准好氧填埋场CH_4减排和加速稳定化的微生物学机制研究
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摘要
本研究利用现代分子生物学技术,研究了准好氧和厌氧填埋填埋场渗滤液中细菌、准好氧填埋场不同填埋时期固体垃圾中甲烷氧化菌的数量、群落组成与多样性,准好氧填埋场长期降解和稳定化特征,主要得到以下研究结果：
(1)不同填埋工艺对细菌丰度存在明显影响。准好氧填埋场渗滤液中细菌16srRNA基因拷贝数在填埋初期和填埋2年期没有明显变化。厌氧填埋场渗滤液中细菌16srRNA基因拷贝数在填埋初期显著大于填埋稳定期。准好氧填埋场渗滤液中pmoA基因拷贝数分别是厌氧填埋场的48和59倍左右,显著高于厌氧填埋场。
填埋场渗滤液中细菌群落结构及多样性对不同填埋工艺和不同填埋时期的变化反应敏感。准好氧填埋场的两种渗滤液样品细菌群落多样性高于厌氧填埋场。准好氧填埋场(初期和稳定期)渗滤液样品和厌氧填埋场初期的细菌群落结构具有较高的相似性,而与厌氧填埋场稳定阶段渗滤液中细菌群落结构存在明显差异。此外,通过克隆测序研究发现,填埋场渗滤液中细菌群落结构分为7大类群：Proteobacteria (34.8%), Spirochaetes (23.9%), Firmicutes (23.9%), Bacteroidetes (8.7%),Thermotoge (6.5%)和不可分类(2.2%)。准好氧填埋场的Proteobacteria和Bacteroidetes高于厌氧填埋场。准好氧填埋场渗滤液之间细菌群落结构具有较高的相似性,但是厌氧填埋场之间差异显著。甲烷氧化菌(Methylocaldum sp.05J-I-7)仅仅在准好氧填埋场渗滤液样品中发现。因此,填埋工艺造成的填埋场环境的不同是影响细菌丰度和群落结构的重要因素。
(2)填埋时间影响垃圾样品中CH4氧化菌丰度,但对群落组成与多样性影响不显著。准好氧填埋场固体垃圾中的CH4氧化菌以填埋时间最早的1“填埋区pmoA基因拷贝数最高,填埋龄为1年左右的准好氧填埋场固体垃圾中的CH4氧化菌丰度随着填埋时间的增加有增加的趋势,表现出一定的正相关关系。导气管周围垃圾中的CH4氧化菌数量均要高于距离导气管15m处的垃圾。不同填埋时期填埋时间最长的准好氧填埋区甲烷氧化菌多样性高于填埋时间较短的填埋区,且分配更均匀。同一填埋时期的甲烷氧化菌群落结构随距离导气管的远近相似性有所差异。但填埋时间最短的4“填埋区相似性很高。根据序.列的系统发育分析,准好氧填埋场甲烷氧化菌群落结构归为个甲烷氧化菌属：Methylocaldum、Methylobacter、Methylocystis。
(3)经过5年的填埋处置,准好氧填埋体的沉降量和减容率均显著高于厌氧填埋体,同时渗滤液回灌比清水回灌更容易引起填埋体的不均匀沉降。准好氧填埋体填埋气不同时期的CH4含量均显著低于厌氧填埋体,而O2含量则显著高于厌氧填埋体,陈腐垃圾中VS和TOC含量较新鲜垃圾显著降低,同时准好氧填埋体陈腐垃圾的VS和TOC含量均低于厌氧填埋体。渗滤液和清水回灌造成了填埋体表面明显的不均匀沉降。
准好氧填埋体陈腐垃圾的BDM值显著低于厌氧填埋体,BDM值随着导气管距离的增加逐渐升高；随着填埋深度的增加而逐渐升高,且距离导气管距离越大,层问BDM值差异越大,陈腐垃圾的腐殖质提取率略低于厌氧填埋体；同时,陈腐垃圾的HA/FA相对较高,但两者之间的HA/FA差异不显著。
In this paper, molecular biological techniques of real-time polymerase chain reaction and denaturing gradient gel electrophoresis and cloning analysis of 16S rRNA and pmoA gene were used to describe community composition and abundance of bacteria in leachate of semi-aerobic and anaerobic landfills, methanotrophs in waste of semi-aerobic landfill. The results as follows:
Firstly, the bacterial anoudance and composition in leachate were effceted by different landfill method, the bacterial 16S rRNA copy numbers of the leachate had no significant reduction in initial and stable period. In aerobic landfill, the highest bacterial 16S rRNA gene copy numbers in leachate were found at initial stage, but reduced significantly at stable period. The MOB populations in stabilized leachate were one order of magnitude lower than that of initial ones both in semi-aerobic and anaerobic landfills. More MOB populations existed in semi-aerobic landfill leachate than that of the anaerobic ones.
The bacterial diversities of two leachate samples in semi-aerobic landfills were significantly higher than that in anaerobic landfill sites. In addition, bacterial communities in semi-aerobic landfills leachate (both in initial and stable periods) and initial anaerobic leachate had a relatively higher homology with each other, but the bacterial communities in stable anaerobic leachate showed obvious distinguishability.
