源自生物絮团产絮凝剂的异养硝化-好氧反硝化菌xt1的鉴定及其脱氮特性
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摘要
为了高效进行水体脱氮,本实验从形成于凡纳滨对虾(Litopenaeus vannamei)养殖水体的生物絮团中分离到一株具产絮能力的脱氮菌xt1,经16S r RNA基因测序与生理生化分析确定菌株xt1为短小芽孢杆菌(Bacillus pumilus)。在此基础上,本文研究了该菌的脱氮特性。结果表明:菌株xt1最佳碳源为葡萄糖,以其为底物对氨氮、硝态氮去除率分别达95.56%和57.40%。以蔗糖为碳源亦具较高脱氮率,对氨氮、硝态氮去除率分别达69.95%和49.50%;该菌能利用有机氮加速生长,添加0.25%、0.5%、1%和2%的蛋白胨能促进OD600,分别达到0.925、1.034、1.103和1.314,均高于未加蛋白胨下的生长,且氨氮去除率均超过90%,硝态氮去除率均超过88%;该菌能适应20—200mg/L无机氮浓度;该菌能以NH4+-N、NO2–-N或NO3–-N为唯一氮源进行异养硝化-好氧反硝化,反应84h去除率分别达到94.16%、47.60%和91.17%。其中,该菌的硝化形式是将氨氮转化为气态氮脱除,其硝态氮反硝化形式是先将硝态氮转化为亚硝氮,再以气态氮脱除。在进行异养硝化-好氧反硝化同时,菌株xt1体现絮凝特性,絮凝率最高分别达到82.28%、73.15%和75.60%;此外,添加该菌于养殖水体中能加速生物絮团形成,同时提高脱氮率。各项结果表明,菌株xt1可作为水产养殖水体脱氮的备选菌株。
A nitrogen removal strain xt1 with flocculation ability was isolated from biofloc in aquaculture wastewater of Litopenaeus vannamei. The results of molecular biological, physiological, and biochemical identification showed that the strain xt1 was Bacillus pumilus. On this basis, the nitrogen removal characteristics of this strain were studied. The results show that the best carbon source of the strain xt1 was glucose. The removal rate of ammonia and nitrate was 95.56% and 57.40% with glucose as substrate, and when sucrose was used as the carbon source, the removal rates of ammonia and nitrate reached 69.95% and 49.50%, respectively. The strain could use organic nitrogen to accelerate the growth. When 0.25%, 0.5%, 1% and 2% peptone were added, OD600 was increased to 0.925, 1.034, 1.103 and 1.314, respectively, in which the removal rate of ammonia was more than 90%, the removal rate of nitrate was over 88%. The strain could adapt to the concentration of 20—200 mg/L inorganic nitrogen. The strain could use NH4+-N, NO2–-N or NO3-N as a sole nitrogen source, and the removal efficiency reached 94.16%, 47.60% and 91.17% within 84 h, respectively. Among them, the nitrifying form of the strain was the conversion of ammonia into gaseous nitrogen. The form of nitrate denitrification was converted to nitrate into nitrite, and then to gaseous nitrogen. In the process of heterotrophic nitrification-aerobic denitrification, the strain xt1 showed flocculation characteristics, and the highest flocculation rate reached 82.28%, 73.15%, and 75.60%, respectively. Besides, the addition of strain xt1 in the wastewater could accelerate the formation of biofloc and increase the rate of nitrogen removal. Therefore, strain xt1 can be used as an alternative strain for nitrogen removal from aquaculture wastewater.
A nitrogen removal strain xt1 with flocculation ability was isolated from biofloc in aquaculture wastewater of Litopenaeus vannamei. The results of molecular biological, physiological, and biochemical identification showed that the strain xt1 was Bacillus pumilus. On this basis, the nitrogen removal characteristics of this strain were studied. The results show that the best carbon source of the strain xt1 was glucose. The removal rate of ammonia and nitrate was 95.56% and 57.40% with glucose as substrate, and when sucrose was used as the carbon source, the removal rates of ammonia and nitrate reached 69.95% and 49.50%, respectively. The strain could use organic nitrogen to accelerate the growth. When 0.25%, 0.5%, 1% and 2% peptone were added, OD600 was increased to 0.925, 1.034, 1.103 and 1.314, respectively, in which the removal rate of ammonia was more than 90%, the removal rate of nitrate was over 88%. The strain could adapt to the concentration of 20—200 mg/L inorganic nitrogen. The strain could use NH4+-N, NO2–-N or NO3-N as a sole nitrogen source, and the removal efficiency reached 94.16%, 47.60% and 91.17% within 84 h, respectively. Among them, the nitrifying form of the strain was the conversion of ammonia into gaseous nitrogen. The form of nitrate denitrification was converted to nitrate into nitrite, and then to gaseous nitrogen. In the process of heterotrophic nitrification-aerobic denitrification, the strain xt1 showed flocculation characteristics, and the highest flocculation rate reached 82.28%, 73.15%, and 75.60%, respectively. Besides, the addition of strain xt1 in the wastewater could accelerate the formation of biofloc and increase the rate of nitrogen removal. Therefore, strain xt1 can be used as an alternative strain for nitrogen removal from aquaculture wastewater.
