填埋垃圾生物反应器反硝化性能及反硝化微生物研究
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摘要
卫生填埋技术是目前我国生活垃圾处理处置最主要的方法。但填埋过程中产生的大量成分复杂、性质多变的难降解渗滤液已成为制约我国生活垃圾填埋场可持续发展的主要因素之一。《生活垃圾填埋场污染控制标准》(GB16889-2008)的颁布,大幅度提高了渗滤液外排要求,尤其氨氮,规定其外排浓度≤25mgL-1。而采用现有的场外运行工艺处理含高浓度氨氮渗滤液,其处理成本高达30-45元／吨,为绝大多数填埋场所难以承受。因此,开发经济高效的渗滤液脱氮技术具有重要意义。而在渗滤液回灌基础上,结合垃圾堆体本身为厌氧环境的特性,开发出的生物反应器垃圾堆体渗滤液原位脱氮技术已成为目前的研究热点。本论文以从杭州市固体废弃物处理有限公司天子岭填埋场采集的不同填埋龄垃圾为试材,利用系列自制生活垃圾生物反应器模拟装置,通过回灌硝酸盐溶液和含硝酸盐渗滤液,探明了不同填埋龄垃圾在碳源不足及碳源充足情况下的反硝化性能特征,确定了反硝化作用所需最适C/N；并以nirS为分子标记建立基因文库的方法阐明了生物反应器内反硝化微生物种群多样性变化规律,从一定程度上揭示了反硝化微生物学脱氮机理。研究结果为生物反应器垃圾堆体渗滤液原位脱氮技术的进一步研发提供了重要理论依据,并为同类研究提供了可借鉴的研究方法。具体研究结果如下：
从杭州市固体废弃物处理有限公司天子岭填埋场采集了1-12年填埋龄垃圾,通过对其基本理化性质的分析发现：①不同填埋龄垃圾中以类似腐殖质的混合物为主,其次为塑料类；混合类物质含量随垃圾填埋龄的增加而上升。②由于填埋场含水率低,pH、TN(总氮)、BDM(生物可降解物)、HE(腐殖质可提取物)等指标在垃圾填埋4年后基本趋于稳定,垃圾进入降解缓慢期。这表明填埋场的传统操作模式不利于垃圾稳定化,开发以渗滤液回灌为特征的生物反应器填埋技术具有现实意义。
将从天子岭填埋场采集的1-12年填埋龄垃圾装填于系列自制生活垃圾生物反应器模拟装置中(相应的命名为R1、R2……R12),回灌硝酸盐溶液,考察间歇回灌模式下不同填埋龄垃圾反硝化性能,结果发现：①当垃圾堆体自身降解的有机物量充足时,1-12年垃圾的反硝化性能均较强,且差异不大；所有反应器在最初的1小时内硝酸盐去除率最高,达70-92%。②当垃圾堆体自身降解的有机物量不足时,不同填埋龄垃圾生物反应器反硝化性能出现差异。Rl、R6的硝酸盐还原速率显著高于R11,其速率分别为6.80 mg NO3--N kg-TSwaste-1 h-1.3.00 mgNO3--N kg-TSwaste-1 h-1和1.10 mg NO3--N kg-TSwaste-1 h-1。③不同反应器间的反硝化终产物也存在差异。R1、R6的反硝化终产物以N2为主,其最终浓度分别为82.5%和80.2%；而在R11中则以N2O为主,累积浓度高达19.3%。推断得垃圾的低C/N易导致反硝化终产物以N20为主。因此,综合考虑反硝化速率和反硝化终产物,当碳源不足时,填埋龄短的垃圾更适合作为反硝化介质用于渗滤液脱氮。
根据上述试验结果,选择R6为试验对象,以采自天子岭填埋场的新鲜渗滤液为回灌液,通过调节不同C/N,考察有机物浓度对生物反应器反硝化性能的影响,探讨反硝化最适C/N。结果表明：R6硝酸盐还原速率随着回灌入垃圾堆体中渗滤液COD浓度的提高而提高。当C/N为3.11和5.03时,反应器内反硝化作用不显著,硝酸盐还原速率仅为1.14 mg NO3--N kg-TSwaste-1 h-1和1.24 mgNO3--N kg-TSwaste-1 h-1;但当C/N调节为9.08和13.08时,反应器内硝酸盐还原迅速,其速率分别高达6.33 mg NO3--N kg-TSwaste-1 h-1和11.40 mg NO3--N kg-TSwaste-1 h-1。综合分析得当COD/NO3-N达到6.0(g/g)时,可保证反应器内反硝化过程的快速、稳定进行,即最适C/N为6.0。因此,C/N可作为渗滤液硝酸盐完全反硝化作用的一个特征性参数。
在此基础上,选择R4、R8和R12为试验对象,根据最适C/N调节回灌入垃圾堆体的渗滤液有机物含量,考察了不同填埋龄垃圾在自身降解产生的有机物量不足时的反硝化特征。结果发现：R4、R8、R12均表现出较强的反硝化性能,且差异不大,每个反应器均可在70h左右将500 mgL-1的硝酸盐还原完全。这表明在回灌渗滤液C/N适宜的条件下,所有垃圾堆体均可作为厌氧系统进行反硝化作用。
