共生细菌对盐生小球藻亚砷酸盐代谢的影响
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摘要
为探讨共生细菌对盐生小球藻(Chlorella salina)亚砷酸盐(As(Ⅲ))富集和形态转化的影响,从C. salina培养液中分离培养出一株共生细菌,并采用抗生素处理C. salina获得无菌藻,设置0,75,150,300,750μg/L As(Ⅲ)溶液处理带菌和无菌的C. salina,7d后测定C. salina对As(Ⅲ)的吸收、吸附和富集量,以及培养液和藻细胞内As的形态,计算带菌和无菌C.salina对As(Ⅲ)的氧化率和去除率;使用不同浓度的As(Ⅲ)处理共生细菌7d,并以300μg/L的As(Ⅲ)处理共生细菌不同时间,计算两种情况下共生细菌对培养液中As(Ⅲ)的氧化率和去除率.结果表明:C.salina的共生细菌为根癌农杆菌(AgrobacteriumtumefaciensWB-1);与无菌C.salina相比,带菌C.salina生长更快、对As(Ⅲ)的耐性更强.带菌C.salina对As(Ⅲ)的富集量为103.10～448.12mg/kg、氧化率为78.93%～96.88%、去除率为18.92%～55.21%,显著高于无菌C. salina的富集量(11.68～91.39mg/kg)、氧化率(14.46%～26.39%)和去除率(12.82%～29.15%),也高于A. tumefaciens WB-1对As(Ⅲ)的氧化率(4.51%～30.61%)和去除率(1.86%～16.19%).此外,带菌C. salina胞内还检测到少量的As(Ⅲ)和甲基砷,而在无菌C. salina胞内没有甲基砷存在.共生细菌促进了盐生小球藻对As(Ⅲ)的富集和形态转化.
In order to investigate the effects of a symbiotic bacterium on arsenite(As(Ⅲ)) accumulation and transformation of C. salina, one symbiotic bacterial strain was isolated from culture medium of C. salina. Antibiotics were added to C. salina to obtain the axenic microalga, and a series of As(Ⅲ) treatments(0, 75, 150, 300, 750μg/L) were applied to the non-axenic and axenic C. salina. After 7 days, the absorption, adsorption and accumulation of As(Ⅲ) by C. salina were determined. The As speciation in the culture and algal cells were also analyzed and the rates of As(Ⅲ) oxidation and removal by non-axenic and axenic C. salina were calculated. At the same time, the symbiotic bacterium was treated with different concentrations of As(Ⅲ) for 7 days, and with 300μg/L As(Ⅲ) for different time. The oxidation and removal rates of As(Ⅲ) in the bacterial culture medium were then calculated. Results show that the symbiotic bacterium was identified as Agrobacterium tumefacienss WB-1. Compared with the axenic C. salina, the non-axenic C. salina grew faster with higher tolerance to As(Ⅲ). Moreover, in the presence of bacterium, As(Ⅲ) accumulation by C. salina from the culture solution varied from 103.10 to 448.12 mg/kg, the oxidation and removal rates of As(Ⅲ) were 78.93%～96.88% and 18.92%～55.21%, respectively. These values were significantly higher than those of the axenic C. salina, which varied from 11.68 to 91.39 mg/kg, 14.46%～26.39% and 12.82%～29.15%, respectively. The As(Ⅲ) oxidation and removal rates in the non-axenic C. salina culture were also significantly higher than those in the A. tumefaciens cuture(4.51%～30.61% and 1.86%～16.19%, respectively). In addition, a small amounts of As(Ⅲ) and methylated As were detected in the cells of non-axenic C. salina, while no methylated As was present in the axenic C. salina cells. Symbiotic bacteria promote the enrichment and transformation of As(Ⅲ) by Chlorella salina.