引文
中华人民共和国国家质量监督检验检疫总局,中国国家标准化管理委员会,2008.GB17378.4-2007海洋监测规范第4部分:海水分析.北京:中国标准出版社
方圣琼,胡雪峰,巫和昕,2004.水产养殖废水处理技术及应用.环境污染治理技术与设备,5(9):51-55
方海洋,王智,李建华等,2015.异养硝化-好氧反硝化菌粪产碱杆菌的脱氮特性.环境工程学报,9(2):983-988
王骁静,于德爽,李津等,2017.海洋异养硝化-好氧反硝化菌y6同步脱氮除碳特性.中国环境科学,37(2):686-695
东秀珠,蔡妙英,2001.常见细菌系统鉴定手册.北京:科学出版社,409-412
孙振,王秀华,黄倢,2013.一种微生物絮团的生化分析及其对凡纳滨对虾免疫力的影响.水产学报,37(3):473-480
孙庆花,于德爽,张培玉等,2016a.1株海洋异养硝化-好氧反硝化菌的分离鉴定及其脱氮特性.环境科学,37(2):647-654
孙庆花,于德爽,张培玉等,2016b.海洋菌株y3的分离鉴定及其异养硝化-好氧反硝化特性.环境科学,37(3):1089-1097
何霞,赵彬,吕剑等,2007.异养硝化细菌Bacillus sp.LY脱氮性能研究.环境科学,28(6):1404-1408
何腾霞,倪九派,李振轮等,2016.1株Arthrobacter arilaitensis菌的耐冷异养硝化和好氧反硝化作用.环境科学,37(3):1082-1088
余瑞兰,聂湘平,魏泰莉等,1999.分子氨和亚硝酸盐对鱼类的危害及其对策.中国水产科学,6(3):73-77
吴琦,李军,李陈等,2007.一株产胶原蛋白酶短小芽孢杆菌的分离与鉴定.中国皮革,36(17):16-19
吴建江,王兆阳,许培雅,2013.一株高效异养硝化菌的分离、鉴定及其氨氮去除特性.中国环境科学,33(7):1309-1315
宋宇杰,李屹,刘玉香等,2013.碳源和氮源对异养硝化好氧反硝化菌株Y1脱氮性能的影响.环境科学学报,33(9):2491-2497
张列宇,饶本强,熊瑛等,2010.人工湿地黑臭水体处理系统微生物脱氮机理研究.水生生物学报,34(2):256-261
张庆华,封永辉,王娟等,2011.地衣芽孢杆菌对养殖水体氨氮、残饵降解特性研究.水生生物学报,35(3):498-503
张家顺,苏真真,薛菲菲等,2015.2株氨氮去除菌的分离鉴定及去除率影响因素分析.水产学报,39(10):1549-1557
杜怡青,丁海涛,赵宇华,2011.芽孢杆菌葡萄糖脱氢酶结构与功能研究进展.科技通报,27(1):50-56
杨章武,杨铿,张哲等,2015.基于宏基因组测序技术分析凡纳滨对虾育苗中生物絮团细菌群落结构.福建水产,37(2):91-97
辛玉峰,曲晓华,袁梦冬等,2011.一株异养硝化-反硝化不动杆菌的分离鉴定及脱氮活性.微生物学报,51(12):1646-1654
罗国芝,陈家捷,于文杰等,2016.一株新型异养硝化细菌处理养殖水的效果.环境工程学报,10(8):4206-4212
信欣,姚力,鲁磊等,2014.耐高氨氮异养硝化-好氧反硝化菌TN-14的鉴定及其脱氮性能.环境科学,(10):3926-3932
郝红红,陈浚,程鳌等,2013.一株好氧反硝化菌的筛选鉴定及固定化研究.环境科学学报,33(11):3017-3024
郭婧,王娟,宋增福等,2013.短小芽孢杆菌X93及胞外产物抑菌活性的研究.水产学报,37(10):1564-1571
梁贤,任勇翔,杨垒等,2015.异养硝化-好氧反硝化菌YL的脱氮特性.环境科学,36(5):1749-1756
董双林,2011.高效低碳--中国水产养殖业发展的必由之路.水产学报,35(10):1595-1600
蒋鹏,赵春贵,杨素萍,2014.小分子有机碳、氮源对海洋着色菌(Marichromatium gracile)生长和去除高浓度无机三态氮的影响.海洋与湖沼,45(6):1218-1224
谢凤行,张峰峰,周可等,2012.一株解淀粉芽胞杆菌的分离鉴定及水质净化效果的研究.环境科学学报,32(11):2781-2788
Buchanan R E,Gibbons N E,1984.伯杰细菌鉴定手册.中国科学院微生物研究所《伯杰细菌鉴定手册》翻译组译.北京:科学出版社
Avnimelech Y,1999.Carbon/nitrogen ratio as a control element in aquaculture systems.Aquaculture,176(3-4):227-235
Avnimelech Y,Schryver P D,Emmereciano M et al,2012.Biofloc Technology.Baton Rouge:The World Aquaculture Society,37,50,146,201
Crab R,Avnimelech Y,Deeoirdt T et al,2007.Nitrogen removal techniques in aquaculture fora sustainable production.Aquaculture,270(1-4):1-14
