高盐含酚废水生物处理及微生物群落结构研究
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摘要
含酚废水来源广泛、成分复杂,如焦化废水、医药废水、化工及印染废水等,这些废水中既含有高浓度的酚类化合物,又含有大量的盐份(Cl~-、Na~+、Ca~(2+)等),会对微生物的生长产生抑制作用,从而增加生物处理的难度,成为现阶段国内外含酚废水处理技术领域亟待解决的一个难题。为了解决含酚废水中高盐含量对传统生物处理系统的抑制问题,本文以模拟含酚废水为基质,构建既能耐受高盐度、又能高效降解酚类化合物的生物处理体系,并在不同条件下对其降解特性进行详细的分析,同时利用RISA指纹、AFDRA功能基因分析、16S rDNA测序、FISH以及实时荧光定量PCR等分子生物技术对生物处理体系的群落结构进行解析,阐明宏观功能变化与微观结构之间的内在联系,为高盐含酚废水的生物处理提供一定的理论依据和技术支持。
高盐条件下,构建的活性污泥体系能够处理不同组分的含酚废水,其中邻苯二酚和水杨酸存在时对活性污泥体系的总酚去除速率影响较小,而对甲基酚存在时总酚去除速率明显降低。盐度和pH冲击对活性污泥体系降解性能影响较大,盐度在50g/L NaCl以下时去除速率随盐度的增加而增加,但盐度达100 g/L NaCl以上时,去除速率明显下降。进水pH 7.0～9.0范围内活性污泥去除速率比较稳定。同时利用RISA(核糖体基因间隔序列分析)指纹技术,并借助16S rDNA序列分析对活性污泥体系进行群落动态解析,进而揭示系统微观结构与宏观功能之间的内在联系。结果表明,苯酚单独作为基质时,活性污泥体系的微生物群落多样性明显增加,而混合酚作为基质时,系统微生物群落多样性变化较小。盐度冲击过程中活性污泥体系微生物群落结构发生了剧烈的变化,而pH冲击过程中系统微生物群落结构比较稳定。遗传分析表明,盐度冲击过程中盐单胞菌属Halomonas和海洋杆菌属Marinobacter菌株代谢旺盛,说明这两类菌属菌株具有较强的耐盐能力;而pH和底物浓度冲击使海洋杆菌属Marinobacter和粪产碱菌属Alcaligenes faecalis菌株在处理系统中大量生长,逐渐成为优势菌株。在进化分析中还发现一少部分序列归属于希瓦氏菌属Shewanella和节杆菌属Arthrobacter等。
本实验还采用扩增功能DNA限制性分析(AFDRA)的方法对不同条件下活性污泥体系苯酚羟化酶基因的变化进行了分析,与RISA指纹分析相互补充,在基因水平上揭示了活性污泥体系微生物群落结构和功能的变化。研究结果表明,活性污泥体系中降酚菌群的苯酚羟化酶功能基因的多样性因处理系统的底物种类、底物浓度、盐度和pH而有明显差异。苯酚羟化酶基因的变化可以反映生物系统内微生物群落的动态变化,而微生物群落的组成又会直接影响功能基因的多样性。
从活性污泥中分离到三株耐盐苯酚降解菌,经形态观察、生理生化分析以及16SrDNA或26S rDNA分析分别鉴定为Arthrobacter sp.W1、Rodococcus sp.W2和Candidasp.W3,其序列的GenBank登陆序列号分别为EU339930、EU339931和EU349016。考察了3株菌的生长及降解酚类化合物的基本特性,为耐盐酚类降解复合菌体系的构建奠定了基础。3株菌均能在温度20～30℃、pH 5.0～9.0和盐度10～100 g/L NaCl范围内以苯酚为唯一碳源进行生长,能够利用苯甲酸、水杨酸、对甲基酚、氨基酚、对苯二酚等多种芳香化合物。高盐条件下,3株菌能够在细胞内积累相容性物质(主要是四氢嘧啶和甜菜碱)抵御外界高盐环境,并且相容性物质的含量随盐度升高而增加,在培养基中外加相容性物质能够促进菌株的生长,并且增强菌株的耐盐能力。
通过正交实验确定菌株W1、W2和W3以1:1:2比例混合构建高效的耐盐酚类降解复合菌群,该复合菌与3株单菌相比,对环境的适应能力更强,能在温度10～40℃、pH4.0～12.0和盐度10～150 g/L NaCl范围内以苯酚为唯一碳源进行生长,在最佳降解条件下140 h即可完全降解2000 mg/L苯酚,降解酚类化合物范围更广。模拟SBR工艺对不同条件下复合菌群处理高盐混合酚废水进行了研究,结果表明,在3个月运行期内,复合菌群处理效果稳定,能够耐受一定范围的盐度(10～150 g/L NaCl)、pH(5.0～12.0)和底物浓度(200～1500 mg/L)的冲击。与活性污泥体系相比,复合菌体系抗冲击能力更强,去除速率更高。
采用荧光原位杂交技术(HSH)对复合菌群内W1、W2和W3菌株的变化进行分析,结果表明,复合菌群中的W1菌株具有较强的抗盐度和底物浓度冲击的能力,而3株菌对pH冲击均有较好的耐受性。进一步采用实时荧光定量PCR技术对复合菌群中的优势菌株W1进行定量分析,结果表明,NaCl浓度由10 g/L增加到100 g/LNaCl时,细胞数量由1.16×10~(13)增加到5.71×10×~(13)细胞/每克干重;底物浓度冲击过程中细胞数量变化与盐度冲击过程细胞数量呈现相同的变化趋势,细胞数量在1.47×10~(13)和5.53×10~(13)细胞/每克干重之间变化;而在pH冲击条件下,整个过程中细胞数量基本保持在一个稳定水平。由此推断,W1菌株可能是复合菌群处理高盐混合酚废水的重要的功能菌,在环境条件波动时稳定存在,并且保持较高的代谢活性。
The source of phenolic wastewater is extensive and the compositions are complicated, such as coking,pharmaceutical,chemical and dying wastewatwer.The existence of high concentration of phenolics and salts in wastewater will inhibit the growth of the microorganism and brings more difficulty in biological treatment,which has become a difficult problem in domestic and international wastewater treatment field and is urgent to be solved currently.To solve the problem of inhibitory effect of high salt on traditional biological treatment