INTRODUCTION

Arsenic(As),from geogenic and anthropogenic sources,is a toxic metalloid widely distributed in the water,soil,and air(Oremland and Stolz,2005).Although it exists in four oxidation states,arsenite(As(III))and arsenate(As(V))are the two predominant forms of inorganic As found in nature(Smedley and Kinniburgh,2002).Among the two forms,As(III)is 50–100 times more toxic than As(V)(Hu et al.,2012),because As(III)is difficult to remove from water owing to its higher solubility than As(V)(Clifford,1990;Rhine et al.,2006).Arsenic contamination is currently considered as a major public health concern(Rosen,1999;Nordstrom,2002;Smith et al.,2002;Achour et al.,2007)with about 100 million people at risk from drinking groundwater contaminated with 10µg L−1As,which is above the permissible limit prescribed by World Health Organization(Fendorf et al.,2010;Hu et al.,2012).Among these people at risk,5 million are from West Bengal,India alone(Meharg,2005).Groundwater from shallow tube wells is used as the major source of irrigation for crop fields in West Bengal.The irrigation water with severe As contamination leads to the deposition of As in soil throughout the year.Eventually,As-contaminated irrigation water resulted in elevated levels of As in the top soil,causing subsequent accumulation of As in crops and its entry into food chain(Roychowdhury et al.,2005;Rahaman et al.,2013).Nine districts of West Bengal are considered to be severely As contaminated(Uchino et al.,2006),with people exposed to water containing＞50µg L−1As(Chakraborti et al.,2003),and this has become a serious health issue for people of West Bengal(Acharyya et al.,1999;Nayak et al.,2008).

Microbes are ubiquitous;in the As-contaminated habitat,the adaptive microbial As resistance leads to the evolution of an As-resistance system(Cervantes et al.,1994;Mukhopadhyay et al.,2002;Zhu et al.,2014;Paul and Mukherjee,2016).Microbes can grow in As-stressed environment by deriving energy from the oxidation of As(III)to As(V)as a strategy for survival(Santini et al.,2000;Garcia-Dominguez et al.,2008).Two types of As(III)oxidizers are found in nature∶autotrophic and heterotrophic As(III)oxidizers.Under the role of autotrophic As(III)oxidizers,As(III)acts as the electron donor,oxygen as electron acceptor,and carbon dioxide as the carbon source.Heterotrophic As(III)oxidizers can be utilized for pre-oxidation of As(III)during remediation of As-contaminated gro-undwater(Bahar et al.,2012),in which As(III)is catalyzed by arsenite oxidase to form As(V)(Muller et al.,2003).The As(III)oxidation potential of bacteria from different environmental samples is well documented(Green,1918;Ilyaletdinov and Abdrashitova,1981;Weeger et al.,1999;Santini et al.,2000;Salmassi et al.,2002;Liao et al.,2011;Bahar et al.,2012;Govarthanan et al.,2015b,2016),suggesting the wide distribution of As(III)-oxidizing bacteria that could play a critical role in environmental As remediation.Thus,eco-friendly and efficient remediation of As-contaminated groundwater for irrigation could be achieved by microbemediated pre-oxidation of As(III),which is converted to less toxic As(V),followed by microbial As adsorption.

矿石中主要的碲、铋矿物为碲银矿、碲铋矿、辉铋矿。碲银矿，晶体呈叶、粒、块状，粒度较细，多呈包裹体包裹于黄铁矿中，经能谱分析，该矿中主元素银含量为58.30%～62.42%，平均为61.29%，碲含量为37.58%～41.70%，平均为38.51%。碲铋矿，晶体呈叶、粒、块状，铅灰色，粒度较细，多呈包裹体包裹于黄铁矿中。辉铋矿，晶体呈长柱状或针状，集合体为致密粒状。该矿中主元素铋含量平均为79.10%。由于Te在元素周期表中与Au具有相近的化学性质，基于碲与金的密切关系，经分析发现矿区成矿提供充足的碲元素可能性不大，对金成矿的贡献也不大。

Arsenic-resistant plant-growth-promoting rhizobacterial strains,isolated from As-contaminated soil,were previously reported for their possible As decontaminating ability in fields(Cavalca et al.,2010;Mallick et al.,2014).The treatment of As-contaminated groundwater is now a serious environmental and health issue.Pretreatment of contaminated water for possible As decontamination by intervening in the As redox state seems to be a promising option in sustainable As bioremediation.The aims of the present study were to isolate an As-resistant microbial strain and to evaluate its possible use in environmental As bioremediation.

MATERIALS AND METHODS

Soil sample

Single soil sample,belonging to the soil order Inceptisol,was collected in March,2013 from the paddy fields of Phulia Village in Nadia District,West Bengal,India(23°13′13.85′′N,88°30′29.43′′E).The sampling site was reported to be a highly As-contaminated region(Chakraborti et al.,2009;Mallick et al.,2014).The soil sample was collected in a sterilized container and then air-dried,ground,sieved,and finally stored at−20°C in a laboratory freezer.For the determination of pH,100 mL CaCl2solution(0.01 mol L−1)was added to 50 g of air-dried soil and incubated overnight(Scho field and Taylor,1955).The pH of the supernatant was measured using a pH meter(Fisher Scientific,USA)without disturbing the precipitated soil.After acid digestion with 4∶1(volume∶volume)HNO3and HClO4(Sinha and Mukherjee,2008),total As content of the soil was estimated by atomic absorption spectrometer(iCE 3000 Series AA spectrometer,Thermo Scientific,USA).The soil sample had a pH of 7.1 and a total As content of 12.5 mg kg−1.

Isolation,characterization,and identification of Asresistant bacterial strain

The soil sample was serially diluted up to 10−6fold using sterilized phosphate buffer(pH 7.2)and inoculated using the pour plate method on nutrient agar(Hi-Media,India)containing 0.5–15 mmol L−1As(III)in the form of NaAsO2(HiMedia,India).The plates were incubated at 37°C for 24 h(Mallick et al.,2014).The bacterial colony that showed maximum tolerance to As was selected and designated as KUMAs15.Pure culture of the isolate was established by single colony isolation and transferred to minimal salt broth(MSB;pH 7.2,KH2PO40.3 g,Na2HPO40.6 g,NaCl 0.5 g,NH4Cl 0.2 g,MgSO40.1 g,and glucose 0.8 g dissolved in 100 mL Milli-Q water)(Millipore Co.,USA)supplemented with 2 mmol L−1As(III).This step ensured the stability of As resistance in KUMAs15 and maintained it for further analysis.

