利用16S rDNA克隆文库研究猪场污水微藻净化后菌群的变化
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摘要
为促进猪场污水治理,研究微藻净化对猪场污水菌群的影响,将小球藻接种于含60%沼液与40%水的猪场污水进行批式模式培养,循环三次后将小球藻液离心,检测猪场污水、小球藻液以及离心后上清液中的水质指标及菌群变化情况。菌群采用16S rDNA克隆文库方法检测。结果显示,和猪场污水相比,小球藻液及上清液中的化学耗氧量(COD)、总氮、氨氮、总磷、铜、锌等含量极显著降低(P<0.01)。除未知菌群外,猪场污水中的主要菌群为Firmicutes群和Bellilinea群,含量分别为12.25%和11.22%;小球藻液中主要菌群为小球藻和Cytophaga群,含量分别为42.42%和12.12%;上清液中的主要菌群为Dyadobacter、Cytophaga、Algoriphagus群和Pedobacter群,含量分别为25.00%、14.00%、11.00%和10.00%。猪场污水和小球藻液中未发现共存的微生物,小球藻液与上清液中皆含有Cytophaga群和Dyadobacter、Pseudomonas、Flavobacterium、Algoriphagus、Flexibacter群。表明接种小球藻可以净化猪场污水中的氨氮、总磷以及重金属,改变污水中的菌群,污水净化可能是小球藻和其共存菌共同作用的结果。
In order to promote wastewater treatment,the effects of purification with microalgae on microflora of swine wastewater were studied. The Chlorella vulgaris was inoculated in swine wastewater with 60% of biogas slurry and 40% of water,and then the chlorella solution was centrifuged after cycling culturing for three times with batch mode. The water quality indices were measured in swine wastewater,chlorella solution and supernatant solution after centrifugation,and the microflora changes were detected with 16 S r DNA clone library technology. The results showed that compared with swine wastewater,the chemical oxygen demand( COD),total nitrogen( TN),ammonia nitrogen( NH3-N),total phosphor( TP),copper and zinc concentrations significantly decreased in chlorella solution and supernatant solution( P < 0. 01). Besides uncultured microflora,theprincipal microflora in swine wastewater were Firmicutes bacterium( 12. 25%) and Bellilinea sp.( 11. 22%),those in chlorella solution were Chlorella( 42. 42%) and Cytophaga sp.( 12. 12%),and those in supernatant solution were Dyadobacter sp.( 25. 00%),Cytophaga sp.( 14. 00%),Algoriphagus sp.( 11. 00%) and Pedobacter sp.( 10. 00%). There was no coexisting microflora in swine wastewater and chlorella solution,while Cytophaga sp.,Dyadobacter sp.,Pseudomonas sp.,Flavobacterium sp.,Algoriphagus sp. and Flexibacter sp.existed in both chlorella solution and supernatant solution. In summary,inoculating Chlorella could decrease the NH3-N,TP and heavy metal contents in swine wastewater and change the microflora in wastewater. The purification of swine wastewater might be the result of Chlorella and its coexisting microflora.
In order to promote wastewater treatment,the effects of purification with microalgae on microflora of swine wastewater were studied. The Chlorella vulgaris was inoculated in swine wastewater with 60% of biogas slurry and 40% of water,and then the chlorella solution was centrifuged after cycling culturing for three times with batch mode. The water quality indices were measured in swine wastewater,chlorella solution and supernatant solution after centrifugation,and the microflora changes were detected with 16 S r DNA clone library technology. The results showed that compared with swine wastewater,the chemical oxygen demand( COD),total nitrogen( TN),ammonia nitrogen( NH3-N),total phosphor( TP),copper and zinc concentrations significantly decreased in chlorella solution and supernatant solution( P < 0. 01). Besides uncultured microflora,theprincipal microflora in swine wastewater were Firmicutes bacterium( 12. 25%) and Bellilinea sp.( 11. 22%),those in chlorella solution were Chlorella( 42. 42%) and Cytophaga sp.( 12. 12%),and those in supernatant solution were Dyadobacter sp.( 25. 00%),Cytophaga sp.( 14. 00%),Algoriphagus sp.( 11. 00%) and Pedobacter sp.( 10. 00%). There was no coexisting microflora in swine wastewater and chlorella solution,while Cytophaga sp.,Dyadobacter sp.,Pseudomonas sp.,Flavobacterium sp.,Algoriphagus sp. and Flexibacter sp.existed in both chlorella solution and supernatant solution. In summary,inoculating Chlorella could decrease the NH3-N,TP and heavy metal contents in swine wastewater and change the microflora in wastewater. The purification of swine wastewater might be the result of Chlorella and its coexisting microflora.
