反硝化基因工程菌株YP4S的构建及对污水除氮特性研究
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摘要
从养殖场污泥中筛选出菌株YP4,经16S rDNA分子发育树的同源序列比对,确定为克雷伯什菌属(Klebsiella sp.)。由NCBI数据库查编码亚硝酸还原酶(Nir)的基因nirS序列,设计引物,以铜绿假单胞菌PAOI基因组DNA为模板,应用PCR技术扩增目的片段nirS,经过双酶切、克隆和转化,得到重组质粒pYP4S,然后转化野生菌株YP4,构建反硝化基因工程菌YP4S。菌株生长曲线测定表明,工程菌株YP4S与YP4的生长特性基本一致。工程菌株YP4S对模拟污水COD、TN、NH_4~+-N和NO_3~--N具有较高的去除率,YP4S与YP4相比,对NO_2~--N积累的减少量为(32.44±3.96)%,明显减少了NO_2~--N的积累。通过正交试验获得工程菌株YP4S在C/N=10、T=30℃、r=200 r/min和pH=7.0的最佳组合条件下,对模拟污水TN去除率较高。应用工程菌株YP4S处理猪场沉淀池的实际污水,COD、TN、TP、NH_4~+-N和NO_3~--N去除率分别为(95.87±0.82)%、(76.38±3.84)%、(97.13±0.54)%和(75.35±2.57)%,NO_2~--N积累量为(3.31±1.24) mg/L,表明工程菌株YP4S具有较好反硝化作用,对含氮量高的实际污水修复具有潜在的应用前景。
The strain YP4 was screened from the farm sludge. It was identified as Klebsiella sp. through the homologous sequence alignment with a 16 S r DNA phylogenetic tree. The gene sequence of nirS encoding nitrite reductase(Nir) was obtained by searching NCBI database, and the primers were designed. Taking the genomic DNA of Pseudomonas aeruginosa PAOI as template, the target fragment nirS was amplified by PCR, and the recombinant plasmid pYP4 S was obtained by double enzyme digestion, cloning and transformation. Finally, wild strain YP4 was transformed to construct denitrifying genetic engineering strain YP4 S. The identification result of strain growth curve showed that the growth characteristics of engineering strain YP4 S and YP4 were basically the same. The engineering strain YP4 S had a high removal rate of COD, TN, NH_4~+-N, NO_3~--N in simulated wastewater. Compared with strain YP4, stain YP4 S could significantly reduce NO_2~--N accumulation by(32.44±3.96)%, which significantly reduced the accumulation of NO_2~--N. Stain YP4 S, which was obtained by means of orthogonal test, had a higher removal rate of TN in simulated wastewater under the optimum conditions with C/N=10, T=30℃, r=200 r/min and pH =7. Applying the engineering strain YP4 S to treat the actual wastewater in pig farm sedimentation tank, the removal rates of COD, TN, TP, NH_4~+-N, NO_3~--N of strain YP4 S were(95.87±0.82)%,(76.38±3.84)%,(97.13±0.54)%,(75.35±2.57)% respectively. The accumulation of NO_2~--N was(3.31±1.24) mg/L, indicating that the engineering strain YP4 S had a high-efficient denitrification and it had potential application prospects for the actual sewage remediation with high nitrogen content.
