【作者】 李蒙英；

【导师】 李顺鹏；

【作者基本信息】 南京农业大学 ， 微生物学， 2007， 博士

【摘要】 自然环境中能降解硝基芳香烃等异型生物质的微生物种类和数量很少，向污染水体投加有强降解能力的土著或外来野生型或基因工程茵的生物强化研究在实验室反应器中取得了一定的成功，但在实际废水处理中常因降解茵不易定殖而无法长期维持处理效果。本文研究了成膜力较强的细菌和降解茵混合培养的成膜特性，旨在探索促进降解细菌定殖于废水处理系统中的新策略．一、硝基芳香烃降解茵的分离筛选及降解特性以含多种硝基芳香烃的合成废水为进水设置反应器，投加长期受硝基芳香烃和其他化学品污染的环境样品，经敞开式大通量方法富集一段时间，从反应器中分离到一株3,5-二硝基苯甲酸(3,5-DNBA)降解菌株(A3);根据其形态特征、生理生化特征及16SrDNA同源性比较，初步鉴定为睾丸酮丛毛单胞菌(Comamonas testosteroni)．进一步研究了A3的降解特性与生物学特性，A3能以200mg/L 3，5-二硝基苯甲酸为唯一碳源，12h的降解率可达95％以上,24h可溶性有机碳(DOC)由72.5mgC/L降至10.2mgC／L，将3,5-二硝基苯甲酸降解矿化；A3以200mg／L 3,5-二硝基苯甲酸为唯一碳、氮源时将底物转化为一种黄色产物，培养液颜色在以后的几天中始终未消退，24h时DOC为56.5mgC／L，3，5-二硝基苯甲酸未完全降解；A3降解3,5-二硝基苯甲酸的适宜温度为20—30℃，适宜pH为5～9．二、生物膜上细菌的分离鉴定及细菌生物学特性对生物膜形成贡献的评价从废水处理系统、生活污水下水口等多处生物膜上共分离获得18株细菌，异位测定结果显示它们的生物膜形成能力有很大的差异，成膜力最强的与最弱的相差28倍，对其中有较强成膜力的7株细菌进行了鉴定，它们分别为Pseudomonas sp．M8、Pseudomonas putida M9、Aeromonas caviae M1O、Bacillus cereus M19、Pseudomonasplecoglossicida M21、Aeromonas hydrophila M22和C.testosterone A3；进一步测定了18株细菌可能与成膜力有关的5个生物学特性，采用相关分析和通径分析评估了这5个细茵生物学特性对生物膜形成的贡献，它们对生物膜形成影响的相对重要性依次为：胞外多糖>鞭毛>AHL群体感应信号分子>胞外蛋白>群游力。三、2种功能菌株荧光蛋白基因的标记为了了解降解菌和生物膜形成菌混合培养或投加到废水处理系统后2种菌在生物膜中的数量分布和A3定殖情况,对降解菌株A3和有较强成膜能力的M9和M22进行了荧光标记,采用三亲接合法和电转化法成功地将带有绿色荧光蛋白基因的质粒pTR102gtp转入了C.testosteroni A3、P.putida M9和A.AJdrophila M22菌株,将带有红色荧光蛋白基因的质粒pJZ402rfp转入了C.testosteroni A3。荧光检测显示,细菌个体和群落都显示出明亮的绿色或红色荧光。四、不同生物膜形成菌对降解菌定殖的影响将有较强成膜能力的5株细菌与降解菌组合,研究了降解菌株A3分别与5株成膜力较强的菌株M8、M9、M19、M21和M22混合培养时的降解能力、生物膜形成量和生物膜抗冲击能力等指标,结果显示混合培养时的生物膜形成量是降解菌A3单独培养的1.76～5.95倍,其中M9和M22与A3混合培养不但促进了生物膜的大量形成且生物膜附着牢固,培养24h形成的双菌生物膜比A3单菌和其他3种双菌生物膜表现出更强的抗3,5-二硝基苯甲酸冲击能力,每次更换合成废水6h后3,5-二硝基苯甲酸降解率即分别达63.3～91.6%和70.7～89.4%;连续冲击前后A3M9和A3M22双菌生物膜中的总菌数和A3菌数比A3单菌和其他3种双菌生物膜高2～3个数量级,M9和M22促进了降解菌在生物膜中的定殖,说明某些生物膜形成菌强化降解菌固定于生物膜中的这种自固定作用可能会促使处理系统保持更持久稳定的降解能力。五、不同营养条件对生物膜形成影响的研究研究了不同营养物对A3与M9和A3与M22混合培养的影响,表明3,5-二硝基苯甲酸基础盐合成废水(DCMM)中添加不同浓度的LB培养液对反应系统降解3,5-二硝基苯甲酸的降解效率没有显著影响,但容易被细菌利用的LB培养液含量的增加对生物膜形成量有很强的促进作用;用流式细胞仪测定了A3与P.putida M9(pTR102gfp)和A3与A.Aydrophila M22(pTR102gfp)在不同营养条件下混合培养时2种功能菌的比例,随DCMM中LB培养液含量从0%增加至5%生物膜上P.putida M9(pTR102gfp)和A.AyJrophila M22(pTR102gfp)所占的比例分别从43.8%增加至82.3%和从39.2%增加至79.6%。采用激光共聚焦显微镜观察了A3M9gfp和A3rfpM22gfp生物膜结构和生物膜中2种菌的数量分布情况,生物膜三维结构显示A3被均匀地固定于生物膜内部;从生物膜形成量、生物膜中降解菌数和生物膜质量综合评价添加2%LB的DCMM合成废水中不但能形成较大量的生物膜,固定于生物膜中的降解菌数量也较高;实验室条件下对2种功能菌混合培养形成生物膜情况的研究结果对实际废水处理中调节废水营养成分强化降解菌定殖具有一定的指导意义。六、生物膜法废水处理中2种功能菌的生物强化作用具有较强成膜力的细菌如能促进降解菌定殖于生物膜内,使其通过自固定作用固定于载体表面,将有可能长期保持生物强化降解效果。采用8L反应器测定了2种功能菌在生物接触氧化工艺中的生物强化作用,结果显示投加A3和M9的1号反应器和投加A3的2号反应器能在24h内快速降解3,5-二硝基苯甲酸,并且在以后35d的运行过程中保持了良好的降解效果,DCMM选择平板培养和DGGE检测结果都证明反应器内生物膜中有降解菌A3的存在;只接种城市污水处理厂活性污泥的3号反应器在前2周的运行过程中基本不降解3,5-二硝基苯甲酸,但15d后降解率快速增加,21d后对3,5-二硝基苯甲酸的降解效果与1号和2号反应器无明显差异,3号反应器生物膜的DGGE图谱中未发现与A3菌株相似的条带,说明土著微生物经2～3周的驯化也可降解3,5-二硝基苯甲酸;运行35d时未投加生物膜形成菌株M9的2个反应器生物膜DGGE图谱中也发现与M9菌株相似的条带,说明M9可能存在于接种污泥或反应器运行环境中并且容易形成生物膜。更多还原

【Abstract】 Microorganisms that can degrade xenobiotic,such as nitroaromatic compounds are quite scarce in natural environment.The study of bioaugmentation of adding indigenous or exogenous wild type microorganisms or genetically engineered microorganisms into contaminated waters has made some achievements in laboratory scale.But in reality,very often,due to the flowing-off of degrading bacteria,the bioaugmentation can not be remained.This paper is to explore the bioaugmentation strategies with the mixed inoculant of biofilm-forming bacteria and degrading bacteria to approach new ways of immobilizing degrading bacteria into the wastewater treatment system.The soil and sediments contaminated by nitroaromatic compounds and some other chemicals for a long time were inoculated into a bioreactor full of synthetic wastewater with