引文
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[10]冯蕾,陈竞,代金平,等.两种胶浓度PCR-DGGE方法对不同处理羊粪低温沼气发酵优势细菌结构多样性的分析[J].中国沼气,2014,32(6):9-15.
[11]张小妹.以含氮杂环化合物为碳源短程反硝化厌氧产甲烷及复合工艺的特征分析与性能研究[D].太原:太原理工大学,2015.
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[13]Guo Z,Tong Y W.The interactions between Chlorella vulgaris and algal symbiotic bacteria under photoautotrophic and photoheterotrophic conditions[J].Journal of Applied Phycology,2014,26(3):1483-1492.
[14]Yang T,Bu X,Han Q,et al.A small periplasmic protein essential for Cytophaga hutchinsonii cellulose digestion[J].Applied Microbiology and Biotechnology,2016,100(4):1935-1944.
[15]Logemann S,Schantl J,Bijvank S,et al.Molecular microbial diversity in a nitrifying reactor system without sludge retention[J].FEMS Microbiology Ecology,1998,27(3):239-249.
[16]苗祯,杜宗军,李会荣,等.5株北极微藻藻际环境的细菌多样性[J].生态学报,2015,35(5):1587-1600.
[17]毛丹丹.生活污水活性污泥中菌群结构分析及功能菌的分离与培养[D].杭州:浙江工业大学,2012.
[18]张艳丽.滇池浮游细菌的时空分布及优势菌对铅、镉的吸附效果[D].昆明:云南大学,2012.
[19]Mujtaba G,Rizwan M,Lee K.Simultaneous removal of inorganic nutrients and organic carbon by symbiotic co-culture of Chlorella vulgaris,and Pseudomonas putida[J].Biotechnology&Bioprocess Engineering,2015,20(6):1114-1122.
[20]Strom E V,Dinarieva T Y,Netrusov A I.The cytochrome cbo from the obligate methylotroph Methylobacillus flagellatus KT is a cytochrome c oxidase[J].Microbiology,2004,73(2):124-128.
[21]Muntyan M S,Dinarieva T Y,Baev M V,et al.Effect of growth conditions on the synthesis of terminal oxidases in Methylobacillus flagellatus KT[J].Archives of Biochemistry and Biophysics,2002,398(1):118-124.
[22]But S Y,Khmelenina V N,Reshetnikov A S,et al.Bifunctional sucrose phosphate synthase/phosphatase is involved in the sucrose biosynthesis by Methylobacillus flagellatus KT[J].FEMS Microbiology Letters,2013,347(1):43-51.
[23]Su Y,Mennerich A,Urban B.Municipal wastewater treatment and biomass accumulation with a wastewater-born and settleable algal-bacterial culture[J].Water Research,2011,45(11):3351-3358.
[2]Renju G L,Kurup G M,Bandugula V R.Effect of lycopene isolated from Chlorella marina on proliferation and apoptosis in human prostate cancer cell line PC-3[J].Tumor Biology,2014,35(11):10747-10758.
[3]Shimada M,Hasegawa T,Nishimura C,et al.Anti-hypertensive effect of gamma-aminobutyric acid(GABA)-rich Chlorella on high-normal blood pressure and borderline hypertension in placebo-controlled double blind study[J].Clinical and Experimental Hypertension,2009,31(4):342-354.
