一株贫营养异养硝化-好氧反硝化菌的筛选及脱氮特性
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摘要
为了探究并优化菌剂应用于微污染水源水体修复的机制和条件,主要针对水库沉积物内筛选出的贫营养好氧反硝化菌进行了菌种鉴定及脱氮特性研究,考察菌株在不同环境条件下的脱氮效果,明确了该菌株的最适宜生长条件,并基于水库水体中贫营养条件对菌株进行水源水库原水的驯化培养试验研究,以期实现该菌株对微污染水源水库原水中氮源污染物的脱除,为原位投菌技术实际工程应用提供理论依据。从微污染水源水库沉积物中驯化筛分出一株高效异养硝化-好氧反硝化菌A14,通过扫描电镜观察、生理生化特征、16S r RNA基因测序和Biolog GenⅢ鉴定,确定该菌株为革兰氏阴性短杆菌,鉴定为皮特不动杆菌(Acinetobacter pittii)。在好氧条件下,菌株细胞内表达反硝化功能基因nap A,以NO3-为唯一氮源进行反硝化作用时,36 h时NO3-去除率为78.89%。以NH4+为唯一氮源时,48 h NH4+去除率为95.25%,TN去除率达80.42%,TOC去除率达98.30%,表明该菌株具有异养硝化-好氧反硝化特性。在改变环境条件过程中,该菌株在以乙酸钠为碳源,温度为30℃,C/N为12,p H为7,接种量为10%时,NO3-去除率最高为86.62%,并且在10℃下脱氮率达到40.18%。在水源水库原水脱氮实验中,接种处理TN去除率为50.95%,NO3-去除率为80.25%。结果表明,菌株A14在微污染水源水体菌剂脱氮修复中具有良好的应用潜力。
To investigate the feasibility that the strain can used to treat micro-polluted water, the research is mainly about the identification and the denitrification characteristics of the aerobic-denitrification strain that was isolated from sediment in water reservoir and the effects of nitrogen removal under different environmental factors. The optimal conditions of the strain can be determined. Through the method of cultivation and acclimatization to make the strain adapt to the oligotrophic condition and make effects on nitrogen removal, which all prove a theoretical basis for the practical application of onsite biological treatment in the future. A high efficient heterotrophic nitrification-denitrification aerobic bacteria(A14) was isolated from sediment in water reservoir. Based on its microscopic observation, biochemical/morphological characteristics, its 16 S r RNA sequence homologic analysis and Biolog GenⅢ identification, this strain was a gram-negative bacillus and was identified as Acinetobacter pittii. The efficient nitrogen removal in 36 hours can be 78.89% when nitrogen as the nitrogen source and the strain's partial nap A functional gene was cloned. When ammonia as the nitrogen source, TN removal rate can be 80.42%, ammonia removal rate can be 95.25%, TOC removal rate can be 98.3%. The highest nitrite removal rate reaches 86.62% when carbon resource is Na Ac, temperature=30 ℃, C/N=12, p H=7, inoculating ratio is 10%. In addition, the nitrite removal rate can reach 40.18% under 10 ℃. When the strain is cultivated and acclimated in reservoir raw water, TN removal can be 50.95%, the nitrogen removal can be 80.25%. It is therefore demonstrated that the strain A14 can be used to treat micro polluted water.
To investigate the feasibility that the strain can used to treat micro-polluted water, the research is mainly about the identification and the denitrification characteristics of the aerobic-denitrification strain that was isolated from sediment in water reservoir and the effects of nitrogen removal under different environmental factors. The optimal conditions of the strain can be determined. Through the method of cultivation and acclimatization to make the strain adapt to the oligotrophic condition and make effects on nitrogen removal, which all prove a theoretical basis for the practical application of onsite biological treatment in the future. A high efficient heterotrophic nitrification-denitrification aerobic bacteria(A14) was isolated from sediment in water reservoir. Based on its microscopic observation, biochemical/morphological characteristics, its 16 S r RNA sequence homologic analysis and Biolog GenⅢ identification, this strain was a gram-negative bacillus and was identified as Acinetobacter pittii. The efficient nitrogen removal in 36 hours can be 78.89% when nitrogen as the nitrogen source and the strain's partial nap A functional gene was cloned. When ammonia as the nitrogen source, TN removal rate can be 80.42%, ammonia removal rate can be 95.25%, TOC removal rate can be 98.3%. The highest nitrite removal rate reaches 86.62% when carbon resource is Na Ac, temperature=30 ℃, C/N=12, p H=7, inoculating ratio is 10%. In addition, the nitrite removal rate can reach 40.18% under 10 ℃. When the strain is cultivated and acclimated in reservoir raw water, TN removal can be 50.95%, the nitrogen removal can be 80.25%. It is therefore demonstrated that the strain A14 can be used to treat micro polluted water.
