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

Abstract

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 $85^{\circ}$ 32'E and latitude $25^{\circ}$ 11'N, was initially examined. Arsenic tolerant bacteria from the rhizosphere of Amaranthas viridis were found and identified as Bacillus 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 licheniformis were then assessed. The minimal inhibitory concentrations (MIC) for As(V) and As(III) was determined to be 10 and 7 mM, respectively. At 3 mM 100% As(V) was uptaken by the bacteria with the liberation of 42% As(III) into the medium, whereas at 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 consumed 100%, whereas at 6 mM, the As(III) consumption was only 40%. The role of pH was significant for the speciation, availability 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.

Introduction

Arsenic is an ubiquitous metalloid and its compounds are common in many environmental compartments near toxic level. Natural sources of arsenic are minerals, found commonly concentrated around sulphide bearing minerals and hydrous iron oxides [24]. Other sources are reported from anthropogenic activities like use of pesticides, burning of coal, industrial metal smelting etc. In environment, inorganic 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 pentavalent forms of arsenic are most common but As(III) is most toxic.

Arsenic is considered the most significant potential threat to human health due to its ubiquity and toxicity. Hence removal of arsenic from environment is of great importance for human welfare. To trace out, the occurrence and distribution of native flora capable of arsenic tolerance is very important in bacterial biotechnology. It makes us to understand the extent of potential of such flora in detoxifying arsenic 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 is taken by crops and from this trophic level arsenic enters into food chain and cause many health hazards. Rapid and huge withdrawal of water creates an opportunity to release the arsenic from its chelated sources. Hence it is of immediate importance that natural bacterial flora need to be isolated and its potential for remediation and biotransformation need to be work out.

Relative abundance of As(V) and As(III) in soil environment is influenced by microbial transformations. Bacteria developed various strategies to resist arsenic toxicity either by extrusion after oxidation or reduction of arsenic species or by intracellular chelations.

For uptake and transport by bacteria, both As(V) and As(III) are available at neutral pH [44]. As(V) are found in ionised form as an oxyanion (AsO43−) at neutral pH and are transported into bacterial cell by phosphate (PO4 3−) transporters due to chemical analogy with phosphate. As(V) can also easily incorporated into metabolic pathway of an organism as a substitute of phosphate [46]. This substitution for phosphate and inhibition of oxidative phosphorylation is the main toxic effects of As(V) [17]. As(III) are found mostly unionised as As(OH)3 at neutral pH and are transported into cells by aqua glycoporins (glycerol transport proteins) [36, 38]. As(III) has high affinity for cells proteinthiol so it can easily chelated with protein and result toxicity.

There are two systems in cell, which help in tolerating arsenate As(V). The first system is to uptake As(V) with the help of transporters and reduce into As(III) with the help of enzyme arsenate reductase. The second system help to extrude reduced As(III) from cell. Arsenic reducing bacteria contain arsenic resistance gene (ars) either on chromosome or on plasmid, which confers arsenic resistance by encoding two necessary components (a) reductase enzyme (ArsC), for reducing arsenic(V) to arsenic(III). (b) an efflux pump (ArsB) that extrudes As(III) from the cytoplasm for lowering intracellular concentration of toxic arsenic [37]. As(III) uptake occurs by glycerol transporter and get chelated with intracellular protein by thiols.

Protein content of bacteria under different stress are variable [41]. The goal of present study is to isolate and identify the arsenic resistant bacteria and evaluation of the potential to tolerate As(V) and As(III). The other objective is to determine the potential of reduction of As(V) to As(III). The effects of pH on growth and uptake have also been determined, total protein content of bacterial cell has estimated to check the level of stress.

 

Material and Methods

Isolation of bacteria

In natural habitat, bacteria grow together containing many species and to study on the individual bacteria one need a pure culture of a single strain. Spread plate technique [35] had been used to obtain a pure culture of a single strain of bacteria. Soil was isolated from rhizosphere of Amaranthas viridis, which was grown on arsenic contaminated area. 1 mg of soil sample was taken and diluted with sterile water. 1 ml of diluted soil was put on tryptone yeast extract glucose (TYEG) [33]. As living cell, generally exposed to arsenic in the form of arsenate and arsenite in which As(V) are most available, hence arsenate has been used for isolation of arsenic resistant bacteria. Agar plates amended with 10 μM, 100 μM, 1 mM and 10 mM of arsenate (Sodium arsenate Na2HAsO4·7H2O) and spread evenly over the surface with the help of sterile L-shaped glass rod. Plates were incubated at 37℃ for 24 h. Many visible bacterial colonies were grown on plates; only 10 mM plates contain no colony. Hence one of the isolate, which was able to grow on 1 mM As (V) amended TYEG plate was considered as more tolerant species and was selected for further study.