The strain YP4 was screened from the farm sludge. It was identified as Klebsiella sp. through the homologous sequence alignment with a 16 S r DNA phylogenetic tree. The gene sequence of nirS encoding nitrite reductase(Nir) was obtained by searching NCBI database, and the primers were designed. Taking the genomic DNA of Pseudomonas aeruginosa PAOI as template, the target fragment nirS was amplified by PCR, and the recombinant plasmid pYP4 S was obtained by double enzyme digestion, cloning and transformation. Finally, wild strain YP4 was transformed to construct denitrifying genetic engineering strain YP4 S. The identification result of strain growth curve showed that the growth characteristics of engineering strain YP4 S and YP4 were basically the same. The engineering strain YP4 S had a high removal rate of COD, TN, NH_4~+-N, NO_3~--N in simulated wastewater. Compared with strain YP4, stain YP4 S could significantly reduce NO_2~--N accumulation by(32.44±3.96)%, which significantly reduced the accumulation of NO_2~--N. Stain YP4 S, which was obtained by means of orthogonal test, had a higher removal rate of TN in simulated wastewater under the optimum conditions with C/N=10, T=30℃, r=200 r/min and pH =7. Applying the engineering strain YP4 S to treat the actual wastewater in pig farm sedimentation tank, the removal rates of COD, TN, TP, NH_4~+-N, NO_3~--N of strain YP4 S were(95.87±0.82)%,(76.38±3.84)%,(97.13±0.54)%,(75.35±2.57)% respectively. The accumulation of NO_2~--N was(3.31±1.24) mg/L, indicating that the engineering strain YP4 S had a high-efficient denitrification and it had potential application prospects for the actual sewage remediation with high nitrogen content.
引文
Azziz G.,Monza J.,Etchebehere C.,and Irisarri P.,2017,Nir S-and nirK-type denitrifier communities are differentially affected by soil type,rice cultivar and water management,European Journal of Soil Biology,78:20-28
Bai Y.H.,Liu R.P.,Liang J.S.,and Qu J.H.,2013,Integrated metagenomic and physiochemical analyses to evaluate the potential role of microbes in the sand filter of a drinking water treatment system,PLoS One,8:1006-1011
Barnard R.,Leadley P.W.,and Hungate B.A.,2005,Global change,nitrification,and denitrification:A review,Global Biogeochemical Cycles,19(1):564-569
Beuckels A.,Smolders E.,and Muylaert K.,2015,Nitrogen availability influences phosphorus removal in microalgaebased wastewater treatment,Water Research,77:98-106
Bi H.Z.,2012,The fast method to determine the chemical oxygen demand(COD)for water quality,Huanbao Keji(Environmental Protection and Technology),18(1):31-33(毕惠珍,2012,水质化学需氧量(COD)的快速测定的方法,环保科技,18(1):31-33)
Casciottik K.L.,and Ward B.B.,2001,Dissimilatory nitrite re ductase genes from autotrophic ammonia-Oxidizing bacteria,Applied&Environmental Microbiology,67(5):2213-2221
Chen Q.,Ni J.,Ma T.,Liu T.,and Zheng M.S.,2015,Bioaugmentation treatment of municipal wastewater with heterotrophic-aerobic nitrogen removal bacteria in a pilot-scale SBR,Bioresource Technology,183:25-32
Choi K.H.,and Schweizer H.P.,2006,Mini-Tn7 insertion in bacteria with single attTn7 sites:example Pseudomonas aeruginosa,Nature Protocols,1:153-161
Duan J.,Fang H.,Su B.,Chen J.F.,and Lin J.,2015,Characterization of a halophilic heterotrophic nitrification-aerobic denitrification bacterium and its application on treatment of saline wastewater,Bioresource Technology,179:421-428
