水平潜流人工湿地氮循环微生物效应及生态模型研究
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摘要
本文以水平潜流芦苇砾石床人工湿地系统为研究对象,以防治水污染为目的,开展了以下研究内容:水平潜流湿地氮去除速率及主控因素分析;水平潜流湿地氮形态分布及转化规律的研究;水平潜流湿地氮转化微生物沿程分布和种群分析;水平潜流人工湿地总细菌及氨氧化细菌多样性研究;特征脱氮菌种筛选、鉴定及生理生化特性分析;水平潜流湿地氮循环生态动力学模型研究。得出主要研究结论如下:
(1)在平均水力停留时间3.85h的条件下,通过对水平潜流人工湿地氮素去除效率分析,水平潜流人工湿地系统表现出一定的除污能力,进水COD、NH_4~+-N、NO_3~--N、Org-N和TN浓度为分别7.8~12.7mg·L~(-1)、0.05~15.0mg·L~(-1)、1.3~5.2mg·L~(-1)、0.66~5.18mg·L~(-1)、2.2~20.0mg·L~(-1),芦苇砾石床系统的年平均去除率为25.8%、10.0%、42%、31.2%和18.9%。季节变化对污染组分的去除效率影响非常显著,在寒季系统对COD、NH_4~+-N、NO_3~--N、Org-N和TN的去除效率有所下降,各组分的平均去除率约为11.2%、6.5%、31.0%、20.5%和15%左右。
(2)季节变化对温度和污染负荷率两个因子的影响显著。进水温度在0.9~24.8℃之间变化时,各形态氮素的体积去除速率常数K_v与温度呈正相关,NH_4~+-N、NO_3~--N、Org-N和TN的K_v值分别为0.01~0.2d~(-1)、0.03~0.3d~(-1)、0.07~0.3d~(-1)和0.02~0.15d~(-1);温度在20℃时的体积去除速率常数K_(v20)分别为0.0769、0.1673、0.1508和0.0732 d~(-1),说明该湿地系统对NO_3~--N、Org-N的消减速率较NH_4~+-N大。NH_4~+-N和Org-N的去除速率与负荷率呈对数正相关,当NH_4~+-N的负荷率为15gN·m~(-2)·d~(-1)时,其最大去除速率为1.02gN·m~(-2)·d~(-1);当Org-N的负荷率为3.2gN·m~(-2)·d~(-1)时,其最大去除速率可达到0.36gN·m~(-2)·d~(-1)。
(3)通过对16项进出水理化指标的主因素分析,确立了5个主因素,主因素1与进出水温度呈负相关,关系相对最为密切;主因素2和3分别与湿地进水NO_3~--N和NH_4~+-N负荷呈正相关;主因素4和5分别与进水DO和ORP关系最为密切,且均呈负相关。在16项进出水指标的分析中主因素的排列顺序是:温度>NO_3~--N负荷>NH_4~+-N负荷>DO>ORP,与5项出水指标主因素分析排列顺序略有不同,其主要原因是人工湿地作为一个独立的生态系统,影响因素比较复杂、具有不确定性,但温度作为主要的影响因素地位明显高于其它因素。
(4)比较了根际与非根际的硝化/反硝化强度及呼吸作用强度,研究发现根际效应显著强于非根际。通过对氨化细菌、亚硝化细菌、硝化细菌及反硝化细菌等四种脱氮种群的研究发现,氨化细菌的数量级在10~8~10~(10)cfu之间,其沿湿地空间位置(长度)数量分布无规律性变化;亚硝化细菌的数量级在10~3~10~7MPN之间,从湿地进水端至尾端亚硝化细菌的数量逐渐减少,尤其在暖季变化显著;硝化细菌的数量级在10~5~10~7MPN之间,其分布无显著变化,暖季时硝化细菌的数量是前端、中部高于尾端;反硝化细菌的数量级在10~4~10~7MPN之间,湿地系统尾端数量略高于前端。
(5)运用PCR-DGGE技术,比较真实的反应了水平潜流人工湿地系统中微生物种群结构及分布特征。水平潜流人工湿地中微生物种群多样性随着水流方向呈现先逐渐减少的趋势。通过湿地系统沿程COD与Shannon指数相关性分析,发现芦苇砾石系统沿程COD变化与Shannon指数相关性较好,不同季节湿地系统中微生物多样性的空间分布特征对处理有机物效能存在差异。对氨氧化菌群落结构多样性的研究表明:不同季节水平潜流人工湿地中氨氧化菌的时空变化显著,并分析得出了湿地系统的优势氨氧化菌种,可为系统氨氮去除途径的分析提供微生物学标识。
(6)筛选出9株反硝化细菌均具有一定的脱氮能力,脱氮反应主要发生在菌体生长的对数生长期,脱氮速率随着生物量的增加逐渐加快。脱氮能力对比结果显示菌株DF2和DF3脱氮效率最高,均能在72h内将90%的TN去除,最终脱氮率维持在95%左右。特别重点考察了菌株DF2的脱氮特性,利用16SrDNA技术对菌株DF2进行了鉴定,通过与参比菌株的比较,确定了该菌株的系统发育地位,结果表明该菌株位于德克斯氏菌属,至今未发现类似的报道。