nitroaromatic compounds.One 3,5-dinitrobenzoic acid-degrading bacterium A3, isolated from the wastewater treatment system which operated one month,was identified preliminarily as Comamonas testosteroni based on its morphological characters, physiological and biochemical analyses and 16S rDNA series same source analysis.This bacterium degradation rate of 12h could reach above 95%,using 200mg/L 3,5-dinitrobenzoic acid as sole carbon source.24h dissolved organic carbon(DOC) was reduced from 72.5mgC/L to 10.2mgC/L,indicated that 3,5-dinitrobenzoic acid was degraded through mineralization but not transformed into other organic matter.But the strain A3 transformed 3,5-dinitrobenzoic acid into a yellow product,cultivated liquor was not discolored all along in the next several days,when 200mg/L 3,5-dinitrobenzoic acid was used as the sole carbon and nitrogen sources.DOC being 56.5mgC/L in 24h,indicated that the yellow product was not further degraded easily.The suitable temperature for A3 degradation of 3,5-dinitrobenzoic acid was 20-30"C and pH value was 5-9.A total number of 18 bacterial isolates were obtained from the biofilms of wastewater treatment systems and of little carpolite in soil.Results of their in vivo detection showed a great difference in their abilities to form biofilms:the bacteria with the strongest biofilm-forming capacity can form biofilms biomass 28 times stronger than that formed by the weakest.The phylogeny affiliation of those isolates showing high biofilm formation capacity has been determined through a 16s rDNA sequencing and they were grouped into 7 bacterial species including Pseudomonas sp.,Pseudomonas putida,Aeromonas caviae, Bacillus cereus,Pseudomonas plecoglossicida,Aeromonas hydrophila and Comamonas testosteroni.Selected biological characteristics that are potentially related to biofilm forming capacity were investigated and the relative importance of these biological properties in biofilm formation was statistically assessed by path analysis.According to the coefficient of determination,the relative importance of the five biological characteristics to biofilm formation was in the order from greatest to least:exopolysaccharide＞flagella＞N-acyl-homoserine lactones(AHLs) signaling molecules＞extracellular protein＞swarming motility.In order to investigate the distribution of the degrading bacteria and biofilm-forming bacteria in a dual-species biofilms or in the biofilm of the wastewater treatment system and immobilization of A3 in biofilms,C.testosteroni A3,P.putida M9 and A.hydrophila M22 were tagged using fluorescence labeling,pTR102gfp plasmid in E.coli DH5a was introduced into C.testosteroni A3,P.putida M9 and A.hydrophila M22 by triparental mating and pJZ402rfp plasmid was introduced into C.testosteroni A3 by electroporation. Fluorescent measurement shows that both the bacteria and the colonies have bright green or red fluorescence.The degrading ability,the biofilm biomass,the resistance of biofilm to shock loading and the fxation of A3 were investigated in a bioreactor with mixed inoculation of high biofilm-forming bacteria and degrading bacterium A3.The results showed that the biofilm biomass formed by all the mixed inoculation was 1.76-5.95 times higher than that of the single inoculation of A3.The dual-species biofilms,A3 with M9 and A3 with M22,had a strong resistance to 3,5-DNBA shock loading during successive replacement of DCMM2 synchetic wastewater(3,5-dinitrobenzoic acid mineral medium containing 2%LB broth), and degradation rate reached 63.3～91.6%and 70.7～89.4%,in 6 h after each time of