Growth phase study and minimum inhibitory concentration (MIC)

Overnight grown culture was prepared in 50 ml of TYEG broth. For subculturing, into test and control tubes 0.5 ml of bacterial culture was aseptically transferred into culture tubes containing 4.5 ml of TYEG broth, which had been previously amended with arsenic (Control no added arsenic).

The culture tubes were then incubated at 37℃ and 150 rpm in shaker incubator. At different time interval, from 0, 1, 2, 4, 6, 8, 10, 24, 26, 28, 30 h, cultures were taken for turbidity measurement. Absorbance was measured at 600 nm in (Double beam spectrophotometer, Systronics, India); uninnoculated broth was served as a blank. In one set of experiment, broth amended by one particular concentration of As(V) or As(III) and the same procedure was repeated for various concentration until the MIC obtained.

Estimation of concentration of As(V) and As(III) in residual media and As in biomass

Bacterial cultures prepared for turbidity measurement at various time intervals were also taken for estimation of amount of arsenic in residual media. Bacterial culture were taken in clean sterile Eppendorf tubes (1.5 ml) and centrifuged at 10,000 rpm for 2-4 min. Pellet were taken separately for biomass digestion [16] and 1 ml of cell free supernatant 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 estimation of amount of As(III) in residual media by Azure B method [9]. After digestion, sample was further allowed to estimate for total arsenic in residual media by Azure B method with some modifications. Concentration of As(V) obtained by subtracting concentration of As(III) from concentration of total arsenic in medium. Pellet obtained above were washed twice and placed in an oven for drying and dried sample was digested by nitric acid method and then arsenic present in biomass was estimated by Azure B method.

Extraction and estimation of bacterial cell protein

5 ml bacterial broth culture was prepared in culture tubes amended with arsenate As(V),and As(III) with acidic and basic pH. It was observed that pH of normal TYEG broth was 7.2 i.e. approximately 7, pH of arsenate/As(V) and arsenite/As(III) enriched media was 7.8 and 8.7, respectively. Hence pH 6 as (acidic pH) and pH 9 and 10 as (basic pH) taken for arsenate and arsenite stress, respectively. Bacterial cultures at different time intervals were taken for protein extraction. Bacterial culture was taken in clean sterile Eppendorf tubes (1.5 ml) and centrifuged at 12,000 rpm for 10 min at 10℃. Cell free supernatant was discarded and to the pellet 50 μl of lysozyme (10 mg/ml) was added and kept for incubation at 37℃ for 20 min [45] after that cells 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. 5 ml of Coomassie reagent was added into each test tube and after incubation reading was taken at 595 nm [7] in Double beam Spectrophotometer. Amount of protein calculated with the help of protein SDR prepared according to Bradford protocol [7].

Identification of isolates based on 16S rRNA gene sequence

PCR amplification: Total genomic DNA of the isolate was extracted with Wizard genomic DNA purification kit (Promega, Madison, WI, USA). 16S rRNA gene was amplified using universal bacterial primer 8f (5'-AGA GTT TGA TYM TGG CTC AG- 3' and 1495r (5'-CTA CGG CTA CCT TGT 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 polymerase (Genei, Bangalore). The Polymerase chain reactions were performed on Veriti 96 well Thermal cycler (Applied Biosystem, USA) under the reaction condition of an initial denaturation of 5 min at 95℃ followed by 35 cycles of 1 min at 94℃, 1 min at 51℃, and 1 min at 72℃, with a final extension of 5 min at 72℃. The 16S rRNA gene amplicons were analyzed in 0.8% agarose gel at 5 V cm−1 and visualized under UV light with Alpha imager (Alpha Innotech Corporation, UK).

Sequencing: 16S rRNA genes were purified with Wizard SV gel PCR purification kit (Promega, USA) and quantified using ND-1000 Spectrophotometer (NanoDrop, Wilmington, 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') and 561f (5'-AATTACTGGGCGTAAAG-3') [8] using the BigDye Terminator v3.1 Cycle sequencing kit (Applied Biosystems, Switzerland) in an ABI PrismTM 310 automated DNA Sequencer (Applied Biosystems).