The removal of total As from the MSB medium by KUMAs15 was also determined in each treatment by quantifying the residual amount of As in the culture media.Total As concentration in the supernatant was quantified by an atomic absorption spectrophotometer after centrifugation and acid-digestion as mentioned earlier,and represented as percent removal of the initial concentration.The percentage of As removal(PR)was calculated following the equation∶PR=(C0−Ce)/C0×100,where C0is the initial As concentration and Ceis the equilibrium As concentration.The effects of pH and temperature on As removal were also estimated.

The morphological and physiological characterization of the isolated bacterial colony was carried out following the standard methods(Pelczar et al.,1957).Different biochemical tests(i.e.,carbohydrate and citrate utilization ability;production of enzymes like catalase,oxidase,urease,and nitrate reductase;hydrolysis of gelatin and starch;indole and sul fide production;and methyl red and Voges-Proskauer tests)were considered to characterize the strain(Brown,2007).

The minimum inhibitory concentrations(MICs)of As(V)(Na2HAsO4·7H2O,HiMedia,India)and As(III)(NaAsO2,HiMedia,India)were determined in both nutrient broth(NB)and MSB following the method of Yilmaz(2003).The isolate KUMAs15 was also determined for its resistance to other metals.The MICs of cadmium(CdCl2·H2O),zinc(ZnSO4·7H2O),chromium(K2CrO4),cobalt(CoCl2·6H2O)and nickel(NiCl2·6H2O)for the resistant isolate were determined.

From the pure culture pellet,genomic DNA was isolated using genomic DNA isolation kit(Invitrogen,USA).About 1.5 kb of 16S rDNA gene fragment were amplified from the bacterial genomic DNA using specific forward and reverse primer sets 5′-AGAGTTTGATCMTGGCTCAG-3′and 5′-TACGGYTACCTTGTTACGACTT-3′and sequenced(Amnion Bioscience,India).The sequence data were analyzed by NCBI BLAST algorithm(Altschul et al.,1990)and Ribosomal Database Project(Maidak et al.,1997).

Growth kinetics of isolate KUMAs15

Descriptive statistical analyses of data were performed for each experiment.All experiments were carried out in triplicate.Results represent the means with standard deviations.The statistical evaluation was carried out by analysis of variance(ANOVA)at P＜0.05.The GraphPad Prism software(version 5.0)was used for all the statistical analyses.

As oxidation and reduction assay

我院2013－2016年耐碳青霉烯类铜绿假单胞菌医院感染的危险因素与临床结局分析 …………………… 罗赛赛等（5）：667

(2)2005～2009年城市化与生态环境耦合协调度增长速率有所提高，表明城市化子系统和生态环境子系统进一步协调，协调类型逐步过渡为低水平协调阶段。这一时期的城市化综合水平持续增加，城市经济发展保持稳定速率，而这一时期的生态环境综合水平从2005年开始回升。通过查看各项指标情况可以发现，这一时期在环保等投资与GDP的比值，生活垃圾无害化处理率，人均公共绿地面积，工业固体废弃物综合利用率这部分生态环境响应指标的带动下，生态环境质量有较明显的改善，这些变化与湖南省的工作计划也密切相关，计划中提出发展循环经济，倡导生态文明，在发展中保护环境，实现速度与质量、效益的有机统一。

To determine cellular As accumulation in KUMAs15,equal amount of cell suspension from 16-h grown culture was inoculated in MSB supplemented with 0.5,1,2,and 4 mmol L−1As(III)(NaAsO2)and incubated at 37°C on a rotary shaker.Bacterial cell pellets harvested by centrifugation from different treatments were acid-digested followed by quantification of the total As using an atomic absorption spectrometer.The effects of pH and temperature on As accumulation in MSB supplemented with 2 mmol L−1As were also determined,due to the maximum As accumulation observed in this concentration at 37 °C for 24–72 h.Control sets of respective treatments without bacterial inoculation were maintained to monitor the artefacts resulting from abiotic chelation of As.

To determine the effect of pH on As redox reactions,the pH of culture medium was adjusted to different values with filter sterilized NaOH and HCl.The effect of pH was assessed by following the same procedure used in the oxidation and reduction experiment.The As(III)oxidation by KUMAs15 was also investigated with other carbohydrate sources(dextrose,fructose,maltose,sucrose,and mannitol)in the minimal culture medium following the above mentioned procedure for initial pH 7 at 37°C for 48 h(Bahar et al.,2012).

As accumulation and removal assay

where Asoxidized,Asuntreated,and Asreducedare the As concentrations determined in the oxidized,untreated,and reduced treatments,respectively.

优水优用，优化调用各类水源［8］，实现环境良性循环，形成分质分管道供水系统。可考虑三级引用水的标准，即优质水（能够直接饮用，大肠杆菌等主要水质指标为零）、普通水（能够达到生活饮用水标准）以及洗涤水（可用以满足基本的冲刷、洗菜功能）［3］，分别铺设两条管线，做到优质水和洗涤水分开输送。

Cellular localization of As accumulation

The cellular localization of accumulated As was determined through the fractionation of As(III)-treated bacterial cells after incubation for 72 h.Treated cell mass was kept in 40 mL mixture of 0.2 mol L−1Tris-HCl buffer(pH 7.1)and 40%(weight∶volume)sucrose for osmotic stress for an equilibration period of 30 min.Subsequently,the cells were treated with 0.5 mmol L−1 MgCl2at 4°C and shocked.The homogenate was centrifuged at 90000×g for 1 h to separate total membrane component and cytosol following the established protocols(Fricke and Aurich,1993;Sinha and Mukherjee,2009).The total As content was determined by an atomic absorption spectroscopy.