以nirS基因为分子标记,采用“PCR-克隆-测序”并建立基因文库的方法,探讨了填埋垃圾生物反应器内反硝化微生物多样性特征。结果发现：①在R6的nirS基因文库中,检测到的微生物主要归属于变形菌门(Proteobacteria)的β-变形菌纲(Betaproteobacteria);但当C/N为5.03-9.08时,种群结构较接近,而低C/N(3.11)和高C/N(13.08)时种群结构存在显著性差异。Thiobacillus denitrificans和Azoarcus tolulyticus始终是反应器内优势反硝化菌,但随着回灌渗滤液中C/N的提高,Thiobacillus denitrificans和Azoarcus tolulyticus呈现一定的演替规律：Thiobacillus denitrificans的克隆丰富度变化情况为77.22%、15.38%、17.39%和72.46%；而Azoarcus tolulyticus的克隆丰富度变化情况则刚好相反,分别为10.13%、75%、59.42%和7.25%。②R4、R8和R12的nirS基因文库中,最相似序列均可归属于两大类：变形菌门(Proteobacteria)细菌和未培养微生物,但各反应器间的群落结构仍存在一定差异。R4内的反硝化种群以Thiobacillus denitrificans、Azoarcus tolulyticus和GenBank为AY078277.1的未培养微生物为王;R8 nirS文库中以Azoarcus tolulyticus为主；而R12的优势种群则为Azoarcus tolulyticus和GenBank为AY078277.1的未培养微生物。综合以上试验结果表明,填埋垃圾生物反应器内,反硝化菌种群结构相对单一,多样性偏低,但Thiobacillus denitrificans和Azoarcus tolulyticus是垃圾堆体中生长较稳定的种群,猜测在渗滤液反硝化脱氮中发挥着重要作用。
根据实验室研究结果,在杭州市固体废弃物处理有限公司开展了填埋垃圾生物反应器反硝化性能中试研究,结果表明硝化渗滤液经生物反应器处理后,出水水质稳定,硝酸盐、亚硝酸盐可被基本去除完。结合实验室研究结果进一步表明开发生物反应器垃圾堆体渗滤液原位脱氮技术具有现实可行性。
Landfilling is one of the most important methods for refuse disposal in China. However, leachate produced from the landfill is rich in various contaminants, especially ammonia and if not managed well can pose serious threat to the environment. Recently, the Chinese government published a new standard for pollution control of municipal solid waste landfills, which prescribes that the emission concentration of ammonia in leachate must be lower than 25 mg L"1. At present, ammonia removal is mostly practiced ex situ. However, this treatment approach requires large spatial and financial investment, which restricts its utilization. Therefore, development of an in situ nitrogen removal technique would be an attractive alternative. This technique involves ex situ nitrification of leachate which is coupled with the use of the landfill as a bioreactor for denitrification. In this thesis, refuse at different landfill ages were sampled from Hangzhou Tianziling Landfill. The denitrification capacities of bioreactors filled with refuse at different landfill ages were studied. nirS gene libraries were constructed with a PCR-based clone approach in order to study the structure and diversity of the denitrifying bacteria. The information derived from this research will help other researchers carry out further studies. Some principle results of this thesis are summaried as follows.