replacement.It was found that the colony forming units(CFUs) the total and A3 cells of biofilms formed by A3 with M22 or M9,were significantly higher than those of other three dual-species biofilms.The relatively high quantity of degrading strains in biofilm also demonstrated that strain A3 can be well maintained in these two dual-specises biofllms over time.Thus,strain M9 and M22 did enhance the degrading strain A3 fixed.Therefore,it seems feasible to use some specific biofilm-forming bacteria as an viable option for bioaugmentation to enhance immobilization of degrading bacteria in biofilm in an engineering setting,and the self-immobilization may also help maintain a durable and stable degradation capacity.Investigation of the effects of different nutrient status on the mixed inoculation of A3 with M9 or M22 showed that different amounts of LB broth added into 3,5-dinitrobenzoic acid mineral medium(DCMM) had no significant effects on the degradation rates of 3,5-DNBA,but the increase of LB broth from 0%to 5%,the nutriment enhanced the formation of biofilms greatly.The results from flow cytometer showed tnat the ratios of fluorescing bacteria in dual-specises biofilm of A3 with P.putida M9(pTR102gfp) and A3 with A.hydrophila M22(pTR102gfp) enhanced with the increase of LB broth.The P. putida M9(pTR102gfp) and the A.hydrophila M22(pTR102gfp) on the biofilms increased from 43.8%to 82.3%and from 39.2%to 79.6%,respectively.The distribution of the two bacteria within the biofilms and the three-dimensional differentiation observed by confocal laser scanning microscopy(CLSM) showed that A3 was homogeneously fixed in the biofilms;The overall evaluation indicated that 2%LB of addition to DCMM wastewater was the best to obtain a higher biofilm biomass and a higher percentage of degrading bacteria immobilized in the biofilms.The above investigation on the biofilm formation by the mixed culture of two functional bacteria carded out in the laboratory conditions was of significance in practical treatment of the wastewater by regulating the nutrient composition to promote immobilization of the degrading bacteria.If the bacteria with high biofilm-forming capability are able to enhance the self-immobilization of the degrading bacteria,this immobilization might make the degrading abilities run in a long term.The bioaugmentation of derading bacterium A3 and biofilm-forming bacteria M9 was detected in an 8L bioreactors.The results show that 3.5-DNBA can be quickly degraded in the reactor 1 with A3 and M9 and the reactor 2 with only A3,and in the following 35 days,the degradation is well remained.The results from the culture of selecting agar plates and DGGE detection demonstrated the existence of degrading bacterium A3 within the biofilm.In the reactor 3 with the only activated sludge from the municipal wastewater treatment factories,almost none of 3,5-DNBA has been degraded within first two weeks.However,after 15 days the degradation rate of 3,5-DNBA was greatly increased.And after 21 days the degrading rate from reactor 3 was no more different from the ones from reactor 1 and reactor 2.DGGE profles of the biofilm of reactor 3 are lack of the band similar to Strain A3,demonstrating that the aboriginal microorganisms can degrade 3,5-DNBA if they had been cultured for 2～3 weeks.On the 35th day,on the DGGE pattern of biofilms in those two reactors into which M9 had not been added,bands that were similar to Strain M9 were found.And this showed that M9 might exist in inoculated sludge or environment and it had quite high capability to form biofilm.更多还原