多氯联苯高效脱氯培养物的富集及特征研究
发布时间：2018-08-04 13:52
【摘要】：多氯联苯(Polychlorinated biphenyls,PCBs)是典型的环境持久性有机污染物。PCBs污染的微生物修复是一种有前景的修复技术,高氯代PCBs厌氧脱氯是其中的关键。PCBs厌氧脱氯菌极难分离,对富集厌氧脱氯培养物的研究有助于我们更好地富集稳定的混合培养物、分离高效PCBs脱氯菌。本文以水稻土为富集源,以典型高氯代PCBs混合物Aroclor1260为电子受体,添加乳酸钠为碳源和电子供体,进行富集培养,考察培养过程中PCBs脱氯特征变化、PCBs脱氯菌和脱氯基因变化及微生物群落结构的演替,并对高效稳定的富集脱氯培养物PSCJ-V进行表征。六代富集培养物的培养过程可分为初始、过渡和稳定三个阶段。主要研究结果如下:(1)随着富集过程的进行,培养物中的土壤颗粒逐渐减少至无颗粒物,脱氯延滞期和脱氯周期逐渐缩短,氯原子去除速率逐步加快,脱氯程度与脱氯途径基本无变化;脱氯效果在PSCJ-V中达到最优。与此同时,PCBs脱氯菌Dehalococcoides和脱氯基因A4、A5、Prd11随着多次的传代培养逐步增加,且四者有相似的变化,A1、Prd8基因随着多次的传代培养逐渐减少,被逐步淘汰。(2)经过长期的富集培养,本文得到一个高效的Aroclor1260脱氯培养物PSCJ-V。该培养物可以在40天左右的周期内,以5.14～6.01μmol·L-1·day-1的速率,按途径N和途径LP共同对Aroclor1260脱氯,得到的产物单位联苯上仅有4.3～5.2个氯原子。该培养物在脱氯程度、脱氯周期、氯原子去除速率方面均优于多数培养物,是目前最高效的Aroclor1260脱氯培养物之一。(2)培养物PSCJ-V中存在与Dehalococcoides mccartyi CG4和Dehalococcoides mccartyi195亲缘关系极近的PCBs脱氯菌PSCJ-Dhc,其细胞浓度达到3.98×107 cells/mL左右,是目前脱氯菌细胞浓度最高的Aroclor1260脱氯培养物之一,仅次于JN培养物。该培养物中某些脱氯菌携带了与pcbA5基因序列相似度为98.7%的假定脱氯基因pcbPSCJ,两者蛋白质仅有11个氨基酸的差异。(3)培养物PCSJ-V中古菌丰度为30.45%,以产甲烷菌为主;细菌丰度为69.55%,以产酸发酵菌为主;主要存在以下菌属:脱氯菌Dehalococcoides,产氢产乙酸菌Candidatus Cloacamonas、Smithella、Aminivibrio、Thermovirga、Proteiniphilum、Syntrophomonas、Sphaerochaeta、Youngiibacter、Desulfovibrio、Methanosarcina,产类咕啉菌Methanobacterium、Methanoculleus、Methanothrix、Desulfovibrio、Methanosarcina,以及其他菌属Methanomassiliicoccus。
[Abstract]:Polychlorinated biphenyls (Polychlorinated) is a typical environmental persistent organic pollutant. Microbial remediation of PCBs is a promising remediation technique. Anaerobic dechlorination of high chlorinated PCBs is the key. The study on the enrichment of anaerobic dechlorination culture is helpful for us to better enrich the stable mixed culture and isolate the high efficiency PCBs dechlorination bacteria. In this paper, paddy soil was used as enrichment source, Aroclor1260 as electron acceptor, sodium lactate as carbon source and electron donor. The characteristics of dechlorination in PCBs were investigated. The changes of dechlorination bacteria and dechlorination genes and the succession of microbial community structure were investigated. The highly efficient and stable enrichment dechlorination culture (PSCJ-V) was characterized. The culture process of six generations of enriched culture can be divided into three stages: initial stage, transition stage and stabilization stage. The main results are as follows: (1) with the enrichment process, soil particles in the culture gradually decreased to no particles, dechlorination delay period and dechlorination cycle gradually shortened, chlorine atom removal rate gradually accelerated. The dechlorination degree and dechlorination pathway had no change, and the dechlorination effect was optimal in PSCJ-V. At the same time, the number of Dehalococcoides and the dechlorination gene A4A5 / Prd11 gradually increased with the passage of several times, and the same changes were observed in the four groups, and the gene of A1 / Prd8 was gradually decreased and phased out with the repeated passage culture. (2) after a long period of enrichment culture, A high efficiency Aroclor1260 dechlorination culture PSCJ-V was obtained. The culture could dechlorinate Aroclor1260 at the rate of 5.14 ~ 6.01 渭 mol L ~ (-1) day-1 at the rate of 5.14 ~ 6.01 渭 mol 路L ~ (-1) day-1 for about 40 days, and only 4.3 ~ 5.2 chlorine atoms could be obtained on the unit biphenyls of the product according to pathway N and path LP. The dechlorination degree, dechlorination period and chlorine atom removal rate of the culture were better than that of most cultures. It is one of the most efficient Aroclor1260 dechlorination cultures at present. (2) there is PSCJ-Dhc, which is closely related to Dehalococcoides mccartyi CG4 and Dehalococcoides mccartyi195 in PSCJ-V, and its cell concentration is about 3.98 脳 10 ~ 7 cells/mL, which is one of the highest Aroclor1260 dechlorination cultures. Second only to JN culture. Some dechlorinated bacteria in the culture carried a hypothetical dechlorination gene pcbPSCJ whose similarity to pcbA5 gene sequence was 98.7%, and the difference between the two proteins was only 11 amino acids. (3) the abundance of ancient bacteria in PCSJ-V was 30.45%, mainly methanogenic bacteria. The bacterial abundance is 69.55, mainly acid-producing bacteria, mainly in the following genera: Dehalococcus coidesi, Candidatus Cloacamonas Smithellaa, Aminivibrilaa Thermovirgaa Proteiniphila Syntrophia sphaerochaeta Youngiibacterium Desulfoviochaeta, Methanobacterium-like bacteria Methanobacterium Methanothrix Desulfovibrio Methanoscinara, and other bacteria, Methanobacteriumum Methanobacteriumum method ocululus Methanothrix Desulfovibrio Methanoscinara.