[4]Kim S B,Lee S J,Yoon B D,et al.Selection of microalgae for advanced treatment of swine wastewater and optimization of treatment condition[J].Microbiology and Biotechnology Letters,1998,26(1):76-82.
[5]简恩光.小球藻和菌藻共生系统综合处理养猪业沼液研究[D].南昌:南昌大学,2013.
[6]毕向东.小球藻与优势共栖异养细菌间的相互作用及其对细菌群体感应信号分子的响应[D].青岛:中国海洋大学,2013.
[7]Madelaine Q E.Selecting and characterizing bacterial consortia with the potential of fixing CO2and removing H2S in a biogas atmosphere[J].Water Air&Soil Pollution,2014,225(5):1-9.
[8]Huang X P,Guo J S,Chen C L,et al.Control the pollution of pig farm’s manure wastewater and resource to cultivate microalgae[J].Advanced Materials Research,2014,1015:725-728.
[9]黄魁.藻类去除污水中氮磷及其机理的研究[D].南昌:南昌大学,2007.
[10]冯蕾,陈竞,代金平,等.两种胶浓度PCR-DGGE方法对不同处理羊粪低温沼气发酵优势细菌结构多样性的分析[J].中国沼气,2014,32(6):9-15.
[11]张小妹.以含氮杂环化合物为碳源短程反硝化厌氧产甲烷及复合工艺的特征分析与性能研究[D].太原:太原理工大学,2015.
[12]Dellagreca M,Zarrelli A,Fergola P,et al.Fatty acids released by Chlorella vulgaris and their role in interference with Pseudokirchneriella subcapitata:experiments and modelling[J].Journal of Chemical Ecology,2010,36(3):339-349.
[13]Guo Z,Tong Y W.The interactions between Chlorella vulgaris and algal symbiotic bacteria under photoautotrophic and photoheterotrophic conditions[J].Journal of Applied Phycology,2014,26(3):1483-1492.
[14]Yang T,Bu X,Han Q,et al.A small periplasmic protein essential for Cytophaga hutchinsonii cellulose digestion[J].Applied Microbiology and Biotechnology,2016,100(4):1935-1944.
[15]Logemann S,Schantl J,Bijvank S,et al.Molecular microbial diversity in a nitrifying reactor system without sludge retention[J].FEMS Microbiology Ecology,1998,27(3):239-249.
[16]苗祯,杜宗军,李会荣,等.5株北极微藻藻际环境的细菌多样性[J].生态学报,2015,35(5):1587-1600.
[17]毛丹丹.生活污水活性污泥中菌群结构分析及功能菌的分离与培养[D].杭州:浙江工业大学,2012.
[18]张艳丽.滇池浮游细菌的时空分布及优势菌对铅、镉的吸附效果[D].昆明:云南大学,2012.
[19]Mujtaba G,Rizwan M,Lee K.Simultaneous removal of inorganic nutrients and organic carbon by symbiotic co-culture of Chlorella vulgaris,and Pseudomonas putida[J].Biotechnology&Bioprocess Engineering,2015,20(6):1114-1122.
[20]Strom E V,Dinarieva T Y,Netrusov A I.The cytochrome cbo from the obligate methylotroph Methylobacillus flagellatus KT is a cytochrome c oxidase[J].Microbiology,2004,73(2):124-128.
[21]Muntyan M S,Dinarieva T Y,Baev M V,et al.Effect of growth conditions on the synthesis of terminal oxidases in Methylobacillus flagellatus KT[J].Archives of Biochemistry and Biophysics,2002,398(1):118-124.
[22]But S Y,Khmelenina V N,Reshetnikov A S,et al.Bifunctional sucrose phosphate synthase/phosphatase is involved in the sucrose biosynthesis by Methylobacillus flagellatus KT[J].FEMS Microbiology Letters,2013,347(1):43-51.
[23]Su Y,Mennerich A,Urban B.Municipal wastewater treatment and biomass accumulation with a wastewater-born and settleable algal-bacterial culture[J].Water Research,2011,45(11):3351-3358.