Isolation of total RNA and semi-quantitative reversetranscription of As-resistant genes

Total RNA was isolated using TRIzol reagent(Invitrogen,USA)from the cells grown in MSB supplemented with 2 mmol L−1As(III)(NaAsO2)following the standard protocol(Rio et al.,2010).The RNA pellet was eluted with 20µL of RNase-free water(Thermo Scientific,USA)and quantified at 260 nm with an Evolution 201 UV-Vis spectrophotometer.The cDNA was synthesized by reverse transcription,using the total RNA as template with RevertAidTMFirst Strand cDNA synthesis kit(Thermo Scientific,USA)according to the manufacturer’s protocol,followed by the polymerase chain reaction(PCR)amplification of the genes of interest using the cDNA as template.The PCR was carried out using the reported primers for the arsenite oxidase gene aoxB(Rhine et al.,2007),arsenite transporter gene arsB(Chang et al.,2008),and arsenate reductase gene arsC(Bachate et al.,2009).The PCR products were subjected to electrophoresis and staining with ethidium bromide,and then were analyzed using the image analysis software for gel documentation(Gel DocTMXR+system,BioRad,USA).The expression of the genes of interest was compared with the constitutive expression of the housekeeping gene 16S rRNA as a control.

Cellular surface adsorption of As in KUMAs15

To investigate the cellular surface adsorption of As in isolate KUMAs15,1%(volume∶volume)16-h grown isolate culture was inoculated in MSB supplemented with 2 mmol L−1of As(III)and incubated for 72 h at 37°C.Cells were harvested by centrifugation and washed thrice with phosphate buffer saline(pH 7.4).The bacterial smear was prepared on a cover glass,treated with piranha solution(H2SO4∶H2O2=3∶1,volume∶volume),and heat fixed over a flame for 1 to 2 s followed by fixation of the smear by 2.5%(volume∶volume)glutaraldehyde for 45 min(Chao and Zhang,2011).The slides were then dehydrated by passing through a series of 50%–90%of alcohol solutions and finally through absolute alcohol for 5 min each.The bacteria on the cover glass were gold coated,and they were observed under a scanning electron microscope(EvoLS10,Zeiss,USA)coupled with an energy dispersive X-ray spectroscopy(SEM-EDX)to con firm As-specific peak on the cellular surface of the isolate.

Statistical analysis

Growth kinetics of isolate KUMAs15 was determined under the aerobic culture conditions in the pre-sence of different concentrations of As(III)or As(V)in both NB and MSB,while the respective medium without As(III)and As(V)supplementation was considered as the control.Effect of different As concentrations on the isolate was ascertained by inoculating equal volumes of 16-h grown cultures in MSB,which was supplemented separately with 2,4,8,and 10 mmol L−1As(III)or As(V).A range of different incubation temperatures were used for studying the growth response of KUMAs15;the results con firmed 37°C as the optimum incubation temperature.Therefore,further studies were performed at 37°C.Viable cell count as colony forming units(CFUs)was obtained following conventional dilution plate count method.Growth response of KUMAs15 was also determined at different pH values from acidic(pH 4)to alkaline(pH 9)in MSB supplemented with 2 mmol L−1of either As(V)or As(III)to determine the optimal culture conditions for the strain.

RESULTS

Characterization and identification of the strain KUMAs15

The primary characterization of the isolated Asresistant strain KUMAs15 was performed on the basis of colony morphology,cellular morphology,and Gram staining characteristics.The colony morphology of the strain was circular,pinheaded with convex margins.The isolate was microscopically studied and observed to be round shaped.The Gram staining revealed that the strain was a Gram-positive bacterial strain.The biochemical properties of the strain KUMAs15 are listed in Table I.The 16S rDNA gene sequence of the As-resistant bacterial strain KUMAs15 was submitted to NCBI databank(NCBI GenBank accession No.KP455684).The homology search based on BLAST algorithm followed by phylogenetic analyses(Fig.1)and Ribosomal Database Project indicated that the strain belonged to the genus Micrococcus.

Tolerance of KUMAs15 to As and other metals

The obtained MICs revealed that the isolated strain KUMAs15 showed variable resistance to different heavy metals including the metalloid As(Table II).The strain could tolerate up to 25 and 20 mmol L−1of As(III)in NB and MSB,respectively,whereas it could tolerate higher level of As(V),as evidenced by the MICs of 400 and 375 mmol L−1in NB and MSB,respectively.The result showed that As(III)inhibited bacterial growth more severely than As(V).The MICs of As and other heavy metals indicated that the isolated Micrococcus sp.KUMAs15 had a metal tolerance in the order of As(v)＞As(III)＞Cr＞Ni＞Cd＞Zn＞ Co.The tolerance of KUMAs15 to different heavy metals was higher in NB than in MSB,probably due to the masking effect of undefined components abundant in NB.

TABLE I Biochemical properties of As-resistant strain KUMAs15

Biochemical test Remark Biochemical test Remark Catalase Positive Urease Positive Indole production Negative Sul fide production Negative Methyl red test Negative Carbohydrate utilization(sucrose,lactose, Positive Voges-Proskauer test Negative dextrose,fructose,maltose,mannitol)Citrate utilization Positive Gelatin hydrolysis Positive Nitrate reductase Negative Gram staining Positive Oxidase Positive Starch hydrolysis Positive

Fig.1 Neighbor-joining tree based on the 16S rDNA gene sequence obtained for As-resistant strain KUMAs15 and representative species of the genus Micrococcus.The numbers at the branch nodes are bootstrap values based on 1000 replicates.

TABLE II Minimal inhibitory concentrations of different tested metals for As-resistant strain KUMAs15,incubated in nutrient broth and minimal salt broth

Metal tested Nutrient broth Minimal salt broth mmol L−1 As(III) 25 20 As(V) 400 375 Cd(II) 3 1 Zn(II) 1.0 0.5 Cr(VI) 11 7 Co(II) 0.6 0.1 Ni(II) 7 4

Growth kinetics of KUMAs15

The growth response of KUMAs15 was determined both in complex NB and defined MSB supplemented with 2 mmol L−1As(III)and As(V)separately(Fig.2a,b).Bacterial growth was inhibited in the presence of both forms of As and showed lengthened lag phase;the growth inhibition was greater in MSB than in NB,which corroborated the earlier findings obtained while determining MICs.The effect of different concentrations of As(III)and As(V)on the growth was also assessed in MSB(Fig.2c,d).Irrespective of As concentration,the maximum growth was observed after 28–30 h of incubation,and the growth declined up to 96 h of incubation,with non-significant alteration among the pHs of culture media(Fig.2e,f).Among the different As(III)and As(V)concentrations,2 mmol L−1of As(III)and As(V)were observed to be least effective on the growth of the isolated strain KUMAs15.Optimum temperature and pH for the growth of KUMAs15 in the presence of As(III)and As(V)were found to be 37 °C and 7.0,respectively(Fig.2e–h).