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X592;X172
本文编号：2164087
[Abstract]:Polychlorinated biphenyls (Polychlorinated) is a typical environmental persistent organic pollutant. Microbial remediation of PCBs is a promising remediation technique. Anaerobic dechlorination of high chlorinated PCBs is the key. The study on the enrichment of anaerobic dechlorination culture is helpful for us to better enrich the stable mixed culture and isolate the high efficiency PCBs dechlorination bacteria. In this paper, paddy soil was used as enrichment source, Aroclor1260 as electron acceptor, sodium lactate as carbon source and electron donor. The characteristics of dechlorination in PCBs were investigated. The changes of dechlorination bacteria and dechlorination genes and the succession of microbial community structure were investigated. The highly efficient and stable enrichment dechlorination culture (PSCJ-V) was characterized. The culture process of six generations of enriched culture can be divided into three stages: initial stage, transition stage and stabilization stage. The main results are as follows: (1) with the enrichment process, soil particles in the culture gradually decreased to no particles, dechlorination delay period and dechlorination cycle gradually shortened, chlorine atom removal rate gradually accelerated. The dechlorination degree and dechlorination pathway had no change, and the dechlorination effect was optimal in PSCJ-V. At the same time, the number of Dehalococcoides and the dechlorination gene A4A5 / Prd11 gradually increased with the passage of several times, and the same changes were observed in the four groups, and the gene of A1 / Prd8 was gradually decreased and phased out with the repeated passage culture. (2) after a long period of enrichment culture, A high efficiency Aroclor1260 dechlorination culture PSCJ-V was obtained. The culture could dechlorinate Aroclor1260 at the rate of 5.14 ~ 6.01 渭 mol L ~ (-1) day-1 at the rate of 5.14 ~ 6.01 渭 mol 路L ~ (-1) day-1 for about 40 days, and only 4.3 ~ 5.2 chlorine atoms could be obtained on the unit biphenyls of the product according to pathway N and path LP. The dechlorination degree, dechlorination period and chlorine atom removal rate of the culture were better than that of most cultures. It is one of the most efficient Aroclor1260 dechlorination cultures at present. (2) there is PSCJ-Dhc, which is closely related to Dehalococcoides mccartyi CG4 and Dehalococcoides mccartyi195 in PSCJ-V, and its cell concentration is about 3.98 脳 10 ~ 7 cells/mL, which is one of the highest Aroclor1260 dechlorination cultures. Second only to JN culture. Some dechlorinated bacteria in the culture carried a hypothetical dechlorination gene pcbPSCJ whose similarity to pcbA5 gene sequence was 98.7%, and the difference between the two proteins was only 11 amino acids. (3) the abundance of ancient bacteria in PCSJ-V was 30.45%, mainly methanogenic bacteria. The bacterial abundance is 69.55, mainly acid-producing bacteria, mainly in the following genera: Dehalococcus coidesi, Candidatus Cloacamonas Smithellaa, Aminivibrilaa Thermovirgaa Proteiniphila Syntrophia sphaerochaeta Youngiibacterium Desulfoviochaeta, Methanobacterium-like bacteria Methanobacterium Methanothrix Desulfovibrio Methanoscinara, and other bacteria, Methanobacteriumum Methanobacteriumum method ocululus Methanothrix Desulfovibrio Methanoscinara.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X592;X172
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