Potential for the Uptake and Removal of Arsenic [as (v) And

8/15/2019 Potential for the Uptake and Removal of Arsenic [as (v) And

1/11

http://dx.doi.org/10.4014/kjmb.1401.01004

Korean J. Microbiol. Biotechnol. (2014), 42(3), 238–248http://dx.doi.org/10.4014/kjmb.1401.01004pISSN 1598-642X eISSN 2234-7305

Korean Journal of Microbiology and Biotechnology

Potential for the Uptake and Removal of Arsenic [As (V) andAs (III)] and the Reduction of As (V) to As (III) by Bacilluslicheniformis (DAS1) under Different Stresses

Kumari Tripti 1 , D. Sayantan 1 , Shardendu Shardendu 1 * , Durgesh Narain Singh 2 , and Anil K. Tripathi 2

1 Laboratory of Environment and Biotechnology, Department of Botany, Patna Science College, Patna University,Patna 800005, India2 School of Biotechnology, Banaras Hindu University, Varanasi 221005, India

Received: January 27, 2014 / Revised: April 3, 2014 / Accepted: May 22, 2014

Introduction

Arsenic is an ubiquitous metalloid and its compounds are

common in many environmental compartments near toxiclevel. Natural sources of arsenic are minerals, found com-monly concentrated around sulphide bearing minerals andhydrous iron oxides [24]. Other sources are reported fromanthropogenic activities like use of pesticides, burning of coal, industrial metal smelting etc. In environment, inor-ganic arsenic occurs in many oxidations states those are

As(V) arsenate, As(III) arsenite, As(0) elemental arsenic, As(-III) arsenide. Among them trivalent and pentavalentforms of arsenic are most common but As(III) is most toxic.

Arsenic is considered the most significant potential threatto human health due to its ubiquity and toxicity. Henceremoval of arsenic from environment is of great importancefor human welfare. To trace out, the occurrence and distri-bution of native flora capable of arsenic tolerance is veryimportant in bacterial biotechnology. It makes us to under-stand the extent of potential of such flora in detoxifyingarsenic through oxidation, reduction or methylation [1].

Indo-Gangetic plain of Bengal delta, which includes Bihar is a principle arsenic sink. Agriculture is the major source of livelihood for majority of population of this region. Arsenic

contaminated ground water is used for the irrigation, which

The metalloid arsenic ( Z = 33) is considered to be a significant potential threat to human health due to its ubiquity and toxicity,even in rural regions. In this study a rural region contaminated with arsenic, located at longitude 85º 32'E and latitude 25º 11'N,

was initially examined. Arsenic tolerant bacteria from the rhizosphere of Amaranthas viridis were found and identified as Bacil-lus licheniformis through 16S rRNA gene sequencing. The potential for the uptake and removal of arsenic at 3, 6 and 9 mM[As(V)], and 2, 4 and 6 mM [As(III)], and for the reduction of the above concentrations of As(V) to As(III) by the Bacillus licheni-formis were then assessed. The minimal inhibitory concentrations (MIC) for As(V) and As(III) was determined to be 10 and 7mM, respectively. At 3 mM 100% As(V) was uptaken by the bacteria with the liberation of 42% As(III) into the medium, whereasat 6 mM As(V), 76% AS(V) was removed from the media and 56% was reduced to As(III). At 2 mM As(III), the bacteria con-sumed 100%, whereas at 6 mM, the As(III) consumption was only 40%. The role of pH was significant for the speciation, avail-ability and toxicity of the arsenic, which was measured as the variation in growth, uptake and content of cell protein. Both As(V)and As(III) were most toxic at around a neutral pH, whereas both acidic and basic pH favored growth, but at variable levels.Contrary to many reports, the total cell protein content in the bacteria was enhanced by both As(V) and As(III) stress.