The mixtures which had similar performance with soil comprised the highest proportion in the refuse of different landfill ages, followed by plastics. Refuse in the landfill became relatively stable but not fully aged after being placed for four years primarily due to low water content. Therefore, in order to accelerate refuse decomposition, a reasonable approach is to develop bioreactor landfills characterized by leachate recirculation.
In this study, nitrate solution was injected intermittently. Refuse at different landfill ages all had a considerably high denitrification capacity when the refuse body itself could provide enough organic carbon for denitrification. And the nitrate reduction rates could reach up to 70-92% in the first hour. However, when the refuse contained low organic matter, refuse at different landfill ages possessed different denitrification capacity. R1, R6 reduced nitrate much more quickly than R11, and the nitrate reduction rates were 6.80 mg NO3--N kg-TSwaste-1 h'1、3.00 mg NO3--N kg-TSwaste-1 h-1 and 1.10 mg NO3--N kg-TSwaste-1 h-1, respectively. Moreover, the final products in R1 and R6 were N2 and the concentrations were 82.5% and 80.2%, respectively. On the contrary, N2O accumulated in R11 and the final concentration reached up to 19.3%. These results suggested that organic matter played an important role in nitrate reduction and partly-degraded refuse was more suitable to use as denitrification medium.
The effects of different ratios of chemical oxygen demand (COD) and nitrate concentrations in the injected leachate on the denitrification capacity of refuse were evaluated. It was found that nitrate reduction rate increased with the increasing COD concentration in the injected leachate. Nitrate reduction rates in tests 1 to 4 was 1.14 mg NO3--N kg-TSwaste-1 h-1,1.24 mg N03--N kg-TSwaste-1 h-1,6.33 mg NO3--N h-1 and 11.40 mg NO3--N kg-TSwaste-1 h-1, respectively. And for rapid denitrification, COD/NO3--N ratio in the initial leachate should be adjusted to higher than 6.0.
According to the optimum ratio of COD/NO3--N, R4, R8 and R12 were used to investigate the denitrification capacity when the refuse could not provide enough organic matter. And the results showed that refuse at different landfill ages possessed a high denitrification capacity when the injection contained enough organic matter. Moreover, their nitrate reduction rates were almost the same.
The analysis of nirS clone libraries indicated that the denitrifying bacteria fell into Proteobacteria (Betaproteobacteria) in R6. When the COD/NO3--N ratio was between 5.03-9.08, the diversities of denitrifying bacteria were almost the same. But the structures of denitrifying bacteria were quite different when the ratios of COD/NO3--N were 3.11 and 13.08. Moreover, Thiobacillus denitrificans and Azoarcus tolulyticus were dominant species in this reactor. But the clone abundance of Thiobacillus denitrificans was contrary to that of Azoarcus tolulyticus. The clone abundance of Thiobacillus denitrificans was 77.22%、15.38%、17.39% and 72.46%, and that of Azoarcus tolulyticus was 10.13%、75%、59.42%和7.25%. On the contrary, the denitrifying bacteria in R4, R8 and R12 fell into two lineages: Proteobacteria and Uncultured bacterium. The structures and diversities of the denitrifying bacteria in these reactors were different. The dominant species in R4 were Thiobacillus denitrificans、Azoarcus tolulyticus and the uncultured bacterium named AY078277.1. In R8, only Azoarcus tolulyticus were dominant species and in R12, Azoarcus tolulyticus and the uncultured bacterium named AY078277.1 predominated.
