扇贝内脏团中耐镉菌株的分离及其吸附镉机理
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摘要
为探讨扇贝内脏团中微生态系统与扇贝高能力富集镉(Cd)的关系,从自然环境中采集的栉孔扇贝内脏团中分离、纯化了2株耐Cd细菌(编号为菌株A和B),运用16S rDNA基因序列进行分子鉴定,通过金属吸附实验研究了耐Cd菌株对Cd的吸附能力及其对Cd的吸附特性,并利用扫描电镜、透射电镜相结合的方法研究了Cd胁迫条件下耐镉菌株细胞形态与结构变化,探讨了其对Cd的吸附机理。结果显示,2株菌株(A和B)在固体培养基上能耐受Cd的浓度分别为100和80 mg/L。经16S rDNA测序鉴定菌株A与Nitratireductor sp.亲源关系最近,菌株B与Ruegeria sp.亲源关系最近。2株菌株对Cd的吸附率远高于铜(Cu)、锰(Mn)、锌(Zn)和铅(Pb)等重金属。在50 mg/L Cd浓度的液体培养基中,菌株A、B对Cd富集量分别为48.57和42.14 mg/g,富集系数分别为971.4和842.8。电镜观察结果显示,经过Cd处理后,2菌株数量均有所减少,细胞中出现空泡,菌株A细胞外沉淀增多,菌株B表面变得粗糙且出现凹陷,表明胞外沉积作用可能是耐Cd菌株对Cd富集作用的重要途径。
In order to explore the relationship between the role of bacteria community and the high capacity for accumulation of Cd in the digestive tissues in scallops, two cadmium(Cd)-resistant bacterial strains(named A and B)were isolated from the digestive gland of scallop which were collected from the natural marine environment. Two bacterial strains were classified using 16 S rDNA sequence analysis. And the ability of two bacterial strains for the accumulation of Cd and their adsorption characteristics of Cd were researched by the metal adsorption experiments. In addition, the combined technique of scanning electron microscopy(SEM) observation and transmission electron microscopy(TEM) observation was used to observe the cell morphology and inside structure of the bacterial strains under Cd exposure, and the cadmium precipitation and distribution outside and inside the cell were studied to explore the adsorption mechanism of Cd for two bacterial strains. Results showed that the two bacterial strains(A and B) could tolerate Cd2+ up to 100 mg/L and 80 mg/L in solid medium respectively. Based on 16 S rDNA homology analysis, bacterial A sequences shared high similarities with Nitratireductor sp. and bacterial B was classified as Ruegeria sp. Their adsorption ability of 5 kinds of heavy metal ions(Cd2+, Cu2+, Mn2+, Zn2+ and Pb2+) were examined and both of them had higher adsorption of Cd than that of other four kinds of metals. In addition, bacterial strain A and B could accumulate Cd to 48.57 mg/g and 42.14 mg/g respectively in 50 mg/L liquid mediums and the accumulation factor for Cd was 971.4 and 842.8 respectively. Scanning electron microscopy(SEM) indicated that the amount of two strains both decreased and empty cavity occurred in some cells after Cd exposure. Obvious Cd precipitates appeared on A strain and more rough surface and irregular convex also appeared on B strain; therefore, extracellular precipitation might be an important way for two bacterial strains to resist and accumulate Cd.
