【作者】 刘畅；

【导师】 马放；

【作者基本信息】 哈尔滨工业大学 ， 环境科学与工程， 2012， 博士

【摘要】 绿色生物制剂生物破乳剂以其低毒、无二次污染、可生物降解等独特的环境友好特性，逐渐成为该领域的研究热点。原油采出液因为使用水驱、蒸汽驱以及聚合物驱、碱驱、三元复合驱等强化驱油技术导致温度偏高，pH值变化范围大，使其破乳脱水增加了难度。运用生物破乳剂替代大量使用的化学破乳剂，对提高乳状液脱水率，降低环境污染风险具有深远意义。但随着生物破乳剂研究的不断深入，野生菌的产量或性能难以满足工业生产的要求、生物破乳剂的使用受环境条件制约以及生产成本高等问题，已成为阻碍其大规模生产和推广的关键。本课题围绕以上问题开展相关研究，采用诱变方法改良生物破乳剂产生菌菌种的破乳能力，通过环境条件定向筛选的方法改良菌种极端环境适应能力；从蛋白质组学角度解析环境条件定向筛选对耐高温、耐碱和耐酸突变株的作用机制；初步开展破乳菌复配培养产复合型生物破乳剂的研究，为今后实际生产获得高效、稳定、广谱，廉价的生物破乳剂奠定理论基础。为了提高野生菌株的破乳能力，本文对野生破乳菌XH1(Bacillus sp.)进行诱变育种。通过比较不同的诱变因子对破乳菌XH1的诱变效应，确定最佳诱变方式为紫外线+亚硝基胍（NTG）的复合诱变，筛选获得的最佳破乳突变株XN5，与出发菌株相比破乳能力提高15.12%。优化突变株XN5的培养条件，确定最佳培养条件为：培养温度35℃、摇床转数140min、培养基初始pH值7.0、培养时间21h。考察复合诱变对突变株XN5破乳性能的影响，发现与原始菌株XH1相比，诱变后所产生物破乳剂的投加量减少，破乳速率提高，但环境温度和pH值适应范围无显著变化。通过诱变后进行环境条件定向筛选分别获得了耐高温、耐酸和耐碱的突变株XT3、XP3-3和XP11-2。突变株与原始菌株XH1相比在目的环境条件下的破乳性能大幅提高。利用SDS-PAGE、2-DE和nanoESI-Q-TOF MS/MS质谱结合的蛋白质组学研究手段，对原始菌株和突变株在相应条件下获得的差异表达蛋白质进行分离、鉴定和功能解析，从蛋白质组层面对环境条件定向筛选对与生物破乳剂产生菌的作用机制进行初步的分析。采用生物表面活性剂检测方法及破乳试验相结合的筛选方法，从太平污水处理厂曝气池污泥泡沫中筛选出1株以液体石蜡为碳源的高效、稳定的破乳菌（24h排油率98.5%），实验室编号L1，经鉴定为Gordonia sihwensis。该菌全培养液的破乳能力不受升温处理，高压灭菌和反复冻融的影响(24h排油率>90%）。在无机盐培养基(MSM)中考察培养条件对破乳菌L1破乳效能的影响，获得最佳培养条件为：培养温度30℃、摇床转数140min、培养基初始pH值7.0、培养时间60h。为了摸索复配培养产复合型生物破乳剂的最优培养条件，选择破乳菌XH1与L1在初始无葡萄糖的改进无机盐培养基(MMSM)中进行培养，利用响应面法(RSM)对复配培养条件进行优化，经模型的分析与验证，确定最佳培养条件为：种子液比例(L1:XH1)为3:2、葡萄糖投加时间为第4d，液体石蜡含量3.6%(v/v)，投加葡萄糖后再培养21h，获得复合型生物破乳剂排油率>95%(24h)。与单株菌培养相比，表现出投加量少、破乳接触时间短、对温度和pH的适应性高的优势。同时双株破乳菌复配培养有效的提高了培养中主要营养物质的利用率，生产每吨破乳粗产品与单独培养XH1相比可节约72.6%底物所需的成本。更多还原