As(III)oxidation by KUMAs15

The quantitative analyses of As(III)oxidation by the resistant strain demonstrated that significant As(III)oxidation occurred when the strain was exposed to 2 mmol L−1As(III)under different culture pH values and incubation temperatures.The maximum As(III)oxidation(39.22%)was observed in MSB medium inoculated with strain KUMAs15 for 48 h in neutral pH at 37°C(Table III).The pattern of As(III)oxidation over a range of pHs showed that the extent of As(III)oxidation by the isolate KUMAs15 was biomass dependent.

The isolated strain KUMAs15 was also observed to utilize different carbohydrates as their carbon sources during As(III)oxidation.Comparative analysis of di-fferent carbohydrates as MSB components showed that As(III)oxidation was maximum when fructose(0.5%)was present in the MSB medium as a carbon source.The As(III)oxidation ability of KUMAs15 utilizing di-fferent carbohydrates as a carbon source was observed to be in the order of fructose＞mannitol＞glucose＞maltose＞sucrose＞dextrose(Table IV).No growth was observed when the strain was cultured with As(III)as a sole energy source,indicating that it is a hetero-trophic oxidizer.

Fig.2 Growth kinetics of the As-resistant strain KUMAs15 in nutrient broth(a)and minimal salt broth(MSB)(b)supplemented with 2 mmol L−1As(III)and As(V);growth response of KUMAs15 to different concentrations of As(III)(c)and As(V)(d)in MSB;growth response of KUMAs15 under pH gradient in MSB supplemented with 2 mmol L−1As(III)(e)and As(V)(f);and growth of KUMAs15 at different incubation temperatures in MSB supplemented with 2 mmol L−1As(III)(g)and As(V)(h).Vertical bars indicate standard deviations of the means(n=3).CFUs=colony forming units.

TABLE III As(III)oxidation under pH gradient by the As-resistant strain KUMAs15,incubated in minimal salt broth supplemented with 2 mmol L−1As(III)

*,**Significant at P＜0.05 and P＜0.01,respectively.a)Colony forming units. b)Means±standard deviations(n=3).

pH Incubation time As(III)oxidized Viable cell count lg(CFUsa)mL−1)6.0 24 14.25±0.018b) 6.21±0.008 48 19.01±0.008* 7.35±0.008 72 13.01±0.003* 5.92±0.010 7.0 24 20.79±0.003 7.44±0.003 48 39.22±0.384** 8.91±0.040 72 18.24±0.024* 6.10±0.057 8.0 24 19.32±0.008 7.20±0.033 48 27.91±0.006* 8.22±0.010 72 17.21±0.010* 6.13±0.003 h %

As accumulation and removal efficiency by KUMAs15

The strain KUMAs15 was capable of accumulating As when exposed to a wide range of As concentrations under aerobic culture conditions(Fig.3).Among the different experimental As concentrations,As accumulation was observed to be maximum when the strain was incubated with 2 mmol L−1As(III)for 72 h(Fig.3a).The cellular As accumulation in KUMAs15 was found to be about 55 mg L−1.The As accumulation decreased both at the lower and higher As concentrations studied.The accumulation of As was also found to be maximum at pH 7.0(Fig.3b),although suboptimal accumulation was also observed at both acidic and alkaline pH.The accumulation study with different incubation temperatures showed that the isolated strain KUMAs15 could accumulate As optimally at 37°C(Fig.3c).

The expression of aoxB gene was significantly increased in KUMAs15 when incubated in the presence of 0.5–2 mmol L−1As(III).The maximum aoxB expression was observed in the strain incubated with 2 mmol L−1As(III)at 37 °C and pH 7.0 for 48 h.The expression was observed to decrease in the strain incubated with 4 mmol L−1As(III),probably due to decreased cellular biomass at the higher concentration.Transcriptional expression of the arsB gene,encoding As(III)passive efflux pump,showed no signi ficant change compared to the negative control group,when exposed to different As(III)concentrations in MSB.The significant cellular As-retention ability of KUMAs15,con firmed by the As accumulation study,may be explained by this observation.The arsenate reductase gene arsC showed no significant alterations when incubated with different As concentrations and compared with the untreated group(Fig.4).

The phenomenon of As immobilization by the isolate KUMAs15 was con firmed by quantifying total As content in the cell-free supernatant of the MSB culture medium.Cells of Micrococcus sp.KUMAs15 could significantly accumulate or adsorb As from MSB under aerobic culture,resulting in lower total As content in the cell-free culture medium.The maximum As removal was found to be 36.65%by the strain KUMAs15 incubated with 2 mmol L−1As(III)for 72 h.The optimal temperature and pH for maximum removal on exposure to 2 mmol L−1As(III)were observed to be 37 °C and 7,respectively(Fig.3d–f).

To determine As(III)-oxidation and As(V)-reduction ability of the isolated strain,1%(volume∶volume)16-h grown KUMAs15 culture was inoculated in MSB containing 2 mmol L−1of either As(III)or As(V)and incubated at 37°C.The cell-free supernatant at different time intervals was obtained by centrifugation at 5000× g and 4°C for 10 min and used to quantify either As(V)or As(III)produced through the microbial transformations.The quantification of the As species was carried out following colorimetric method of As speciation analysis described by Hu et al.(2012).The cell-free extract was acidified with 1%HCl and 10 µmol L−1K2HPO4(Mallick et al.,2014).Supernatant was divided into three parallel subsamples treated with 1 mL oxidizing reagent KMnO4(oxidized treatment),reducing reagent NH4N2S(reduced treatment),or deionized water(untreated treatment).Each treatment was incubated for 30 min at room temperature except the sample treated with reducing agent,which was incubated at 80°C and cooled to room temperature.After 30 min,1 mL color reagent,a mixture of C6H8O6,(NH4)6Mo7O24·4H2O,C8H4K2O12Sb2·3H2O,and H2SO4(Hu et al.,2012),was added to each treatment,and the absorbance was measured after 5 min at 880 nm using an Evolution 201 UV-Vis spectrophotometer(Thermo Scientific,USA).The As(III)and As(V)concentrations were calculated using the equations according to Hu et al.(2012)∶

Cellular localization of As accumulation in KUMAs15

The cellular localization of As accumulation was determined through cell fractionation of the isolated strain KUMAs15(Table V).The maximum As accumulation at different As concentrations was found to occur in the cytosol,although As accumulation in the membrane was also evident.The cytosolic accumulation of As was observed to decrease in comparison with membrane accumulation at higher As concentrations(Table V).