In order to explore the relationship between the role of bacteria community and the high capacity for accumulation of Cd in the digestive tissues in scallops, two cadmium(Cd)-resistant bacterial strains(named A and B)were isolated from the digestive gland of scallop which were collected from the natural marine environment. Two bacterial strains were classified using 16 S rDNA sequence analysis. And the ability of two bacterial strains for the accumulation of Cd and their adsorption characteristics of Cd were researched by the metal adsorption experiments. In addition, the combined technique of scanning electron microscopy(SEM) observation and transmission electron microscopy(TEM) observation was used to observe the cell morphology and inside structure of the bacterial strains under Cd exposure, and the cadmium precipitation and distribution outside and inside the cell were studied to explore the adsorption mechanism of Cd for two bacterial strains. Results showed that the two bacterial strains(A and B) could tolerate Cd2+ up to 100 mg/L and 80 mg/L in solid medium respectively. Based on 16 S rDNA homology analysis, bacterial A sequences shared high similarities with Nitratireductor sp. and bacterial B was classified as Ruegeria sp. Their adsorption ability of 5 kinds of heavy metal ions(Cd2+, Cu2+, Mn2+, Zn2+ and Pb2+) were examined and both of them had higher adsorption of Cd than that of other four kinds of metals. In addition, bacterial strain A and B could accumulate Cd to 48.57 mg/g and 42.14 mg/g respectively in 50 mg/L liquid mediums and the accumulation factor for Cd was 971.4 and 842.8 respectively. Scanning electron microscopy(SEM) indicated that the amount of two strains both decreased and empty cavity occurred in some cells after Cd exposure. Obvious Cd precipitates appeared on A strain and more rough surface and irregular convex also appeared on B strain; therefore, extracellular precipitation might be an important way for two bacterial strains to resist and accumulate Cd.
引文
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[15] Wang W X. Metal bioaccumulation in bivalve mollusks:recent progress[M]//Villalba A, Reguera B,Romalde J L, et al. Molluscan Shellfish Safety. Spain:Intergovernmental Oceanographic Commission of UNESCO and Conselleria de Pesca e Asuntos Maritimos da Xunta de Galicia, 2003:503-520.
[16] Wang W X,Rainbow P S. Comparative approaches to understand metal bioaccumulation in aquatic animals[J].Comparative Biochemistry and Physiology Part C:Toxicology&Pharmacology, 2008, 148(4):315-323.
[17]梁淑君,王雅玲,孙力军,等.海洋耐镉微生物筛选及其生物学特性[J].海洋环境科学,2012, 31(4):488-491.Liang S J, Wang Y L, Sun L J, et al. Screen and biological characteristics of marine resistance cadmium microbe[J]. Marine Environmental Science,2012, 31(4):488-491(in Chinese).
[18]许钦坤,赵翠燕.耐镉菌株的筛选及生物学特性[J].江苏农业科学,2015, 43(2):317-318.Xu Q K, Zhao C Y. Screening and biological characteristics of cadmium resistant strains[J]. Jiangsu Agricultural Sciences, 2015, 43(2):317-318(in Chinese).
[19]沈秋悦,曹志强,朱月芳,等.一株耐镉细菌的分离鉴定及其吸附条件的优化[J].土壤,2016,48(3):615-620.Shen Q Y, Cao Z Q, Zhu Y F, et al. Isolation of a cd-resistant bacterium and optimization of its bio-accumulation condition[J]. Soils,2016, 48(3):615-620(in Chinese).
[20]周丽英,叶仁杰,林淑婷,等.水稻根际耐镉细菌的筛选与鉴定[J].中国生态农业学报,2012, 20(5):597-603.Zhou L Y, Ye R J, Lin S T, et al. Screening and identification of cadmium-tolerant bacteria from rhizosphere soils under rice[J]. Chinese Journal of Eco-Agriculture,2012, 20(5):597-603(in Chinese).
[21]刘爱民,黄为一.耐镉菌株的分离及其对Cd2+的吸附富集[J].中国环境科学,2006, 26(1):91-95.Liu A M, Huang W Y. Separation of tolerant cadmium bacterium strain and its accumulation adsorption of Cd2+[J]. China Environmental Science, 2006, 26(1):91-95(in Chinese).
[22]吴海江.耐Cd细菌的筛选及抗性机理研究[D].成都:西南交通大学,2009.Wu H J. Screening and resistance mechanism study on a cadmium resistant strain[D]. Chengdu:Southwest Jiaotong University, 2009(in Chinese).
