【作者】 吴涛；

【导师】 依艳丽；

【作者基本信息】 沈阳农业大学 ， 土壤学， 2013， 博士

【摘要】 土壤石油污染对人类健康和生态系统安全构成了严重威胁,是目前亟待解决的重要环境问题。在我国,大部分陆上石油生产基地的土壤均有不同程度的盐渍化,石油在开采、加工和利用等过程中容易造成盐渍化土壤的石油污染,形成盐渍化石油污染土壤。生物修复是治理石油污染土壤的一种有效、经济、环保的方法,关于生物修复石油污染土壤的成功实例,国内外已有很多报道,但对于盐渍化石油污染土壤修复的研究罕见报道,如何有效修复该类土壤是迫切需要解决的重要任务。本文从黄河三角洲长期受盐渍化石油污染土壤中筛选出1株高效耐盐石油降解菌BM38和1株产生物表面活性剂耐盐菌BF40,通过液体培养试验,研究了BM38耐盐、产生物表面活性剂、降解石油特性,考察了不同因子对BF40产生物表面活性剂的影响以及BF40产生物表面活性剂的动力学特征,并对其产物进行了理化性质分析和初步鉴定；通过室内模拟石油污染土壤修复试验,研究了BM38、BF40及其产物对盐渍化石油污染土壤的强化修复作用,以及土壤环境条件对接种BM38、同时添加BF40产生的表面活性剂强化修复石油污染土壤的影响；通过种子发芽、植物修复试验筛选出1种适合修复石油污染土壤的耐盐植物,构建了盐渍化石油污染土壤的植物-微生物联合修复系统。研究成果将为盐渍化石油污染土壤的生物修复提供支持。本文主要研究结果如下：1.从黄河三角洲盐渍化石油污染土壤中分离出54株细菌,经液体培养初筛和土壤培养复筛试验,得到1株高效耐盐石油降解菌BM38。通过形态特征、生理生化特征和16SrDNA序列分析,确定该菌为恶臭假单胞菌(Pseudomonas putida)。耐盐试验表明,BM38在含0.5%-6%NaCl液体培养基中生长良好,属中度耐盐菌。BM38能产生一种生物乳化剂,其发酵液最大乳化能力出现在生长稳定期,NaCl浓度对发酵液的乳化能力影响较大。BM38能够利用环己烷、甲苯、异辛烷、菲和正十六烷为唯一碳源生长,其中对正构烷烃和芳烃具有较强的利用能力。在含1%、3%和5%NaCl的培养基中BM38对原油降解曲线符合一级反应动力学模型。在中性以及偏碱性的环境中利于BM38对原油的降解,在含0～3%NaCl液体培养基中BM38对原油降解率较高。温度、原油浓度和接种量对BM38降解原油的能力影响较大。2.通过原油平板法从黄河三角洲盐渍化石油污染土壤分离出41株产生物表面活性剂细菌,通过测定发酵液排油活性、表面张力和乳化值,确定了一株高效产生物表面活性剂耐盐菌BF40。BF40可将发酵液表面张力降低到28.4mN·m-1,乳化值(E124)达到96.8%。经鉴定BF40为沙雷氏菌(Serratia sp.)。BF40耐受盐度范围为0.5-7%NaCl,属中度耐盐菌。BF40在含1%NaCl液体培养基中,培养7d,原油降解率达到56.7%。BF40合成生物表面活性剂与其利用的碳氮源类型有关,其中以牛肉膏为碳源,氯化铵为氮源,产生的生物表面活性剂活性最高。在30～37℃,中性及偏碱性环境中BF40产生物表面活性剂较多。NaCl浓度对BF40产生物表面活性剂影响较大,其中对发酵液表面张力的影响程度小于乳化性能。BF40产生物表面活性剂的方式为生长相关型。经薄层层析、GC-MS分析初步鉴定BF40产生的生物表面活性剂(BS40)为脂肽类。BS40水溶液的临界胶束浓度为32.8mg·L-1,具有良好的耐高温、耐盐性和耐酸碱性,对二甲苯、柴油、液体石蜡和橄榄油具有较强的乳化能力。3.石油污染土壤修复试验结果表明,BM38分泌的生物乳化剂(BS38)对盐渍化土壤中石油污染物生物降解强化作用不明显,接种外源菌(BF40、BM38、BM38+BF40)或添加BF40产生的生物表面活性剂(BS40)能促进土壤中石油污染物的降解,其中添加BS40、同时接种BM38修复效果最好,修复60d,石油降解率达到48.3%。石油族组分分析和气相色谱分析表明,添加BS40、同时接种BM38更能有效促进土壤中饱和烃、芳香烃和沥青质的降解,其中正构烷烃几乎全被降解,对姥鲛烷和植烷降解效果明显。接种外源菌(BF40、BM38、BM38+BF40)或添加生物表面活性剂(BS40)、生物乳化剂(BS38)对土壤土壤水溶液表面张力、土壤脱氢酶活性影响不同,表明接种外源菌和添加生物表面活性剂对促进盐渍化石油污染土壤的生物修复存在不同作用机制。4.高盐环境抑制土著微生物降解土壤中石油污染物能力,土壤中接种BM38、同时添加BS40能明显促进不同盐度土壤中石油污染物的降解。在中性偏碱的土壤中接种BM38、同时添加BS40强化石油污染物降解效果较好。在中、高度石油污染水平下,接种BM38、同时添加BS40能明显提高石油污染物的降解效率。添加玉米芯对接种BM38、同时添加BS40强化修复盐渍化石油污染土壤效果好于棉籽壳、麦糠和玉米杆。5.从黄河三角洲筛选出一种对石油污染物耐性强,生物量较高,强化土壤中石油污染物的降解效果好的本土植物虎尾草。在盐渍化石油污染土壤中接种BM38、添加BS40和玉米芯、种植虎尾草组成的植物-微生物复合修复系统与单一处理相比土壤总异养细菌和石油降解细菌数量最多,石油污染物降解率最大。在复合修复系统中BM38和BS40对土壤饱和烃和芳香烃的降解起主要作用,虎尾草则更能有效去除土壤中胶质。土壤脱氢酶活性与土壤中微生物的数量呈显著的正相关关系。接种BM38、同时添加BS40在修复后期对提高土壤脱氢酶活性作用很有限,而种植虎尾草在修复过程中能保持较高的土壤脱氢酶活性。更多还原

【Abstract】 Petroleum contamination, which has significantly threatened human health and ecosystem security, is an important environmental problem that should be solved quickly. In China, the soils of many oil production bases are various degree of salinity. The saline soil is easy to be contaminated when the petroleum is exploited, processed and utilized, finally turned into petroleum contaminated saline soil. Bioremediation is an effective, economic and environmental way to eliminate petroleum contaminated saline soil. There are many studies reported the bioremediation of petroleum contaminated soil, but the bioremediation of petroleum contaminated saline soil, which has received less attention in the literature, is an important environmental task that should be solved quickly. In this study, one efficient halotolerant petroleum-degrading bacteria (BM38) and an high-efficient biosurfactant-producing bacteria (BF40) were screened from the long term petroleum contaminated