Analysis of 16S rRNA gene sequence: 16S rRNA gene sequences were edited using Bioedit software version 3.1 to make a complete sequence. The almost complete sequence was compared with the nucleotide sequences present in the NCBI database using the standard nucleotide BLAST search.

Gene Bank accession numbers: The nucleotide sequence of 16S rRNA gene of isolate has been deposited in Gene Bank under the accession number KF664027.

Data analysis: All the data in the figures are the means ± standard errors of three replicates. The correlation between As(V) uptake and As(V) reduction to As(III) were expressed as scattered plot. The significance of effect of pH on uptake/removal of As(V) and As(III) was calculated by Student’s t-test. The significance of variation of cell protein content of bacterial cell in different stress was also calculated by Student’s t-test. The statistical evaluation were performed by the software STATISTICA𝑣5.52.164.0 and the graphs were drawn using MICROSOFT EXCEL 2003, 2007.

 

Results and Discussion

Isolation of arsenic resistant bacteria

Arsenic contamination is intensified by change in geochemical cycles resulted from anthropogenic activities [43] and also increased by microbial metabolism [15, 22]. Above phenomena resulted the release of arsenic into drinking water in shallow wells. Ground water of Indo-Gangetic plain of Bengal Delta (Bihar located at 85º 32'E longitude and 25º 11'N latitude on the Earth) has been reported as contaminated by arsenic [10, 11]. Identification of native bacterial flora, with the assessment of their potential of uptake/removal of arsenic species and its biotransformation, will be an addition of novel result in research data as the first this type of report from above biotope of the earth. Bacteria were isolated by agar plating technique, from rhizosphere of A. viridis, which was grown on naturally arsenic 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 Yeast extract Glucose (TYEG) agar plate. Bacterial colonies grown on 1 mM plate was considered as more tolerant, from which one isolate was selected randomly and identified as Bacillus licheniformis by 16SrDNA sequencing. Phylogentic tree (Fig. 1) showing the relationship of B. licheniformis DAS-1 (accession number KF664027) with other member of bacillus species.

Fig. 1.Phylogenetic tree showing relationship of Bacillus licheniformis DAS-1 (KF664027) with the other member of Bacillus sp. 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.

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

B. licheniformis was grown in TYEG broth amended with 3 mM, 6 mM, 9 mM and 10 mM As(V) and 2 mM, 4 mM, 6 mM, 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 arsenic i.e. control, took 2 h for lag phase then up to 24 h as exponential phase and from 26 h onwards it spent in stationary phase (Fig. 2A and Fig. 2B). Growth of B. licheniformis is dependent 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 was reduced 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) concentration in medium, 10 h of long lag phase was observed in 9 mM As(V) enriched culture. 10 mM was determined as the MIC since there was no significant growth at this concentration.

Fig. 2.Growth pattern of Bacillus licheniformis in arsenic containing TYEG broth, over the range of arsenic concentrations (A) Arsenate [As(V)] (3 mM, 6 mM, 9 mM and 10 mM), (B) Arsenite [As(III)] (2 mM, 4 mM, 6 mM and 7 mM). Control cultures with no added arsenic [As(V) or As(III)] i.e. (0 mM) are shown. Change in OD600 (Absorbance) of culture was measured over 34 h. Error bars indicate the standard error of the mean of three experiments.

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 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 lag phase with increasing As(III) concentration, longest lag phase 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 utilize plant associated native bacterial flora for remediation of arsenic contamination will be a better tool to minimise the impact of contamination. Generally we find reports on bacterial species tolerating either As(V) or As(III), like Paracoccus, Alcaligenes and Pseudomonas, Bacillus were reported earlier as As(V) tolerant species [4, 12, 47]. Pseudomonas 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) is 10 mM and for As(III) is 7 mM. This is very significant finding, because here single bacterial species having tendency to tolerate both forms of arsenic.

The significant inhibition in growth of Bacillus licheniformis under arsenic stress is showing the toxic effects of As(V) and As(III). Arsenite (MIC 7 mM) was more toxic than arsenate (MIC 10 mM), which is similar to other reports [23]. It might be due to arsenite, has very high affinity for protein thiols so it readily chelated with intracellular proteins and cause damage to biomolecules of cell [26]. Arsenate act as substitute of phosphate and inhibit oxidative phosphorylation resulting cell toxicity [17, 46].