system,the objectives of this paper are to explore of salt-tolerant biological treatment systems,which are also capable of degrading phenolics,study on the characteristics of treatment systems under different conditions and analyze the community structure during treatment process by molecular biology technology,such as RISA(Ribosomal intergenic sequence analysis),AFDRA(Amplified functional DNA restriction analysis) and 16S rDNA sequencing analysis.The experimental results will clarify the relationship between the microstructure and macrofunction of treatment system and provide a theoretical and technical supports for the biological treatment of phenolic hyper-saline wastewater.
The activated sludge could degrade different phenolic mixtures under high salt conditions. Inhibitory effect of catchol and salicylate on phenol degradation was not obvious,but the removal rate was decreased in the presence of p-cresol.Salinity and pH fluctuation would affact the removal effeciency of activated sludge.When the content of NaCl was lower than 50 g/L, the removal rate of phenolics was increased with the NaCl concentration increasing,but decreased in the presence of more than 100 g/L NaCl.The efficiency of degradation was stable under neutral conditions(pH 7.0-9.0).At the same time,the microbial community structure of activated sludge was analyzed by RISA and 16S rDNA sequencing to clarify the relationship between the microstructure and macrofunction of treatment system.The RISA patterns revealed that the diversity of microorganism was increased in single substrate system,but was little change in binary substrates system.The community structure was drastically change during salt fluctuation process,but remained stable during pH fluctuation.16S rDNA sequence analysis showed that Halomonas sp.and Marinobactergenus sp.were predominant species during salt fluctuation process,which have better ability of salt tolerant.Marinobacter sp.and Alcaligenes faecalis sp.were the main species during the pH and substrate concentration fluctuation process.It was also found that Shewanella sp.and Arthrobacter sp.were existence in treatment system.
The changes of phenol hydroxylase gone of activated sludge was analyzed by AFDRA under different conditions,which revealed the change of microbial community structure and function of activated sludge in molecular level.AFDRA on phenol hydroxylase genes show that the diversity of phenol hydroxylase genes was substantially different in various conditions, such as different substrate,concentration,salinity and pH,and the changes are consistent to that of microbial community.Thus,it was concluded that the changes of functional genes could reflect the dynamics of microbial community,and the microbial community structure could affect the diversity of functional genes.
