工业化厌氧颗粒污泥重要菌群及相关功能基因研究
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摘要
产甲烷菌群是厌氧颗粒污泥中的重要菌群,mcrA、mtr和F420是产甲烷菌中特有的辅酶编码基因。以取自工业化UASB反应器中处理阿维菌素和淀粉废水的厌氧颗粒污泥为研究对象,采用PCR-DGGE、FISH等分子生物学手段,对厌氧颗粒污泥的产甲烷菌群及相关功能基因进行研究,考察了菌群结构的多样性、系统发育关系和相对丰度等微生物学信息,同时考察了抗生素残留对厌氧颗粒污泥产甲烷活性的抑制情况,研究获得以下结果：
基于mcrA和16S rRNA两种目标基因获得的PCR产物的DGGE图谱存在差异,但根据图谱计算所得产甲烷菌群Shannon多样性指数、Margalef丰富度指数和Berger-Parker优势度指数没有差异,表明基于两种目标基因的产甲烷菌群多样性分析基本一致。基于不同目标基因的优势产甲烷菌群系统发育种属的分析结果大体相似,产甲烷杆菌目和产甲烷八叠球菌目是厌氧颗粒污泥样品中的优势产甲烷种群；同时分析结果的差异表明两种目标基因的检测特异性不完全相同,但具有可替换性。
对同一种污泥样品基于不同探针的产甲烷菌FISH杂交结果表明：产甲烷菌分布形态相同,但相对丰度存在差异,相对丰度由小到大的顺序为:mcrA 基于产甲烷菌群分析的结果,考察与阿维菌素结构类似的红霉素和典型抗生素链霉素对处理淀粉废水和处理阿维菌素废水污泥样品的产甲烷活性的影响,结果表明：链霉素对两种污泥样品的产甲烷菌和产酸菌都有明显抑制作用,对产甲烷菌的抑制作用表现为产甲烷过程滞后和产甲烷速率的降低。链霉素浓度不同时,对微生物的抑制程度也不同,对处理淀粉废水和处理阿维菌素废水的污泥样品中产甲烷菌的半抑制浓度分别为2.05 mg·L-1和7.49mg·L-1。红霉素对处理淀粉废水的颗粒污泥样品中的微生物也具有抑制作用,表现为低浓度(<10mg·L-1)下,对产酸菌的抑制作用较强,而在高浓度(>20mg·L-1)条件下,对产甲烷菌的抑制作用较强。由于红霉素与阿维菌素具有相似的结构,因而红霉素对处理阿维菌素废水的污泥样品中的微生物的抑制作用较弱。
Methanogens are considered to be important microbial populations in anaerobic granular sludge. and mcrA、mtr and F420 are the important functional genes in methanogens encoding some unique coenzymes. Methanogenic community and functional genes mentioned above in anaerobic granular sludge samples from full-scale UASB bioreactors treating avermactin or starch wastewater were investigated by PCR-DGGE and FISH analysis. The diversity of methanogenic community, the phylogenetic relationship of dominant methanogeinc populations and the relative abundance of methanogens were discussed and compared when 16S rRNA gene or functional gene was used as target gene. The inhibitory effects of antibiotics on methane-producing capacity of sludge samples were also explored. The results were shown as follows.
The diversitv indices of methanogenic communitv calculated from DGGE band patterns. including Shannon diversity index. Margalef richness index and Berger-Parker dominance index. were no difference between mcrA gene-based and 16S rRNA gene-based PCR products. although their DGGE band patterns were different. implying that the diversity analysis of methanogenic community based on mcrA genes was consistent with 16S rRNA gene. The phylogenetic analysis of dominant methanogenic populations based on these two target genes also showed resemble and methanobacteriales and methanosarcinales were determined to be the main orders of methanogenic populations in anaerobic granular sludge sample treating avermectin wastewater. Then it could be conclude that mcrA gene and 16S rRNA gene could be alternative for methanogenic community analysis. although the difference in phylogenetic analysis suggested simultaneously some group-specific of these two target genes.
The distribution forms of methanogens in FISH analysis based on different target genes were almost identical for one kind of sludge sample except some difference in hybridization areas. The average relative abundance of methanogens based on hybridization area was in the following order:mcrA The inhibitory effect of streptomycin or erythromycin on methanogen and acetogenic bacteria in anaerobic granular sludge treating starch or avermectin wastewater was investigated. The results indicated that the process of methane production was lagged and the rate of methane production decreased in the presence of streptomvcin. The inhibitory effect depended on streptomycin concentration. The half-inhibitory streptomycin concentrations for methane production of sludge samples were obtained as 2.05 mg-L-1 for treating starch wastewater and 7.49 mg·L-1 for treating avermectin wastewater. The inhibitory effect of erythromycin was relatively stronger on methanogen at low erythromycin concentrations (<10mg·L-1) but on acetogenic bacteria at high erythromycin concentrations (>20mg·L-1). The inhibitory effect of erythromycin on methanogens in the sludge sample treating avermectin wastewater seemed weaker. due to similar structure of erythromycin with avermectin probably.
基于mcrA和16S rRNA两种目标基因获得的PCR产物的DGGE图谱存在差异,但根据图谱计算所得产甲烷菌群Shannon多样性指数、Margalef丰富度指数和Berger-Parker优势度指数没有差异,表明基于两种目标基因的产甲烷菌群多样性分析基本一致。基于不同目标基因的优势产甲烷菌群系统发育种属的分析结果大体相似,产甲烷杆菌目和产甲烷八叠球菌目是厌氧颗粒污泥样品中的优势产甲烷种群；同时分析结果的差异表明两种目标基因的检测特异性不完全相同,但具有可替换性。
对同一种污泥样品基于不同探针的产甲烷菌FISH杂交结果表明：产甲烷菌分布形态相同,但相对丰度存在差异,相对丰度由小到大的顺序为:mcrA
Methanogens are considered to be important microbial populations in anaerobic granular sludge. and mcrA、mtr and F420 are the important functional genes in methanogens encoding some unique coenzymes. Methanogenic community and functional genes mentioned above in anaerobic granular sludge samples from full-scale UASB bioreactors treating avermactin or starch wastewater were investigated by PCR-DGGE and FISH analysis. The diversity of methanogenic community, the phylogenetic relationship of dominant methanogeinc populations and the relative abundance of methanogens were discussed and compared when 16S rRNA gene or functional gene was used as target gene. The inhibitory effects of antibiotics on methane-producing capacity of sludge samples were also explored. The results were shown as follows.
The diversitv indices of methanogenic communitv calculated from DGGE band patterns. including Shannon diversity index. Margalef richness index and Berger-Parker dominance index. were no difference between mcrA gene-based and 16S rRNA gene-based PCR products. although their DGGE band patterns were different. implying that the diversity analysis of methanogenic community based on mcrA genes was consistent with 16S rRNA gene. The phylogenetic analysis of dominant methanogenic populations based on these two target genes also showed resemble and methanobacteriales and methanosarcinales were determined to be the main orders of methanogenic populations in anaerobic granular sludge sample treating avermectin wastewater. Then it could be conclude that mcrA gene and 16S rRNA gene could be alternative for methanogenic community analysis. although the difference in phylogenetic analysis suggested simultaneously some group-specific of these two target genes.
The distribution forms of methanogens in FISH analysis based on different target genes were almost identical for one kind of sludge sample except some difference in hybridization areas. The average relative abundance of methanogens based on hybridization area was in the following order:mcrA
引文
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