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

The uptake/removal potential of Bacillus licheniformis was dependent on supplied amount of As(V) in the growth media. The concentration of As(V) left in residual medium and concentration of As(III) formed in residual medium [culture medium was previously enriched with As(V)] and concentration of As estimated in biomass, at different time of growth phase has been depicted in Fig. 3A-C. Hence figures depicting the uptake/removal potential of As(V) and efficiency of reduction of As(V) to As(III). In 3 mM of As(V) enriched media, 100% As(V) uptake/removal potential was observed, as no As(V) was found in residual media at the end of growth experiment. There was gradual decrease in As(V) uptake/removal at 6 and 9 mM of As(V) enriched medium, the uptake/removal percentage were 76 and 35 respectively. As(V)/Arsenate (AsO4 3−) is similar to phosphate (PO4 3−) hence enter into cell through phosphate transport membrane system [38, 46]. Nearly every organism prokaryotes or eukaryotes have natural defence mechanism for arsenic detoxification mostly involved transportation, oxidation/reduction, extrusion and immobilisation [5, 6, 43]. Most of the microorganism in culture shows at least one type of As-transforming mechanism, since As(V) is the predominant in oxidized environment [34] and microbial reduction of As(V) to As(III) is an important factor to increase the mobility 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 reduction of uptaken As(V) into As(III) was also dependent on concentration of As(V) supplied in media. 62% of uptaken As(V) was reduced to As(III) with 0.06 mM per h reduction rate in 9 mM As(V) enriched media, whereas the 56% of uptaken As(V) reduced to As(III) with reduction rate of 0.086 mM per h, in 6 mM As(V) enrichment and 42% of reduction with 0.046 mM per h reduction rate was estimated in 3 mM As(V)-enriched media. It was observed that Bacillus licheniformis has uptaken As(V) first and then reduced to As(III) which was extruded in the growth media, as the concentration of As(V) was gradually decreased and concentration of As(III) was gradually increased in residual media as presented in Fig. 3A-C. It might be explained in the manner that As(V) first enters into bacterial cell with the help of phosphate transporter system, then reduced to As(III) inside the cell with the help of (arsC) gene product arsenate reductase [30]. As(III) accumulated in cell then extruded by (arsB) gene product i.e. an antiporter protein channel [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.

Fig. 3.X-axis represents the concentration of As(V) left in residual media at the same time concentration of As(III) [uptaken As(V) reduced to As(III)] formed in media along with the concentration of total arsenic (As) accumulated by bacteria [As in 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.

In biomass, up to 1 mM arsenic has been detected, which is very less as comparison to As(V) supplied. There is possibility of volatilization of very less amount of arsenic of total supply. Bioaccumulation of arsenic was also reported 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 isolated from natural arsenic contaminated site [21, 25]. To check the percentage uptake/removal of arsenite [As(III)], concentration 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% at lower concentration of supplied As(III) in media (2 mM), whereas uptake/removal potential was 75% at 4 mM and 40% at 6 mM of supplied As(III). As(III)/Arsenite (AsO2 −) 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 to move across the cell [31, 42]. There was no biotransformation of As(III) in other form. The accumulation of arsenic in biomass was up to 2 mM, which was higher as compared to As(V) enriched medium. Very little amount of arsenic was unaccounted, which might be volatilised or converted to other organic forms. Since there is no such report regarding As(III) uptake by bacteria, hence this is new and significant finding of present study.

Fig. 4.X-axis represents the concentration of As(III) left in residual media, depicting the concentration of total arsenic uptaken/removed and accumulated by bacteria [As in biomass] at different time point (Y-axis) of growth phase. (A) 2 mM As(III) enriched media. (B) 4 mM As(III) enriched media. (C) 6 mM As(III) enriched media. Concentration of As(III) gradually decreased in residual media showing removal of As(III) from the medium. All values are mean of three replicates and standard errors (SE) are presented as error bars (±).

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

The pH are highly significant variables controlling arsenic speciation [14]. Availability of arsenic species in growth medium depend on pH. At around neutral pH, As(V) found in ionic form as Arsenate (AsO4 3−) and As(III) found in non ionic condition as As(OH)3. Hence around neutral pH, availability 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 on adding 6 mM As(III). Hence pH 6 and pH 9 was taken as acidic and basic pH change of As(V) stress and pH 6 and pH 10 for As(III) stress. In present study, growth pattern of Bacillus licheniformis in As(V) stressed medium, was affected by variation in pH. In Fig. 5A it was observed that both acidic and basic pH change of As(V) stressed medium, had favoured the growth of bacteria. It might be due to significant decrease in uptake of As(V), in changed acidic and basic pH condition of As(V) supplied medium as shown 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 between uptake 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). Hence it is proved that, availability of As(V) in ionic form, is less at acidic and basic pH, which reduced the uptake. Because As(V) only can be uptaken via phosphate transporters in ionic form (AsO4 3−). It was also observed in Fig. 5A that acidic pH (pH 6) of As(V) stressed medium, was slightly more favourable for growth than basic pH (pH 9). It was due to, at alkaline pH, soluble As(V) was more available than at acidic pH [28]. Since more the availability of soluble As(V), more the uptake of As(V) by bacterial cell, hence more the toxic effect resulting less number of cell in growth phase. There are few reports regarding effect of pH on arsenic uptake [19] one was reported in mutant strain of S. faecalis in which the uptake of As(V) was maximal at neutral pH and was declined at higher pH above 7.