Keywords: Arsenate [As(V)], arsenite [As(III)], Bacillus licheniformis , reduction, toxicity, uptake

*Corresponding author Tel: +919473240391, Fax: +916122670877E-mail: [email protected]© 2014, The Korean Society for Microbiology and Biotechnology


2/11


3/11

240 Tripti et al.


amount of arsenic in residual media. Bacterial culture weretaken in clean sterile Eppendorf tubes (1.5 ml) and centri-fuged at 10,000 rpm for 2-4 min. Pellet were taken sepa-rately for biomass digestion [16] and 1 ml of cell freesupernatant was taken into clean culture tubes with 9 ml of distilled water and subjected to digest by nitric acid method[2]. Before digestion, diluted supernatant was taken for esti-mation of amount of As(III) in residual media by Azure Bmethod [9]. After digestion, sample was further allowed toestimate for total arsenic in residual media by Azure Bmethod with some modifications. Concentration of As(V)obtained by subtracting concentration of As(III) from con-centration of total arsenic in medium. Pellet obtained above

were washed twice and placed in an oven for drying anddried sample was digested by nitric acid method and thenarsenic present in biomass was estimated by Azure Bmethod.

Extraction and estimation of bacterial cell protein5 ml bacterial broth culture was prepared in culture tubes

amended with arsenate As(V),and As(III) with acidic andbasic pH. It was observed that pH of normal TYEG broth

was 7.2 i.e. approximately 7, pH of arsenate/As(V) andarsenite/As(III) enriched media was 7.8 and 8.7, respec-tively. Hence pH 6 as (acidic pH) and pH 9 and 10 as (basicpH) taken for arsenate and arsenite stress, respectively.Bacterial cultures at different time intervals were taken for protein extraction. Bacterial culture was taken in clean ster-ile Eppendorf tubes (1.5 ml) and centrifuged at 12,000 rpmfor 10 min at 10ºC. Cell free supernatant was discardedand to the pellet 50 µl of lysozyme (10 mg/ml) was addedand kept for incubation at 37ºC for 20 min [45] after thatcells were vortexed and again centrifuged at 12,000 rpm for 10 min. Supernatant was transferred into clean test tubes

and volume was make up with distilled water upto 1 ml. 5ml of Coomassie reagent was added into each test tubeand after incubation reading was taken at 595 nm [7] inDouble beam Spectrophotometer. Amount of protein calcu-lated with the help of protein SDR prepared according toBradford protocol [7].

Identification of isolates based on 16S rRNA genesequence

PCR amplification: Total genomic DNA of the isolate was extracted with Wizard genomic DNA purification kit

(Promega, Madison, WI, USA). 16S rRNA gene was ampli-fied using universal bacterial primer 8f (5'-AGA GTT TGATYM TGG CTC AG- 3' and 1495r (5'-CTA CGG CTA CCTTGT TAC G-3') [18]. A 50 µl reaction mixture was prepared,

which include 50 ng of bacterial DNA as template, 200-250 µM of each primer, and 1.0 unit of Taq DNA poly-merase (Genei, Bangalore). The Polymerase chain reac-tions were performed on Veriti 96 well Thermal cycler (Applied Biosystem, USA) under the reaction condition of an initial denaturation of 5 min at 95 o C followed by 35cycles of 1 min at 94 o C, 1 min at 51 o C, and 1 min at 72 o C,

with a final extension of 5 min at 72 o C. The 16S rRNA geneamplicons were analyzed in 0.8% agarose gel at 5 V cm −1

and visualized under UV light with Alpha imager (AlphaInnotech Corporation, UK).

Sequencing: 16S rRNA genes were purified with WizardSV gel PCR purification kit (Promega, USA) and quantifiedusing ND-1000 Spectrophotometer (NanoDrop, Wilming-ton, USA). Direct sequencing was performed at SBT, BHU

with three primers 8f (5'-AGA GTT TGA TYM TGG CTC AG-3'), 1495r (5'-CTA CGG CTA CCT TGT TAC G -3') and561f (5'-AATTACTGGGCGTAAAG-3') [8] using the BigDyeTerminator v3.1 Cycle sequencing kit (Applied Biosystems,Switzerland) in an ABI Prism TM 310 automated DNASequencer (Applied Biosystems).

Analysis of 16S rRNA gene sequence: 16S rRNAgene sequences were edited using Bioedit software version3.1 to make a complete sequence. The almost completesequence was compared with the nucleotide sequencespresent in the NCBI database using the standard nucleo-tide BLAST search.

Gene Bank accession numbers: The nucleotidesequence of 16S rRNA gene of isolate has been depositedin Gene Bank under the accession number KF664027.