Forster C F,Dallas Newton J,1980.Activated sludge on settlement some suppositions and suggestion.Water Pollution Control,79(3):338-351
Hargreaves J A,2006.Photosynthetic suspended-growth systems in aquaculture.Aquaculture Engineering,34(3):344-363
Lu Lu,Tan H X,Luo G Z et al,2012.The effects of Bacillus subtilis on nitrogen recycling from aquaculture solid waste using heterotrophic nitrogen as simulation in sequencing batch reactors.Bioresource Technology,124:180-185
Richardson D J,Wehrfritz J M,Keech A et al,1998.The diversity of redox proteins involved in bacterial heterotrophic nitrification andaerobic denitrification.Biochemical Society Transactions,26(3):401-408
Yang X P,Wang S M,Zhang D W et al,2011.Isolation and nitrogen removal characteristics of an aerobic heterotrophic nitrifying-denitrifying bacterium,Bacillus subtilis A1.Bioresource Technology,102(2):854-862
Zhao P,Huang J,Wang X H et al,2012.The application of bioflocs technology in high-intensive,zero exchange farming systems of Marsupenaeus japonicus.Aquaculture,354-355:97-106
方圣琼,胡雪峰,巫和昕,2004.水产养殖废水处理技术及应用.环境污染治理技术与设备,5(9):51-55
方海洋,王智,李建华等,2015.异养硝化-好氧反硝化菌粪产碱杆菌的脱氮特性.环境工程学报,9(2):983-988
王骁静,于德爽,李津等,2017.海洋异养硝化-好氧反硝化菌y6同步脱氮除碳特性.中国环境科学,37(2):686-695
东秀珠,蔡妙英,2001.常见细菌系统鉴定手册.北京:科学出版社,409-412
孙振,王秀华,黄倢,2013.一种微生物絮团的生化分析及其对凡纳滨对虾免疫力的影响.水产学报,37(3):473-480
孙庆花,于德爽,张培玉等,2016a.1株海洋异养硝化-好氧反硝化菌的分离鉴定及其脱氮特性.环境科学,37(2):647-654
孙庆花,于德爽,张培玉等,2016b.海洋菌株y3的分离鉴定及其异养硝化-好氧反硝化特性.环境科学,37(3):1089-1097
何霞,赵彬,吕剑等,2007.异养硝化细菌Bacillus sp.LY脱氮性能研究.环境科学,28(6):1404-1408
何腾霞,倪九派,李振轮等,2016.1株Arthrobacter arilaitensis菌的耐冷异养硝化和好氧反硝化作用.环境科学,37(3):1082-1088
余瑞兰,聂湘平,魏泰莉等,1999.分子氨和亚硝酸盐对鱼类的危害及其对策.中国水产科学,6(3):73-77
吴琦,李军,李陈等,2007.一株产胶原蛋白酶短小芽孢杆菌的分离与鉴定.中国皮革,36(17):16-19
吴建江,王兆阳,许培雅,2013.一株高效异养硝化菌的分离、鉴定及其氨氮去除特性.中国环境科学,33(7):1309-1315
宋宇杰,李屹,刘玉香等,2013.碳源和氮源对异养硝化好氧反硝化菌株Y1脱氮性能的影响.环境科学学报,33(9):2491-2497
张列宇,饶本强,熊瑛等,2010.人工湿地黑臭水体处理系统微生物脱氮机理研究.水生生物学报,34(2):256-261
张庆华,封永辉,王娟等,2011.地衣芽孢杆菌对养殖水体氨氮、残饵降解特性研究.水生生物学报,35(3):498-503
张家顺,苏真真,薛菲菲等,2015.2株氨氮去除菌的分离鉴定及去除率影响因素分析.水产学报,39(10):1549-1557
杜怡青,丁海涛,赵宇华,2011.芽孢杆菌葡萄糖脱氢酶结构与功能研究进展.科技通报,27(1):50-56