Based on the sequences and phylogenetic analysis, bacterial communities in the tests were classified into six groups:Proteobacteria (about 34.8%), Spirochaetes (about 23.9%), Firmicutes (about 23.9%), Bacteroidetes (about 8.7%) and Thermotoge (about 6.5%). The semi-aerobic leacates had the dominantly higher proportions of Proteobacteria and Bacteroidetes (the contributors to biogeochemical processes) than that in anaerobic ones. Between semi-aerobic leachates, the high similarities of microbial composition were found, but a significantly difference was shown between anaerobic ones. The methanotrophic microbe (Methylocaldum sp.05J-I-7) was only found in semi-aerobic landfills.
Secondly, aboudance of MOB was effected by lanfill time, but there was no signicantly inpaction to compostion and divertisity.1# which had the longest landfill time had the highest copy numbers of pmoA genes. The results showed that the quantity of MOB populations had an increasing trend with the landfill time in one-year old wastes, and expressed positive relationship to some extent. It was found MOB of waste around gas pipe all higher than 15m far from gas pipe, It was found MOB of waste around gas pipe all higher than 15m far from gas pipe.
The MOB composition and diversity in long landfill time waste higher than short one in different landfill time. There was some difference in one landfill time with the distance to gas pipe, but 4# landfill had a higher similarity. Based on the sequences and phylogenetic analysis, methanotrophic communities in the tests were classified into three groups:Methylocaldum、Methylobacter、Methylocystis.
Thirdly, pile surface settlements and volume reduction rate of the semi-aerobic landfills were significantly higher than the anaerobic ones after 5 year landfill disposal.Moreover, the treatments with leachate recycling were tend to have the larger uneven settlements than the ones with water recycling. In different landfill periods, the concentrations of O2 in semi-aerobic landfill piles were significantly higher than anaerobic ones, while the concentrations of CH4 in former were dramatically lower than the latter. VS and TOC content of aged-refuse were obviously lower than the ones in fresh refuse. Moreover, VS and TOC content of aged-refuse in semi-aerobic landfills were significantly lower than that in anaerobic ones, BDM value of aged-refuse in semi-aerobic landfills had a significant reductions comparing with that in anaerobic ones. Furthermore, BDM value of aged-refuse in semi-aerobic landfills increased with air pipe distances and depth increasing, and the greater the air pipe distances, the bigger the difference of BDM value between the layers. In addition, total extraction ratios of humic matters in semi-aerobic aged-refuse were slightly lower than anaerobic ones, while the HA/FA in semi-aerobic and anaerobic aged-refuse were relatively high, and no significant differences were found.
(1)不同填埋工艺对细菌丰度存在明显影响。准好氧填埋场渗滤液中细菌16srRNA基因拷贝数在填埋初期和填埋2年期没有明显变化。厌氧填埋场渗滤液中细菌16srRNA基因拷贝数在填埋初期显著大于填埋稳定期。准好氧填埋场渗滤液中pmoA基因拷贝数分别是厌氧填埋场的48和59倍左右,显著高于厌氧填埋场。
填埋场渗滤液中细菌群落结构及多样性对不同填埋工艺和不同填埋时期的变化反应敏感。准好氧填埋场的两种渗滤液样品细菌群落多样性高于厌氧填埋场。准好氧填埋场(初期和稳定期)渗滤液样品和厌氧填埋场初期的细菌群落结构具有较高的相似性,而与厌氧填埋场稳定阶段渗滤液中细菌群落结构存在明显差异。此外,通过克隆测序研究发现,填埋场渗滤液中细菌群落结构分为7大类群：Proteobacteria (34.8%), Spirochaetes (23.9%), Firmicutes (23.9%), Bacteroidetes (8.7%),Thermotoge (6.5%)和不可分类(2.2%)。准好氧填埋场的Proteobacteria和Bacteroidetes高于厌氧填埋场。准好氧填埋场渗滤液之间细菌群落结构具有较高的相似性,但是厌氧填埋场之间差异显著。甲烷氧化菌(Methylocaldum sp.05J-I-7)仅仅在准好氧填埋场渗滤液样品中发现。因此,填埋工艺造成的填埋场环境的不同是影响细菌丰度和群落结构的重要因素。
(2)填埋时间影响垃圾样品中CH4氧化菌丰度,但对群落组成与多样性影响不显著。准好氧填埋场固体垃圾中的CH4氧化菌以填埋时间最早的1“填埋区pmoA基因拷贝数最高,填埋龄为1年左右的准好氧填埋场固体垃圾中的CH4氧化菌丰度随着填埋时间的增加有增加的趋势,表现出一定的正相关关系。导气管周围垃圾中的CH4氧化菌数量均要高于距离导气管15m处的垃圾。不同填埋时期填埋时间最长的准好氧填埋区甲烷氧化菌多样性高于填埋时间较短的填埋区,且分配更均匀。同一填埋时期的甲烷氧化菌群落结构随距离导气管的远近相似性有所差异。但填埋时间最短的4“填埋区相似性很高。根据序.列的系统发育分析,准好氧填埋场甲烷氧化菌群落结构归为个甲烷氧化菌属：Methylocaldum、Methylobacter、Methylocystis。
(3)经过5年的填埋处置,准好氧填埋体的沉降量和减容率均显著高于厌氧填埋体,同时渗滤液回灌比清水回灌更容易引起填埋体的不均匀沉降。准好氧填埋体填埋气不同时期的CH4含量均显著低于厌氧填埋体,而O2含量则显著高于厌氧填埋体,陈腐垃圾中VS和TOC含量较新鲜垃圾显著降低,同时准好氧填埋体陈腐垃圾的VS和TOC含量均低于厌氧填埋体。渗滤液和清水回灌造成了填埋体表面明显的不均匀沉降。
准好氧填埋体陈腐垃圾的BDM值显著低于厌氧填埋体,BDM值随着导气管距离的增加逐渐升高；随着填埋深度的增加而逐渐升高,且距离导气管距离越大,层问BDM值差异越大,陈腐垃圾的腐殖质提取率略低于厌氧填埋体；同时,陈腐垃圾的HA/FA相对较高,但两者之间的HA/FA差异不显著。
