复合膜生物反应器地下水脱氮技术研究
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摘要
地下水是我国华北地区重要的饮用水水源,特别是华北农村生活饮用水几乎全部来自地下水。然而,由于不合理的使用化肥、污水灌溉及污水渗漏等原因,地下水受到了不同程度的硝酸盐污染,并存在日益恶化的趋势。饮用硝酸盐含量超标的地下水容易导致高铁血红蛋白症,三个月以下的婴儿受此危害最大；此外,亚硝酸盐还能使人致癌。
去除地下水中硝酸盐的方法主要有离子交换、反渗透和生物反硝化脱氮等。虽然离子交换和反渗透脱除硝酸盐的效果很好,但会附产再生废液或浓缩液,需要以较高的费用作进一步处置。生物反硝化是脱除地下水中硝酸盐最经济有效的方法,然而以制取饮用水为目的的地下水常规异养反硝化工艺存在两个弊端：(1)反硝化过程中投加的有机碳源会有部分残留在出水中；(2)反硝化菌会引起出水的微生物污染。
本课题将细胞固定化技术与膜分离技术有机地结合在一起,研究开发了三种复合膜生物反应器,分别为两室、三室以及多室复合膜生物反应器。在复合膜生物反应器中,微孔滤膜和膜状固定化细胞构成的复合膜把地下水(简称水室)和反硝化碳源(简称碳室)分隔开,硝酸盐和乙醇分别从水室和碳室扩散进入固定化细胞中,其中的反硝化菌利用乙醇作为电子供体,将硝酸盐还原为氮气。一方面平板状固定化细胞把要处理的地下水与反硝化所需的有机碳源分隔开,可以避免出水被反硝化残留碳源所污染；另一方面覆盖在固定化细胞表面的微孔滤膜,可以防止从固定化细胞中脱落下来的细菌对出水造成微生物污染。
本研究首先将两室复合膜生物反应器(DCMBR)用于地下水反硝化脱氮,初步探索了膜分离技术与细胞固定化技术相结合进行地下水反硝化脱氮的可行性及复合膜生物反应器控制出水中有机物污染的效果。然后,通过正交实验及单因素实验对固定化细胞的制备条件及反硝化脱氮的主要影响因素进行优化。结果表明,复合膜生物反应器中平板状固定化细胞适宜的制备条件是厚度为4mm,15%PVA溶液中细菌包埋量为2%(湿重,W/V)；地下水反硝化脱氮适宜的碳氮比、温度和pH值分别为6：1、30℃、7-8；适宜的反硝化碳源是乙醇；复合膜生物反应器脱氮速率与N03-N初始浓度成正比,地下水中的溶解氧对固定化细胞反硝化脱氮的影响比对游离细胞小。
然后在两室复合膜生物反应器研究的基础上,开发了更加高效的三室复合膜生物反应器(TCMBR)及多室复合膜生物反应器(MCMBR),并对其反硝化特性进行研究。TCMBR的研究结果表明,相同初始硝酸盐浓度下脱氮速率低于DCMBR,但单位容积的产水率却比DCMBR高。对于MCMBR,进水N03-N浓度在100 mg.L1以内时,进水最大负荷可达4.97 gN.m2.d-1,MCMBR连续运行6个多月,脱氮活性没有下降,显示了长期运行的稳定性。出水中的N03--N、NO2--N、CODMn以及主要生物学指标均符合生活饮用水卫生标准。
扩散研究表明固定化细胞对扩散会产生显著的阻碍作用,硝酸盐及乙醇在空白PVA膜中的扩散系数分别为0.707×10-9m2.s-1和0.640×10-9m2.s-1,分别为其在纯水中扩散系数的37.2%和58.2%。同时,包埋的微生物量、冷冻一解冻次数及PVA溶液浓度的增加均会不同程度地增大扩散阻力,微孔滤膜的使用也会使扩散阻力增大,进而使扩散系数减小。PVA浓度为15%、微生物包埋量为20 g.L-1时制备的厚度约为4mm的固定化细胞用于反硝化脱氮时,反硝化脱氮过程主要受内扩散控制。
对处理后出水中有机物的分子量分布及荧光特性进行分析,结果显示出水中主要以小于1000Dalton的小分子物质为主,且主要为腐殖酸类物质。处理后出水无生物毒性。
为了揭示复合膜生物反应器反硝化过程中微生物的多样性,从接种污泥、驯化污泥、固定化细胞膜生物反应器连续运行2个月和6个月的四个不同阶段提取污泥样品,通过提取总DNA,进行PCR-DGGE分析。微生物群落的DGGE指纹图谱分析表明,接种污泥与不同阶段的固定化细胞中既存在着共同的微生物种属也有各自特异的种属。克隆得到10个优势条带,测序和系统发育分析表明,其中两株分别属于Diaphorobacter属和Bacillus属,具有反硝化功能,而其它8株菌则基本可以判定为新菌种；相同的微生物种群在不同阶段中的优势地位不同,各自特有的微生物群落是在培养驯化过程中逐步演替而来,整体的变化趋势是微生物多样性趋向单一化。
Groundwater is the most important source of drinking water in northern China; in some rural areas, it is the only readily available source of drinking water. As a result of excessive use of urea and/or other nitrogen fertilizers, wastewater irrigation and leakage, nitrate contamination of the groundwater has become a common issue. Once consumed, nitrate may be converted to nitrite which is responsible for methemoglobinemia (the blue baby syndrome) in infants and other health problems including cancer.