杨章武,杨铿,张哲等,2015.基于宏基因组测序技术分析凡纳滨对虾育苗中生物絮团细菌群落结构.福建水产,37(2):91-97
辛玉峰,曲晓华,袁梦冬等,2011.一株异养硝化-反硝化不动杆菌的分离鉴定及脱氮活性.微生物学报,51(12):1646-1654
罗国芝,陈家捷,于文杰等,2016.一株新型异养硝化细菌处理养殖水的效果.环境工程学报,10(8):4206-4212
信欣,姚力,鲁磊等,2014.耐高氨氮异养硝化-好氧反硝化菌TN-14的鉴定及其脱氮性能.环境科学,(10):3926-3932
郝红红,陈浚,程鳌等,2013.一株好氧反硝化菌的筛选鉴定及固定化研究.环境科学学报,33(11):3017-3024
郭婧,王娟,宋增福等,2013.短小芽孢杆菌X93及胞外产物抑菌活性的研究.水产学报,37(10):1564-1571
梁贤,任勇翔,杨垒等,2015.异养硝化-好氧反硝化菌YL的脱氮特性.环境科学,36(5):1749-1756
董双林,2011.高效低碳--中国水产养殖业发展的必由之路.水产学报,35(10):1595-1600
蒋鹏,赵春贵,杨素萍,2014.小分子有机碳、氮源对海洋着色菌(Marichromatium gracile)生长和去除高浓度无机三态氮的影响.海洋与湖沼,45(6):1218-1224
谢凤行,张峰峰,周可等,2012.一株解淀粉芽胞杆菌的分离鉴定及水质净化效果的研究.环境科学学报,32(11):2781-2788
Buchanan R E,Gibbons N E,1984.伯杰细菌鉴定手册.中国科学院微生物研究所《伯杰细菌鉴定手册》翻译组译.北京:科学出版社
Avnimelech Y,1999.Carbon/nitrogen ratio as a control element in aquaculture systems.Aquaculture,176(3-4):227-235
Avnimelech Y,Schryver P D,Emmereciano M et al,2012.Biofloc Technology.Baton Rouge:The World Aquaculture Society,37,50,146,201
Crab R,Avnimelech Y,Deeoirdt T et al,2007.Nitrogen removal techniques in aquaculture fora sustainable production.Aquaculture,270(1-4):1-14
Forster C F,Dallas Newton J,1980.Activated sludge on settlement some suppositions and suggestion.Water Pollution Control,79(3):338-351
Hargreaves J A,2006.Photosynthetic suspended-growth systems in aquaculture.Aquaculture Engineering,34(3):344-363
Lu Lu,Tan H X,Luo G Z et al,2012.The effects of Bacillus subtilis on nitrogen recycling from aquaculture solid waste using heterotrophic nitrogen as simulation in sequencing batch reactors.Bioresource Technology,124:180-185
Richardson D J,Wehrfritz J M,Keech A et al,1998.The diversity of redox proteins involved in bacterial heterotrophic nitrification andaerobic denitrification.Biochemical Society Transactions,26(3):401-408
Yang X P,Wang S M,Zhang D W et al,2011.Isolation and nitrogen removal characteristics of an aerobic heterotrophic nitrifying-denitrifying bacterium,Bacillus subtilis A1.Bioresource Technology,102(2):854-862
Zhao P,Huang J,Wang X H et al,2012.The application of bioflocs technology in high-intensive,zero exchange farming systems of Marsupenaeus japonicus.Aquaculture,354-355:97-106