Transcriptional expression of aoxB,arsB,and arsCgenes in KUMAs15

北运河流域面积涉及东城、西城、昌平、海淀、朝阳、顺义、通州、石景山、丰台、大兴、门头沟、延庆和怀柔等13个区县，承担着全市81%以上的人口、80%的经济总量和中心城区90%的排水任务，是北京市人口最集中、产业最聚集、城市化水平最高的流域。

计算得出纯防洪库容3.039亿m3，纯兴利库容1.590亿m3，从防洪限制水位到正常蓄水位之间的重复库容4.753 亿 m3。

Cellular surface adsorption of As in KUMAs15

The scanning electron micrograph of Micrococcus sp.KUMAs15 showed clear difference between Astreated and control groups(Fig.5).The micrograph also showed cellular aggregating tendencies when trea-ted with 2 mmol L−1As(III).The cells of the strain KUMAs15 were further analyzed by EDX,which confirmed the cellular surface adsorption of As in the Astreated strain as indicated by the As-specific peak.

TABLE IV As(III)oxidation ability of the As-resistant strain KUMAs15 utilizing different carbohydrates(0.5%,weight:volume)as a carbon source

**Significant at P＜0.01. a)Means±standard deviations(n=3).

Dextrose Fructose Maltose Sucrose Mannitol%24.80±0.05a)** 46.90±0.10** 36.94±0.59** 33.10±0.08** 40.70±0.08**

Fig.3 Cellular As accumulation in the As-resistant strain KUMAs15 grown in minimal salt broth(MSB)at different As concentrations(a),pH values(b),and incubation temperatures(c)under aerobic culture conditions for different incubation time and As removal from MSB by KUMAs15 grown at different As concentrations(d),pH values(e),and incubation temperatures(f)under aerobic culture conditions for different incubation time.Vertical bars indicate standard deviations of the means(n=3).The asterisks*and**indicate significant difference at P ＜ 0.05 and P ＜ 0.01,respectively.

TABLE V Cellular localization of As accumulation in the As-resistant strain KUMAs15 grown at different concentrations of As under aerobic culture conditions for 72 h

a)Means±standard deviations(n=3).

As concentration Cytosol Membrane mmol L−1mg L−1 0.5 63.163±0.029a) 36.843±0.037 1 61.087±0.009 38.890±0.030 2 57.933±0.029 42.070±0.026 4 58.060±0.027 41.947±0.040

DISCUSSION

Microbe-mediated As(III)oxidation to As(V),followed by microbe-assisted attenuation of the metalloid from the As-contaminated groundwater or irrigation water,could be a promising strategy for environmental As decontamination.This study reported the isolation of an As(III)-oxidizing and As-accumulating gram-positive bacterial strain,belonging to the genus Micrococcus,from the As-contaminated soil in Nadia,West Bengal.The strain had high MIC values for As and other heavy metals,indicating that this strain could be potentially utilized for microbe-assisted remediation of As.

The isolated strain,Micrococcus sp.KUMAs15,could oxidize As(III)from minimal media to As(V).Although higher amounts of As(III)oxidation was reported earlier by other isolates(Salmassi et al.,2002;Yoon et al.,2009),the isolated strain KUMAs15 could oxidize environmentally relevant concentrations of As,showing its high As(III)oxidation capacity.The bacterial strain could oxidize substantial amounts of As(III)over a range of pH values and temperatures,with a optimum oxidation at pH 7 and 37°C.The optimum pH and temperature for microbe-mediated As(III)oxidation were earlier reported to be dependent on the bacterial strains and growth conditions(Battaglia-Brunet et al.,2002;Bachate et al.,2012).The strain utilized glucose and other carbohydrates as carbon sources to oxidize As(III).Culture medium lacking glucose or other carbohydrate sources did not support the growth of KUMAs15,indicating that KUMAs15 was a hete-rotrophic As(III)-oxidizer.This heterotrophic As(III)oxidizer was also reported elsewhere to successfully decontaminate As-contaminated soil(Bahar et al.,2012).Strain KUMAs15 showed growth at neutral pH range,similar to that shown by certain bacteria reported in literature(Osborne and Ehrlich,1976;Suttigarn and Wang,2005).

医学本科生科研能力的培养能够促进其学业和职业的发展[13]。通过参与科研训练，能够提高他们主动学习和应用知识的能力，从而使得学习效果得到大幅度提升，并能学以致用[14]。对于毕业后要深造的学生来说，本科阶段参加过科研训练的学生不仅更受导师青睐，还为其研究生阶段的科研工作打下了良好的基础。当前就业形势日益严峻，研究表明具有创新思维的高校毕业具有较强的就业竞争能力[15]。除此之外，通过科研训练，可以增强医学本科生的观察能力、动手能力，锻炼吃苦耐劳、不怕困难的精神，培养严谨一丝不苟的工作态度，提高工作和学习的效率，而这些都是促进其迅速成才的优秀品质，对个人的发展大有裨益。

Fig.4 Reverse transcription-polymerase chain reaction analysis of the expression of arsenite oxidase gene(aoxB),arsenite transporter gene(arsB),and arsenate reductase gene(arsC)in the As-resistant strain KUMAs15 grown under different concentrations of As at pH 7.0 and 37°C for 72 h and densitometric analyses performed for the expression of aoxB,arsB,and arsC genes.Vertical bars indicate standard deviations of the means(n=3).The asterisks*,**,and***indicate significant difference at P ＜ 0.05,P ＜ 0.01,and P＜0.001,respectively.

Fig.5 Phenomena of cellular surface adsorption of As in the As-resistant strain KUMAs15,con firmed by the scanning electron microscope-energy dispersive X-ray spectroscopy analysis of KUMAs15 cells grown for 72 h in minimal salt broth without(a)or with 2 mmol L−1As(III)(b).The letters K and L following element indicate the atomic shells where the analysis has been done by energy dispersive X-ray spectroscopy.