Arthrobacter sp.W1,Rodococcus sp.W2 and Candida sp.W3 was isolated from the activated sludge and could utilize phenol as sole carbon source under high salt conditions, which was identified on the basis of physiological and biochemical tests and 16S rDNA and 26S rDNA sequence analysis.The sequence of strains were submitted to GenBank with the number EU339930,EU339931 and EU349016.The degradation and growth conditions of three strains were as follows:temperature 20-30℃,pH 5.0-9.0 and salinity 10-100 g/L NaCl. They could utilize benzoic,salicylate,p-cresol,aminophenol and hydroquinone as the carbone source.Under high salt conditions,strains were mainly accumulated compatible solutes(ectoine and betaine) in cells to resist the extracellular hyper-saline environment. Adding compatible solutes such as ectoine,betaine and glutamic acid could improve the growth of strains under high salt conditions and enhance the salt-tolerance ability.
A defined microbial consortium was constructed through orthogonal experiments by using of strain W1,W2 and W3.The adamptation conditons for the phenol degradation by the consortium were as followed:temperature 10-40℃,pH 4.0-12.0,salinity 10-150 g/L NaCl. Compared to other three pure strains,the consortium could degrade phenol under severe conditions and had a wider substrates range.During degradation of mixed phenolics by the consortium in SBR,the treatment efficiency was stable under different conditions in 3 monthes.Compared with the activated sludge system,the consortium showed higher removal efficiency and resistant ability of environmental fluctuation more powerful.
Fluorescence in situ hybridization(FISH) analysis indicates that the abundance and intesnsity of specific probe of Arthrobacter sp.W1 was more than other two strains when salinity and substrate concentration fluctuation.During pH fluctuation process,there was lilltle differences of abundance and intensity of specific probe among three strains.It means that Arthrobacter sp.W1 has resistant ability of salinity and substrate concentration fluctuation more powerful,and three strains are insensitive to pH fluctuation.The results of real-time quantitative PCR showed that the cell numbers of Arthrobacter sp.W1 was increased fromel.16×10~(13) to 5.71×10~(13) cell per g of dry weight when NaCl concentration raised from 10 to 100 g/L.The cell number was change between 1.47×10~(13) to 5.53×10~(13) cell per g of dry weight when substrated concentration fluctuation process,but the cell numbers always remained stable during pH fluctuation process.Thus,it was concluded that W1 was the most important microorganism of the consortium during degradation phenolic hyper-saline wastewater process,which maintained higher metabolic activity under fluctuate conditions.
