【摘要】：近年來,我國青藏高原凍土區(qū)石油開采、運輸、加工等過程中事故頻發(fā),對凍土土壤環(huán)境造成嚴重污染。本文對青藏高原凍土區(qū)土壤石油污染微生物多樣性進行研究,對北麓河地區(qū)土壤溫度連續(xù)監(jiān)測,富集篩分石油土著低溫菌,通過高通量測序技術,分析石油污染下凍土區(qū)微生物的種群結構變化以及混合石油菌的生物種群結構,結合實驗室低溫模擬凍土石油污染修復試驗,研究土著低溫石油菌在不同實驗條件下對土壤石油污染物修復效果,旨在為凍土區(qū)石油類污染生物修復工程提供技術支持。本文主要成果為以下幾個方面:(1)青藏高原北麓河地區(qū)夏季7月中旬至9月上旬土壤溫度最高,為微生物活性最高時間段,是青藏高原石油污染微生物修復的關鍵季節(jié)。北麓河土壤顯弱酸,有機質含量較高,石油污染微生物修復所需的營養(yǎng)元素在北麓河區(qū)域土壤中含量較高,總氮、總磷等基本可以滿足低濃度石油污染生物修復的需求,對于高濃度石油污染修復,則需采用添加營養(yǎng)劑的方法滿足修復過程中對營養(yǎng)鹽的需求。(2)石油污染影響土壤微生物種群結構,高通量測序顯示北麓河地區(qū)未污染土壤(表層、下層)、柴油污染土、機油污染土中優(yōu)勢菌群存在較大差異,反映了油類污染對土壤微生物結構產(chǎn)生了較大的影響,改變了原有群落結構,致使部分微生物消亡,同時對適應以油類為C源的降解菌群所占比重提高,現(xiàn)場油類污染有利于高效油類降解菌的實驗室篩選馴化。(3)恒溫震蕩培養(yǎng)箱對土著低溫石油菌進行富集篩選,利用微生物平板法結合電子顯微鏡進行檢驗,顯示柴油、機油菌液生長形態(tài)以桿狀為主。低溫營養(yǎng)搖瓶條試驗顯示:柴油低溫降解菌17天對柴油的降解率達60%以上;機油低溫降解菌30天對機油的降解率達54.7%。(4)高通量測序分析顯示:土著低溫柴油降解菌、機油降解菌在種群結構上存在一定的相似性,主要為變形菌門。土著低溫柴油降解菌測序顯示為71個菌種,優(yōu)勢菌群為Alkanindiges(54.4%)和Pedobacter(18.2%);土著低溫機油降解菌測序顯示為91個菌種,優(yōu)勢菌群為PYR10d3_norank(23.0%)、Sulfuritalea(19.4)、Parvibaculum(10.94%)和Sphingobium(10.89%)。(5)柴油降解土壤試驗顯示噬冷柴油降解菌液對柴油污染具有較好的降解效果,提高菌液配比、添加營養(yǎng)鹽和表面活性劑可提高其降解效率;在不同柴油污染強度下,菌液對土壤柴油降解的效率具有顯著差異,低污染強度下降解效率遠高于高污染強度。
[Abstract]:In recent years, oil mining, transportation, processing and other accidents occurred frequently in the frozen soil area of Qinghai-Tibet Plateau, which caused serious pollution to the frozen soil environment. The microbial diversity of soil oil pollution in frozen soil of Qinghai-Xizang Plateau was studied in this paper. The soil temperature was continuously monitored and screened by high-throughput sequencing technique. This paper analyzes the changes of microbial population structure in frozen soil under petroleum pollution and the biological population structure of mixed petroleum bacteria, and combines with laboratory experiments to simulate oil pollution in frozen soil at low temperature. In order to provide technical support for bioremediation of petroleum pollution in frozen soil, the remediation effect of indigenous low temperature petroleum bacteria on soil oil pollutants under different experimental conditions was studied. The main achievements of this paper are as follows: (1) the soil temperature is the highest from mid-July to early September in the northern part of Qinghai-Xizang Plateau in summer, which is the most active period of microorganism, which is the key season for microbial remediation of oil pollution in Qinghai-Tibet Plateau. The soil of the Beilu River shows weak acid, the content of organic matter is high, the nutrient elements needed for the microbial remediation of petroleum pollution are higher in the soil of the Beilu River region, the total nitrogen and total phosphorus can basically meet the demand of bioremediation with low concentration of petroleum pollution. For the remediation of high concentration oil pollution, it is necessary to use the method of adding nutrients to meet the demand for nutrients. (2) Oil pollution affects the structure of soil microbial population, and high-throughput sequencing shows that the soil is not contaminated in the Beilu River region. In the lower layer, the dominant microflora in diesel-contaminated soil and oil-contaminated soil is different, which reflects that oil pollution has a great influence on soil microbial structure, which changes the original community structure and results in the extinction of some microbes. At the same time, the proportion of biodegradable bacteria adapted to C source was increased, and field oil pollution was beneficial to the laboratory screening and domestication of high-efficiency oil-degrading bacteria. (3) enrichment and screening of indigenous low-temperature petroleum bacteria in constant temperature shock incubator. The microbiological plate method combined with electron microscope showed that the growth of diesel oil and oil was mainly rod-like. The low temperature nutrition flask test showed that the degradation rate of diesel oil was over 60% in 17 days and 54.7% in 30 days. (4) High throughput sequencing analysis showed that the degradation rate of diesel oil was 60% in 17 days, and 54.7% in 30 days. (4) the results of high throughput sequencing showed that the degradation rate of diesel oil was 60% in 17 days, and 54.7% in 30 days. There was some similarity in the population structure of oil degrading bacteria, mainly Proteus. Alkanindiges (54.4%) and Pedobacter (18.2%) were dominant strains in 71 strains of native low-temperature diesel oil degrading bacteria and 91 strains of native low-temperature oil degrading bacteria were sequenced. The results showed that PYR10d3_norank (23.0%) Sulfuritalea (19.4) Parvibaculum (10.94%) and Sphingobium (10.89%). (5) had better degradation effect on diesel oil pollution. Under different pollution intensity of diesel oil, the degradation efficiency of bacteria solution on soil diesel oil was significantly different, and the degradation efficiency of low pollution intensity was much higher than that of high pollution intensity.
【學位授予單位】：蘭州交通大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：X53;X172

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