土生拉乌尔菌TGRB3的生物学特性及其不同氧气浓度条件下的汞甲基化
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摘要
从三峡库区消落带土壤中分离、纯化得到1株具有汞甲基化能力的细菌,对其形态、生理生化特征及16S rRNA基因序列进行分析后,鉴定为土生拉乌尔菌,并命名为RaouLtella terrigena TGRB3.研究发现,菌株TGRB3具有强酸碱和盐度适应能力,为中高温型菌,在pH=4～9和盐度0.2%～4.0%范围内均能正常生长,最适生长温度为25℃.在Hg~(2+)浓度为1000μg·L~(-1)时,菌株生长受到显著抑制.此外,在初始Hg~(2+)浓度为300 ng·L~(-1)的条件下探讨了该菌在不同氧气浓度(0、7%、14%和21%)下的生长情况及汞甲化能力.结果表明,该菌的生长不受氧气浓度的制约,但在有氧条件下生长更好,且在厌氧或低氧浓度条件下具有更强且稳定的汞甲基化能力.在氧气浓度为14%和21%的条件下,该菌的最大甲基汞含量分别为(0.54±0.01)和(1.62±0.08) ng·L~(-1),试验后期表现出较强的去甲基化现象,培养54 h后的甲基汞含量显著降低为(0.08±0.02)和(0.05±0.00) ng·L~(-1);而在氧气浓度为0和7%的条件下,最大甲基汞含量分别为(6.75±1.75)和(3.24±0.74) ng·L~(-1),且在试验周期内甲基汞持续产生,平均浓度分别为(3.23±1.39)和(1.66±0.71) ng·L~(-1).本试验结果可望为三峡库区消落带土壤汞生物甲基化机理的深入研究及汞污染风险评价等提供全新的试验材料.
A bacterial strain capable of Hg methylation was isolated from the soil of the water-level-fluctuation-zone(WLFZ) of the Three Gorge Reservoir, in Shibaozhai, Zhongxian Country, Chongqing, Southwest China. Through morphological and physiological characterization, and 16 S rRNA gene sequence analysis, this isolate was identified as RaouLtella terrigena sp. and named as R. terrigena TGRB3(accession number: MK102091). Strain TGRB3 has strong acid-base and salinity tolerance, and could survive under pH from 4 to 9, and 0.2%～4.0% of salinity. It is a mesophilic strain, with the optimum growth temperature of 25 ℃. When Hg~(2+) concentration was 1000 μg·L~(-1), the growth of strain TGRB3 was significantly inhibited. In addition, its Hg methylation ability under different oxygen concentrations(0, 7%, 14% and 21%) was studied at the initial Hg~(2+) concentration of 300 ng·L~(-1). Results show that the growth of strain TGRB3 was not restricted by oxygen concentration, but grew better under aerobic conditions. Besides, it had a stronger and more stable Hg methylation ability under anaerobic and low oxygen concentration. Under the oxygen concentration of 14% and 21%, the maximum MeHg levels were(0.54±0.01) and(1.62±0.08) ng·L~(-1), which decreased significantly to(0.08±0.02) and(0.05±0.00) ng·L~(-1) at the 54 h, respectively. When the oxygen concentration was 0 and 7%, MeHg were produced continuously during the whole incubation period, reaching the maximum MeHg levels at(6.75±1.75) and(3.24±0.74) ng·L~(-1), respectively, and(3.23±1.39) and(1.66±0.71) ng·L~(-1), in average. This study might be expected to provide new experimental data for in-depth study of the Hg bio-methylation mechanism and the risk assessment of Hg pollution in the WLFZ of the Three Gorge Reservoir.
