【作者】 叶峰；

【导师】 陈建孟； 张丽丽；

【作者基本信息】 浙江工业大学 ， 环境工程， 2009， 硕士

【摘要】 本研究将定向驯化后具备高效降解苯系物能力的活性污泥作为种泥,在特定的载体上实施高密度发酵扩大培养,制成复合微生物菌剂。菌剂中的有效活菌数>10⁹CFU/g,含水量<5%,可替代活性污泥直接投加,具有同驯化的活性污泥相当的降解性能。分别考察了pH值、烘干温度、载体比例对菌剂降解性能的影响,获得了菌剂制备的最佳工艺条件:pH9.0,烘干温度35℃,麦麸:纤维素:稳定剂比例为85%:10%:5%。该菌剂具有较好的稳定性,可在4℃稳定保存6个月,仍保持对BTX较高的去除效率,相对活性污泥更适合保存。从用于制备上述复合微生物菌剂的高活性混合菌种中,选育到4株能同时高效降解BTEX混合物的菌株,分别命名为苯2-2、甲苯3-1、甲苯3-2、H1-4,基于菌株的形态与生理生化特征分析,及16S rDNA序列系统学分析和Biolog鉴定结果,可确定这4株菌株分别为Microbacterium oxydans（氧化微杆菌）、Burkholderia cepacia（伯克霍尔氏菌）、Mycobacterium cosmeticum（分枝杆菌）、Bacillusamyloliquefaciens（解淀粉芽孢杆菌）,经查阅相关文献,可确定上述菌株为降解BTX的新性能物种。菌株苯2-2、甲苯3-1、甲苯3-2、H1-4的最适生长温度都为30℃,最佳pH分别为8、6、7、8,此时菌株的生长和对苯系物的降解能力都达到最高。当苯、甲苯、乙苯和邻二甲苯的浓度分别低于500mg/L、300mg/L、100mg/L和125mg/L时,菌株生长良好,且苯系物在120h内大致被降解完全。菌株对单一底物和混合底物的降解特性表明,其在混合底物的情况下生长与降解能力要稍弱于对单一底物的降解能力,菌株苯2-2、甲苯3-1、甲苯3-2、H1-4对4种苯系化合物的降解难易程度分别为邻二甲苯>乙苯>甲苯>苯、邻二甲苯>乙苯>苯>甲苯、邻二甲苯>乙苯>苯>甲苯、邻二甲苯>乙苯>甲苯>苯。基于上述菌株的降解特性制备的菌剂,其对苯系物的降解性能与对应菌株降解性能相似,表明上述菌株在本试验的制备工艺中均可实现高密度发酵,而保留其对目标污染物的高效降解性能,可用于高效菌剂的制备。对选育的野生型菌株进行⁶⁰C_Oγ射线诱变,成功获得两株对BTX降解能力得到进一步提升的正突变株1-1000-4、7-1000-1。正突变菌株1-1000-4相对于原始野生型菌株苯2-2,对苯、甲苯、邻二甲苯的降解能力可分别提高41.6%、31.6%、33%左右;突变菌株7-1000-1相对于原始菌株H1-4,其降解能力的提高更为明显,对苯、甲苯、邻二甲苯的降解能力分别提高了70%、31.4%、33.3%。同时这两株菌株具有良好的的传代稳定性,其诱变获得的优良性状不会因传代而丢失,可为特效菌剂制备奠定基础。更多还原

【Abstract】 The activated sludge with high efficiency for the removal of benzene, toluene,and o-xylene（BTX） was inoculated on a specific carrier and enriched via a fermentation process.Thus,the composite microorganism agent applied for BTX degradation was successfully prepared.In the microorganism agent,the active organisms was more than 10⁹ CFU /g,and water content was less than 5%.The microorganism agent,instead of the acclimated activated sludge,could be employed for the start-up of the reactor to purify BTX without substantial loss of removal capacity.The effect of several operating parameters,including pH,drying temperature and the carrier proportion,on the performance of microorganism agent was investigated in detail.With an initial pH of 9.0,drying temperature of 35℃,and the carrier proportion of 85%:10%:5%,the best microorganism agent were obtained.Compared to the activated sludge,the microorganism agent has high stability,which could be stored for six months or more at 4℃.Four microbial strains capable of degrading BTEX were screened from the high-efficiency complex seeding sludge,which were named for Benzene 2-2,Toluene 3-1,Toluene 3-2,and H1-4,respectively.Basing on analysis of the Strains’ morphological characteristics,physiological and biochemical characteristics,16S rDNA sequences and the Biolog identification,these four strains belonged to Microbacterium oxydans, Burkholderia cepacia,Mycobacterium cosmeticum,and Bacillus amyloliquefaciens respectively.These four strains were proposed as new species which can efficiently degrade BTX according to the relevant literatures.These four strains Benzene 2-2,Toluene 3-1,Toluene 3-2,and H1-4 could grow between 15-35℃with the optimum growth temperature of 30℃,and the optimum growth pH were 8,6,7,and 8 respectively.Strains growed well and BTEX could be degraded in 120h when the concentration of benzene,toluene,ethylbenzene,o-xylene were lower than 500mg/L, 300mg/L,l00mg/L and 125mg/L,respectively.These microbial strains could grow well and had higher removal efficiency for single substrate than for the BTEX mixtures.BTEX as substrates could be degraded by Strain Benzene 2-2,Toluene 3-1,Toluene 3-2,H1-4 in the following order: o-xylene＞ethylbenzene＞toluene>benzene,oxylene＞ethylbenzene＞benzen e＞toluene,o-xylene＞ethylbenzene＞benzene＞toluene,and oxylene＞ethyl benzene＞toluene＞benzene,respectively.The experimental results indicated these four strains could be successfully prepared for microorganism agent without substantial loss of removal capacity for BTX.Two positive mutant strains named as 1-1000-4 and 7-1000-1 were selected via an induction by ⁶⁰C_oγGamma rays for the original and wild-type strains.Compared to its original strain Benzene 2-2,strain 1-1000-4’ removal capacities for BTX could be enhanced by 41.6%, 31.6%and 33%;while that of strain 7-1000-1 were enhanced by 70%, 31.4%,33.3%.,and these two strains have high passage stability, indicating their potential for the preparation of high-efficiency microbial agent.更多还原