The isolated strain KUMAs15 can accumulate As from the minimal salt medium,and maximum accumulation was observed when incubated with 2 mmol L−1 As(III)for 72 h.The phenomenon of As accumulation by KUMAs15 was also con firmed by the quantification of As removal from the minimal medium.The maximum As removal by KUMAs15 was observed to be 36.65%under similar conditions as those for maximum As accumulation.Thus,the isolate could remove about 733 µmol L−1of As(III),approximately 54.97 mg L−1 of As,from MSB under aerobic culture conditions.The similar results obtained from the accumulation and removal studies demonstrated that As removal by KUMAs15 was due to As uptake by the strain,which can be achieved either by active uptake(bioaccumulation)or passive uptake(surface adsorption).While bioaccumulation could be executed only by living cells,adsorption can be mediated by dead cells or even by fragments of cells(Ayangbenro and Babalola,2017).The bioaccumulation of As by Micrococcus sp.KUMAs15 seemed to be due to incorporation of As into both cytosolic and membrane fractions;however,the major portions of As were found to be accumulated in the cytosol.The strain KUMAs15 was also capable of As adsorption,which was con firmed by the SEM-EDX analysis.Heavy metal removal by microbial surface adsorption has been previously reported in Bacillus sp.(Garc´ıa et al.,2016),where＞90%of the adsorbed metals were distributed in cell wall and cell membrane,as well as in Brevibacillus sp.(Mallick et al.,2014)and others.The passive uptake of heavy metals could be dependent on physical conditions,and it was reported to be greater in stationary phase than in exponential phase due to charge distribution on the cell surface(Walker et al.,2005).However,it could also be due to extracellular material-mediated complexation,which might be responsible for metal remediation even by the dead microbial mass(Gupta and Diwan,2017).Considering the above facts,it seems plausible that the cellular surface adsorption of As in Micrococcus sp.KUMAs15 could be due to pure physical means or by extracellular material-mediated complexation with the metalloid(Anyanwu and Ugwu,2010).Elucidation of the detailed mechanisms of cellular surface adsorption of As in KUMAs15 needs further investigation.

Arsenic contaminant habitats present high As stress to the inhabiting microbial community,leading to the selection and maintenance of an array of Asdetoxifying mechanisms bypassing the restriction for growth(Huang et al.,2010).The As resistance mechanisms of microbes can be plasmid associated(Tsai et al.,1997),or by ars operon containing the genes arsRBC(Carlin et al.,1995).Bacterial As(III)efflux pump encoding gene arsB helps in the extrusion of As(III)from the cell(Rosen,1999;Poirel et al.,2013).The present study demonstrated that arsB displayed no significant alteration in As-stressed cells of the Asresistant strain of Micrococcus sp.The absence of significant up-regulation of arsB gene in the isolated bacterium can be correlated with the significant Asaccumulation capability of the strain,as up-regulation of ArsB efflux pump protein would confer great As extrusion.The transcriptional expression of aoxB gene,encoding arsenite oxidase enzyme,showed significant up-regulation under 2 mmol L−1As stress.The aoxB gene might be responsible for the As(III)-oxidizing capacity of KUMAs15,as evident from the As(III)oxidation assay.The isolated strain KUMAs15 could oxidize As(III)to As(V)and,therefore,demonstrated higher As resistance owing to its higher tolerance of As(V)than As(III),as evident from the MIC values.

3)对杨庄路口两侧行人过街，其信号灯放行时序与上游阜石路路口东西直行相位相同，此时南北方车辆因处于排队状态，因此行人过街安全性得到保证；同时当南北向车流获得通行权，行人过街处于红灯状态，因此减缓了行人过街对直行车辆造成的影响.

Arsenic-resistant microbes,having arsenate reductase and arsenite oxidase for As(V)reduction and As(III)oxidation,respectively,are often reported to oxidize and reduce As as the strategy for As resistance(Banerjee et al.,2011;Mallick et al.,2014).The bacterial isolate KUMAs15 was able to oxidize As(III)to less toxic form As(V)and showed only the oxidase activity.Govarthanan et al.(2015a)reported the microbial reduction of As(V)to more toxic As(III)as a strategy for As resistance by bacteria isolated from metalcontaminated soil.Analysis of transcriptional expression of arsenate reductase gene arsC in the isolated strain KUMAs15 did not show any significant alteration in As stress condition.The lack of response in the expression pattern of arsC indicated that the activity of ArsC protein,responsible for As(V)reduction,was probably lacking,limiting the possibility of extrusion of toxic As(III)by KUMAs15 into the environment.Therefore,the As-resistant Micrococcus sp.KUMAs15 could be an eco-friendly candidate for As decontamination in the environment conducive for its growth with suitable temperature and pH as mentioned.

混凝土抹面与填充混凝土的界面应采用与混凝土配合比一致的水泥砂浆，并设置5mm左右的浆料以确保粘结密度。在安装上部胶合板之前，需在胶合底部开孔，孔之间的距离在模板的上方和下方同10cm。钻孔后，应安装长度为400mm的12mm限位螺栓（不在模板的接合处）。应安装止水板，防止止水栓渗水。

CONCLUSIONS

This study reported the isolation of an As-resistant strain KUMAs15 of Micrococcus sp.,which showed high As tolerance with a MIC of 400 mmol L−1for As(V),from the As-contaminated soil in West Bengal,India.This work also presented the As(III)oxidation ability of the isolated strain KUMAs15 and the role of the arsenite oxidase gene(aoxB)in the oxidation process in Micrococcus sp.The strain KUMAs15 could be utilized to pre-oxidize As(III)to As(V)in groundwater,followed by the attenuation of As by adsorption processes.The strain could also accumulate about 55 mg L−1As from the aerobic culture medium.Together,the As(III)oxidation ability,high As tolerance(high MIC value),and As accumulation ability indicated the potential of KUMAs15 to serve as a biodecontaminating agent for environmental As pollution.

ACKNOWLEDGEMENT

This study was supported by the financial grant received from Department of Biotechnology,Government of India(No.BT/PR4693/BCE/8/894/2012).

REFERENCES

Acharyya S K,Chakraborty P,Lahiri S,Raymahashay B C,Guha S,Bhowmik A.1999.Arsenic poisoning in the Ganges delta.Nature.401:545.

Achour A R,Bauda P,Billard P.2007.Diversity of arsenite transporter genes from arsenic-resistant soil bacteria.Res Microbiol.158:128–137.

Altschul S F,Gish W,Miller W,Myers E W,Lipman D J.1990.Basic local alignment search tool.J Mol Biol.215:403–410.Anyanwu C U,Ugwu C E.2010.Incidence of arsenic resistant bacteria isolated from a sewage treatment plant.Int J Basic Appl Sci.10:64–78.