saline soil samples in Yellow River Delta. A series of liquid incubation experiments were conducted to researched the halotolerance, producing biosurfactants and characteristics of petroleum-degradating of BM38, discussed the effect of different factors to the biosurfactant and dynamics of a salt-tolerant Serratia BF40, analyzed and identified the product of BF40based on the morphological, physiochemical characteristics. Laboratory simulation test of petroleum contaminated soil remediation were also studied to discuss the effects of BM38, BF40and their products to the petroleum degradation in saline soil, the effects of different soil conditions to the accelerating of saline soil remediation by BM38and BF40inoculating and biosurfactant application. A halophyte that was suitable for petroleum contaminated soil remediation was screened by seed germination experiments and plant remediation texts. A phyto-microbial remediation system in petroleum contaminated saline soil was structured. The results will provide support for the bioremediation of petroleum contaminated saline soil. The main result of the study could be seen as below:1.54bacteria strains were isolated from the petroleum contaminated saline soil samples in Yellow River Delta, One bacterium (strain BM38) were found to efficiently degrade crude oil in highly saline environments based on a series of liquid and soil incubation experiments. According to its morphology, physiochemical characteristics and16S rDN A sequence analysis, this strain was identified as Pseudomonas putida. The salt resistant test demonstrated that strain BM38grew well at NaCl concentrations ranging from0.5%to6.0%. Strain BM38could produce a bioemulsifier in a liquid culture medium. The highest emulsifying capacity of fermentation broth comes out in the steady growth stage. But NaCl concentration had the significant effect on the emulsifying capacity of fermentation broth. This strain was able to grow in mineral liquid media amended with hexadecane, toluene, phenanthrene, isooctane and cyclohexane as the sole carbon sources. Among these hydracarbons, strain BM38showed the higher ability to degrade n-alkanes and aromatic hydracarbons. The crude oil degradation curve of BM38conforms to the first-order reaction kinetic model in the medium containing1%,3%and5%NaCl. The neutral and alkaline environments are suitable for degradating of BM38to crude oil. The degradating rate was higher at NaCl concentrations ranging from0%to3%. The temperature, concentration of crude oil and the inoculation amounts have significant effects to the degradating capacity of BM38.2.41microbial strains were isolated from saline soil contaminated by crude oil of the Yellow River Delta through plate culture method, using crude oil as C and energy source. BF40strain with salt-tolerant ability was screeded in this study, based on culture solution of degreasing, biosurfacial and emulsifying activities from different strains. Surface tension of BF40culture solution reduced to28.4mN·m-1, and emulsifying index was96.8%. BF40strain was identified as Serratia sp. It can grow in the saline condition ranged from0.5～7%of NaCl, and its salt-tolerant ability reached moderate level. After incubation of7d,56.7%of crude oil in BF40liquid culture with1.0%of NaCl was degraded. Biosurfactant production of