The high NO3--N concentration can be reduced by ion exchange (Ⅸ), reverse osmosis (RO) and biological denitrification. AlthoughⅨor RO treatment may be effective, it however will result in a concentrate stream of waste by-product that requires further treatment or final disposal at a high cost. Biological denitrification is the most cost-effective process for nitrate removal in groundwater. However, the unresolved issues of poor retention of both the microbial biomass and the electron donor have delayed their full-scale applications.
To overcome such stated disadvantages of the groundwater denitrification processes, research was conducted in our lab to develop three types of composite membrane bioreactors which integrates immobilized cells technique with membrane separation technology for groundwater denitrification, including double-compartments composite membrane bioreactor (DCMBR), three-compartments composite membrane bioreactor (TCMBR) and multiple-compartments composite membrane bioreactor (MCMBR). The groundwater compartment (water compartment for short) and carbon source compartment (carbon compartment for short) were separated by the composite membrane consisting of a microporous membrane facing the influent and an immobilized cells membrane facing the ethanol solution. Molecules of nitrate and ethanol diffused from the respective compartments into the immobilized cells membrane where nitrate was reduced to gaseous nitrogen (N2) by the denitrifying bacteria present there with ethanol as carbon source. On the one hand, the tabulate immobilized cells can prevent product water contaminated by carbon source through separating groundwater and carbon source; On the other hand, the microporous membrane attached to one side of immobilized cells to separate product water from an immobilized cells or to provide effective retention of the biomass.
The main objectives of this research were therefore to investigate the possibility of DCMBR for groundwater denitrification and the efficiency of controlling contamination of the product water by added organic carbon source. The optimal preparation conditions of immobilized cells and denitrification conditions were obtained by orthogonal and single factor experiments, respectively. The results showed that the optimum conditions of immobilized cells preparation were 4 mm thickness, embedding denitrifying bacteria in 15% PVA solution was 20g (wet weight per litter.; The optimum carbon to nitrogen ratio, temperature and pH were 6:1,30℃and 7-8, respectively. And ethanol was a proper carbon source.The denitrification rate of DCMBR was proportional to the initial concentration of NO3--N. The effect of dissolved oxygen in groundwater on the denitrification of immobilized cells was little than that of free cells.