Data analysis: All the data in the figures are themeans ± standard errors of three replicates. The correlationbetween As(V) uptake and As(V) reduction to As(III) wereexpressed as scattered plot. The significance of effect of pH on uptake/removal of As(V) and As(III) was calculatedby Student’s t -test. The significance of variation of cell pro-tein content of bacterial cell in different stress was also cal-culated by Student’s t -test. The statistical evaluation wereperformed by the software STATISTICA υ 5.52.164.0 and thegraphs were drawn using MICROSOFT EXCEL 2003,2007.


4/11

Uptake/removal and Reduction of As Species by B. licheniformis 241

September 2014 | Vol. 42 | No. 3

Results and Discussion

Isolation of arsenic resistant bacteria Arsenic contamination is intensified by change in geo-

chemical cycles resulted from anthropogenic activities [43]and also increased by microbial metabolism [15, 22].

Above phenomena resulted the release of arsenic intodrinking water in shallow wells. Ground water of Indo-Gan-getic plain of Bengal Delta (Bihar located at 85º 32'E longi-tude and 25º 11'N latitude on the Earth) has been reportedas contaminated by arsenic [10, 11]. Identification of nativebacterial flora, with the assessment of their potential of uptake/removal of arsenic species and its biotransforma-tion, will be an addition of novel result in research data asthe first this type of report from above biotope of the earth.Bacteria were isolated by agar plating technique, fromrhizosphere of A. viridis, which was grown on naturallyarsenic contaminated site. Isolated bacteria were further allowed to grow on different concentration of arsenate

As(V) that were 10 µM, 100 µM, 1 mM on Tryptone Yeastextract Glucose (TYEG) agar plate. Bacterial coloniesgrown on 1 mM plate was considered as more tolerant,from which one isolate was selected randomly and identi-fied as Bacillus licheniformis by 16SrDNA sequencing. Phy-logentic tree (Fig. 1) showing the relationship of B.licheniformis DAS-1 (accession number KF664027) with

other member of bacillus species.

Effects of arsenate [As(V)] and arsenite [As(III)] ongrowth of bacteria and MIC

B. licheniformis was grown in TYEG broth amended with 3mM, 6 mM, 9 mM and 10 mM As(V) and 2 mM, 4 mM, 6mM, 7 mM As(III) to find out the MIC and growth pattern.The turbidity of cultures was measured as absorbance(O.D.) at 600 nm at different time points of growth phase. B.licheniformis grown in TYEG broth without added arsenici.e. control, took 2 h for lag phase then up to 24 h as expo-nential phase and from 26 h onwards it spent in stationaryphase (Fig. 2A and Fig. 2B). Growth of B. licheniformis isdependent on concentration of supplied As(V) in medium.There was gradual decrease in cell growth by increasing

As(V) concentration in medium. About 20% cell growth wasreduced in 3 mM As(V), 40% in 6 mM As(V), 61% in 9 mM

As(V) and 98% in 10 mM As(V). The duration of lag phase was also gradually increased on increasing As(V) concen-tration in medium, 10 h of long lag phase was observed in 9mM As(V) enriched culture. 10 mM was determined as theMIC since there was no significant growth at this concentra-tion.

The growth pattern of B. licheniformis in arsenite (AsIII)enriched media has been depicted in Fig. 2B. Cell growth

was gradually reduced on increasing As(III) concentration

Fig. 1. Phylogenetic tree showing relationship of Bacillus licheniformis DAS-1 (KF664027) with the other member of Bacillussp. in the NCBI database constructed by neighbour-joining method. Accession numbers of selected sequences are given in

parentheses. Scale bar represents 0.002 substitution per nucleotide position.


5/11

242 Tripti et al.


in medium. 24% of reduction was observed at 2 mM As(III),32% at 4 mM As(III), 55% at 6 mM and 90% at 7 mM As(III)

enrichment. There was gradual increase in duration of lagphase with increasing As(III) concentration, longest lagphase i.e. of 12 h was observed in 6 mM As(III) enrichment.