In order to investigate the effects of a symbiotic bacterium on arsenite(As(Ⅲ)) accumulation and transformation of C. salina, one symbiotic bacterial strain was isolated from culture medium of C. salina. Antibiotics were added to C. salina to obtain the axenic microalga, and a series of As(Ⅲ) treatments(0, 75, 150, 300, 750μg/L) were applied to the non-axenic and axenic C. salina. After 7 days, the absorption, adsorption and accumulation of As(Ⅲ) by C. salina were determined. The As speciation in the culture and algal cells were also analyzed and the rates of As(Ⅲ) oxidation and removal by non-axenic and axenic C. salina were calculated. At the same time, the symbiotic bacterium was treated with different concentrations of As(Ⅲ) for 7 days, and with 300μg/L As(Ⅲ) for different time. The oxidation and removal rates of As(Ⅲ) in the bacterial culture medium were then calculated. Results show that the symbiotic bacterium was identified as Agrobacterium tumefacienss WB-1. Compared with the axenic C. salina, the non-axenic C. salina grew faster with higher tolerance to As(Ⅲ). Moreover, in the presence of bacterium, As(Ⅲ) accumulation by C. salina from the culture solution varied from 103.10 to 448.12 mg/kg, the oxidation and removal rates of As(Ⅲ) were 78.93%～96.88% and 18.92%～55.21%, respectively. These values were significantly higher than those of the axenic C. salina, which varied from 11.68 to 91.39 mg/kg, 14.46%～26.39% and 12.82%～29.15%, respectively. The As(Ⅲ) oxidation and removal rates in the non-axenic C. salina culture were also significantly higher than those in the A. tumefaciens cuture(4.51%～30.61% and 1.86%～16.19%, respectively). In addition, a small amounts of As(Ⅲ) and methylated As were detected in the cells of non-axenic C. salina, while no methylated As was present in the axenic C. salina cells. Symbiotic bacteria promote the enrichment and transformation of As(Ⅲ) by Chlorella salina.
引文
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[19]Duncan E G,Maher W A,Foster S D,et al.The influence of arsenate and phosphate exposure on arsenic uptake,metabolism and species formation in the marine phytoplankton Dunaliella tertiolecta[J].Marine Chemistry,2013,157:78-85.
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[24]Mallick I,Bhattacharyya C,Mukherji S,et al.Effective rhizoinoculation and biofilm formation by arsenic immobilizing halophilic plant growth promoting bacteria(PGPB)isolated from mangrove rhizosphere:A step towards arsenic rhizoremediation[J].Science of The Total Environment,2018,610-611:1239-1250.
[25]Levy J L,Stauber J L,Adams M S,et al.Toxicity,biotransformation,and mode of action of arsenic in two freshwater microalgae(Chlorella sp.and Monoraphidium arcuatum)[J].Environmental Toxicology&Chemistry,2010,24(10):2630-2639.
[26]Howard A G,Comber S D W,Kifle D,et al.Arsenic speciation and seasonal changes in nutrient availability and micro-plankton Abundance in Southampton Water,U.K.[J].Estuarine Coastal and Shelf Science,1995,40(4):435-450.
[27]王振红,张汉鹏,罗专溪.不同磷源下铜绿微囊藻的生长差异及对砷酸盐的响应[J].环境科学,2016,37(7):2570-2576.Wang ZH H,Zhang H P,Luo ZH X.Growth difference of Microcystis aeruginosa under different phosphorus sources and response to arsenate[J].Environment Science,2016,37(7):2570-2576.
[28]王振红,罗专溪,车霏霏,等.不同磷水平下铜绿微囊藻对砷酸盐的吸收和净化[J].中国环境科学,2015,35(2):533-538.Wang ZH H,Luo ZH X,Che F F,et al.Absorption and purification of arsenate by Microcystis aeruginosa under different phosphorus levels[J].China Environmental Science,2015,35(2):533-538.
[29]Wang N X,Huang B,Xu S,et al.Effects of nitrogen and phosphorus on arsenite accumulation,oxidation,and toxicity in Chlamydomonas reinhardtii[J].Aquatic Toxicology,2014,157(7):167-174.
[30]Wang Y,Zhang C H,Lin M M,et al.A symbiotic bacterium differentially influences arsenate absorption and transformation in Dunaliella salina under different phosphate regimes[J].Journal of Hazardous Materials,2016,318:443-451.
[31]郭盾,张艮林,金军.水体砷污染微生物修复的研究进展[J].环境保护科学,2015,(6):44-49.Guo D,Zhang G L,Jin J.Research progress on microbial remediation of arsenic pollution in water[J].Environment Protection Science,2015,(6):44-49.
[32]Shi K,Fan X,Qiao Z,et al.Arsenite oxidation regulator Aio Rregulates bacterial chemotaxis towards arsenite in Agrobacterium tumefaciens GW4[J].Scientific Reports,2017,7:43252.
[33]Tokmina-Lukaszewska M,Shi Z,Tripet B,et al.Metabolic response of Agrobacterium tumefaciens 5A to arsenite[J].Environmental Microbiology,2017,19(2):710-721.