引文
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DUAN J M,FANG H D,SU B,et al.2015.Characterization of a halophilicheterotrophic nitrification-aerobic denitrification bacterium and itsapplication on treatment of saline wastewater[J]BioresourceTechnology,179:421-428.
HOLT J G,KRIEG N R,SNEATH PHA,et al.1994.Berger’s Manual ofDeteminative Bacteriology,9th Ed[M].Baltimore:The Williams andWilkins Co.
KYOUNG J P,KYOUNG S C,SOO W N,et al.2005.Aerobicnitrification-denitrification by heterotrophic Bacillus strains[J].Bioresource Technology,96(17):1897-1906.
MAKOTO S,YOICHI I.2014.Heterotrophic nitrification and aerobicdenitrification of high-strength ammonium in anaerobically digestedsludge by Alcaligenes faecalis strain No.4[J].Journal of Bioscienceand Bioengineering,117(6):737-741.
MIYAHARA M,KIM S W,ZHOU S,et al.2012.Survival of the aerobicdenitrifier Pseudomonas stuzeri strain TR2 during co-culture withactivated sludge under denitrifying conditions[J].Bioscience,Biotechnology,and Biochemistry,76(3):495-500.
RICHARDSON L A,CORNELISSE R,DE V P,et al.1989.Aerobicdenitrification in various heterotrophic nitrifiers[J].Antonic VanLeeuwenhoek,56(4):289-299.
TIMMERMANS P,VAN HAUTE A.1983.Denitrification with methanol:Fundamental study of the growth and denitrification capacity ofHyphomicrobium sp[J].Water Research,17(10):1249-1255.
WANG X,LI T L,DONG M Y,et al.2011.Reducing nitrate ingroundwater by stabilized iron nanoparticles and denitrifyingbacteria[J].Journal of Agro-Environment Science,30(4):739-745.
YANG Y,ZHU H,COLVIN V L,et al.2011.Cellular and transcriptionalresponse of Pseudomonas stutzeri to quantum dots under aerobic anddenitrifying conditions[J].Environmental Science&Technology,45(11):4988-4994.
REN Y X,LEI Y,LIANG X.2014.The characteristics of a novelheterotrophic nitrifying and aerobic denitrifying bacterium,Acinetobacter junii YB[J].Bioresource Technology,171:1-9.
ZHOU J Z,BRUNS M A,TIEDJE J M.1996.DNA recovery from soil ofdiverse composition[J].Applied and Environmental Microbiology,62(2):316-322.
陈茂霞,王欢,周后珍,等.2013.异养硝化-好氧反硝化菌HN-02的筛选及其特性[J].应用与环境生物学报,19(4):688-693.
韩永和,章文贤,庄志刚,等.2013.耐盐好氧反硝化菌A13菌株的分离鉴定及其反硝化特性[J].微生物学报,53(1):47-58.
何斐,李磊,徐炎华.2008.微污染水源水处理技术研究进展[J].安徽农业科学,36(11):4672-4685.
黄廷林,刘婷婷,张海涵,等.2013.水库贫营养好氧反硝化细菌分离及其对水体微生物群落影响[J].西安建筑科技大学学报:自然科学版,44(6):876-882.
李雯,阎爱华,黄秋娴,等.2014.尾矿区不同植被恢复模式下高效固氮菌的筛选及Biolog鉴定[J].生态学报,34(9):2329-2337.
林娜,郭楚玲,郭延萍,等.2012.红树林湿地中好氧-厌氧反硝化菌脱氮特性及其种群结构分析[J].环境科学学报,32(1):173-181.
刘燕,甘莉,黄哲强,等.2010.脱氮副球菌YF1的反硝化特性研究[J].水处理技术,36(10):61-65.
乔森,刘雪洁,周集体,等.2014.异养硝化-好氧反硝化在生物脱氮方面的研究进展[J].安全与环境学报,14(2):128-135.