The three-compartments composite membrane bioreactor (TCMBR) and multiple-compartments composite membrane bioreactor (MCMBR) were developed and their characteristics of denitrification for groundwater were investigated. The results showed that the denitrification rate of TCMBR was low than that of DCMBR, but the producing water quantity per reactor volume was higher than that of DCMBR. For the MCMBR, the maximum influent loading reached up to 4.97 gN·m2·d-1 as the influent nitrate nitrogen concentration was lower than 100 mg·L-1. The MCMBR demonstrated the excellent operational stability without decreasing of denitrificaton activity after running over 6 months. The quality of the product water was fit to standards for drinking water quality.
The study showed that the immobilized cells could obviously block the diffusion process. The diffusion coefficients of nitrate and ethanol were 0.707×10-9m2·s-1 and 0.640×10-9m2·s-1 in PVA gel without bacteria respectively, which were 37.2% and 58.2% of their diffusion coefficients in the pure water. And embedding cells in PVA gel, number of freezing and thawing, PVA solution concentration and use of microporous membrane would affect the diffusion coefficient. The groundwater denitrification rate was controlled by the internal mass transfer as the immobilized cells was prepared with 15% PVA contain 20 g·L-1 bacteria, and the immobilized cells was 4 mm thickness.
The determination of molecular weight distribution and fluorescence properties of the dissolved organic matter (DOM) in product water showed that the small moleculars below 1000 Da were the main part in the product water using fluorescence excitation-matrix(EEM) spectroscopy. Most of the small molecular were humus without biotoxicity in product water.
In order to investigate the microbial diversity during denitrification process of MCMBR, four microbial samples were taken to total microbial DNA extraction and polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis from the initial inoculation sludge, acclimation sludge and immobilized cells running for two and six months. The DGGE profile analysis showed that the same and different species existed in inoculation sludge and immobilized cells in different stage. Sequencing and phylogenetic analysis of 10 dominant bands in the DGGE profile indicated that 2 of strains belonged to Diaphorobacter sp. and Bacillus sp. with function of denitrification and other 8 strains were new strains. The dominant status of same microbial species in different stage was different and special microbial community was due to the succession of long term. The total change trend of microbial diversity tended to simplifying.