BF40was affected by the forms of C and N sources. The greatest production was observed in the treatment using beef extract as C source and NH4Cl as N source. The higher production also founded in neutral or weak alkaline condition under temperature ranged from30to37℃. Biosurfacial activity of BF40was affected by salinity obviously. The lower effect of salinity on biosurfacial activity was founded than that on emulsifying activity. Biosurfacial activity of BF40was proposed to be the model of grow correlated. Biosurfactant (BS40) from BF40was determined to be lipopeptides by thin layer chromatography and GC-MS analysis. The critical micelle concentration of BS40solution was32.8mg·L-1. BS40possessed high temperature, salt, and acid-base tolerant properties. Higher emulsifying activity also observed for p-xylene, diesel, liquid paraffin and olive oil.3. Results of soil remediation experiment showed that biosurfactant (BS38) from BM38increased crude oil biodegradation insignificantly. Inoculation with exogenous strains (BF40, BM38, and BM38+BF40) or biosurfactant of BS40can increase crude oil degradation, and the greatest degradation rate was founded in the treatment with BS40addition and BM38inoculation, showing48.3%of degradation rate after60d incubation. Combination of BS40addition and BM38inoculation can effectively enhance saturated and aromatic hydrocarbon and asphalt degradation through GC analysis, and n-alkanes was almost degraded completely. The improvement of pristine and phytane degradation was also significant. Different effects on surface tension of soil solution and soil dehydrogenase activity were observed among exogenous strain inoculation, BS40addition, and BS38addition, which indicated that exogenous strain inoculation and biosurfactant addition improve soil remediation through different mechanisms.4. High salinity inhibited crude oil degradation in soil. Combination of BM38seeding and BS40addition can increase crude oil degradation in soils with different salinity. In weak alkaline soil, the greater crude oil dissipation was in the treatment with combination of BM38seeding and BS40addition. In the soil contaminated with high and moderate levels of crude oil, combination of BM38seeding and BS40addition can significantly increase crude oil degradation rate. Corncob addition increased crude oil degradation better than that did by cottonseed hull, wheat bran and cornstalk addition.5. Phytoremediation experiment showed that Alopecurus pratensis Swartz. possessed high crude oil tolerant ability, biomass, and the greatest remediation efficiency. In plant-microbial systems, Alopecurus pratensis Swartz. cultivation in combination of BM38seeding and BS40and corncob addition treatment, significantly increased population of total heterotrophic bacteria and crude oil degrading bacteria, which in turn enhance degradation. In combined remediation system, saturated and aromatic hydrocarbon degradation was mainly caused by BM38seeding and BS40addition, and gelatine degradation was mainly caused by Alopecurus pratensis Swartz. cultivation. Soil dehydrogenase activity positively correlated with microbial population. Combination of BM38seeding and BS40addition increased soil dehydrogenase activity insignificantly, while Alopecurus pratensis Swartz. cultivation increased significantly.更多还原