Ammonia nitrogen removal by nitrifying bacteria from different habitats
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摘要: 硝化细菌是微生物脱氮的关键功能菌群之一，筛选优质硝化细菌对于强化微生物脱氮具有重要意义。将河流沉积物、土壤自然环境和市场售人为环境3种生境来源的硝化细菌功能进行比较研究，并利用分子生物学手段分析了硝化细菌群落结构。结果表明，沉积物生境中硝化细菌的活性以及耐氨氮、pH和盐度等环境因子的能力高于土壤和市售硝化细菌。利用MPN-PCR法和克隆文库分析法对各生境硝化细菌丰度及群落结构组成进行分析，发现沉积物生境中硝化螺旋菌(Nitrospira)生物量较高，促使该生境硝化细菌氨氮去除率较高。沉积物生境中硝化细菌的高耐氨氮和耐pH能力与群落中存在硝化杆菌(Nitrobacter)有关，而高耐盐性可能是由于该生境中存在耐盐或适度嗜盐特异基因型硝化细菌，运用Blast程序进行序列比对发现，这些特异基因型硝化细菌为不可培养微生物。Abstract: Nitrifying bacteria are one of the key functional groups in microbial nitrogen removal, and the screening of high-quality nitrifying bacteria is beneficial to enhancing nitrogen removal. The functions of nitrifying bacteria from river sediment, soil natural environment and artificial habitat in the market were compared, and their community structures were analyzed using molecular biological methods. The results showed that the activities of nitrifying bacteria from the sediment habitat and their resistance abilities to ammonia nitrogen, pH and salinity were higher than those from the other two habitats. The abundance and community structure analysis of nitrifying bacteria in each habitat by MPN-PCR and clone library profiling showed that the biomass of Nitrospira in the sediment habitat was high, which could promote the ammonia nitrogen removal rate of nitrifying bacteria in this habitat. For sediment nitrifying bacteria, the higher resistance to ammonium and pH was due to the presence of Nitrobacter, while the higher resistance ability to salinity was possibly due to the presence of salt-tolerant species or moderately halophilic specific genotypes of nitrifying bacteria in the habitat. In addition, these specific genotypes of nitrifying bacteria were identified as uncultured microbes by sequence alignment by Blast program.
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Key words:
- habitat /
- nitrifying bacteria /
- MPN-PCR /
- clone library profiling /
- sequence alignment
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图 1 不同生境来源硝化细菌硝化速率差异性
Figure 1. Differences in nitrification rates of nitrifying bacteria from different habitats
图 2 不同生境来源硝化细菌硝化速率对氨氮初始浓度的响应
Figure 2. Nitrification rate response of denitrifying bacteria from different habitats to initial ammonium concentrations
图 3 不同生境来源硝化细菌硝化速率对环境温度的响应
Figure 3. Nitrification rate response of denitrifying bacteria from different habitats to temperature
图 4 不同生境来源硝化细菌硝化速率对环境pH的响应
Figure 4. Nitrification rate response of denitrifying bacteria from different habitats to pH
图 5 不同生境来源硝化细菌硝化速率对环境盐度的响应
Figure 5. Nitrification rate response of denitrifying bacteria from different habitats to salinity
图 6 不同生境来源硝化细菌MPN-PCR产物的琼脂糖凝胶电泳图谱
Figure 6. Agarose gel electrophoresis for MPN-PCR products of denitrifying bacteria from different habitats
图 7 Nitrobacter和Nitrospira在硝化细菌中所占比例
Figure 7. Percentages of Nitrobacter and Nitrospira in nitrifying bacteria
图 8 基于amoA基因序列所构建的NJ树
注：CJ、TR和SS分别代表沉积物、土壤和市售生境硝化细菌克隆子；节点处数值为分支检测得到，仅显示大于50%的数值。
Figure 8. NJ tree ofamoA genes
图 9 amoA基因克隆子中Nitrosospira和Nitrosomona占比
Figure 9. The abundances of Nitrosospira and Nitrosomona in amoA gene clones
表 1 各生境中Nitrobacter的基因型分析
Table 1. Genotype analysis of Nitrobacter from different habitats
沉积物生境 土壤生境 市售生境 基因型 克隆子 基因型 克隆子 基因型 克隆子 基因型1 CJ1、CJ2、CJ3、CJ5、CJ8、CJ9、CJ13、CJ22、CJ25、CJ26、CJ31、CJ34、CJ36、CJ37、CJ38、CJ39、CJ43、CJ70、CJ71 基因型1 TR5、TR6、TR8、TR13、TR15、TR18、TR20、TR21、TR22、TR29、TR31、TR32、TR35、TR38、TR40、TR41、TR42、TR43、TR44、TR45、TR46 基因型1 SS4、SS6、SS7、SS13、SS14、SS15、SS16、SS19、SS20、SS21、SS22、SS27、SS29、SS30、SS35、SS36、SS37、SS38、SS39、SS43、SS45、SS46、SS51、SS52、SS54、SS55、SS56、SS57、SS58、SS68、SS69、SS70、SS71、SS72 基因型2 CJ67 基因型3 TR14 基因型2 SS44 基因型4 TR71 基因型5 SS5 基因型6 SS8 基因型7 SS28 基因型8 SS42 
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表 2 各生境中Nitrospira基因型分析
Table 2. Genotype analysis of Nitrospira from different habitats
沉积物生境 土壤生境 市售生境 基因型 克隆子 基因型 克隆子 基因型 克隆子 基因型1 CJ5、CJ12、CJ13、CJ14、CJ17、CJ18、CJ19、CJ25、CJ30、CJ53、CJ56、CJ57、CJ64、CJ66 基因型1 TR7、TR8、TR9、TR24、TR29、TR41、TR42、TR44、TR69 基因型1 SS3、SS4、SS8、SS42、SS43、
SS59、SS62、SS64、SS67基因型2 CJ6、CJ7、CJ10、CJ15、CJ20、
CJ24、CJ39、CJ41、CJ54基因型2 TR26、TR28、TR33、TR39 基因型2 SS17、SS38 基因型3 CJ40 基因型3 TR21、TR72 基因型3 SS5、SS19、SS35、SS63、SS68 基因型5 CJ42 基因型4 TR2、TR16、TR35、TR40 基因型4 SS41、SS44 基因型8 CJ8 基因型5 TR10、TR14、TR66、TR68 基因型6 SS57 … … 基因型6 TR67、TR71 基因型7 SS10、SS60 基因型22 CJ65 基因型23 TR1 基因型36 SS6 … … … … 基因型35 TR43 基因型51 SS66
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