[34]Shi K,Wang Q,Fan X,et al.Proteomics and genetic analyses reveal the effects of arsenite oxidation on metabolic pathways and the roles of Aio R in Agrobacterium tumefaciens GW4[J].Environmental Pollution,2018,235:700-709.
[2]Ma Z,Lin L,Wu M,et al.Total and inorganic arsenic contents in seaweeds:absorption,accumulation,transformation and toxicity[J].Aquaculture 2018,497:49-55.
[3]Mandal B K,Suzuki K T.Arsenic round the world:a review[J].Talanta,2002,58(1):201-235.
[4]Cai L,Liu G,Rensing C,et al.Genes involved in arsenic transformation and resistance associated with different levels of arsenic-contaminated soils[J].Bmc Microbiology,2009,9(1):164-174.
[5]Dhankher O P,Rosen B P,Mckinney E C,et al.Hyperaccumulation of arsenic in the shoots of Arabidopsis silenced for arsenate reductase(ACR2)[J].Proceedings of the National Academy of Sciences of the United States of America,2006,103(14):5413-5418.
[6]王长海.微藻与微藻生物技术[J].渔业现代化,2006,(1):20-22.Wang C H.Micro-algae and micro-algal Biotechnology[J].Fishery Modernization,2006,(1):20-22.
[7]Wang Y,Zhang C,Zheng Y,et al.Bioaccumulation kinetics of arsenite and arsenate in Dunaliella salina under different phosphate regimes[J].Environmental Science&Pollution Research,2017,24(26):21213-21221.
[8]王亚,张春华,王淑,等.带菌盐藻对不同形态砷的富集和转化研究[J].环境科学,2013,34(11):4257-4265.Wang Y,Zhang CH H,Wang S,et al.Accumulation and transformation of different arsenic species in nonaxenic Dunaliella salina[J].Environment science,2013,34(11):4257-4265.
[9]Qin J,Lehr C R,Yuan C,et al.Biotransformation of arsenic by a Yellowstone thermoacidophilic eukaryotic alga[J].Proceedings of the National Academy of Sciences,2009,106(13):5213-5217.
[10]张兵,王利红,徐玉新,等.集胞藻(Synechocystis sp.PCC6803)对砷吸收转化特性的初步研究[J].生态毒理学报,2011,06(6):629-633.Zhang B,Wang L H,Xu Y X,et al.Preliminary study on the absorption and transformation characteristics of arsenic by Synechocystis(Synechocystis sp.PCC6803)[J].Asian Journal of Ecotoxicology,2011,06(6):629-633.
[11]Yin X X,Wang L H,Bai R,et al.Accumulation and transformation of arsenic in the blue-green alga Synechocysis sp.PCC6803[J].Water Air and Soil Pollution,2012,223(3):1183-1190.
[12]Murray L A,Raab A,Marr I L,et al.Biotransformation of arsenate to arsenosugars by Chlorella vulgaris[J].Applied Organometallic Chemistry,2010,17(9):669-674.
[13]Levy J L,Stauber J L,Wakelin S A,et al.The effect of bacteria on the sensitivity of microalgae to copper in laboratory bioassays[J].Chemosphere,2009,74(9):1266-1274.
[14]Karadjova I B,Slaveykova V I,Tsalev D L.The biouptake and toxicity of arsenic species on the green microalga Chlorella salina in seawater[J].Aquatic Toxicology,2008,87(4):264-271.
[15]Levy J L,Stauber J L,Wakelin S A,et al.The effect of field-collected biofilms on the toxicity of copper to a marine microalga(Tetraselmis sp.)in laboratory bioassays[J].Marine and Freshwater Research,2011,62:1362-1372.
[16]Amin S A,Hmelo L R,Van Tol H M,et al.Interaction and signalling between a cosmopolitan phytoplankton and associated bacteria[J].Nature,2015,522(7554):98-101.
[17]廖敏,谢正苗,王锐.菌藻共生体去除废水中砷初探[J].环境污染与防治,1997,(2):11-12.Liao M,Xie ZH M,Wang R.Preliminary study on the removal of arsenic from wastewater by bacterial algae[J].Environmental Pollution&Control,1997,(2):11-12.
[18]许平平,刘聪,王亚,等.共生细菌对盐生小球藻富集和转化砷酸盐的影响[J].环境科学,2016,37(9):3438-3446.Xu P P,Liu C,Wang Y,et al.Effects of a symbiotic bacterium on the accumulation and transformation of arsenate by Chlorella salina[J].Environment science,2016,37(9):3438-3446.