去除地下水中硝酸盐的方法主要有离子交换、反渗透和生物反硝化脱氮等。虽然离子交换和反渗透脱除硝酸盐的效果很好,但会附产再生废液或浓缩液,需要以较高的费用作进一步处置。生物反硝化是脱除地下水中硝酸盐最经济有效的方法,然而以制取饮用水为目的的地下水常规异养反硝化工艺存在两个弊端：(1)反硝化过程中投加的有机碳源会有部分残留在出水中；(2)反硝化菌会引起出水的微生物污染。
本课题将细胞固定化技术与膜分离技术有机地结合在一起,研究开发了三种复合膜生物反应器,分别为两室、三室以及多室复合膜生物反应器。在复合膜生物反应器中,微孔滤膜和膜状固定化细胞构成的复合膜把地下水(简称水室)和反硝化碳源(简称碳室)分隔开,硝酸盐和乙醇分别从水室和碳室扩散进入固定化细胞中,其中的反硝化菌利用乙醇作为电子供体,将硝酸盐还原为氮气。一方面平板状固定化细胞把要处理的地下水与反硝化所需的有机碳源分隔开,可以避免出水被反硝化残留碳源所污染；另一方面覆盖在固定化细胞表面的微孔滤膜,可以防止从固定化细胞中脱落下来的细菌对出水造成微生物污染。
本研究首先将两室复合膜生物反应器(DCMBR)用于地下水反硝化脱氮,初步探索了膜分离技术与细胞固定化技术相结合进行地下水反硝化脱氮的可行性及复合膜生物反应器控制出水中有机物污染的效果。然后,通过正交实验及单因素实验对固定化细胞的制备条件及反硝化脱氮的主要影响因素进行优化。结果表明,复合膜生物反应器中平板状固定化细胞适宜的制备条件是厚度为4mm,15%PVA溶液中细菌包埋量为2%(湿重,W/V)；地下水反硝化脱氮适宜的碳氮比、温度和pH值分别为6：1、30℃、7-8；适宜的反硝化碳源是乙醇；复合膜生物反应器脱氮速率与N03-N初始浓度成正比,地下水中的溶解氧对固定化细胞反硝化脱氮的影响比对游离细胞小。
然后在两室复合膜生物反应器研究的基础上,开发了更加高效的三室复合膜生物反应器(TCMBR)及多室复合膜生物反应器(MCMBR),并对其反硝化特性进行研究。TCMBR的研究结果表明,相同初始硝酸盐浓度下脱氮速率低于DCMBR,但单位容积的产水率却比DCMBR高。对于MCMBR,进水N03-N浓度在100 mg.L1以内时,进水最大负荷可达4.97 gN.m2.d-1,MCMBR连续运行6个多月,脱氮活性没有下降,显示了长期运行的稳定性。出水中的N03--N、NO2--N、CODMn以及主要生物学指标均符合生活饮用水卫生标准。
扩散研究表明固定化细胞对扩散会产生显著的阻碍作用,硝酸盐及乙醇在空白PVA膜中的扩散系数分别为0.707×10-9m2.s-1和0.640×10-9m2.s-1,分别为其在纯水中扩散系数的37.2%和58.2%。同时,包埋的微生物量、冷冻一解冻次数及PVA溶液浓度的增加均会不同程度地增大扩散阻力,微孔滤膜的使用也会使扩散阻力增大,进而使扩散系数减小。PVA浓度为15%、微生物包埋量为20 g.L-1时制备的厚度约为4mm的固定化细胞用于反硝化脱氮时,反硝化脱氮过程主要受内扩散控制。
对处理后出水中有机物的分子量分布及荧光特性进行分析,结果显示出水中主要以小于1000Dalton的小分子物质为主,且主要为腐殖酸类物质。处理后出水无生物毒性。
为了揭示复合膜生物反应器反硝化过程中微生物的多样性,从接种污泥、驯化污泥、固定化细胞膜生物反应器连续运行2个月和6个月的四个不同阶段提取污泥样品,通过提取总DNA,进行PCR-DGGE分析。微生物群落的DGGE指纹图谱分析表明,接种污泥与不同阶段的固定化细胞中既存在着共同的微生物种属也有各自特异的种属。克隆得到10个优势条带,测序和系统发育分析表明,其中两株分别属于Diaphorobacter属和Bacillus属,具有反硝化功能,而其它8株菌则基本可以判定为新菌种；相同的微生物种群在不同阶段中的优势地位不同,各自特有的微生物群落是在培养驯化过程中逐步演替而来,整体的变化趋势是微生物多样性趋向单一化。
Groundwater is the most important source of drinking water in northern China; in some rural areas, it is the only readily available source of drinking water. As a result of excessive use of urea and/or other nitrogen fertilizers, wastewater irrigation and leakage, nitrate contamination of the groundwater has become a common issue. Once consumed, nitrate may be converted to nitrite which is responsible for methemoglobinemia (the blue baby syndrome) in infants and other health problems including cancer.
The high NO3--N concentration can be reduced by ion exchange (Ⅸ), reverse osmosis (RO) and biological denitrification. AlthoughⅨor RO treatment may be effective, it however will result in a concentrate stream of waste by-product that requires further treatment or final disposal at a high cost. Biological denitrification is the most cost-effective process for nitrate removal in groundwater. However, the unresolved issues of poor retention of both the microbial biomass and the electron donor have delayed their full-scale applications.