A bacterial strain capable of Hg methylation was isolated from the soil of the water-level-fluctuation-zone(WLFZ) of the Three Gorge Reservoir, in Shibaozhai, Zhongxian Country, Chongqing, Southwest China. Through morphological and physiological characterization, and 16 S rRNA gene sequence analysis, this isolate was identified as RaouLtella terrigena sp. and named as R. terrigena TGRB3(accession number: MK102091). Strain TGRB3 has strong acid-base and salinity tolerance, and could survive under pH from 4 to 9, and 0.2%～4.0% of salinity. It is a mesophilic strain, with the optimum growth temperature of 25 ℃. When Hg~(2+) concentration was 1000 μg·L~(-1), the growth of strain TGRB3 was significantly inhibited. In addition, its Hg methylation ability under different oxygen concentrations(0, 7%, 14% and 21%) was studied at the initial Hg~(2+) concentration of 300 ng·L~(-1). Results show that the growth of strain TGRB3 was not restricted by oxygen concentration, but grew better under aerobic conditions. Besides, it had a stronger and more stable Hg methylation ability under anaerobic and low oxygen concentration. Under the oxygen concentration of 14% and 21%, the maximum MeHg levels were(0.54±0.01) and(1.62±0.08) ng·L~(-1), which decreased significantly to(0.08±0.02) and(0.05±0.00) ng·L~(-1) at the 54 h, respectively. When the oxygen concentration was 0 and 7%, MeHg were produced continuously during the whole incubation period, reaching the maximum MeHg levels at(6.75±1.75) and(3.24±0.74) ng·L~(-1), respectively, and(3.23±1.39) and(1.66±0.71) ng·L~(-1), in average. This study might be expected to provide new experimental data for in-depth study of the Hg bio-methylation mechanism and the risk assessment of Hg pollution in the WLFZ of the Three Gorge Reservoir.
引文
Bisogni J J,Lawrence A W.1975.Kinetics of mercury methylation in aerobic and anaerobic aquatic environments[J].Journal of Water Pollution Control Federation,47(1):135-152
Bowles K C,Apte S C,Maher W A,et al.2001.Bioaccumulation and biomagnification of mercury in Lake Murray,Papua New Guinea[J].Journal Canadien Des Sciences Halieutiques Et Aquatiques,58(58):888-897
Brunberg A K,Blomqvist P.2001.Quantification of anthropogenic threats to lakes in a lowland county of central Sweden[J].Ambio,30(3):127-134
Delaune R D,Jugsujinda A,Devai I,et al.2004.Relationship of sediment redox conditions to methyl mercury in surface sediment of Louisiana Lakes[J].Environmental Letters,39(8):1925-1933
丁振华,王文华,庄敏.2005.汞的界面地球化学研究进展[J].海洋科学,29(10):54-57
Fleming E J,Mack E E,Green P G,et al.2006.Mercury methylation from unexpected sources:molybdate-inhibited freshwater sediments and an iron-reducing bacterium[J].Applied and Environmental Microbiology,72(1):457-464
冯新斌.2011.水库汞的生物地球化学循环研究进展[J].环保科技,17(1):1-5
冯新斌,洪业汤.1997.汞的环境地球化学研究进展[J].地球与环境,32(4):503-530
高亚辉,荆红梅,黄德强,等.2002.海洋微藻胞外产物研究进展[J].海洋科学,26(3):35-38
Hamdy M K,Noyes O R.1975.Formation of methyl mercury by bacteria[J].Appl Microbiol,30(3):424-432
Hamelin S,Amyot M,Barkay T,et al.2011.Methanogens:principal methylators of mercury in Lake Periphyton[J].Environmental Science & Technology,45(18):7693-7700