全向春,岑艳,钱殷,等.2013.2株好氧反硝化菌的筛选及其强化贫营养生物膜脱氮效果[J].环境科学,34(7):2862-2868.
苏俊峰,杨少斐,黄廷林,等.2013.低温贫营养好氧反硝化细菌SY13的反硝化特性[J].城市环境与城市生态,26(6):39-42.
苏婉昀,高俊发,赵红梅,等.2013.异养硝化-好氧反硝化菌的研究进展[J].工业水处理,33(12):1-5.
孙雪梅,李秋芬,张艳,等.2012.一株海水异养硝化-好氧反硝化菌系统发育及脱氮特性[J].微生物学报,52(6):687-695.
王弘宇,马放,苏俊峰,等.2007.好氧反硝化菌株的鉴定及其反硝化特性研究[J].环境科学,28(7):1548-1552.
王景峰,金敏,谌志强,等.2011.1株好氧脱氮菌的筛选与脱氮特性研究[J].环境科学,32(8):2409-2413.
王兆阳,陈国耀,姜珂,等.2014.1株耐冷兼性嗜碱好氧反硝化菌的分离鉴定及反硝化特性[J].环境科学,35(6):2341-2347.
魏巍,黄廷林,苏俊峰,等.2010.1株贫营养好氧反硝化菌的分离鉴定及其脱氮特性[J].生态环境学报,19(9):2166-2171.
肖继波,江惠霞,褚淑祎,等.2012.好氧反硝化菌Defluvibacterlusatiensisstr.DN7的分离鉴定和异养硝化性能[J].应用生态学报,23(7):1979-1984.
修海峰,朱仲元,丁爱中,等.2011.好氧反硝化菌种DF2的分离鉴定及生理生化特性分析[J].生态环境学报,20(8/9):1307-1314.
赵丹,于德爽,于津,等.2013.菌株ZD8的分离鉴定及其异养硝化和缺氧/好氧反硝化特性研究[J].环境科学学报,33(11):3007-3016.
周莉,李正魁,王易超,等.2013.纯种氨氧化菌短程反硝化特性[J].环境工程学报,7(4):1219-1224.
周迎芹,信欣,姚力,等.2013.一株高效异养硝化-好氧反硝化菌的分离鉴定及脱氮性能[J].环境工程学报,7(10):4127-4132.
CINDY J S,DAVID B N,LIANG F D,et al.2007.Diversity andabundance of nitrate reductase genes(nar G and nap A),Nitritereductase genes(nir S and nrf A),and their transcripts in estuarinesediments[J].Applied and Environmental Microbiology,73(11):3612-3622.
DUAN J M,FANG H D,SU B,et al.2015.Characterization of a halophilicheterotrophic nitrification-aerobic denitrification bacterium and itsapplication on treatment of saline wastewater[J]BioresourceTechnology,179:421-428.
HOLT J G,KRIEG N R,SNEATH PHA,et al.1994.Berger’s Manual ofDeteminative Bacteriology,9th Ed[M].Baltimore:The Williams andWilkins Co.
KYOUNG J P,KYOUNG S C,SOO W N,et al.2005.Aerobicnitrification-denitrification by heterotrophic Bacillus strains[J].Bioresource Technology,96(17):1897-1906.
MAKOTO S,YOICHI I.2014.Heterotrophic nitrification and aerobicdenitrification of high-strength ammonium in anaerobically digestedsludge by Alcaligenes faecalis strain No.4[J].Journal of Bioscienceand Bioengineering,117(6):737-741.
MIYAHARA M,KIM S W,ZHOU S,et al.2012.Survival of the aerobicdenitrifier Pseudomonas stuzeri strain TR2 during co-culture withactivated sludge under denitrifying conditions[J].Bioscience,Biotechnology,and Biochemistry,76(3):495-500.
RICHARDSON L A,CORNELISSE R,DE V P,et al.1989.Aerobicdenitrification in various heterotrophic nitrifiers[J].Antonic VanLeeuwenhoek,56(4):289-299.
TIMMERMANS P,VAN HAUTE A.1983.Denitrification with methanol:Fundamental study of the growth and denitrification capacity ofHyphomicrobium sp[J].Water Research,17(10):1249-1255.
WANG X,LI T L,DONG M Y,et al.2011.Reducing nitrate ingroundwater by stabilized iron nanoparticles and denitrifyingbacteria[J].Journal of Agro-Environment Science,30(4):739-745.