In general, the diversities of denitrifying bacteria in bioreactor landfill were relatively low. Although the community structures were different, Thiobacillus denitrificans and Azoarcus tolulyticus could be found in all the reactors. This indicated that Thiobacillus denitrificans and Azoarcus tolulyticus grew stablely in refuse and they might play important roles in nitrogen removal.
Based on the lab researches, pilot scale study was conducted in Hangzhou Tianziling Landfill. And the results showed that the bioreactor had good effect on nitrate reduction and developing a new way for nitrogen removal with the use of the landfill as a bioreactor for denitrification had certain feasibility.
从杭州市固体废弃物处理有限公司天子岭填埋场采集了1-12年填埋龄垃圾,通过对其基本理化性质的分析发现：①不同填埋龄垃圾中以类似腐殖质的混合物为主,其次为塑料类；混合类物质含量随垃圾填埋龄的增加而上升。②由于填埋场含水率低,pH、TN(总氮)、BDM(生物可降解物)、HE(腐殖质可提取物)等指标在垃圾填埋4年后基本趋于稳定,垃圾进入降解缓慢期。这表明填埋场的传统操作模式不利于垃圾稳定化,开发以渗滤液回灌为特征的生物反应器填埋技术具有现实意义。
将从天子岭填埋场采集的1-12年填埋龄垃圾装填于系列自制生活垃圾生物反应器模拟装置中(相应的命名为R1、R2……R12),回灌硝酸盐溶液,考察间歇回灌模式下不同填埋龄垃圾反硝化性能,结果发现：①当垃圾堆体自身降解的有机物量充足时,1-12年垃圾的反硝化性能均较强,且差异不大；所有反应器在最初的1小时内硝酸盐去除率最高,达70-92%。②当垃圾堆体自身降解的有机物量不足时,不同填埋龄垃圾生物反应器反硝化性能出现差异。Rl、R6的硝酸盐还原速率显著高于R11,其速率分别为6.80 mg NO3--N kg-TSwaste-1 h-1.3.00 mgNO3--N kg-TSwaste-1 h-1和1.10 mg NO3--N kg-TSwaste-1 h-1。③不同反应器间的反硝化终产物也存在差异。R1、R6的反硝化终产物以N2为主,其最终浓度分别为82.5%和80.2%；而在R11中则以N2O为主,累积浓度高达19.3%。推断得垃圾的低C/N易导致反硝化终产物以N20为主。因此,综合考虑反硝化速率和反硝化终产物,当碳源不足时,填埋龄短的垃圾更适合作为反硝化介质用于渗滤液脱氮。
根据上述试验结果,选择R6为试验对象,以采自天子岭填埋场的新鲜渗滤液为回灌液,通过调节不同C/N,考察有机物浓度对生物反应器反硝化性能的影响,探讨反硝化最适C/N。结果表明：R6硝酸盐还原速率随着回灌入垃圾堆体中渗滤液COD浓度的提高而提高。当C/N为3.11和5.03时,反应器内反硝化作用不显著,硝酸盐还原速率仅为1.14 mg NO3--N kg-TSwaste-1 h-1和1.24 mgNO3--N kg-TSwaste-1 h-1;但当C/N调节为9.08和13.08时,反应器内硝酸盐还原迅速,其速率分别高达6.33 mg NO3--N kg-TSwaste-1 h-1和11.40 mg NO3--N kg-TSwaste-1 h-1。综合分析得当COD/NO3-N达到6.0(g/g)时,可保证反应器内反硝化过程的快速、稳定进行,即最适C/N为6.0。因此,C/N可作为渗滤液硝酸盐完全反硝化作用的一个特征性参数。
在此基础上,选择R4、R8和R12为试验对象,根据最适C/N调节回灌入垃圾堆体的渗滤液有机物含量,考察了不同填埋龄垃圾在自身降解产生的有机物量不足时的反硝化特征。结果发现：R4、R8、R12均表现出较强的反硝化性能,且差异不大,每个反应器均可在70h左右将500 mgL-1的硝酸盐还原完全。这表明在回灌渗滤液C/N适宜的条件下,所有垃圾堆体均可作为厌氧系统进行反硝化作用。
以nirS基因为分子标记,采用“PCR-克隆-测序”并建立基因文库的方法,探讨了填埋垃圾生物反应器内反硝化微生物多样性特征。结果发现：①在R6的nirS基因文库中,检测到的微生物主要归属于变形菌门(Proteobacteria)的β-变形菌纲(Betaproteobacteria);但当C/N为5.03-9.08时,种群结构较接近,而低C/N(3.11)和高C/N(13.08)时种群结构存在显著性差异。Thiobacillus denitrificans和Azoarcus tolulyticus始终是反应器内优势反硝化菌,但随着回灌渗滤液中C/N的提高,Thiobacillus denitrificans和Azoarcus tolulyticus呈现一定的演替规律：Thiobacillus denitrificans的克隆丰富度变化情况为77.22%、15.38%、17.39%和72.46%；而Azoarcus tolulyticus的克隆丰富度变化情况则刚好相反,分别为10.13%、75%、59.42%和7.25%。