Hamelin S,Planas D,Amyot M.2015.Mercury methylation and demethylation by periphyton biofilms and their host in a fluvial wetland of the St.Lawrence River (QC,Canada)[J].Science of the Total Environment,512-513:464-471
Heyes A,Mason R P,Kim E H,et al.2006.Mercury methylation in estuaries:Insights from using measuring rates using stable mercury isotopes[J].Marine Chemistry,102(1):134-147
贺春凤.2013.三峡库区消落带土壤淹水过程汞释放与甲基化特征的模拟研究[D].重庆:西南大学
胡海燕.2012.厌氧微生物与汞的相互作用:结合、氧化、还原和甲基化[D].北京:中国科学院大学
何熙.2013.三峡库区消落带土壤(沉积物)总汞及甲基汞变化特征[D].重庆:西南大学
Jackson T A.2016.Historical variations in the stable isotope composition of mercury in a sediment core from a riverine lake:Effects of dams,pulp and paper mill wastes,and mercury from a chlor-alkali plant[J].Applied Geochemistry,71(2016):86-98
Janssen S E,Schaefer J K,Barkay T,et al.2016.Fractionation of mercury stable isotopes during microbial methylmercury production by iron- and sulfate-reducing bacteria[J].Environmental Science & Technology,50(15):8077
蒋红梅,冯新斌,梁琏,等.2004.蒸馏-乙基化GC-CVAFS法测定天然水体中的甲基汞[J].中国环境科学,24(5):568-571
Kearns K D,Hunter M D.2010.Green algal extracellular products regulate antialgal toxin production in a cyanobacterium[J].Environmental Microbiology,2(3):291-297
Kumano S,Sugiyama M,Yamada M,et al.2013.Development of a portable mass spectrometer characterized by discontinuous sample gas introduction,a low-pressure dielectric barrier discharge ionization source,and a vacuumed headspace technique[J].Analytical Chemistry,85(10):5033-5039
康义,郭泉水,程瑞梅,等.2010.三峡库区消落带土壤物理性质变化[J].林业科学,46(6):1-5
Lindberg S E,Kim K H,Meyers T P,et al.1995.Micrometeorological gradient approach for quantifying air/surface exchange of mercury vapor:Tests over contaminated soils[J].Environmental Science and Technology,29(1):126-135
Lu X,Liu Y,Johs A,et al.2016.Anaerobic mercury methylation and demethylation by Geobacter bemidjiensis Bem[J].Environmental Science & Technology,50(8):4366-4373
Miller T L,Wolin M J.1974.A serum bottle modification of the Hungate technique for cultivating obligate anaerobes[J].Applied microbiology,27(5):985-987
马力,于瑞林,李杰,等.2018.基于时间序列分析并预测三峡库区局地环境温度的变化[J].中国农业气象,39(1):9-17
马利民,张明,滕衍行,等.2008.三峡库区消落区周期性干湿交替环境对土壤磷释放的影响[J].环境科学,29(4):1035-1039
马诗淳,罗辉,杨丽丽,等.2009.中温厌氧纤维素菌的分离鉴定,系统发育学分析及其酶学性质的研究[J].中国沼气,27(3):3-7
孟紫强.2000.环境毒理学[M].北京:中国环境科学出版社
Nalewajko C,Fay K L.1980.Significance of algal extracellular products to bacteria in Lakes and in Cultures[J].Microbial Ecology,6(3):199-207
邱权,陈雯莉.2013.三峡库区小江流域消落区土壤微生物多样性[J].华中农业大学学报,32(3):15-20
Ramamoorthy S,Cheng T C,Kushner D J.1982.Effect of microbial life stages on the fate of methylmercury in natural waters[J].Bulletin of Environmental Contamination & Toxicology,29(2):167-173
R.E.布坎南.1984.伯杰细菌鉴定手册[M].北京:科学出版社
陶兰兰,向玉萍,王定勇,等.2016.1株兼具好、厌氧汞甲基化能力细菌的分离鉴定[J].环境科学,37(11):4389-4394
Vonk J W,Sijpesteijn A K.1973.Studies on the methylation of mercuric chloride by pure cultures of bacteria and fungi[J].Antonie Van Leeuwenhoek,39(1):505-513
肖国生,胡廷章,唐华丽,等.2011.三峡水库消落带淹没前后土壤微生物生态分布及优势菌群的鉴定[J].江苏农业科学,39(4):493-496
Yang H J,Yue Q.2012.The modification of glucose levels and N source in the Hungate′s medium to stimulate the production of fibrolytic enzymes of Anaeromyces sp.YQ3 grown on corn stalks[J].Animal Feed Science & Technology,171(2/4):146-153