There are various studies of plants utilized for uptake of metal, metalloid including arsenic [3, 39, 40]. But to utilizeplant associated native bacterial flora for remediation of arsenic contamination will be a better tool to minimise theimpact of contamination. Generally we find reports onbacterial species tolerating either As(V) or As(III), likeParacoccus , Alcaligenes and Pseudomonas, Bacillus werereported earlier as As(V) tolerant species [4, 12, 47]. Pseu-

domonas and Corynebacterium were reported as As(III)

tolerant [1, 32]. Reports on single species having both[As(V) and As(III)] tolerance capability is rare and very few[5]. Bacillus licheniformis had tolerated both As(V) and

As(III) at different concentration with MIC for As(V) is10 mM and for As(III) is 7 mM. This is very significant find-ing, because here single bacterial species having tendencyto tolerate both forms of arsenic.

The significant inhibition in growth of Bacillus licheniformisunder arsenic stress is showing the toxic effects of As(V)and As(III). Arsenite (MIC 7 mM) was more toxic than arse-nate (MIC 10 mM), which is similar to other reports [23]. Itmight be due to arsenite, has very high affinity for proteinthiols so it readily chelated with intracellular proteins andcause damage to biomolecules of cell [26]. Arsenate act assubstitute of phosphate and inhibit oxidative phosphoryla-tion resulting cell toxicity [17, 46].

Potential of uptake/removal of As(V) and its reduction toAs(III)

The uptake/removal potential of Bacillus licheniformis was dependent on supplied amount of As(V) in the growthmedia. The concentration of As(V) left in residual mediumand concentration of As(III) formed in residual medium [cul-ture medium was previously enriched with As(V)] and con-centration of As estimated in biomass, at different time of growth phase has been depicted in Fig. 3A-C. Hence fig-ures depicting the uptake/removal potential of As(V) andefficiency of reduction of As(V) to As(III). In 3 mM of As(V)enriched media, 100% As(V) uptake/removal potential wasobserved, as no As(V) was found in residual media at theend of growth experiment. There was gradual decrease in

As(V) uptake/removal at 6 and 9 mM of As(V) enrichedmedium, the uptake/removal percentage were 76 and 35respectively. As(V)/Arsenate (AsO 4 3 −) is similar to phos-

phate (PO 43 −

) hence enter into cell through phosphatetransport membrane system [38, 46]. Nearly every organ-ism prokaryotes or eukaryotes have natural defence mech-anism for arsenic detoxification mostly involved transportation,oxidation/reduction, extrusion and immobilisation [5, 6, 43].Most of the microorganism in culture shows at least onetype of As-transforming mechanism, since As(V) is the pre-dominant in oxidized environment [34] and microbial reduc-tion of As(V) to As(III) is an important factor to increase themobility and bioavailability of As [20, 27]. In present study

we found the reduction of uptaken As(V) to As(III) and

extrusion of reduced As(III) in growth media. Efficiency of

Fig. 2. Growth pattern of Bacillus licheniformis in arseniccontaining TYEG broth, over the range of arsenic concen-trations (A) Arsenate [As(V)] (3 mM, 6 mM, 9 mM and10 mM), (B) Arsenite [As(III)] (2 mM, 4 mM, 6 mM and7 mM). Control cultures with no added arsenic [As(V) or As(III)]i.e. (0 mM) are shown. Change in OD 600 (Absorbance) of cul-ture was measured over 34 h. Error bars indicate the standarderror of the mean of three experiments.


6/11



reduction of uptaken As(V) into As(III) was also dependenton concentration of As(V) supplied in media. 62% of uptaken As(V) was reduced to As(III) with 0.06 mM per hreduction rate in 9 mM As(V) enriched media, whereas the56% of uptaken As(V) reduced to As(III) with reduction rateof 0.086 mM per h, in 6 mM As(V) enrichment and 42% of reduction with 0.046 mM per h reduction rate was esti-mated in 3 mM As(V)-enriched media. It was observed thatBacillus licheniformis has uptaken As(V) first and thenreduced to As(III) which was extruded in the growth media,as the concentration of As(V) was gradually decreased andconcentration of As(III) was gradually increased in residualmedia as presented in Fig. 3A-C. It might be explained inthe manner that As(V) first enters into bacterial cell with thehelp of phosphate transporter system, then reduced to

As(III) inside the cell with the help of (arsC) gene productarsenate reductase [30]. As(III) accumulated in cell then

extruded by (arsB) gene product i.e. an antiporter proteinchannel [29]. Fig. 3D represents the correlation between

As(V) uptake and As(III) formation, with r = 0.98 and p <0.001, showing the positive and very significant correlation.

