽孢杆菌属(Bacillus)6株,假单胞菌属(Pseudomonas)3株,鞘脂菌属(Sphingobium) 2株,弧菌属(Vibrio)2株,土芽孢杆菌属(Geobacillus)1株,短芽孢杆菌属(Brevibacillus)1株,短杆菌属(Brevibacterium)1株,亚硫酸盐杆菌属(Sulfitobacter)1株,副球菌属(Paracoccus)1株,不动杆菌属(Acinetobacter)1株；其中4株为新种,有待于进一步的研究。并测得18℃、28℃和30℃下细菌的生长曲线和对柴油的降解率,可以看出：所有菌株生长趋势均为“细菌型曲线生长,分为调整期、对数生长期、稳定期和衰退期。在28℃和30℃下细菌的调整期、对数生长期均比18℃下的时间短,细菌数量多。三种温度下细菌对柴油降解率均在30%以上,不同菌株在不同温度下对柴油的降解率略有差异。(2)同时本试验对大连、石岛、深圳地区添加1%(V/V)柴油前后的水样进行高通量测序,可以看出：添加柴油前后的水样中共有OTU含量仅为10%左右,且细菌的总量、种类都发生了显著差异。大连水样添加柴油前约有221属,添加柴油后减少为173个属；石岛水样添加柴油前约有321属,添加柴油之后减少为196属,深圳水样在添加柴油前有241属,添加柴油后变为203属。其中深圳水样中菌落结构变化最少,大连次之,石岛水样中菌群差异最为显著,这和采样水域石油污染程度不同一致。假螺菌属(Pseudospirillum)在三个地区均相对丰富,添加柴油之后出现不同程度的减少；科尔韦尔氏菌属(Colwellia)为石岛和深圳水样的优势种,在大连地区却未发现,添加柴油之后明显减少,几乎不能适应新环境；食烷菌属(Alcanivorax)和单胞菌属(Alteromonas)在添加柴油之后的三组水样中显著增加,体现了这两种菌对柴油的偏好性。总体来说,柴油对海洋生态环境菌群的结构和数量均有较大影响。
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：X172;X55

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