[23]陈美标,郭建华,姚青,等.大宝山矿区耐Cd2+细菌的分离鉴定及其生物学特性[J].微生物学通报,2012,39(12):1720-1733.Chen M B, Guo J H, Yao Q, et al. Isolation and identification, and biological characteristics of cadmium-tolerant bacteria from the Dabao Mine[J]. Microbiology China, 2012, 39(12):1720-1733(in Chinese).
[2]林晓燕,牟仁祥,曹赵云,等.耐镉细菌菌株的分离及其吸附镉机理研究[J].农业环境科学学报,2015,34(9):1700-1706.Lin X Y, Mou R X, Cao Z Y, et al. Isolation and cadmium adsorption mechanisms of cadmium-resistant bacteria strains[J]. Journal of Agro-Environment Science,2015, 34(9):1700-1706(in Chinese).
[3]杨彩霞.两个养殖海区及栉孔扇贝消化盲囊细菌群落多样性分析[D].青岛:中国海洋大学,2011.Yang C X. Seasonal changes of bacterial community composition in two culture area and digestive diverticula of scallops, Chlamys farreri[D]. Qingdao:Ocean University of China, 2011(in Chinese).
[4]Miller B S. Mussels as biomonitors of point and diffuse sources of trace metals in the Clyde sea area,Scotland[J]. Water Science&Technology,1999, 39(12):233-240.
[5]Romeo M, Frasila C, Gnassia-Barelli M, et al. Biomonitoring of trace metals in the Black Sea(Romania)using mussels Mytilus galloprovincialis[J]. Water Research-2005, 39(4):596-604.
[6] Bustamante P, Miramand P. Interspecific and geographical variations of trace element concentrations in Pectinidae from European waters[J]. Chemosphere,2004,57(10):1355-1362.
[7]赵艳芳,宁劲松,翟毓秀,等.栉孔扇贝不同组织中镉的微区分布特征及其变化规律[J].水产学报,2016,40(8):1203-1210.Zhao Y F, Ning J S, Zhai Y X, et al. Research on the subcellular fate and transformation of cadmium in different tissues of the scallop Chlamys farreri[J]. Journal of Fisheries of China, 2016,40(8):1203-1210(in Chinese).
[8]潘风山,陈宝,马晓晓,等.一株镉超积累植物东南景天特异内生细菌的筛选及鉴定[J].环境科学学报,2014, 34(2):449-456.Pan F S, Chen B, Ma X X, et al. Isolation and characterization of a specific endophytic from the Cd hyperaccumulator Sedum alfredi Hance[J]. Acta Scientiae Circumstantiae,2014, 34(2):449-456(in Chinese).
[9]杨彩霞,李赞,张婧宇,等.栉孔扇贝消化盲囊细菌群落组成的季节变化分析[J].水产学报,2012, 36(10):1579-1584.Yang C X, Li Y, Zhang J Y, et al. Seasonal changes of bacterial community composition in digestive diverticula of scallops Chlamys farreri[J]. Journal of Fisheries of China, 2012, 36(10):1579-1584(in Chinese).
[10]许强,杨红生,王红,等.桑沟湾养殖栉孔扇贝食物来源研究——脂肪酸标志法[J].海洋科学,2007, 31(9):78-84.Xu Q, Yang H S, Wang H, et al. Food sources of cultured scallop Chlamys farreri in Sanggou bay:Indicated by fatty acid biomarkers[J]. Marine Sciences, 2007,31(9):78-84(in Chinese).
[11]肖建青,朱泓溢,刘祝祥,等.硇洲岛潮汐带牡蛎相关可培养细菌多样性[J].微生物学通报,2013, 40(6):939-950.Xiao J Q, Zhu H Y, Liu Z X, et al. Biodiversity of culturable bacteria associated with Crassostrea hongkongensis from the tidal flat of Naozhou Island in the South China Sea[J]. Microbiology China,2013, 40(6):939-950(in Chinese).