(7)水平潜流人工湿地系统硝化与反硝化作用是氮元素转化的主要途径之一,其速率分别达到了0.791gN·m~(-2)·d~(-1)和0.823gN·m~(-2)·d~(-1);其次植物对氮的吸收0.747 gN·m~(-2)·d~(-1);沉淀和再生速率也分别达到了0.514gN·m~(-2)·d~(-1)和0.446gN·m~(-2)·d~(-1);其它贡献的途径如植物腐败0.282gN·m~(-2)·d~(-1)和矿化0.0126gN·m~(-2)·d~(-1)。在整个系统氮转化和去除的主要途径递减顺序为反硝化、植物吸收、沉淀。其中,反硝化作用占总氮去除的60.7%、植物吸收占34.3%、沉淀作用占5.0%,模型在预测结果方面具有一定的准确性和合理性。
The performance and mechanisms of horizontal subsurface flow contructed wetlands reed/gravel bed system of Guanting Reservoir were studied for the preventive treatment of the pollution of potable water sources. The study includes:(1) The Nitrogen removal rate and main factor analysis of horizontal subsurface flow constructed wetlands;(2) The forms distribution and transformation rule;(3) The analysis of the frictional distribution and population of Nitrogen transformation microbes in the system;(4) The microbial diversity research in the system;(5) The featured denitrifiers were screened,the physiological and biochemical characteristics were analyzed and identified;(6) Modeling of nitrogen cycles in horizontal subsurface flow contructed wetlands.The major research findings are as follows:
(1) On the condition that the average hydraulic stayed for 3.85 hours, through Nitrogen removal analysis of horizontal subsurface flow constructed wetlands. The system exhibits certain decontamination ability. The influent COD、NH_4~+-N、NO_3~--N、Org-N and TN are 7.8~12.7mg·L~(-1)、0.05~15.0mg·L~(-1)、1.3~5.2mg·L~(-1)、0.66~5.18mg·L~(-1)、2.2~20.0mg·L~(-1). The annual average removal rate of the gravel bed below the reed wetlands system are 25.8%、10.0%、42%、31.2% and 18.9%.Seasonal variation has a significant effect on the removal rate of the polluted elements. System removal rate has a little decrease in the elements of COD、NH_4~+-N、NO_3~--N、Org-N and TN in cold season. Removal rate of every elements are about 11.2%、6.5%、31.0%、20.5% and 15%.