Fig. 5.Growth pattern and uptake of Bacillus licheniformis affected by changed pH condition of arsenic enriched media. (A) 9 mM As(V) enriched media. Three control (no added arsenate) with pH 7.2, 6 and 9, medium enriched with 9 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 the growth. (B) X-axis represents the concentration of As(V) uptaken, from 9 mM As(V) enriched media with pH 7.8, 6 and 9. Uptake reduced on changing pH of As(V) stress.

Growth of bacteria in As(III) stress was also increased on exposing to acidic and basic pH change Fig. 6A. There was also 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 in basic pH. It was due to less availability of As(III) in the form of non-ionic [As(OH)3], for uptake via glycerol transport at changed pH condition than at normal pH. There was also one more interesting observation, that arsenic was found as favourable factor for growth of Bacillus licheniformis, in acidic and basic pH condition of TYEG medium, as found in Fig. 5A and 6A Growth in acidic and basic pH condition of control medium was very less as compared to medium with arsenic supplied at same pH.

Fig. 6.(A) 6 mM As(III) enriched media. Three control (no added 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 represents the concentration of As(III) uptaken/removed, from 6 mM As(III) enriched media with pH 8.7, 6 and 10. As(III) uptake reduced in changed pH condition of As(III) stress. All values are mean of three replicates and standard errors(SE) are presented as error bars (±).

Variation in total protein content of bacterial cell in different stress

Under different growth condition/stress the cell size and thus protein content can change to several folds [41]. Hence study of variation in level of total cell protein can measure the level of stress. Cell protein was extracted from the four set of culture tubes that were control culture (pH 7.2), As(V) stressed culture (pH 7.8) and As(V) stress exposed to acidic and alkaline pH (6 and 9) at different time point 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 quantified and represented in graph in the form of amount (μg) of proprotein 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, respectively) variation was observed in amount of protein content of bacterial cell in different stress condition. It might be explained as due to stress the cell division was inhibited but there was enlargement of cell size/volume, hence the level of protein per cell was increased The highest amount of protein was determined in As(V) (9 mM) stress condition and lowest amount in control (no added arsenic) as shown in Fig. 7A. Protein content in As(V) stress with changed acidic and basic pH condition was significantly lower (p = 0.0032 for acidic change and p = 0.0094 for basic change) than only As(V) stress. This result suggests the change in pH of arsenic stress, modifies/lessen the level of arsenic stress [13]. Similar pattern of variation in protein content was also obtained in As(III) stress in Fig. 7B (p = 0.0153 for acidic change and p = 0.0204 for basic change of As(III) stress) explaining the same phenomena.

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

In conclusion, B. licheniformis was isolated from arsenic contaminated region located at 85º 32'E longitude and 25º 11'N latitude on the Earth. Isolated bacteria tolerated both As(V) [MIC 10 mM] as well as As(III) [MIC 7 mM], which is rare to find that single species tolerating both forms of arsenic. Uptake/removal potential of B. licheniformis was dependent on supplied amount of As(V) and As(III) in the growth media and 100% uptake/removal was determined in lower concentration of As(V) and As(III) (3 mM and 2 mM, respectively). B. licheniformis was also capable of reducing uptaken As(V) into As(III), and its potential of reduction was also dependent on concentration of supplied As(V). It was also capable of accumulating little amount of arsenic in biomass. The potential of uptake, reduction and growth pattern were also affected by variation in pH of arsenic stress. Variation in pH mitigates the arsenic stress, as different level of cell’s protein (showing level of stress) was determined at different pH of arsenic stress. Hence native bacteria can be utilized in minimizing the arsenic contamination and its efficiency of uptake/removal can also be modify by changing pH of system, so it can help to avoid the entry of the arsenic in human food chain.