【Abstract】 Development and application of green biological agents in environmentalprotection fields has been taken more seriously. Bio-demulsifier has graduallybecome a hot research, because of its environmentally friendly property includinglow toxicity, non-secondary pollution and biodegradation. Bio-demulsifier can beused to treat the petroleum field emulsions and other industrial emulsionsin stead ofchemical demulsifier widely used, which has profound meaning for enhancing thedehydration rate of the emulsions and decreasing the environmental risks. However,with deep bio-demulsifier research, some significant problems have become theobstructions of large scale production and promotion. For example, bio-demulsifieryields and performance of wild strains cannot meet the requirements of industrialproduction, application of bio-demulsifier is restricted by environmental conditionsand incubation way is sole.This paper conducts correlative studies according to above problems forfilling the gaps of this fields and improving application. That is mainly includingusing the mutation breeding approaches to improve the demulsifying ability andextreme environment adaptation of bio-demulsifier producing bacteria; based onproteomics analysis of environmental adaptation mutation mechanism ofthermotolerant, acid resistance and alkali resistance mutants; selection of highefficiency demulsifying new strains used to study complex culture and compoundbio-demulsifier development.In order to enhance the demulsifying ability of wild strains, wild demulsifyingstrain XH1(Bacillus mojavensis) was proceed mutation breeding. Comparing themutation effect of the different mutation factors on demulsifying strain XH1, it isdetermined that the optimal mutation approach was UV+nitrosoguanidine(NTG) ofcompound mutation. The mutant with the highest demulsifying ability was strainXN5, which exhibited a15.12%increase of demulsifying ratios compared withoriginal strain XH1. Through study on the effect of cultivation condition on growthand demulsifying ability of mutant XN5, it obtained the optimal cultivationconditions were as follows:35℃cultivation temperature,140r/min shaking tablespeed,7.0initial pH-value,21h incubation time.The correlative techniques of proteomics including SDS-PAGE,2-DE andnanoESI-Q-TOF MS/MS were used to analysis of environmental adaptationmutation mechanism of temperature-fast, acid-fast and alkali-fast mutants. Thedifferential expression protein was separated, identified and function analyzedwhich was from the original strain XH1grown on routine conditions, the mutant isolated by temperature grown on50℃incubation temperature, the mutant isolatedby acid environment grown on medium with pH3, the mutant isolated by alkalienvironment grown on medium with pH11, respectively. The results show thatsignificant variation of the numbers of the differential expression protein spots fromthermotolerant, acid resistance and alkali resistance mutants were14,8and9.Through identification and function analysis of differential expression protein ofdemulsifying strain XH1produced by mutation under the different environmentalconditions, the mutation mechanism of demulsifying bacteria was preliminaryanalyzed on the proteomics levels.Combination the detection methods ofbiosurfactants and demulsifying test was used to isolate the demulsifying bacteriafrom the activated sludge in aeration tank of Taiping waste water treatment plant.One strain with high and steady demulsifying characteristics (98.5%of24hdemulsifying ratios), named as L1, identified as Gordonia sihwensis. Thedemulsifying activity of bio-demulsifier produced by L1could not be influenced bythe temperature treatment, autoclaved sterilization and freeze thawing. Throughresearch on the effects of the cultivation conditions on demulsifying efficiency ofstrain L1grown on mineral salt medium(MSM), it obtained that the optimalcultivation conditions were as follows:30℃cultivation temperature,140minshaking table speed,7.0initial pH-value,60h incubation time.In order to study on the optimal cultivation conditions of mixed cultureproducing compound bio-demulsifier, strain L1and XH1selected were incubated inmodified mineral salt medium(MMSM) without glucose. Then the response surfacemethodology (RSM) was used to optimize mixed culture conditions. According tothe results of the model analysis and verification, it was determined that the optimalconditions were follows:3:2inoculum proportion (strain L1:XH1),3.6%(v/v) liquidparaffin contents, the fourth day fed glucose, continue incubating21h after feedingglucose. Compared with single strain culture, the compound bio-demulsifierproduced by complex culture of L1and XH1exhibited a decrease of dose, anincrease of stability and a shortened demulsifying time. And the complex cultureincreased the utilization ratio of main nutrients in the medium, and the per tonbio-demulsifier cost decreased by72.6%.更多还原