高盐条件下,构建的活性污泥体系能够处理不同组分的含酚废水,其中邻苯二酚和水杨酸存在时对活性污泥体系的总酚去除速率影响较小,而对甲基酚存在时总酚去除速率明显降低。盐度和pH冲击对活性污泥体系降解性能影响较大,盐度在50g/L NaCl以下时去除速率随盐度的增加而增加,但盐度达100 g/L NaCl以上时,去除速率明显下降。进水pH 7.0～9.0范围内活性污泥去除速率比较稳定。同时利用RISA(核糖体基因间隔序列分析)指纹技术,并借助16S rDNA序列分析对活性污泥体系进行群落动态解析,进而揭示系统微观结构与宏观功能之间的内在联系。结果表明,苯酚单独作为基质时,活性污泥体系的微生物群落多样性明显增加,而混合酚作为基质时,系统微生物群落多样性变化较小。盐度冲击过程中活性污泥体系微生物群落结构发生了剧烈的变化,而pH冲击过程中系统微生物群落结构比较稳定。遗传分析表明,盐度冲击过程中盐单胞菌属Halomonas和海洋杆菌属Marinobacter菌株代谢旺盛,说明这两类菌属菌株具有较强的耐盐能力;而pH和底物浓度冲击使海洋杆菌属Marinobacter和粪产碱菌属Alcaligenes faecalis菌株在处理系统中大量生长,逐渐成为优势菌株。在进化分析中还发现一少部分序列归属于希瓦氏菌属Shewanella和节杆菌属Arthrobacter等。
本实验还采用扩增功能DNA限制性分析(AFDRA)的方法对不同条件下活性污泥体系苯酚羟化酶基因的变化进行了分析,与RISA指纹分析相互补充,在基因水平上揭示了活性污泥体系微生物群落结构和功能的变化。研究结果表明,活性污泥体系中降酚菌群的苯酚羟化酶功能基因的多样性因处理系统的底物种类、底物浓度、盐度和pH而有明显差异。苯酚羟化酶基因的变化可以反映生物系统内微生物群落的动态变化,而微生物群落的组成又会直接影响功能基因的多样性。
从活性污泥中分离到三株耐盐苯酚降解菌,经形态观察、生理生化分析以及16SrDNA或26S rDNA分析分别鉴定为Arthrobacter sp.W1、Rodococcus sp.W2和Candidasp.W3,其序列的GenBank登陆序列号分别为EU339930、EU339931和EU349016。考察了3株菌的生长及降解酚类化合物的基本特性,为耐盐酚类降解复合菌体系的构建奠定了基础。3株菌均能在温度20～30℃、pH 5.0～9.0和盐度10～100 g/L NaCl范围内以苯酚为唯一碳源进行生长,能够利用苯甲酸、水杨酸、对甲基酚、氨基酚、对苯二酚等多种芳香化合物。高盐条件下,3株菌能够在细胞内积累相容性物质(主要是四氢嘧啶和甜菜碱)抵御外界高盐环境,并且相容性物质的含量随盐度升高而增加,在培养基中外加相容性物质能够促进菌株的生长,并且增强菌株的耐盐能力。
通过正交实验确定菌株W1、W2和W3以1:1:2比例混合构建高效的耐盐酚类降解复合菌群,该复合菌与3株单菌相比,对环境的适应能力更强,能在温度10～40℃、pH4.0～12.0和盐度10～150 g/L NaCl范围内以苯酚为唯一碳源进行生长,在最佳降解条件下140 h即可完全降解2000 mg/L苯酚,降解酚类化合物范围更广。模拟SBR工艺对不同条件下复合菌群处理高盐混合酚废水进行了研究,结果表明,在3个月运行期内,复合菌群处理效果稳定,能够耐受一定范围的盐度(10～150 g/L NaCl)、pH(5.0～12.0)和底物浓度(200～1500 mg/L)的冲击。与活性污泥体系相比,复合菌体系抗冲击能力更强,去除速率更高。
采用荧光原位杂交技术(HSH)对复合菌群内W1、W2和W3菌株的变化进行分析,结果表明,复合菌群中的W1菌株具有较强的抗盐度和底物浓度冲击的能力,而3株菌对pH冲击均有较好的耐受性。进一步采用实时荧光定量PCR技术对复合菌群中的优势菌株W1进行定量分析,结果表明,NaCl浓度由10 g/L增加到100 g/LNaCl时,细胞数量由1.16×10~(13)增加到5.71×10×~(13)细胞/每克干重;底物浓度冲击过程中细胞数量变化与盐度冲击过程细胞数量呈现相同的变化趋势,细胞数量在1.47×10~(13)和5.53×10~(13)细胞/每克干重之间变化;而在pH冲击条件下,整个过程中细胞数量基本保持在一个稳定水平。由此推断,W1菌株可能是复合菌群处理高盐混合酚废水的重要的功能菌,在环境条件波动时稳定存在,并且保持较高的代谢活性。
The source of phenolic wastewater is extensive and the compositions are complicated, such as coking,pharmaceutical,chemical and dying wastewatwer.The existence of high concentration of phenolics and salts in wastewater will inhibit the growth of the microorganism and brings more difficulty in biological treatment,which has become a difficult problem in domestic and international wastewater treatment field and is urgent to be solved currently.To solve the problem of inhibitory effect of high salt on traditional biological treatment system,the objectives of this paper are to explore of salt-tolerant biological treatment systems,which are also capable of degrading phenolics,study on the characteristics of treatment systems under different conditions and analyze the community structure during treatment process by molecular biology technology,such as RISA(Ribosomal intergenic sequence analysis),AFDRA(Amplified functional DNA restriction analysis) and 16S rDNA sequencing analysis.The experimental results will clarify the relationship between the microstructure and macrofunction of treatment system and provide a theoretical and technical supports for the biological treatment of phenolic hyper-saline wastewater.