In biomass, up to 1 mM arsenic has been detected, which is very less as comparison to As(V) supplied. Thereis possibility of volatilization of very less amount of arsenicof total supply. Bioaccumulation of arsenic was alsoreported in few species [16].

Uptake/removal potential of As(III)B. licheniformis has tolerated upto 6 mM As(III) (MIC

7 mM), which might be considered as hypertolerant for As(III). Because in maximum reports only 1-5 mM As(III)tolerant rhizospheric bacterial species had been isolatedfrom natural arsenic contaminated site [21, 25]. To checkthe percentage uptake/removal of arsenite [As(III)], concen-

Fig. 3. X-axis represents the concentration of As(V) left in residual media at the same time concentration of As(III) [uptakenAs(V) reduced to As(III)] formed in media along with the concentration of total arsenic (As) accumulated by bacteria [Asin biomass], at different time point (Y-axis) of growth phase. (A) 3 mM As(V) enriched media. (B) 6 mM As(V) enriched media.(C) 9 mM As(V) enriched media. All values are mean of three replicates and standard errors (SE) are presented as error bars ( ±).

(D) correlation between As(V) uptake and As(III) formation, with r = 0.98 and p < 0.001.


7/11

244 Tripti et al.


tration of As(III) left in residual media and concentration of arsenic in biomass was estimated at different time point of growth phase as represented in Fig. 4A-C As(III) uptake/removal potential of Bacillus licheniformis was 100% atlower concentration of supplied As(III) in media (2 mM),

whereas uptake/removal potential was 75% at 4 mM and40% at 6 mM of supplied As(III). As(III)/Arsenite (AsO 2 −)occurs in its hydroxide form as As(OH) 3 at neutral pH,

which is an inorganic equivalent of non-ionized glycerol,hence As(III) uses glycerol membrane transport system tomove across the cell [31, 42]. There was no biotransforma-tion of As(III) in other form. The accumulation of arsenic inbiomass was up to 2 mM, which was higher as comparedto As(V) enriched medium. Very little amount of arsenic

was unaccounted, which might be volatilised or convertedto other organic forms. Since there is no such reportregarding As(III) uptake by bacteria, hence this is new andsignificant finding of present study.

Effects of pH on growth and uptake/removal of Bacilluslicheniformis in arsenate As(V) and arsenite As(III)stress

The pH are highly significant variables controlling arsenicspeciation [14]. Availability of arsenic species in growthmedium depend on pH. At around neutral pH, As(V) foundin ionic form as Arsenate (AsO 4 3 −) and As(III) found in nonionic condition as As(OH) 3 . Hence around neutral pH, avail-ability of As(V) and As(III) is higher for uptake by bacteria[44]. It was observed in this study, that pH of TYEG broth or control medium (without added arsenic) was 7.2, which

was increased to 7.8 on adding 9 mM As(V) and to 8.7 onadding 6 mM As(III). Hence pH 6 and pH 9 was taken asacidic and basic pH change of As(V) stress and pH 6 andpH 10 for As(III) stress. In present study, growth pattern of

Bacillus licheniformis in As(V) stressed medium, wasaffected by variation in pH. In Fig. 5A it was observed thatboth acidic and basic pH change of As(V) stressedmedium, had favoured the growth of bacteria. It might bedue to significant decrease in uptake of As(V), in changedacidic and basic pH condition of As(V) supplied medium asshown in Fig. 5B. Student’s t-test between pair data of uptake at pH 7.8 and at pH 6, with p = 0.0413 and betweenuptake at pH 7.8 and pH 9 with p = 0.038. Here change of uptake in both the case is very significant ( p < 0.05). Henceit is proved that, availability of As(V) in ionic form, is less at

acidic and basic pH, which reduced the uptake. Because

Fig. 4. X-axis represents the concentration of As(III) left inresidual media, depicting the concentration of total arsenicuptaken/removed and accumulated by bacteria [As in bio-mass] at different time point (Y-axis) of growth phase.(A) 2 mM As(III) enriched media. (B) 4 mM As(III) enrichedmedia. (C) 6 mM As(III) enriched media. Concentration of

As(III) gradually decreased in residual media showing removalof As(III) from the medium. All values are mean of three repli-

cates and standard errors (SE) are presented as error bars ( ±).