Ayangbenro A S,Babalola O O.2017.A new strategy for heavy metal polluted environments:A review of microbial biosorbents.Int J Environ Res Public Health.14:94.

Bachate S P,Cavalca L,Andreoni V.2009.Arsenic-resistant bacteria isolated from agricultural soils of Bangladesh and characterization of arsenate-reducing strains.J Appl Microbiol.107:145–156.

Bachate S P,Khapare R M,Kodam K M.2012.Oxidation of arsenite by two β-proteobacteria isolated from soil.Appl Microbiol Biotechnol.93:2135–2145.

Bahar M M,Megharaj M,Naidu R.2012.Arsenic bioremediation potential of a new arsenite-oxidizing bacterium Stenotrophomonas sp.MM-7 isolated from soil.Biodegradation.23:803–812.

Banerjee S,Datta S,Chattyopadhyay D,Sarkar P.2011.Arsenic accumulating and transforming bacteria isolated from contaminated soil for potential use in bioremediation.J Environ Sci Health Part A.46:1736–1747.

Battaglia-Brunet F,Dictor M C,Garrido F,Crouzet C,Morin D,Dekeyser K,Clarens M,Baranger P.2002.An arsenic(III)-oxidizing bacterial population:Selection,characterization,and performance in reactors.J Appl Microbiol.93:656–667.

Brown A E.2007.Benson’s Microbiological Applications:Laboratory Manual in General Microbiology.Short version.10th Edn.McGraw-Hill,New York.

Carlin A,Shi W,Dey S,Rosen B P.1995.The ars operon of Escherichia coli confers arsenical and antimonial resistance.J Bacteriol.177:981–986.

Cavalca L,Zanchi R,Corsini A,Colombo M,Romagnoli C,Canzi E,Andreoni V.2010.Arsenic-resistant bacteria associated with roots of the wild Cirsium arvense(L.)plant from an arsenic polluted soil,and screening of potential plant growth-promoting characteristics.Syst Appl Microbiol.33:154–164.

Cervantes C,Ji G Y,Ramirez J L,Silver S.1994.Resistance to arsenic compounds in microorganisms.FEMS Microbiol Rev.15:355–367.

Chakraborti D,Das B,Rahman M M,Chowdhury U K,Biswas B,Goswami A B,Nayak B,Pal A,Sengupta M K,Ahamed S,Hossain A,Basu G,Roychowdhury T,Das D.2009.Status of groundwater arsenic contamination in the state of West Bengal,India:A 20-year study report.Mol Nutr Food Res.53:542–551.

Chakraborti D,Mukherjee S C,Pati S,Sengupta M K,Rahman M M,Chowdhury U K,Lodh D,Chanda C R,Chakraborti A K,Basu G K.2003.Arsenic groundwater contamination in Middle Ganga Plain,Bihar,India:A future danger?Environ Health Perspect.111:1194–1201.

Chang J S,Ren X H,Kim K W.2008.Biogeochemical cyclic activity of bacterial arsB in arsenic-contaminated mines.J Environ Sci.20:1348–1355.

Chao Y Q,Zhang T.2011.Optimization of fixation methods for observation of bacterial cell morphology and surface ultrastructures by atomic force microscopy.Appl Microbiol Biotechnol.92:381–392.

Clifford D.1990.Ion exchange and inorganic adsorption.In Pontius F(ed.)Water Quality and Treatment.4th Edn.Mc-Graw-Hill,New York.

Fendorf S,Michael H A,Van Geen A.2010.Spatial and temporal variations of groundwater arsenic in South and Southeast Asia.Science.328:1123–1127.

Fricke B,Aurich H.1993.Periplasmic aminopeptidases in Acinetobacter calcoaceticus and Pseudomonas aeruginosa.J Basic Microbiol.33:291–299.

Garc´ıa R,Campos J,Cruz J A,Calder´on M E,Raynal M E,Buitr´on G.2016.Biosorption of Cd,Cr,Mn,and Pb from aqueous solutions by Bacillus sp strains isolated from industrial waste activate sludge.Tip Rev Espec Cienc Qu´ımico-Biol(in Spanish).19:5–14.

Garcia-Dominguez E,Mumford A,Rhine E D,Paschal A,Young L Y.2008.Novel autotrophic arsenite-oxidizing bacteria isolated from soil and sediments.FEMS Microbiol Ecol.66:401–410.

Govarthanan M,Lee S M,Kamala-Kannan S,Oh B T.2015a.Characterization,real-time quantification and in silico modeling of arsenate reductase(arsC)genes in arsenic-resistant Herbaspirillum sp.GW103.Res Microbiol.166:196–204.

Govarthanan M,Mythili R,Selvankumar T,Kamala-Kannan S,Rajasekar A,Chang Y C.2016.Bioremediation of heavy metals using an endophytic bacterium Paenibacillus sp.RM isolated from the roots of Tridax procumbens.Biotech.6:242.

Govarthanan M,Shim J,Kim S A,Kamala-Kannan S,Oh B T.2015b.Isolation and characterization of multi-metal-resistant Halomonas sp.MG from Tamil Nadu magnesite ore soil in India.Curr Microbiol.71:618–623.

Green H H.1918.Description of a bacterium which oxidizes arsenite to arsenate,and of one which reduces arsenate to arsenite,isolated from a cattle-dipping tank.S Afr J Sci.14:465–467.

Gupta P,Diwan B.2017.Bacterial Exopolysaccharide mediated heavy metal removal:A review on biosynthesis,mechanism and remediation strategies.Biotechnol Rep.13:58–71.

Hu S,Lu J S,Jing C Y.2012.A novel colorimetric method for field arsenic speciation analysis.J Environ Sci.24:1341–1346.

Huang A H,Teplitski M,Rathinasabapathi B,Ma L N.2010.Characterization of arsenic-resistant bacteria from the rhizosphere of arsenic hyperaccumulator Pteris vittata.Can J Microbiol.56:236–246.

Ilyaletdinov A N,Abdrashitova S A.1981.Autotrophic oxidation of arsenic by a culture of Pseudomonas arsenitoxidans.Mikrobiologiya(in Russian).50:197–204.

Liao V H C,Chu Y J,Su Y C,Hsiao S Y,Wei C C,Liu C W,Liao C M,Shen W C,Chang F J.2011.Arsenite-oxidizing and arsenate-reducing bacteria associated with arsenic-rich groundwater in Taiwan.J Contam Hydrol.123:20–29.

