垃圾填埋场覆土层植物根围甲烷氧化活性研究
摘要
垃圾填埋场覆土层中的甲烷氧化菌可氧化温室气体甲烷,其氧化活性受地被植物等多种因素影响。采用滚管计数法、气相色谱法以及分子生物学方法PCR-DGGE,研究了阿苏卫、桃树岗、老虎冲和赵家沟四个垃圾填埋场封场覆土层中甲烷氧化菌的数量、种类多样性以及甲烷氧化活性。
研究发现在富集培养过程中甲烷氧化菌的生长繁殖经历了4个时期：调整期,指数期,稳定期以及衰亡期,在培养的第7天左右甲烷氧化菌的密度达到峰值,进入稳定期。垃圾填埋场土壤中甲烷氧化菌的数量范围在107～108 cfu·g-1干土,甲烷氧化速率在10-7～10-8 mol·h-1·g-1之间。各填埋区土样氧化甲烷速率的平均值从高到低的顺序为：阿苏卫垃圾填埋场>桃树岗垃圾填埋场第3期>老虎冲垃圾填埋场>桃树岗垃圾填埋场第2期>桃树岗垃圾填埋场第1期>赵家沟垃圾填埋场。垃圾填埋场的封场时间与甲烷氧化菌数目呈显著负相关,与甲烷氧化速率呈极显著负相关；填埋场植被覆盖率与甲烷氧化菌数量存在极显著负相关,与氧化速率呈显著负相关；覆土层土壤含水率与甲烷氧化速率等具有一定的负相关；本试验结果显示覆土的pH、有机质与铵态氮等指标与甲烷氧化菌数目、甲烷氧化速率均无显著相关性,夏季较春季更有利于甲烷氧化菌的繁殖及氧化甲烷,但季节性差异并不大,年度差异对甲烷氧化菌的影响也较小。甲烷氧化菌数量和甲烷氧化速率呈显著正相关,但二者在数值上并不构成明显的正比例关系,说明甲烷氧化菌的数量并非影响土壤甲烷氧化活性的唯一决定因素。
同一个填埋场各土样之间无论是Ⅰ型还是Ⅱ型甲烷氧化菌,都具有较高的相似性,相似度基本都在60%以上。填埋场覆土层植物根围土样中甲烷氧化菌条带的数量和亮度明显多于无植物区域的空白对照土样,并且植物的根围覆土中甲烷氧化菌群落的多样性总体上大于无植物生长区域土壤。不同植物之间根围土壤中甲烷氧化菌的多样性指数差异不明显。Ⅰ型、Ⅱ型甲烷氧化菌的多样性指数与丰度均存在正相关性,甲烷氧化菌种类的丰度影响了其多样性,但甲烷氧化菌种类的丰度并不能决定其多样性,二者的相关性并不显著。甲烷氧化菌的多样性与其甲烷氧化速率具有一定的正相关性,但二者并不成线性的正比例关系,这说明了多样性指数并非影响甲烷氧化速率的唯一因素,甲烷的氧化也与环境条件等其它因素相关。
植物可促进甲烷氧化菌的数量、多样性及甲烷氧化活性的提高,打碗花、小飞蓬、刺儿菜和鸡眼草等植物对甲烷氧化菌的促进作用更加明显,建议将其作为填埋场封场后的先锋植物种植。
Greenhouse gas, methane, can be oxidized by methane-oxidizing bacteria (methanotrophs) in landfill cover soils. The oxidation activities of these bacteria were affected by several factors, including plant cover. In this study, the population of methanotrophs in landfill cover soil and methane oxidation activities were investigated by using the hungate roll tube method and gas chromatography, and the evolution of methanotrophs community in landfill cover soils were investigated by DGGE of 16S rDNA fragment polymerase chain reaction products.
Methanotroph growth and reproduction, in the enrichment culture process, can be divided into 4 phases:adjustment, exponential, stationary, and decline phase. The first 7 days in culture about the density of methane oxidizing bacteria reached a peak into the stationary phase. The amount of methanotroph in the landfills soil ranges from 107 to 10s cfu·g-1 dry soil, the oxidation rate on methane fluctuates between 10-7～10-8 mol-h-1·g-1. The high-to-low order of the average oxidation rate in the soil samples from the different landfills is, Asuwei landfill> Taoshugang landfill 3> Laohuchong landfill> Taoshugang landfill 2> Taoshugang landfill 1> Zhaojiagou landfill.
The results showed that the shorter closure time for the landfill, the higher methanotroph population and methane oxidation activities; The vegetation coverage were highly significant negative correlations with methanotroph population and significant negative correlations with methane oxidation activities; there was negative correlation between moisture content and methane oxidation activities; The results also showed that there were no significant correlation between pH, organic matter, NH4+ of landfill cover soils and methane oxidation activities. It was better for methanotrophs to reproduce and oxidizing methane in summer than Spring, but the seasonal and year variations were not in relievo. The methane oxidation activities were positively correlated with the population of methanotrophs, but that was not the only factor to influence the activities.
Not the typeⅠmethanotrophs nor the typeⅡmethanotrophs in the cover soils of the same landfill showed high similarity(generally over 60%).The methanotrophs community composition in rhizosphere soil of landfill was higher than bulk soil. The various of Shannon index of methanotrophs in rhizosphere soil of different plants were not obviously, there was a positively correlation between the abundance and Shannon index of both typeⅠand typeⅡmethanotrophs, the abundance of methanotrophs affected the methanotrophs community composition, but not existing significant correlation between them. The methanotrophs community composition was positively correlated with the methane oxidation activities, but was not directly proportional each other, so methanotrophs community composition was not the only factor to influence the methane oxidation activities.