Yu C H,Yu H F,Ho K C,et al.2011.Isolation and identification of a new tetrodotoxin-producing bacterial species,Raoultella terrigena,from Hong Kong marine puffer fish Takifugu niphobles[J].Marine Drugs,9(12):2384-2396
Xiang Y P,Xiang,Du H X,Shen H,et al.2014.Dynamics of total culturable bacteria and its relationship with methylmercury in the soils of the water level fluctuation zone of the Three Gorges Reservoir[J].Science Bulletin,59(24):2966-2972
阴皎阳,尹大强,王锐.2014.沉积物中汞的甲基化研究进展[J].生态毒理学报,9(5):819-831
袁辉,王里奥,詹艳慧,等.2006.三峡库区消落带健康评价指标体系[J].长江流域资源与环境,15(2):249-253
Zhang J,Wang F Y,House J D,et al.2004.Thiols in wetland interstitial waters and their role in mercury and methylmercury speciation[J].Limnology and Oceanography,49(6):2276-2286
张成,陈宏,王定勇,等.2014.三峡库区消落带土壤汞形态分布与风险评价[J].环境科学,35(3):1060-1067
张金洋,王定勇,石孝洪.2004.三峡水库消落区淹水后土壤性质变化的模拟研究[J].水土保持学报,18(6):120-123
曾艳,陈强,王敏,等.2009.一株高抗汞细菌的分离鉴定及其抗性基因的克隆与表达[J].微生物学报,49(12):1628-1633
Bowles K C,Apte S C,Maher W A,et al.2001.Bioaccumulation and biomagnification of mercury in Lake Murray,Papua New Guinea[J].Journal Canadien Des Sciences Halieutiques Et Aquatiques,58(58):888-897
Brunberg A K,Blomqvist P.2001.Quantification of anthropogenic threats to lakes in a lowland county of central Sweden[J].Ambio,30(3):127-134
Delaune R D,Jugsujinda A,Devai I,et al.2004.Relationship of sediment redox conditions to methyl mercury in surface sediment of Louisiana Lakes[J].Environmental Letters,39(8):1925-1933
丁振华,王文华,庄敏.2005.汞的界面地球化学研究进展[J].海洋科学,29(10):54-57
Fleming E J,Mack E E,Green P G,et al.2006.Mercury methylation from unexpected sources:molybdate-inhibited freshwater sediments and an iron-reducing bacterium[J].Applied and Environmental Microbiology,72(1):457-464
冯新斌.2011.水库汞的生物地球化学循环研究进展[J].环保科技,17(1):1-5
冯新斌,洪业汤.1997.汞的环境地球化学研究进展[J].地球与环境,32(4):503-530
高亚辉,荆红梅,黄德强,等.2002.海洋微藻胞外产物研究进展[J].海洋科学,26(3):35-38
Hamdy M K,Noyes O R.1975.Formation of methyl mercury by bacteria[J].Appl Microbiol,30(3):424-432
Hamelin S,Amyot M,Barkay T,et al.2011.Methanogens:principal methylators of mercury in Lake Periphyton[J].Environmental Science & Technology,45(18):7693-7700
Hamelin S,Planas D,Amyot M.2015.Mercury methylation and demethylation by periphyton biofilms and their host in a fluvial wetland of the St.Lawrence River (QC,Canada)[J].Science of the Total Environment,512-513:464-471
Heyes A,Mason R P,Kim E H,et al.2006.Mercury methylation in estuaries:Insights from using measuring rates using stable mercury isotopes[J].Marine Chemistry,102(1):134-147
贺春凤.2013.三峡库区消落带土壤淹水过程汞释放与甲基化特征的模拟研究[D].重庆:西南大学
胡海燕.2012.厌氧微生物与汞的相互作用:结合、氧化、还原和甲基化[D].北京:中国科学院大学
何熙.2013.三峡库区消落带土壤(沉积物)总汞及甲基汞变化特征[D].重庆:西南大学
Jackson T A.2016.Historical variations in the stable isotope composition of mercury in a sediment core from a riverine lake:Effects of dams,pulp and paper mill wastes,and mercury from a chlor-alkali plant[J].Applied Geochemistry,71(2016):86-98
Janssen S E,Schaefer J K,Barkay T,et al.2016.Fractionation of mercury stable isotopes during microbial methylmercury production by iron- and sulfate-reducing bacteria[J].Environmental Science & Technology,50(15):8077