Maidak B L,Olsen G J,Larson N,Overbeek R,McCaughey M J,Woese C R.1997.The RDP(Ribosomal Database Project).Nucleic Acids Res.25:109–111.

Mallick I,Hossain S T,Sinha S,Mukherjee S K.2014.Brevibacillus sp.KUMAs2,a bacterial isolate for possible bioremediation of arsenic in rhizosphere.Ecotoxicol Environ Saf.107:236–244.

Meharg A A.2005.Venomous Earth:How Arsenic Caused the World’s Worst Mass Poisoning.Macmillan,New York.

Mukhopadhyay R,Rosen B P,Phung L T,Silver S.2002.Microbial arsenic:From geocycles to genes and enzymes.FEMS Microbiol Rev.26:311–325.

Muller D,Li`evremont D,Simeonova D D,Hubert J C,Lett M C.2003.Arsenite oxidase aox genes from a metal-resistant β-proteobacterium.J Bacteriol.185:135–141.

Nayak B,Das B,Mukherjee S C,Pal A,Ahamed S,Hossain M A,Maity P,Dutta R N,Dutta S,Chakraborti D.2008.Groundwater arsenic contamination in the Sahibganj district of Jharkhand state,India in the middle Ganga plain and adverse health effects.Toxicol Environ Chem.90:673–694.

Nordstrom D K.2002.Worldwide occurrences of arsenic in ground water.Science.296:2143–2145.

Oremland R S,Stolz J F.2005.Arsenic,microbes and contaminated aquifers.Trends Microbiol.13:45–49.

Osborne F H,Ehrlich H L.1976.Oxidation of arsenite by a soil isolate of Alcaligenes.J Appl Bacteriol.41:295–305.

Paul T,Mukherjee S K.2016.The rise of arsenic resistance(ars)operon.Int J Curr Innov Res.2:350–354.

Pelczar M J Jr,Bard R C,Burnett G W,Conn H J,Demoss R D,Euans E E,Weiss F A,Jennison M W,Meckee A P,Riker A J,Warren J,Weeks O B.1957.Manual of Microbiological Methods.McGraw-Hill,New York.

Poirel J,Joulian C,Leyval C,Billard P.2013.Arsenite-induced changes in abundance and expression of arsenite transporter and arsenite oxidase genes of a soil microbial community.Res Microbiol.164:457–465.

Rahaman S,Sinha A C,Pati R,Mukhopadhyay D.2013.Arsenic contamination:A potential hazard to the affected areas of West Bengal,India.Environ Geochem Health.35:119–132.

Rhine E D,N´ı Chadhain S M,Zylstra G J,Young L Y.2007.The arsenite oxidase genes(aroAB)in novel chemoautotrophic arsenite oxidizers.Biochem Biophys Res Commun.354:662–667.

Rhine E D,Phelps C D,Young L Y.2006.Anaerobic arsenite oxidation by novel denitrifying isolates.Environ Microbiol.8:899–908.

Rio D C,Ares M Jr,Hannon G J,Nilsen T W.2010.Purification of RNA using TRIzol(TRI reagent).Cold Spring Harb Protoc.2010:pdb.prot5439.

Rosen B P.1999.Families of arsenic transporters.Trends Microbiol.7:207–212.

Roychowdhury T,Tokunaga H,Uchino T,Ando M.2005.Effect of arsenic-contaminated irrigation water on agricultural land soil and plants in West Bengal,India.Chemosphere.58:799–810.

Salmassi T M,Venkateswaren K,Satomi M,Newman D K,Hering J G.2002.Oxidation of arsenite by Agrobacterium albertimagni,AOL15,sp.nov.,isolated from hot creek,California.Geomicrobiol J.19:53–66.

Santini J M,Sly L I,Schnagl R D,Macy J M.2000.A new chemolithoautotrophic arsenite-oxidizing bacterium isolated from a gold mine:Phylogenetic,physiological,and preliminary biochemical studies.Appl Environ Microbiol.66:92–97.

Scho field R K,Taylor A W.1955.The measurement of soil pH.Soil Sci Soc Am J.19:164–167.

Sinha S,Mukherjee S K.2008.Cadmium-induced siderophore production by a high Cd-resistant bacterial strain relieved Cd toxicity in plants through root colonization.Curr Microbiol.56:55–60.

Sinha S,Mukherjee S K.2009.Pseudomonas aeruginosa KUCD1,a possible candidate for cadmium bioremediation.Braz J Microbiol.40:655–662.

Smedley P L,Kinniburgh D G.2002.A review of the source,behaviour and distribution of arsenic in natural waters.Appl Geochem.17:517–568.

Smith A H,Lopipero P A,Bates M N,Steinmaus C M.2002.Arsenic epidemiology and drinking water standards.Science.296:2145–2146.

Suttigarn A,Wang Y T.2005.Arsenite oxidation by Alcaligenes faecalis strain O1201.J Environ Eng.131:1293–1301.

Tsai K J,Hsu C M,Rosen B P.1997.Efflux mechanisms of resistance to cadmium,arsenic and antimony in prokaryotes and eukaryotes.Zool Stud.36:1–16.

Uchino T,Roychowdhury T,Ando M,Tokunaga H.2006.Intake of arsenic from water,food composites and excretion through urine,hair from a studied population in West Bengal,India.Food Chem Toxicol.44:455–461.

Walker S L,Hill J E,Redman J A,Elimelech M.2005.In fluence of growth phase on adhesion kinetics of Escherichia coli D21g.Appl Environ Microbiol.71:3093–3099.

Weeger W,Li`evremont D,Perret M,Lagarde F,Hubert J C,Leroy M,Lett M C.1999.Oxidation of arsenite to arsenate by a bacterium isolated from an aquatic environment.Biometals.12:141–149.

Yilmaz E I.2003.Metal tolerance and biosorption capacity of Bacillus circulans strain EB1.Res Microbiol.154:409–415.Yoon I H,Chang J S,Lee J H,Kim K W.2009.Arsenite oxidation by Alcaligenes sp.strain RS-19 isolated from arsenic-contaminated mines in the Republic of Korea.Environ Geochem Health.31:109–117.

Zhu Y G,Yoshinaga M,Zhao F J,Rosen B P.2014.Earth abides arsenic biotransformations.Annu Rev Earth Planet Sci.42:443–467.