(2) Seasonal variation has a significant effect on the factors of temperature and pollutionload rate. If the influent temperature changes from 0.9℃to 24.8℃, a positive corelation is found between the removal rate of various forms of Nitrogen elements’volume and the temperature. The constants K_v of NH_4~+-N、NO_3~--N、Org-N and TN are 0.01~0.2d~(-1)、0.03~0.3d~(-1)、0.07~0.3d~(-1)and 0.02~0.15d~(-1). Meanwhile, the volume removal rate constants K_(v20) are 0.0769、0.1673、0.1508 and 0.0732 d~(-1) when the temperature is 20℃.It interprets that the wetlands have stronger capability to reduce NO_3~--N and Org-N than NH_4~+-N. The removal rate of NH_4~+-N and Org-N are logarithms related to load rate. When the load rate of NH_4~+-N is 15gN·m~(-2)·d~(-1) , its removal rate of 1.02 gN·m~(-2)·d~(-1) is the maximum. At the same time, when the load rate of Org-N is 3.2 gN·m~(-2)·d~(-1) , its removal rate of 0.36 gN·m~(-2)·d~(-1) is the maximum.
(3) Five major factors were established by analyzing principal factors in five outlet water has a negative correlation with water temperature. The 2nd and 3rd have a positive correlation with wetland fill NO_3~--N and NH_4~+-N load. The 4th and 5th factors have closest relations and negative correlation with influent DO and ORP. The sort order of the major factors in the sixteen indicators are: temperature> NO_3~--N load > NH_4~+-N load > DO > ORP. The sequence is slightly different from the five outlet indicators.As an independent eco-system, the effect factors in artificial marshland are more complex and unceratin ; however, temperature is still the major element among all elements.
(4) Having compared the nitrification/denitrification strength and respiration intensity of rhizosphere and non- rhizosphere, the researcher finds rhizosphere worked significantly better than non- rhizosphere. The study on denitrification groups such as ammonifying bacteria, nitrite bacteria, nitrifying bacteria and denitrifying bacteria shows the order of magnitude of ammoniation bacteria is 10~8~ 10~(10)cfu and its distribution regularity change is not found. And it did not necessarily linked wetlands spatial position (length); When the order of magnitude of nitrification bacteria is 10~3~10~7MPN, Bacterial nitrification is reducing at the wetland fill end, especially in warm days. The distribution did not significantly change when the magnitude of nitrifying bacteria is 10~5 ~ 10~7MPN.In warm season, the number of nitrifying bacteria is at the front end and the middle is higher than the tail end. The tail end is slightly higher than the front end of wetlands when the number of denitrifying bacteria level is in 10~4~10~7MPN.
(5) The use of PCR-DGGE technique reflects the real level of the microbial population structure and distribution in subsurface flow constructed wetland system. Microbial diversity in horizontal subsurface flow constructed wetland decreases along the waterflow direction first. Through the correlation analysis between frictional COD in the wetland system and the Shannon index, we found frictional COD change and Shannon index correlated well in the reed-gravel system , and different spatial distribution of microbial diversity have different effectiveness of disposing organic matter in different seasons.
(6) Nine selected denitrifying bacteria showed some ability of denitrification, and the denitrification reation occurs mainly in the logarithmic phase of cell growth, and the reaction rate speeds up gradually with the biomass increases. The comparison of denitrification capacity shows that bacterial strain DF2 and DF3 have the highest deni trification efficiency; they can remove 90% TN in 72h, and the final removal rate remains at about 95%. We focus on the removal characteristics of strain DF2 by using 16SrDNA to identify strain DF2; compared with reference strains,we determined the phylogenetic position of the strain, and the results show that the strain belongs to the Dexter's genus, no similar reports were available until now.
(7) Nitrification and denitrification in horizontal subsurface flow constructed wetland are the main methods to transform nitrogen, the rates respectively reach 0.791gN·m~(-2)·d~(-1) and 0.823gN·m~(-2)·d~(-1); Secondly, plants’absorption of nitrogen is 0.747gN·m~(-2)·d~(-1); precipitation and regeneration rates have reached 0.514gN·m~(-2)·d~(-1) and 0.446gN·m~(-2)·d~(-1); other contributory means are plant-corruption 0.282gN·m~(-2)·d~(-1) and mineralization 0.0126 gN·m~(-2)·d~(-1). The main influencing factors in conversion and removal of nitrogen in the system in decreasing order are denitrification, plant intake and sedimentation. Denitrification takes up 60.7% of the total nitrogen removal, plant uptake accounts for 34.3%, and 5.0% in precipitation, the modelachieved certain accuracy and rationality in predicting the results.