8/11



As(V) only can be uptaken via phosphate transporters inionic form (AsO 4 3 −). It was also observed in Fig. 5A thatacidic pH (pH 6) of As(V) stressed medium, was slightlymore favourable for growth than basic pH (pH 9). It was

due to, at alkaline pH, soluble As(V) was more availablethan at acidic pH [28]. Since more the availability of soluble

As(V), more the uptake of As(V) by bacterial cell, hencemore the toxic effect resulting less number of cell in growthphase. There are few reports regarding effect of pH onarsenic uptake [19] one was reported in mutant strain of S.faecalis in which the uptake of As(V) was maximal at neu-tral pH and was declined at higher pH above 7.

Growth of bacteria in As(III) stress was also increased onexposing to acidic and basic pH change Fig. 6A. There wasalso significant effect of pH on As(III) uptake as shown in

Fig. 6B that, change in pH significantly ( p < 0.05) reduced

the uptake with p = 0.038 in acidic pH and p = 0.035 inbasic pH. It was due to less availability of As(III) in the form

of non-ionic [As(OH) 3 ], for uptake via glycerol transport atchanged pH condition than at normal pH. There was alsoone more interesting observation, that arsenic was foundas favourable factor for growth of Bacillus licheniformis , inacidic and basic pH condition of TYEG medium, as found inFig. 5A and 6A Growth in acidic and basic pH condition of control medium was very less as compared to medium witharsenic supplied at same pH.

Variation in total protein content of bacterial cell in dif-ferent stress

Under different growth condition/stress the cell size and

Fig. 5. Growth pattern and uptake of Bacillus licheniformisaffected by changed pH condition of arsenic enriched

media. (A) 9 mM As(V) enriched media. Three control (noadded arsenate) with pH 7.2, 6 and 9, medium enriched with9 mM As(V) has pH 7.8 which, was changed to [As(V) pH 6]and [As(V) pH 9]. Changed pH of As(V) stress favoured thegrowth. (B) X-axis represents the concentration of As(V)uptaken, from 9 mM As(V) enriched media with pH 7.8, 6 and9. Uptake reduced on changing pH of As(V) stress.

Fig. 6. (A) 6 mM As(III) enriched media. Three control (noadded arsenite) with pH 7.2, 6 and 10, medium enriched with

6 mM As (III) has pH 8.7 which was changed to [As(III) pH 6]and [As(III) pH 10]. Change in pH of As(III) stress reduced the

As(III) toxicity and hence enhanced the growth. (B) X-axis rep-resents the concentration of As(III) uptaken/removed, from6 mM As(III) enriched media with pH 8.7, 6 and 10. As(III)uptake reduced in changed pH condition of As(III) stress. Allvalues are mean of three replicates and standard errors(SE)are presented as error bars ( ±).


9/11

246 Tripti et al.


thus protein content can change to several folds [41].

Hence study of variation in level of total cell protein canmeasure the level of stress. Cell protein was extracted fromthe four set of culture tubes that were control culture (pH7.2), As(V) stressed culture (pH 7.8) and As(V) stressexposed to acidic and alkaline pH (6 and 9) at different timepoint of growth phase. Similar set was prepared for As(III),

which contained, control (pH 7.2), As(III) (pH 8.7) and As(III) with pH (6 and 10). Extracted protein was quantifiedand represented in graph in the form of amount ( µg) of pro-tein per unit absorbance/cell density with time in Fig. 7A for

As(V) stress and in Fig. 7B for As(III) stress. Significant

( p < 0.01 and p < 0.05 for As(V) and As(III) stress, respec-

tively) variation was observed in amount of protein contentof bacterial cell in different stress condition. It might beexplained as due to stress the cell division was inhibited butthere was enlargement of cell size/volume, hence the levelof protein per cell was increased The highest amount of protein was determined in As(V) (9 mM) stress conditionand lowest amount in control (no added arsenic) as shownin Fig. 7A. Protein content in As(V) stress with changedacidic and basic pH condition was significantly lower ( p = 0.0032 for acidic change and p = 0.0094 for basicchange) than only As(V) stress. This result suggests thechange in pH of arsenic stress, modifies/lessen the level of arsenic stress [13]. Similar pattern of variation in proteincontent was also obtained in As(III) stress in Fig. 7B( p = 0.0153 for acidic change and p = 0.0204 for basicchange of As(III) stress) explaining the same phenomena.