[12] Pan K, Wang W X. Allometry of cadmium and zinc concentrations and bioaccumulation in the scallop Chlamys nobilis[J]. Marine Ecology Progress Series, 2008, 365:115-126.
[13] Metian M, Warnau M, Oberhansli F, et al. Interspecific comparison of Cd bioaccumulation in European Pectinidae(Chlamys varia and Pecten maximus)[J]. Journal of Experimental Marine Biology and Ecology, 2007,353(1):58-67.
[14] Chong K, Wang W X. Assimilation of cadmium, chromium, and zinc by the green mussel Perna viridis and the clam Ruditapes philippinarum[J]. Environmental Toxicology and Chemistry, 2000, 19(6):1660-1667.
[15] Wang W X. Metal bioaccumulation in bivalve mollusks:recent progress[M]//Villalba A, Reguera B,Romalde J L, et al. Molluscan Shellfish Safety. Spain:Intergovernmental Oceanographic Commission of UNESCO and Conselleria de Pesca e Asuntos Maritimos da Xunta de Galicia, 2003:503-520.
[16] Wang W X,Rainbow P S. Comparative approaches to understand metal bioaccumulation in aquatic animals[J].Comparative Biochemistry and Physiology Part C:Toxicology&Pharmacology, 2008, 148(4):315-323.
[17]梁淑君,王雅玲,孙力军,等.海洋耐镉微生物筛选及其生物学特性[J].海洋环境科学,2012, 31(4):488-491.Liang S J, Wang Y L, Sun L J, et al. Screen and biological characteristics of marine resistance cadmium microbe[J]. Marine Environmental Science,2012, 31(4):488-491(in Chinese).
[18]许钦坤,赵翠燕.耐镉菌株的筛选及生物学特性[J].江苏农业科学,2015, 43(2):317-318.Xu Q K, Zhao C Y. Screening and biological characteristics of cadmium resistant strains[J]. Jiangsu Agricultural Sciences, 2015, 43(2):317-318(in Chinese).
[19]沈秋悦,曹志强,朱月芳,等.一株耐镉细菌的分离鉴定及其吸附条件的优化[J].土壤,2016,48(3):615-620.Shen Q Y, Cao Z Q, Zhu Y F, et al. Isolation of a cd-resistant bacterium and optimization of its bio-accumulation condition[J]. Soils,2016, 48(3):615-620(in Chinese).
[20]周丽英,叶仁杰,林淑婷,等.水稻根际耐镉细菌的筛选与鉴定[J].中国生态农业学报,2012, 20(5):597-603.Zhou L Y, Ye R J, Lin S T, et al. Screening and identification of cadmium-tolerant bacteria from rhizosphere soils under rice[J]. Chinese Journal of Eco-Agriculture,2012, 20(5):597-603(in Chinese).
[21]刘爱民,黄为一.耐镉菌株的分离及其对Cd2+的吸附富集[J].中国环境科学,2006, 26(1):91-95.Liu A M, Huang W Y. Separation of tolerant cadmium bacterium strain and its accumulation adsorption of Cd2+[J]. China Environmental Science, 2006, 26(1):91-95(in Chinese).
[22]吴海江.耐Cd细菌的筛选及抗性机理研究[D].成都:西南交通大学,2009.Wu H J. Screening and resistance mechanism study on a cadmium resistant strain[D]. Chengdu:Southwest Jiaotong University, 2009(in Chinese).
[23]陈美标,郭建华,姚青,等.大宝山矿区耐Cd2+细菌的分离鉴定及其生物学特性[J].微生物学通报,2012,39(12):1720-1733.Chen M B, Guo J H, Yao Q, et al. Isolation and identification, and biological characteristics of cadmium-tolerant bacteria from the Dabao Mine[J]. Microbiology China, 2012, 39(12):1720-1733(in Chinese).
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