(1)在平均水力停留时间3.85h的条件下,通过对水平潜流人工湿地氮素去除效率分析,水平潜流人工湿地系统表现出一定的除污能力,进水COD、NH_4~+-N、NO_3~--N、Org-N和TN浓度为分别7.8~12.7mg·L~(-1)、0.05~15.0mg·L~(-1)、1.3~5.2mg·L~(-1)、0.66~5.18mg·L~(-1)、2.2~20.0mg·L~(-1),芦苇砾石床系统的年平均去除率为25.8%、10.0%、42%、31.2%和18.9%。季节变化对污染组分的去除效率影响非常显著,在寒季系统对COD、NH_4~+-N、NO_3~--N、Org-N和TN的去除效率有所下降,各组分的平均去除率约为11.2%、6.5%、31.0%、20.5%和15%左右。
(2)季节变化对温度和污染负荷率两个因子的影响显著。进水温度在0.9~24.8℃之间变化时,各形态氮素的体积去除速率常数K_v与温度呈正相关,NH_4~+-N、NO_3~--N、Org-N和TN的K_v值分别为0.01~0.2d~(-1)、0.03~0.3d~(-1)、0.07~0.3d~(-1)和0.02~0.15d~(-1);温度在20℃时的体积去除速率常数K_(v20)分别为0.0769、0.1673、0.1508和0.0732 d~(-1),说明该湿地系统对NO_3~--N、Org-N的消减速率较NH_4~+-N大。NH_4~+-N和Org-N的去除速率与负荷率呈对数正相关,当NH_4~+-N的负荷率为15gN·m~(-2)·d~(-1)时,其最大去除速率为1.02gN·m~(-2)·d~(-1);当Org-N的负荷率为3.2gN·m~(-2)·d~(-1)时,其最大去除速率可达到0.36gN·m~(-2)·d~(-1)。
(3)通过对16项进出水理化指标的主因素分析,确立了5个主因素,主因素1与进出水温度呈负相关,关系相对最为密切;主因素2和3分别与湿地进水NO_3~--N和NH_4~+-N负荷呈正相关;主因素4和5分别与进水DO和ORP关系最为密切,且均呈负相关。在16项进出水指标的分析中主因素的排列顺序是:温度>NO_3~--N负荷>NH_4~+-N负荷>DO>ORP,与5项出水指标主因素分析排列顺序略有不同,其主要原因是人工湿地作为一个独立的生态系统,影响因素比较复杂、具有不确定性,但温度作为主要的影响因素地位明显高于其它因素。
(4)比较了根际与非根际的硝化/反硝化强度及呼吸作用强度,研究发现根际效应显著强于非根际。通过对氨化细菌、亚硝化细菌、硝化细菌及反硝化细菌等四种脱氮种群的研究发现,氨化细菌的数量级在10~8~10~(10)cfu之间,其沿湿地空间位置(长度)数量分布无规律性变化;亚硝化细菌的数量级在10~3~10~7MPN之间,从湿地进水端至尾端亚硝化细菌的数量逐渐减少,尤其在暖季变化显著;硝化细菌的数量级在10~5~10~7MPN之间,其分布无显著变化,暖季时硝化细菌的数量是前端、中部高于尾端;反硝化细菌的数量级在10~4~10~7MPN之间,湿地系统尾端数量略高于前端。
(5)运用PCR-DGGE技术,比较真实的反应了水平潜流人工湿地系统中微生物种群结构及分布特征。水平潜流人工湿地中微生物种群多样性随着水流方向呈现先逐渐减少的趋势。通过湿地系统沿程COD与Shannon指数相关性分析,发现芦苇砾石系统沿程COD变化与Shannon指数相关性较好,不同季节湿地系统中微生物多样性的空间分布特征对处理有机物效能存在差异。对氨氧化菌群落结构多样性的研究表明:不同季节水平潜流人工湿地中氨氧化菌的时空变化显著,并分析得出了湿地系统的优势氨氧化菌种,可为系统氨氮去除途径的分析提供微生物学标识。
(6)筛选出9株反硝化细菌均具有一定的脱氮能力,脱氮反应主要发生在菌体生长的对数生长期,脱氮速率随着生物量的增加逐渐加快。脱氮能力对比结果显示菌株DF2和DF3脱氮效率最高,均能在72h内将90%的TN去除,最终脱氮率维持在95%左右。特别重点考察了菌株DF2的脱氮特性,利用16SrDNA技术对菌株DF2进行了鉴定,通过与参比菌株的比较,确定了该菌株的系统发育地位,结果表明该菌株位于德克斯氏菌属,至今未发现类似的报道。
(7)水平潜流人工湿地系统硝化与反硝化作用是氮元素转化的主要途径之一,其速率分别达到了0.791gN·m~(-2)·d~(-1)和0.823gN·m~(-2)·d~(-1);其次植物对氮的吸收0.747 gN·m~(-2)·d~(-1);沉淀和再生速率也分别达到了0.514gN·m~(-2)·d~(-1)和0.446gN·m~(-2)·d~(-1);其它贡献的途径如植物腐败0.282gN·m~(-2)·d~(-1)和矿化0.0126gN·m~(-2)·d~(-1)。在整个系统氮转化和去除的主要途径递减顺序为反硝化、植物吸收、沉淀。其中,反硝化作用占总氮去除的60.7%、植物吸收占34.3%、沉淀作用占5.0%,模型在预测结果方面具有一定的准确性和合理性。