Hallin S.,and Lindgren P.E.,1999,PCR tection of genes encoding nitrite reductase in denitrifying bacteria,Applied&Environmental Microbiology,65(4):1652-1657
Hasegawa N.,Arai H.,and Igarashi Y.,2001,Two c-type cytochromes,Nir M and Nir C,encoded in the nir gene cluster of Pseudomonas aeruginosa act as electron donors forn nitrite reductase,Biochemical&Biophysical Research Communicate,288(5):1223-1230
He T.,Li Z.,Sun Q.,Xu Y.,and Ye Q.,2016,Heterotrophic nitrification and aerobic denitrification by Pseudomonas tolaasii Y-11 without nitrite accumulation during nitrogen conversion,Bioresource Technology,200:493-499
Huang H.K.,and Tseng S.K.,2001,Nitrate reduction by citrobacter diversus under aerobic environment,Appl.Microbiol.Biotechnol.,55:90-94
Kampman C.,Temmink H.,Hendrickx T.L.,Zeeman G.,and Buisman C.J.,2014,Enrichment of denitrifying methanotrophic bacteria from municipal wastewater sludge in a membrane bioreactor at 20 degrees C,Journal of Hazardous Materials,274:428-435
Liu H.,Sun Y.F.,Jia X.S.,Li J.,Zhou K.Q.,Qu X.D.,Tao X.Q.,and Chen Y.,2013,Identification and metabolic mechanism of non-fermentative short-cut denitrifying phosphorus-removing bacteria,Chinese Journal of Chemical Engineering,21(3):332-340
Peng Z.X.,Peng Y.Z.,Liu X.L.,Yu Z.B.,and Chai T.Z.,2012,Characteristic differences of activated sludge systems between real wastewater and simulated wastewater,Huanjing Gongcheng Xuebao(Chinese Journal of Environmental Engineering),6(3):749-754(彭赵旭,彭永臻,刘旭亮,于振波,柴同志,2012,实际污水与模拟污水活性污泥系统的特性差异,环境工程学报,6(3):749-754)
Xu Z.A.,Wang T.B.,Li C.Y.,Bao L.Y.,Ma Q.M.,and Miao Y.N.,2002,Brief introduction to the orthogonal test design,Keji Qingbao Yu Jingji(Sci/Tech Information Development&Economy),12(5):148-150(徐仲安,王天保,李常英,暴丽艳,马青梅,苗玉宁,2002,正交试验设计法简介,科技情报开发与经济,12(5):148-150)
Yu H.,Zhou J.,and Fang C.Y.,2013,Isolation of heterotrophic nitrifiers to degrade slaughterhouse wastewater and their ammonia nitrogen removal condition,Shuichuli Jishu(Technology of Water Treatment),39(6):41-44(余欢,周健,方春玉,2013,降解屠宰废水异养硝化菌的筛选及其氨氮去除条件,水处理技术,39(6):41-44)
Zheng J.P.,2011,Discussion on the improvement of determination of total-nitrogen in water by potassium persulfate oxidation-UV spectrophotometry,Guangpu Shiyanshi(Chinese Journal of Spectroscopy Laboratory),28(1):210-217(郑京平,2011,关于过硫酸钾氧化-紫外分光光度法测定水中总氮方法改进探讨,光谱实验室,28(1):210-217)
Zhou M.,Ye H.,and Zhao X.,2014,Isolation and characterization of a novel heterotrophic nitrifying and aerobic denitrifying bacterium Pseudomonas stutzeri KTB for bioremediation of wastewater,Biotechnology and Bioprocess Engineering,19(2):231-238
Bai Y.H.,Liu R.P.,Liang J.S.,and Qu J.H.,2013,Integrated metagenomic and physiochemical analyses to evaluate the potential role of microbes in the sand filter of a drinking water treatment system,PLoS One,8:1006-1011
Barnard R.,Leadley P.W.,and Hungate B.A.,2005,Global change,nitrification,and denitrification:A review,Global Biogeochemical Cycles,19(1):564-569
Beuckels A.,Smolders E.,and Muylaert K.,2015,Nitrogen availability influences phosphorus removal in microalgaebased wastewater treatment,Water Research,77:98-106
Bi H.Z.,2012,The fast method to determine the chemical oxygen demand(COD)for water quality,Huanbao Keji(Environmental Protection and Technology),18(1):31-33(毕惠珍,2012,水质化学需氧量(COD)的快速测定的方法,环保科技,18(1):31-33)