Plants could promote the Population, diversity and methane oxidation activity of methanotrophs. The functional plants for methane purification, such as Calystegia hederacea Wall, Conyza Canadensis, Cirsium setosum (Willd.) MB, and Kummerowia striata (Thunb.) Schindl., were suitable as pioneer plant species for revegetation in closed landfills.
研究发现在富集培养过程中甲烷氧化菌的生长繁殖经历了4个时期：调整期,指数期,稳定期以及衰亡期,在培养的第7天左右甲烷氧化菌的密度达到峰值,进入稳定期。垃圾填埋场土壤中甲烷氧化菌的数量范围在107～108 cfu·g-1干土,甲烷氧化速率在10-7～10-8 mol·h-1·g-1之间。各填埋区土样氧化甲烷速率的平均值从高到低的顺序为：阿苏卫垃圾填埋场>桃树岗垃圾填埋场第3期>老虎冲垃圾填埋场>桃树岗垃圾填埋场第2期>桃树岗垃圾填埋场第1期>赵家沟垃圾填埋场。垃圾填埋场的封场时间与甲烷氧化菌数目呈显著负相关,与甲烷氧化速率呈极显著负相关；填埋场植被覆盖率与甲烷氧化菌数量存在极显著负相关,与氧化速率呈显著负相关；覆土层土壤含水率与甲烷氧化速率等具有一定的负相关；本试验结果显示覆土的pH、有机质与铵态氮等指标与甲烷氧化菌数目、甲烷氧化速率均无显著相关性,夏季较春季更有利于甲烷氧化菌的繁殖及氧化甲烷,但季节性差异并不大,年度差异对甲烷氧化菌的影响也较小。甲烷氧化菌数量和甲烷氧化速率呈显著正相关,但二者在数值上并不构成明显的正比例关系,说明甲烷氧化菌的数量并非影响土壤甲烷氧化活性的唯一决定因素。
同一个填埋场各土样之间无论是Ⅰ型还是Ⅱ型甲烷氧化菌,都具有较高的相似性,相似度基本都在60%以上。填埋场覆土层植物根围土样中甲烷氧化菌条带的数量和亮度明显多于无植物区域的空白对照土样,并且植物的根围覆土中甲烷氧化菌群落的多样性总体上大于无植物生长区域土壤。不同植物之间根围土壤中甲烷氧化菌的多样性指数差异不明显。Ⅰ型、Ⅱ型甲烷氧化菌的多样性指数与丰度均存在正相关性,甲烷氧化菌种类的丰度影响了其多样性,但甲烷氧化菌种类的丰度并不能决定其多样性,二者的相关性并不显著。甲烷氧化菌的多样性与其甲烷氧化速率具有一定的正相关性,但二者并不成线性的正比例关系,这说明了多样性指数并非影响甲烷氧化速率的唯一因素,甲烷的氧化也与环境条件等其它因素相关。
植物可促进甲烷氧化菌的数量、多样性及甲烷氧化活性的提高,打碗花、小飞蓬、刺儿菜和鸡眼草等植物对甲烷氧化菌的促进作用更加明显,建议将其作为填埋场封场后的先锋植物种植。
Greenhouse gas, methane, can be oxidized by methane-oxidizing bacteria (methanotrophs) in landfill cover soils. The oxidation activities of these bacteria were affected by several factors, including plant cover. In this study, the population of methanotrophs in landfill cover soil and methane oxidation activities were investigated by using the hungate roll tube method and gas chromatography, and the evolution of methanotrophs community in landfill cover soils were investigated by DGGE of 16S rDNA fragment polymerase chain reaction products.
Methanotroph growth and reproduction, in the enrichment culture process, can be divided into 4 phases:adjustment, exponential, stationary, and decline phase. The first 7 days in culture about the density of methane oxidizing bacteria reached a peak into the stationary phase. The amount of methanotroph in the landfills soil ranges from 107 to 10s cfu·g-1 dry soil, the oxidation rate on methane fluctuates between 10-7～10-8 mol-h-1·g-1. The high-to-low order of the average oxidation rate in the soil samples from the different landfills is, Asuwei landfill> Taoshugang landfill 3> Laohuchong landfill> Taoshugang landfill 2> Taoshugang landfill 1> Zhaojiagou landfill.
The results showed that the shorter closure time for the landfill, the higher methanotroph population and methane oxidation activities; The vegetation coverage were highly significant negative correlations with methanotroph population and significant negative correlations with methane oxidation activities; there was negative correlation between moisture content and methane oxidation activities; The results also showed that there were no significant correlation between pH, organic matter, NH4+ of landfill cover soils and methane oxidation activities. It was better for methanotrophs to reproduce and oxidizing methane in summer than Spring, but the seasonal and year variations were not in relievo. The methane oxidation activities were positively correlated with the population of methanotrophs, but that was not the only factor to influence the activities.
Not the typeⅠmethanotrophs nor the typeⅡmethanotrophs in the cover soils of the same landfill showed high similarity(generally over 60%).The methanotrophs community composition in rhizosphere soil of landfill was higher than bulk soil. The various of Shannon index of methanotrophs in rhizosphere soil of different plants were not obviously, there was a positively correlation between the abundance and Shannon index of both typeⅠand typeⅡmethanotrophs, the abundance of methanotrophs affected the methanotrophs community composition, but not existing significant correlation between them. The methanotrophs community composition was positively correlated with the methane oxidation activities, but was not directly proportional each other, so methanotrophs community composition was not the only factor to influence the methane oxidation activities.
Plants could promote the Population, diversity and methane oxidation activity of methanotrophs. The functional plants for methane purification, such as Calystegia hederacea Wall, Conyza Canadensis, Cirsium setosum (Willd.) MB, and Kummerowia striata (Thunb.) Schindl., were suitable as pioneer plant species for revegetation in closed landfills.
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