In this paper, molecular biological techniques of real-time polymerase chain reaction and denaturing gradient gel electrophoresis and cloning analysis of 16S rRNA and pmoA gene were used to describe community composition and abundance of bacteria in leachate of semi-aerobic and anaerobic landfills, methanotrophs in waste of semi-aerobic landfill. The results as follows:
Firstly, the bacterial anoudance and composition in leachate were effceted by different landfill method, the bacterial 16S rRNA copy numbers of the leachate had no significant reduction in initial and stable period. In aerobic landfill, the highest bacterial 16S rRNA gene copy numbers in leachate were found at initial stage, but reduced significantly at stable period. The MOB populations in stabilized leachate were one order of magnitude lower than that of initial ones both in semi-aerobic and anaerobic landfills. More MOB populations existed in semi-aerobic landfill leachate than that of the anaerobic ones.
The bacterial diversities of two leachate samples in semi-aerobic landfills were significantly higher than that in anaerobic landfill sites. In addition, bacterial communities in semi-aerobic landfills leachate (both in initial and stable periods) and initial anaerobic leachate had a relatively higher homology with each other, but the bacterial communities in stable anaerobic leachate showed obvious distinguishability.
Based on the sequences and phylogenetic analysis, bacterial communities in the tests were classified into six groups:Proteobacteria (about 34.8%), Spirochaetes (about 23.9%), Firmicutes (about 23.9%), Bacteroidetes (about 8.7%) and Thermotoge (about 6.5%). The semi-aerobic leacates had the dominantly higher proportions of Proteobacteria and Bacteroidetes (the contributors to biogeochemical processes) than that in anaerobic ones. Between semi-aerobic leachates, the high similarities of microbial composition were found, but a significantly difference was shown between anaerobic ones. The methanotrophic microbe (Methylocaldum sp.05J-I-7) was only found in semi-aerobic landfills.
Secondly, aboudance of MOB was effected by lanfill time, but there was no signicantly inpaction to compostion and divertisity.1# which had the longest landfill time had the highest copy numbers of pmoA genes. The results showed that the quantity of MOB populations had an increasing trend with the landfill time in one-year old wastes, and expressed positive relationship to some extent. It was found MOB of waste around gas pipe all higher than 15m far from gas pipe, It was found MOB of waste around gas pipe all higher than 15m far from gas pipe.
The MOB composition and diversity in long landfill time waste higher than short one in different landfill time. There was some difference in one landfill time with the distance to gas pipe, but 4# landfill had a higher similarity. Based on the sequences and phylogenetic analysis, methanotrophic communities in the tests were classified into three groups:Methylocaldum、Methylobacter、Methylocystis.
Thirdly, pile surface settlements and volume reduction rate of the semi-aerobic landfills were significantly higher than the anaerobic ones after 5 year landfill disposal.Moreover, the treatments with leachate recycling were tend to have the larger uneven settlements than the ones with water recycling. In different landfill periods, the concentrations of O2 in semi-aerobic landfill piles were significantly higher than anaerobic ones, while the concentrations of CH4 in former were dramatically lower than the latter. VS and TOC content of aged-refuse were obviously lower than the ones in fresh refuse. Moreover, VS and TOC content of aged-refuse in semi-aerobic landfills were significantly lower than that in anaerobic ones, BDM value of aged-refuse in semi-aerobic landfills had a significant reductions comparing with that in anaerobic ones. Furthermore, BDM value of aged-refuse in semi-aerobic landfills increased with air pipe distances and depth increasing, and the greater the air pipe distances, the bigger the difference of BDM value between the layers. In addition, total extraction ratios of humic matters in semi-aerobic aged-refuse were slightly lower than anaerobic ones, while the HA/FA in semi-aerobic and anaerobic aged-refuse were relatively high, and no significant differences were found.
引文
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