蒋红梅,冯新斌,梁琏,等.2004.蒸馏-乙基化GC-CVAFS法测定天然水体中的甲基汞[J].中国环境科学,24(5):568-571
Kearns K D,Hunter M D.2010.Green algal extracellular products regulate antialgal toxin production in a cyanobacterium[J].Environmental Microbiology,2(3):291-297
Kumano S,Sugiyama M,Yamada M,et al.2013.Development of a portable mass spectrometer characterized by discontinuous sample gas introduction,a low-pressure dielectric barrier discharge ionization source,and a vacuumed headspace technique[J].Analytical Chemistry,85(10):5033-5039
康义,郭泉水,程瑞梅,等.2010.三峡库区消落带土壤物理性质变化[J].林业科学,46(6):1-5
Lindberg S E,Kim K H,Meyers T P,et al.1995.Micrometeorological gradient approach for quantifying air/surface exchange of mercury vapor:Tests over contaminated soils[J].Environmental Science and Technology,29(1):126-135
Lu X,Liu Y,Johs A,et al.2016.Anaerobic mercury methylation and demethylation by Geobacter bemidjiensis Bem[J].Environmental Science & Technology,50(8):4366-4373
Miller T L,Wolin M J.1974.A serum bottle modification of the Hungate technique for cultivating obligate anaerobes[J].Applied microbiology,27(5):985-987
马力,于瑞林,李杰,等.2018.基于时间序列分析并预测三峡库区局地环境温度的变化[J].中国农业气象,39(1):9-17
马利民,张明,滕衍行,等.2008.三峡库区消落区周期性干湿交替环境对土壤磷释放的影响[J].环境科学,29(4):1035-1039
马诗淳,罗辉,杨丽丽,等.2009.中温厌氧纤维素菌的分离鉴定,系统发育学分析及其酶学性质的研究[J].中国沼气,27(3):3-7
孟紫强.2000.环境毒理学[M].北京:中国环境科学出版社
Nalewajko C,Fay K L.1980.Significance of algal extracellular products to bacteria in Lakes and in Cultures[J].Microbial Ecology,6(3):199-207
邱权,陈雯莉.2013.三峡库区小江流域消落区土壤微生物多样性[J].华中农业大学学报,32(3):15-20
Ramamoorthy S,Cheng T C,Kushner D J.1982.Effect of microbial life stages on the fate of methylmercury in natural waters[J].Bulletin of Environmental Contamination & Toxicology,29(2):167-173
R.E.布坎南.1984.伯杰细菌鉴定手册[M].北京:科学出版社
陶兰兰,向玉萍,王定勇,等.2016.1株兼具好、厌氧汞甲基化能力细菌的分离鉴定[J].环境科学,37(11):4389-4394
Vonk J W,Sijpesteijn A K.1973.Studies on the methylation of mercuric chloride by pure cultures of bacteria and fungi[J].Antonie Van Leeuwenhoek,39(1):505-513
肖国生,胡廷章,唐华丽,等.2011.三峡水库消落带淹没前后土壤微生物生态分布及优势菌群的鉴定[J].江苏农业科学,39(4):493-496
Yang H J,Yue Q.2012.The modification of glucose levels and N source in the Hungate′s medium to stimulate the production of fibrolytic enzymes of Anaeromyces sp.YQ3 grown on corn stalks[J].Animal Feed Science & Technology,171(2/4):146-153
Yu C H,Yu H F,Ho K C,et al.2011.Isolation and identification of a new tetrodotoxin-producing bacterial species,Raoultella terrigena,from Hong Kong marine puffer fish Takifugu niphobles[J].Marine Drugs,9(12):2384-2396
Xiang Y P,Xiang,Du H X,Shen H,et al.2014.Dynamics of total culturable bacteria and its relationship with methylmercury in the soils of the water level fluctuation zone of the Three Gorges Reservoir[J].Science Bulletin,59(24):2966-2972
阴皎阳,尹大强,王锐.2014.沉积物中汞的甲基化研究进展[J].生态毒理学报,9(5):819-831
袁辉,王里奥,詹艳慧,等.2006.三峡库区消落带健康评价指标体系[J].长江流域资源与环境,15(2):249-253
Zhang J,Wang F Y,House J D,et al.2004.Thiols in wetland interstitial waters and their role in mercury and methylmercury speciation[J].Limnology and Oceanography,49(6):2276-2286
张成,陈宏,王定勇,等.2014.三峡库区消落带土壤汞形态分布与风险评价[J].环境科学,35(3):1060-1067
张金洋,王定勇,石孝洪.2004.三峡水库消落区淹水后土壤性质变化的模拟研究[J].水土保持学报,18(6):120-123
曾艳,陈强,王敏,等.2009.一株高抗汞细菌的分离鉴定及其抗性基因的克隆与表达[J].微生物学报,49(12):1628-1633