②R4、R8和R12的nirS基因文库中,最相似序列均可归属于两大类：变形菌门(Proteobacteria)细菌和未培养微生物,但各反应器间的群落结构仍存在一定差异。R4内的反硝化种群以Thiobacillus denitrificans、Azoarcus tolulyticus和GenBank为AY078277.1的未培养微生物为王;R8 nirS文库中以Azoarcus tolulyticus为主；而R12的优势种群则为Azoarcus tolulyticus和GenBank为AY078277.1的未培养微生物。综合以上试验结果表明,填埋垃圾生物反应器内,反硝化菌种群结构相对单一,多样性偏低,但Thiobacillus denitrificans和Azoarcus tolulyticus是垃圾堆体中生长较稳定的种群,猜测在渗滤液反硝化脱氮中发挥着重要作用。
根据实验室研究结果,在杭州市固体废弃物处理有限公司开展了填埋垃圾生物反应器反硝化性能中试研究,结果表明硝化渗滤液经生物反应器处理后,出水水质稳定,硝酸盐、亚硝酸盐可被基本去除完。结合实验室研究结果进一步表明开发生物反应器垃圾堆体渗滤液原位脱氮技术具有现实可行性。
Landfilling is one of the most important methods for refuse disposal in China. However, leachate produced from the landfill is rich in various contaminants, especially ammonia and if not managed well can pose serious threat to the environment. Recently, the Chinese government published a new standard for pollution control of municipal solid waste landfills, which prescribes that the emission concentration of ammonia in leachate must be lower than 25 mg L"1. At present, ammonia removal is mostly practiced ex situ. However, this treatment approach requires large spatial and financial investment, which restricts its utilization. Therefore, development of an in situ nitrogen removal technique would be an attractive alternative. This technique involves ex situ nitrification of leachate which is coupled with the use of the landfill as a bioreactor for denitrification. In this thesis, refuse at different landfill ages were sampled from Hangzhou Tianziling Landfill. The denitrification capacities of bioreactors filled with refuse at different landfill ages were studied. nirS gene libraries were constructed with a PCR-based clone approach in order to study the structure and diversity of the denitrifying bacteria. The information derived from this research will help other researchers carry out further studies. Some principle results of this thesis are summaried as follows.
The mixtures which had similar performance with soil comprised the highest proportion in the refuse of different landfill ages, followed by plastics. Refuse in the landfill became relatively stable but not fully aged after being placed for four years primarily due to low water content. Therefore, in order to accelerate refuse decomposition, a reasonable approach is to develop bioreactor landfills characterized by leachate recirculation.