YANG Y,ZHU H,COLVIN V L,et al.2011.Cellular and transcriptionalresponse of Pseudomonas stutzeri to quantum dots under aerobic anddenitrifying conditions[J].Environmental Science&Technology,45(11):4988-4994.
REN Y X,LEI Y,LIANG X.2014.The characteristics of a novelheterotrophic nitrifying and aerobic denitrifying bacterium,Acinetobacter junii YB[J].Bioresource Technology,171:1-9.
ZHOU J Z,BRUNS M A,TIEDJE J M.1996.DNA recovery from soil ofdiverse composition[J].Applied and Environmental Microbiology,62(2):316-322.
陈茂霞,王欢,周后珍,等.2013.异养硝化-好氧反硝化菌HN-02的筛选及其特性[J].应用与环境生物学报,19(4):688-693.
韩永和,章文贤,庄志刚,等.2013.耐盐好氧反硝化菌A13菌株的分离鉴定及其反硝化特性[J].微生物学报,53(1):47-58.
何斐,李磊,徐炎华.2008.微污染水源水处理技术研究进展[J].安徽农业科学,36(11):4672-4685.
黄廷林,刘婷婷,张海涵,等.2013.水库贫营养好氧反硝化细菌分离及其对水体微生物群落影响[J].西安建筑科技大学学报:自然科学版,44(6):876-882.
李雯,阎爱华,黄秋娴,等.2014.尾矿区不同植被恢复模式下高效固氮菌的筛选及Biolog鉴定[J].生态学报,34(9):2329-2337.
林娜,郭楚玲,郭延萍,等.2012.红树林湿地中好氧-厌氧反硝化菌脱氮特性及其种群结构分析[J].环境科学学报,32(1):173-181.
刘燕,甘莉,黄哲强,等.2010.脱氮副球菌YF1的反硝化特性研究[J].水处理技术,36(10):61-65.
乔森,刘雪洁,周集体,等.2014.异养硝化-好氧反硝化在生物脱氮方面的研究进展[J].安全与环境学报,14(2):128-135.
全向春,岑艳,钱殷,等.2013.2株好氧反硝化菌的筛选及其强化贫营养生物膜脱氮效果[J].环境科学,34(7):2862-2868.
苏俊峰,杨少斐,黄廷林,等.2013.低温贫营养好氧反硝化细菌SY13的反硝化特性[J].城市环境与城市生态,26(6):39-42.
苏婉昀,高俊发,赵红梅,等.2013.异养硝化-好氧反硝化菌的研究进展[J].工业水处理,33(12):1-5.
孙雪梅,李秋芬,张艳,等.2012.一株海水异养硝化-好氧反硝化菌系统发育及脱氮特性[J].微生物学报,52(6):687-695.
王弘宇,马放,苏俊峰,等.2007.好氧反硝化菌株的鉴定及其反硝化特性研究[J].环境科学,28(7):1548-1552.
王景峰,金敏,谌志强,等.2011.1株好氧脱氮菌的筛选与脱氮特性研究[J].环境科学,32(8):2409-2413.
王兆阳,陈国耀,姜珂,等.2014.1株耐冷兼性嗜碱好氧反硝化菌的分离鉴定及反硝化特性[J].环境科学,35(6):2341-2347.
魏巍,黄廷林,苏俊峰,等.2010.1株贫营养好氧反硝化菌的分离鉴定及其脱氮特性[J].生态环境学报,19(9):2166-2171.
肖继波,江惠霞,褚淑祎,等.2012.好氧反硝化菌Defluvibacterlusatiensisstr.DN7的分离鉴定和异养硝化性能[J].应用生态学报,23(7):1979-1984.
修海峰,朱仲元,丁爱中,等.2011.好氧反硝化菌种DF2的分离鉴定及生理生化特性分析[J].生态环境学报,20(8/9):1307-1314.
赵丹,于德爽,于津,等.2013.菌株ZD8的分离鉴定及其异养硝化和缺氧/好氧反硝化特性研究[J].环境科学学报,33(11):3007-3016.
周莉,李正魁,王易超,等.2013.纯种氨氧化菌短程反硝化特性[J].环境工程学报,7(4):1219-1224.
周迎芹,信欣,姚力,等.2013.一株高效异养硝化-好氧反硝化菌的分离鉴定及脱氮性能[J].环境工程学报,7(10):4127-4132.