The performance and mechanisms of horizontal subsurface flow contructed wetlands reed/gravel bed system of Guanting Reservoir were studied for the preventive treatment of the pollution of potable water sources. The study includes:(1) The Nitrogen removal rate and main factor analysis of horizontal subsurface flow constructed wetlands;(2) The forms distribution and transformation rule;(3) The analysis of the frictional distribution and population of Nitrogen transformation microbes in the system;(4) The microbial diversity research in the system;(5) The featured denitrifiers were screened,the physiological and biochemical characteristics were analyzed and identified;(6) Modeling of nitrogen cycles in horizontal subsurface flow contructed wetlands.The major research findings are as follows:
(1) On the condition that the average hydraulic stayed for 3.85 hours, through Nitrogen removal analysis of horizontal subsurface flow constructed wetlands. The system exhibits certain decontamination ability. The influent COD、NH_4~+-N、NO_3~--N、Org-N and TN are 7.8~12.7mg·L~(-1)、0.05~15.0mg·L~(-1)、1.3~5.2mg·L~(-1)、0.66~5.18mg·L~(-1)、2.2~20.0mg·L~(-1). The annual average removal rate of the gravel bed below the reed wetlands system are 25.8%、10.0%、42%、31.2% and 18.9%.Seasonal variation has a significant effect on the removal rate of the polluted elements. System removal rate has a little decrease in the elements of COD、NH_4~+-N、NO_3~--N、Org-N and TN in cold season. Removal rate of every elements are about 11.2%、6.5%、31.0%、20.5% and 15%.
(2) Seasonal variation has a significant effect on the factors of temperature and pollutionload rate. If the influent temperature changes from 0.9℃to 24.8℃, a positive corelation is found between the removal rate of various forms of Nitrogen elements’volume and the temperature. The constants K_v of NH_4~+-N、NO_3~--N、Org-N and TN are 0.01~0.2d~(-1)、0.03~0.3d~(-1)、0.07~0.3d~(-1)and 0.02~0.15d~(-1). Meanwhile, the volume removal rate constants K_(v20) are 0.0769、0.1673、0.1508 and 0.0732 d~(-1) when the temperature is 20℃.It interprets that the wetlands have stronger capability to reduce NO_3~--N and Org-N than NH_4~+-N. The removal rate of NH_4~+-N and Org-N are logarithms related to load rate. When the load rate of NH_4~+-N is 15gN·m~(-2)·d~(-1) , its removal rate of 1.02 gN·m~(-2)·d~(-1) is the maximum. At the same time, when the load rate of Org-N is 3.2 gN·m~(-2)·d~(-1) , its removal rate of 0.36 gN·m~(-2)·d~(-1) is the maximum.