Casciottik K.L.,and Ward B.B.,2001,Dissimilatory nitrite re ductase genes from autotrophic ammonia-Oxidizing bacteria,Applied&Environmental Microbiology,67(5):2213-2221
Chen Q.,Ni J.,Ma T.,Liu T.,and Zheng M.S.,2015,Bioaugmentation treatment of municipal wastewater with heterotrophic-aerobic nitrogen removal bacteria in a pilot-scale SBR,Bioresource Technology,183:25-32
Choi K.H.,and Schweizer H.P.,2006,Mini-Tn7 insertion in bacteria with single attTn7 sites:example Pseudomonas aeruginosa,Nature Protocols,1:153-161
Duan J.,Fang H.,Su B.,Chen J.F.,and Lin J.,2015,Characterization of a halophilic heterotrophic nitrification-aerobic denitrification bacterium and its application on treatment of saline wastewater,Bioresource Technology,179:421-428
Hallin S.,and Lindgren P.E.,1999,PCR tection of genes encoding nitrite reductase in denitrifying bacteria,Applied&Environmental Microbiology,65(4):1652-1657
Hasegawa N.,Arai H.,and Igarashi Y.,2001,Two c-type cytochromes,Nir M and Nir C,encoded in the nir gene cluster of Pseudomonas aeruginosa act as electron donors forn nitrite reductase,Biochemical&Biophysical Research Communicate,288(5):1223-1230
He T.,Li Z.,Sun Q.,Xu Y.,and Ye Q.,2016,Heterotrophic nitrification and aerobic denitrification by Pseudomonas tolaasii Y-11 without nitrite accumulation during nitrogen conversion,Bioresource Technology,200:493-499
Huang H.K.,and Tseng S.K.,2001,Nitrate reduction by citrobacter diversus under aerobic environment,Appl.Microbiol.Biotechnol.,55:90-94
Kampman C.,Temmink H.,Hendrickx T.L.,Zeeman G.,and Buisman C.J.,2014,Enrichment of denitrifying methanotrophic bacteria from municipal wastewater sludge in a membrane bioreactor at 20 degrees C,Journal of Hazardous Materials,274:428-435
Liu H.,Sun Y.F.,Jia X.S.,Li J.,Zhou K.Q.,Qu X.D.,Tao X.Q.,and Chen Y.,2013,Identification and metabolic mechanism of non-fermentative short-cut denitrifying phosphorus-removing bacteria,Chinese Journal of Chemical Engineering,21(3):332-340
Peng Z.X.,Peng Y.Z.,Liu X.L.,Yu Z.B.,and Chai T.Z.,2012,Characteristic differences of activated sludge systems between real wastewater and simulated wastewater,Huanjing Gongcheng Xuebao(Chinese Journal of Environmental Engineering),6(3):749-754(彭赵旭,彭永臻,刘旭亮,于振波,柴同志,2012,实际污水与模拟污水活性污泥系统的特性差异,环境工程学报,6(3):749-754)
Xu Z.A.,Wang T.B.,Li C.Y.,Bao L.Y.,Ma Q.M.,and Miao Y.N.,2002,Brief introduction to the orthogonal test design,Keji Qingbao Yu Jingji(Sci/Tech Information Development&Economy),12(5):148-150(徐仲安,王天保,李常英,暴丽艳,马青梅,苗玉宁,2002,正交试验设计法简介,科技情报开发与经济,12(5):148-150)
Yu H.,Zhou J.,and Fang C.Y.,2013,Isolation of heterotrophic nitrifiers to degrade slaughterhouse wastewater and their ammonia nitrogen removal condition,Shuichuli Jishu(Technology of Water Treatment),39(6):41-44(余欢,周健,方春玉,2013,降解屠宰废水异养硝化菌的筛选及其氨氮去除条件,水处理技术,39(6):41-44)
Zheng J.P.,2011,Discussion on the improvement of determination of total-nitrogen in water by potassium persulfate oxidation-UV spectrophotometry,Guangpu Shiyanshi(Chinese Journal of Spectroscopy Laboratory),28(1):210-217(郑京平,2011,关于过硫酸钾氧化-紫外分光光度法测定水中总氮方法改进探讨,光谱实验室,28(1):210-217)
Zhou M.,Ye H.,and Zhao X.,2014,Isolation and characterization of a novel heterotrophic nitrifying and aerobic denitrifying bacterium Pseudomonas stutzeri KTB for bioremediation of wastewater,Biotechnology and Bioprocess Engineering,19(2):231-238