The activated sludge could degrade different phenolic mixtures under high salt conditions. Inhibitory effect of catchol and salicylate on phenol degradation was not obvious,but the removal rate was decreased in the presence of p-cresol.Salinity and pH fluctuation would affact the removal effeciency of activated sludge.When the content of NaCl was lower than 50 g/L, the removal rate of phenolics was increased with the NaCl concentration increasing,but decreased in the presence of more than 100 g/L NaCl.The efficiency of degradation was stable under neutral conditions(pH 7.0-9.0).At the same time,the microbial community structure of activated sludge was analyzed by RISA and 16S rDNA sequencing to clarify the relationship between the microstructure and macrofunction of treatment system.The RISA patterns revealed that the diversity of microorganism was increased in single substrate system,but was little change in binary substrates system.The community structure was drastically change during salt fluctuation process,but remained stable during pH fluctuation.16S rDNA sequence analysis showed that Halomonas sp.and Marinobactergenus sp.were predominant species during salt fluctuation process,which have better ability of salt tolerant.Marinobacter sp.and Alcaligenes faecalis sp.were the main species during the pH and substrate concentration fluctuation process.It was also found that Shewanella sp.and Arthrobacter sp.were existence in treatment system.
The changes of phenol hydroxylase gone of activated sludge was analyzed by AFDRA under different conditions,which revealed the change of microbial community structure and function of activated sludge in molecular level.AFDRA on phenol hydroxylase genes show that the diversity of phenol hydroxylase genes was substantially different in various conditions, such as different substrate,concentration,salinity and pH,and the changes are consistent to that of microbial community.Thus,it was concluded that the changes of functional genes could reflect the dynamics of microbial community,and the microbial community structure could affect the diversity of functional genes.
Arthrobacter sp.W1,Rodococcus sp.W2 and Candida sp.W3 was isolated from the activated sludge and could utilize phenol as sole carbon source under high salt conditions, which was identified on the basis of physiological and biochemical tests and 16S rDNA and 26S rDNA sequence analysis.The sequence of strains were submitted to GenBank with the number EU339930,EU339931 and EU349016.The degradation and growth conditions of three strains were as follows:temperature 20-30℃,pH 5.0-9.0 and salinity 10-100 g/L NaCl. They could utilize benzoic,salicylate,p-cresol,aminophenol and hydroquinone as the carbone source.Under high salt conditions,strains were mainly accumulated compatible solutes(ectoine and betaine) in cells to resist the extracellular hyper-saline environment. Adding compatible solutes such as ectoine,betaine and glutamic acid could improve the growth of strains under high salt conditions and enhance the salt-tolerance ability.
A defined microbial consortium was constructed through orthogonal experiments by using of strain W1,W2 and W3.The adamptation conditons for the phenol degradation by the consortium were as followed:temperature 10-40℃,pH 4.0-12.0,salinity 10-150 g/L NaCl. Compared to other three pure strains,the consortium could degrade phenol under severe conditions and had a wider substrates range.During degradation of mixed phenolics by the consortium in SBR,the treatment efficiency was stable under different conditions in 3 monthes.Compared with the activated sludge system,the consortium showed higher removal efficiency and resistant ability of environmental fluctuation more powerful.
Fluorescence in situ hybridization(FISH) analysis indicates that the abundance and intesnsity of specific probe of Arthrobacter sp.W1 was more than other two strains when salinity and substrate concentration fluctuation.During pH fluctuation process,there was lilltle differences of abundance and intensity of specific probe among three strains.It means that Arthrobacter sp.W1 has resistant ability of salinity and substrate concentration fluctuation more powerful,and three strains are insensitive to pH fluctuation.The results of real-time quantitative PCR showed that the cell numbers of Arthrobacter sp.W1 was increased fromel.16×10~(13) to 5.71×10~(13) cell per g of dry weight when NaCl concentration raised from 10 to 100 g/L.The cell number was change between 1.47×10~(13) to 5.53×10~(13) cell per g of dry weight when substrated concentration fluctuation process,but the cell numbers always remained stable during pH fluctuation process.Thus,it was concluded that W1 was the most important microorganism of the consortium during degradation phenolic hyper-saline wastewater process,which maintained higher metabolic activity under fluctuate conditions.
引文
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