(3) Five major factors were established by analyzing principal factors in five outlet water has a negative correlation with water temperature. The 2nd and 3rd have a positive correlation with wetland fill NO_3~--N and NH_4~+-N load. The 4th and 5th factors have closest relations and negative correlation with influent DO and ORP. The sort order of the major factors in the sixteen indicators are: temperature> NO_3~--N load > NH_4~+-N load > DO > ORP. The sequence is slightly different from the five outlet indicators.As an independent eco-system, the effect factors in artificial marshland are more complex and unceratin ; however, temperature is still the major element among all elements.
(4) Having compared the nitrification/denitrification strength and respiration intensity of rhizosphere and non- rhizosphere, the researcher finds rhizosphere worked significantly better than non- rhizosphere. The study on denitrification groups such as ammonifying bacteria, nitrite bacteria, nitrifying bacteria and denitrifying bacteria shows the order of magnitude of ammoniation bacteria is 10~8~ 10~(10)cfu and its distribution regularity change is not found. And it did not necessarily linked wetlands spatial position (length); When the order of magnitude of nitrification bacteria is 10~3~10~7MPN, Bacterial nitrification is reducing at the wetland fill end, especially in warm days. The distribution did not significantly change when the magnitude of nitrifying bacteria is 10~5 ~ 10~7MPN.In warm season, the number of nitrifying bacteria is at the front end and the middle is higher than the tail end. The tail end is slightly higher than the front end of wetlands when the number of denitrifying bacteria level is in 10~4~10~7MPN.
(5) The use of PCR-DGGE technique reflects the real level of the microbial population structure and distribution in subsurface flow constructed wetland system. Microbial diversity in horizontal subsurface flow constructed wetland decreases along the waterflow direction first. Through the correlation analysis between frictional COD in the wetland system and the Shannon index, we found frictional COD change and Shannon index correlated well in the reed-gravel system , and different spatial distribution of microbial diversity have different effectiveness of disposing organic matter in different seasons.
(6) Nine selected denitrifying bacteria showed some ability of denitrification, and the denitrification reation occurs mainly in the logarithmic phase of cell growth, and the reaction rate speeds up gradually with the biomass increases. The comparison of denitrification capacity shows that bacterial strain DF2 and DF3 have the highest deni trification efficiency; they can remove 90% TN in 72h, and the final removal rate remains at about 95%. We focus on the removal characteristics of strain DF2 by using 16SrDNA to identify strain DF2; compared with reference strains,we determined the phylogenetic position of the strain, and the results show that the strain belongs to the Dexter's genus, no similar reports were available until now.
(7) Nitrification and denitrification in horizontal subsurface flow constructed wetland are the main methods to transform nitrogen, the rates respectively reach 0.791gN·m~(-2)·d~(-1) and 0.823gN·m~(-2)·d~(-1); Secondly, plants’absorption of nitrogen is 0.747gN·m~(-2)·d~(-1); precipitation and regeneration rates have reached 0.514gN·m~(-2)·d~(-1) and 0.446gN·m~(-2)·d~(-1); other contributory means are plant-corruption 0.282gN·m~(-2)·d~(-1) and mineralization 0.0126 gN·m~(-2)·d~(-1). The main influencing factors in conversion and removal of nitrogen in the system in decreasing order are denitrification, plant intake and sedimentation. Denitrification takes up 60.7% of the total nitrogen removal, plant uptake accounts for 34.3%, and 5.0% in precipitation, the modelachieved certain accuracy and rationality in predicting the results.
引文
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