Conclusion

B. licheniformis was isolated from arsenic contaminatedregion located at 85º 32'E longitude and 25º 11'N latitudeon the Earth. Isolated bacteria tolerated both As(V) [MIC 10mM] as well as As(III) [MIC 7 mM], which is rare to find thatsingle species tolerating both forms of arsenic. Uptake/removal potential of B. licheniformis was dependent onsupplied amount of As(V) and As(III) in the growth mediaand 100% uptake/removal was determined in lower con-centration of As(V) and As(III) (3 mM and 2 mM, respec-tively). B. licheniformis was also capable of reducinguptaken As(V) into As(III), and its potential of reduction wasalso dependent on concentration of supplied As(V). It wasalso capable of accumulating little amount of arsenic in bio-mass. The potential of uptake, reduction and growth pattern

were also affected by variation in pH of arsenic stress. Vari-

ation in pH mitigates the arsenic stress, as different level of cell’s protein (showing level of stress) was determined atdifferent pH of arsenic stress. Hence native bacteria can beutilized in minimizing the arsenic contamination and its effi-ciency of uptake/removal can also be modify by changingpH of system, so it can help to avoid the entry of the arsenicin human food chain.

Acknowledgments

Author pay their sincere gratitude to University Grants

commission, New Delhi, India [F.No.33-169/2007(SR)] and

Fig. 7. Total protein content of bacterial cell in differentstress condition. X-axis represents the amount ( µg) of proteinper unit absorbance (cell density) at different time point (Y-axis)of growth phase. All values are mean of three replicates andstandard errors (SE) are presented as error bars ( ±). (A) Dif-ferent pH of As(V) [9 mM] stress (B) Different pH of As(III) [6mM] stress.


10/11



Council of Scientific and Industrial Research, New Delhi,India [F.No.38(1165)/07/EMR II], for providing financialassistance for the purchase of instrument utilized for this

work. The author would like to thank the anonymousreviewers for the evaluation of this manuscript.

References

1. Anderson LCD, Bruland KW. 1991. Biogeochemistry of arse-nic in natural waters: importance of methylated species. Envi-ron. Sci. Technol . 25 : 420-427.

2. APHA, AWWA, WEF. 2005. In Eaton AD, Clesceri LS, RiceEW, Greenburg AE (eds.), Standards methods for the exam-

ination of water and waste water. American public healthassociation, American water works association, Water Envi-ronment Federation Joint Publication USA.

3. Azaizeh H, Salhani N, Sabeswary Z, Shardendu S, Emons H.2006. Phytoremediation of Selenium using sub-surface flowconstructed Wetlands. Int. J. Phytorem. 8 : 187-198.

4. Bachate SP, Cavalca L, Andreoni V. 2009. Arsenic resistantbacteria isolated from the agricultural soil of Bangladesh andcharacterization of arsenic reducing strain. J. Appl. Microbiol.107 : 145-156.

5. Banerjee S, Santra SC. 2010. Studies on the role of aerobicsoil bacteria in arsenic transformation and mobilization. Int. Q.J. Life. Sci. 2 : 587-594.

6. Bhattacharjee H, Rosen BP. 2007. Arsenic metabolism in pro-karyotic and eukaryotic microbes. In Nies DHS, Simonies(ed). Mol. Microbiol. Heavy Met . 6 : 371-406.

7. Bradford M. 1976. A rapid and sensitive method for the quan-tification of microgram quantities of protein utilizing the princi-ple of protein-dye binding. Anal. Biochem . 72 : 248-254.

8. Castaldini M, Turrini A, Sbrana C, Benedette A, Marchionni M,Mocali S, et al. 2005. Imapct of Bt corn on rhizopheric and soileubacterial communities and on beneficial Mycorrhizal symbi-osis in experimental microsome. Appl. Environ. Microbiol. 71 :6719.

9. Cherian T, Narayana B. 2005. A new spetrophotometricmethod for determination of arsenic in environmental and bio-logical samples. Anal. Lett. 38 : 2207-2216.