In this study, nitrate solution was injected intermittently. Refuse at different landfill ages all had a considerably high denitrification capacity when the refuse body itself could provide enough organic carbon for denitrification. And the nitrate reduction rates could reach up to 70-92% in the first hour. However, when the refuse contained low organic matter, refuse at different landfill ages possessed different denitrification capacity. R1, R6 reduced nitrate much more quickly than R11, and the nitrate reduction rates were 6.80 mg NO3--N kg-TSwaste-1 h'1、3.00 mg NO3--N kg-TSwaste-1 h-1 and 1.10 mg NO3--N kg-TSwaste-1 h-1, respectively. Moreover, the final products in R1 and R6 were N2 and the concentrations were 82.5% and 80.2%, respectively. On the contrary, N2O accumulated in R11 and the final concentration reached up to 19.3%. These results suggested that organic matter played an important role in nitrate reduction and partly-degraded refuse was more suitable to use as denitrification medium.
The effects of different ratios of chemical oxygen demand (COD) and nitrate concentrations in the injected leachate on the denitrification capacity of refuse were evaluated. It was found that nitrate reduction rate increased with the increasing COD concentration in the injected leachate. Nitrate reduction rates in tests 1 to 4 was 1.14 mg NO3--N kg-TSwaste-1 h-1,1.24 mg N03--N kg-TSwaste-1 h-1,6.33 mg NO3--N h-1 and 11.40 mg NO3--N kg-TSwaste-1 h-1, respectively. And for rapid denitrification, COD/NO3--N ratio in the initial leachate should be adjusted to higher than 6.0.
According to the optimum ratio of COD/NO3--N, R4, R8 and R12 were used to investigate the denitrification capacity when the refuse could not provide enough organic matter. And the results showed that refuse at different landfill ages possessed a high denitrification capacity when the injection contained enough organic matter. Moreover, their nitrate reduction rates were almost the same.
The analysis of nirS clone libraries indicated that the denitrifying bacteria fell into Proteobacteria (Betaproteobacteria) in R6. When the COD/NO3--N ratio was between 5.03-9.08, the diversities of denitrifying bacteria were almost the same. But the structures of denitrifying bacteria were quite different when the ratios of COD/NO3--N were 3.11 and 13.08. Moreover, Thiobacillus denitrificans and Azoarcus tolulyticus were dominant species in this reactor. But the clone abundance of Thiobacillus denitrificans was contrary to that of Azoarcus tolulyticus. The clone abundance of Thiobacillus denitrificans was 77.22%、15.38%、17.39% and 72.46%, and that of Azoarcus tolulyticus was 10.13%、75%、59.42%和7.25%. On the contrary, the denitrifying bacteria in R4, R8 and R12 fell into two lineages: Proteobacteria and Uncultured bacterium. The structures and diversities of the denitrifying bacteria in these reactors were different. The dominant species in R4 were Thiobacillus denitrificans、Azoarcus tolulyticus and the uncultured bacterium named AY078277.1. In R8, only Azoarcus tolulyticus were dominant species and in R12, Azoarcus tolulyticus and the uncultured bacterium named AY078277.1 predominated.
In general, the diversities of denitrifying bacteria in bioreactor landfill were relatively low. Although the community structures were different, Thiobacillus denitrificans and Azoarcus tolulyticus could be found in all the reactors. This indicated that Thiobacillus denitrificans and Azoarcus tolulyticus grew stablely in refuse and they might play important roles in nitrogen removal.
Based on the lab researches, pilot scale study was conducted in Hangzhou Tianziling Landfill. And the results showed that the bioreactor had good effect on nitrate reduction and developing a new way for nitrogen removal with the use of the landfill as a bioreactor for denitrification had certain feasibility.
引文
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