To overcome such stated disadvantages of the groundwater denitrification processes, research was conducted in our lab to develop three types of composite membrane bioreactors which integrates immobilized cells technique with membrane separation technology for groundwater denitrification, including double-compartments composite membrane bioreactor (DCMBR), three-compartments composite membrane bioreactor (TCMBR) and multiple-compartments composite membrane bioreactor (MCMBR). The groundwater compartment (water compartment for short) and carbon source compartment (carbon compartment for short) were separated by the composite membrane consisting of a microporous membrane facing the influent and an immobilized cells membrane facing the ethanol solution. Molecules of nitrate and ethanol diffused from the respective compartments into the immobilized cells membrane where nitrate was reduced to gaseous nitrogen (N2) by the denitrifying bacteria present there with ethanol as carbon source. On the one hand, the tabulate immobilized cells can prevent product water contaminated by carbon source through separating groundwater and carbon source; On the other hand, the microporous membrane attached to one side of immobilized cells to separate product water from an immobilized cells or to provide effective retention of the biomass.
The main objectives of this research were therefore to investigate the possibility of DCMBR for groundwater denitrification and the efficiency of controlling contamination of the product water by added organic carbon source. The optimal preparation conditions of immobilized cells and denitrification conditions were obtained by orthogonal and single factor experiments, respectively. The results showed that the optimum conditions of immobilized cells preparation were 4 mm thickness, embedding denitrifying bacteria in 15% PVA solution was 20g (wet weight per litter.; The optimum carbon to nitrogen ratio, temperature and pH were 6:1,30℃and 7-8, respectively. And ethanol was a proper carbon source.The denitrification rate of DCMBR was proportional to the initial concentration of NO3--N. The effect of dissolved oxygen in groundwater on the denitrification of immobilized cells was little than that of free cells.
The three-compartments composite membrane bioreactor (TCMBR) and multiple-compartments composite membrane bioreactor (MCMBR) were developed and their characteristics of denitrification for groundwater were investigated. The results showed that the denitrification rate of TCMBR was low than that of DCMBR, but the producing water quantity per reactor volume was higher than that of DCMBR. For the MCMBR, the maximum influent loading reached up to 4.97 gN·m2·d-1 as the influent nitrate nitrogen concentration was lower than 100 mg·L-1. The MCMBR demonstrated the excellent operational stability without decreasing of denitrificaton activity after running over 6 months. The quality of the product water was fit to standards for drinking water quality.
The study showed that the immobilized cells could obviously block the diffusion process. The diffusion coefficients of nitrate and ethanol were 0.707×10-9m2·s-1 and 0.640×10-9m2·s-1 in PVA gel without bacteria respectively, which were 37.2% and 58.2% of their diffusion coefficients in the pure water. And embedding cells in PVA gel, number of freezing and thawing, PVA solution concentration and use of microporous membrane would affect the diffusion coefficient. The groundwater denitrification rate was controlled by the internal mass transfer as the immobilized cells was prepared with 15% PVA contain 20 g·L-1 bacteria, and the immobilized cells was 4 mm thickness.
The determination of molecular weight distribution and fluorescence properties of the dissolved organic matter (DOM) in product water showed that the small moleculars below 1000 Da were the main part in the product water using fluorescence excitation-matrix(EEM) spectroscopy. Most of the small molecular were humus without biotoxicity in product water.
In order to investigate the microbial diversity during denitrification process of MCMBR, four microbial samples were taken to total microbial DNA extraction and polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis from the initial inoculation sludge, acclimation sludge and immobilized cells running for two and six months. The DGGE profile analysis showed that the same and different species existed in inoculation sludge and immobilized cells in different stage. Sequencing and phylogenetic analysis of 10 dominant bands in the DGGE profile indicated that 2 of strains belonged to Diaphorobacter sp. and Bacillus sp. with function of denitrification and other 8 strains were new strains. The dominant status of same microbial species in different stage was different and special microbial community was due to the succession of long term. The total change trend of microbial diversity tended to simplifying.
引文
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