10. Chakraborti D, Sengupta MK, Rahman MM, Ahamed S. 2004.Groundwater arsenic contamination and its health effects inthe Ganga-Meghna-Brahmaputra plain . J. Environ. Monit. 6 :74N-83N.

11. Chowdhury R, Sen AK, Karak P, Chatterjee R, Giri AK,Chaudhuri K. 2009. Isolation and characterization of arsenicresistant bacterium from a bore well in west Bengal India.

Ann. Microbiol. 59 : 253-258.12. Clausen CA. 2000. Isolating metal-tolerant bacteria capable of

removing copper chromium and arsenic from treated wood.Waste Managem. 18 : 264-268.

13. Ferjani A, Mustardy L, Sulpice R, Marim K, Suzuki I, Hage-

mann M. et al. 2003. Glucosylglycerol a compatible solute,sustain cell division under salt stress. Plant Physiol. 131 :

1628-1637.14. Fitz WJ, Wenzel WL. 2002. Arsenic transformation in the soil-

rhizosphere-plant system: fundamentals and potential appli-cation to phytoremediation. J. Biotechnol. 99 : 259-278.

15. Frankerberger WT Jr (ed.) 2002. Environmental chemistry of arsenic. Marcel Dekker, Inc., New York.

16. Ghodsi H, Hoodaji M, Tahmourespour A, Gheisari MM. 2011.Investigation of bioremediation of arsenic by bacteria isolatedfrom contaminated soil. Afr. J. Microbiol. Res. 5 : 5889-5895.

17. Goyer RA, Clarkson TW. 2001. Toxic effects of metals. Casa-rett and Doull’s Toxicology: The basis of poison, 6th Ed. McGraw Hill.

18. Grifoni A, Bazzicalupo MC, Serio CD, Fancelli S, Fani R.1995. Identification of Azospirillum strain by restriction frag-ment length polymorphism of 16S-rDNA and of the histidineoperon. FEMS Microbiol. Lett . 127 : 85-91.

19. Harold MF, Harold LR, Abrams A. 1965. A mutant of Strepto-coccus faecalis defective in phosphate uptake. J. Biol. Chem.240 : 3145-3153.

20. Harvey CF, Swartz CH, Badruzzaman ABM, Keon-Blute N,Yu W, Ashraf AM, et al. 2002. Arsenic mobility and groundwa-ter extraction in Bangladesh. Science 298 : 1602-1606.

21. Huang A, Teplitski M, Rathinasabathi B, Ma l. 2010. Charater-ization of arsenic resistant bacteria from the rhizosphere of arsenic hyperaccumulator Pteris vittata . Can. J. Microbiol. 56 :

236-246.22. Islam FS, Gault AG, Boothman C, Polya DA, Charnock JM,

Chatterjee D, et al. 2004. Role of metal-reducingbacteria inarsenic release from Bengal delta sediments. Nature 430 : 68-71.

23. Kaltreider RC, Davis AM, Lariviere JP, Halminton JW. 2001.Arsenic alters the function of glucocorticoids receptor as atranscription factor. Environ. Health Perspect. 109 : 245-251.

24. Kolker A, Koeppen RP. 1998. Arsenic variation in pyrite incoal: Proceeding of the fifteenth international Pittsburg CoalConference, 9 p.

25. Liao VH-C, Chu V-J, Su Y-C, Hsiao S-Y, Wei C-C, Liu C-W, et al . 2011. Arsenite oxidising and arsenate reducing bacteriaassociated with arsenic rich ground water in Taiwan. J. Con-tam. Hydrol . 123 : 20-29.

26. Liu SX, Athar M, Lippai I, Waldren C. 2001. Induction of oxy-radical by arsenic : implication for mechanism of genotoxicity.Proc. Natl. Acad. Sci. 98 : 1643-1648.

27. Macur RE, Wheeler JT, McDermott TR, Inskeep WP, 2001.Microbial populations associated with the reduction andenhanced mobilization of arsenic in mine tailings. Environ.Sci. Technol. 35 : 3676-3682.

28. Masscheleyn PH, Delaune RD, Jr Patrick WH. 1991. Effectsof redox potential and pH on arsenic speciation and solubilityin the contaminated soil. Environ. Sci. Technol. 25 : 1414-

1429.


11/11

Documents

Potential for the Uptake and Removal of Arsenic [as (v) And