[19]Duncan E G,Maher W A,Foster S D,et al.The influence of arsenate and phosphate exposure on arsenic uptake,metabolism and species formation in the marine phytoplankton Dunaliella tertiolecta[J].Marine Chemistry,2013,157:78-85.
[20]Weisburg W G,Barns S M,Pelletier D A,et al.16S ribosomal DNAamplification for phylogenetic study[J].Journal of Bacteriology,1991,173(2):697-703.
[21]Levy J L,Stauber J L,Adams M S,et al.Toxicity,biotransformation,and mode of action of arsenic in two fresh-water microalgae(Chlorella sp.and Monoraphidium arcuatum).Environment.Toxicol.Chem.2005,24:2630-2639.
[22]布坎南R E,吉本斯N E,等编.伯杰细菌鉴定手册(第八版)[M].科学出版社,1984:345-347.R.E.Bu K N,N.E.Ji B S,et al.Berger Bacterial Identification Manual(eighth edition)[M].Science Press,1984:345-347.
[23]王晴晴,高金伟,时晓婷,等.海洋微藻与细菌相互关系的研究[J].中国农业信息,2016,(9):113-114.Wang Q Q,Gao J W,Shi X T,et al.Study on the relationship between marine microalgae and bacteria[J].China Agricultural Information,2016,(9):113-114.
[24]Mallick I,Bhattacharyya C,Mukherji S,et al.Effective rhizoinoculation and biofilm formation by arsenic immobilizing halophilic plant growth promoting bacteria(PGPB)isolated from mangrove rhizosphere:A step towards arsenic rhizoremediation[J].Science of The Total Environment,2018,610-611:1239-1250.
[25]Levy J L,Stauber J L,Adams M S,et al.Toxicity,biotransformation,and mode of action of arsenic in two freshwater microalgae(Chlorella sp.and Monoraphidium arcuatum)[J].Environmental Toxicology&Chemistry,2010,24(10):2630-2639.
[26]Howard A G,Comber S D W,Kifle D,et al.Arsenic speciation and seasonal changes in nutrient availability and micro-plankton Abundance in Southampton Water,U.K.[J].Estuarine Coastal and Shelf Science,1995,40(4):435-450.
[27]王振红,张汉鹏,罗专溪.不同磷源下铜绿微囊藻的生长差异及对砷酸盐的响应[J].环境科学,2016,37(7):2570-2576.Wang ZH H,Zhang H P,Luo ZH X.Growth difference of Microcystis aeruginosa under different phosphorus sources and response to arsenate[J].Environment Science,2016,37(7):2570-2576.
[28]王振红,罗专溪,车霏霏,等.不同磷水平下铜绿微囊藻对砷酸盐的吸收和净化[J].中国环境科学,2015,35(2):533-538.Wang ZH H,Luo ZH X,Che F F,et al.Absorption and purification of arsenate by Microcystis aeruginosa under different phosphorus levels[J].China Environmental Science,2015,35(2):533-538.
[29]Wang N X,Huang B,Xu S,et al.Effects of nitrogen and phosphorus on arsenite accumulation,oxidation,and toxicity in Chlamydomonas reinhardtii[J].Aquatic Toxicology,2014,157(7):167-174.
[30]Wang Y,Zhang C H,Lin M M,et al.A symbiotic bacterium differentially influences arsenate absorption and transformation in Dunaliella salina under different phosphate regimes[J].Journal of Hazardous Materials,2016,318:443-451.
[31]郭盾,张艮林,金军.水体砷污染微生物修复的研究进展[J].环境保护科学,2015,(6):44-49.Guo D,Zhang G L,Jin J.Research progress on microbial remediation of arsenic pollution in water[J].Environment Protection Science,2015,(6):44-49.
[32]Shi K,Fan X,Qiao Z,et al.Arsenite oxidation regulator Aio Rregulates bacterial chemotaxis towards arsenite in Agrobacterium tumefaciens GW4[J].Scientific Reports,2017,7:43252.
[33]Tokmina-Lukaszewska M,Shi Z,Tripet B,et al.Metabolic response of Agrobacterium tumefaciens 5A to arsenite[J].Environmental Microbiology,2017,19(2):710-721.
[34]Shi K,Wang Q,Fan X,et al.Proteomics and genetic analyses reveal the effects of arsenite oxidation on metabolic pathways and the roles of Aio R in Agrobacterium tumefaciens GW4[J].Environmental Pollution,2018,235:700-709.