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Identification and characterization of metabolic properties of bacterial populations recovered from arsenic contaminated ground water of North East India (Assam) Soma Ghosh, Pinaki Sar* Department of Biotechnology, Indian Institute of Technology Kharagpur, 721302 West Bengal, India
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abstract
Article history:
Diversity of culturable bacterial populations within the Arsenic (As) contaminated
Received 4 April 2013
groundwater of North Eastern state (Assam) of India is studied. From nine As contaminated
Received in revised form
samples 89 bacterial strains are isolated. 16S rRNA gene sequence analysis reveals pre-
20 July 2013
dominance of Brevundimonas (35%) and Acidovorax (23%) along with Acinetobacter (10%),
Accepted 17 August 2013
Pseudomonas (9%) and relatively less abundant (90 mg/L) in samples A1, A2, J1, J2 and J3 was noticed. As contamination in groundwater of north-eastern states of India covering a large part of GangaeBrahmaputraeMeghna Basin has been reported first by Singh (2006). The concentration of As as reported there is corroborated well with our findings.
3.2. Isolation, identification and phylogenetic study of culturable bacteria Culturable aerobic bacteria capable of tolerating toxic As were isolated using growth medium supplemented with either As(III) (2.5 mM) or As(V) (25 mM) as well as on As free control plate. A total of 89 bacterial isolates were obtained from nine ground water samples. Based on 16S rRNA gene fingerprinting 34 distinct RFLP groups or Operational Taxonomic Units (OTUs) were obtained. Taxonomic identity of isolated strains as ascertained by analysing 16S rRNA gene sequences revealed predominance of Proteobacteria (93%) within all the samples followed by minor representation of Firmicutes (2%) and Actinobacteria species (1%) and few unidentified members (4%) (Fig. 1). Samples collected from the north of Brahmaputra namely A1, A2, A18, A19, A21 and A22 showed preponderance of a-proteobacterial genera Brevundimonas (47%) followed by g, and b-proteobacterial genera Pseudomonas (12%), Acidovorax (16%). Presence of several other genera of a-, g-, b-Proteobacteria [viz. Undibacterium (4%), Herbaspirillum (4%), Rhizobium (5%), Enterobacter (2%), Rhodococcus (2%), Staphylococcus (1%), Bacillus (1%) and Caulobacter (1%)] with relatively lower abundance was observed. In contrast, samples collected from the
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south of Brahmaputra namely J1, J2 and J3 showed maximum abundance of g-Proteobacterial member Acinetobacter (40%), followed by Acidovorax (35%), Rhizobium (15%) and sparse distribution of Brevundimonas (5%). It is noteworthy that a complete reverse distribution of the bacterial (culturable) community composition was observed at the two banks of river Brahmaputra. Moreover, although Brevundimonas is seen to be present ubiquitously throughout the samples yet, they formed the major population in the highly contaminated groundwater samples from the north of the river such as A1, A2 and A19. Followed by taxonomic identification, phylogenetic lineages of our isolates to closest type strain of the same genera and other members retrieved earlier from As contaminated sites or from ground water was investigated (Fig. 2). As evident from the figure, 16 sequences representing 15 Operational Taxonomic Units (OTUs) and covering 31 isolates affiliated to Brevundimonas formed two distinct clades, and show close lineages with Brevundimonas bullata and Brevundimonas nasdae. Similarity of these isolates is also found with As resistant Brevundimonas spp. reported from As rich environments (Drewniak et al., 2008; Sarkar et al., 2013). Isolates showing similarity with N2 fixing bacteria Rhizobium have been obtained from highly contaminated samples A19, J1, J2, and J3 and represent a distant lineage with type strain Rhizobium aggregatum. Six OTUs representing 20 isolates affiliated to the genus Acidovorax show strong relatedness with previously reported As resistant Acidovorax sp. and type strain Acidovorax delafieldii (Cai et al., 2009; Fan et al., 2008). Members of this genus are found within the less contaminated sites (A21) on the north of Brahmaputra as well as a predominant group in high As contaminated areas on the south of the river. Three isolates of another N2 fixing genus Herbaspirillum bear strong relatedness with the type strain Herbaspirillum seropedicae. Interestingly, presence of this genus in As contaminated sites was not reported before. Predominance of the genus Acinetobacter is found in the southern bank of Brahmaputra. Eight isolates of a single OTU belonging to this genus revealed their relatedness with previously characterized Acinetobacter lwoffii and another Acinetobacter strain obtained from As contaminated groundwater (Nazina et al., 2000). Isolates showing 16S rRNA sequence match with Undibacterium revealed close lineage with Undibacterium pigrum and several other cultured as well as uncultured bacteria retrieved from various groundwater aquifers (Li et al., 2010; Shimizu et al., 2006). Members of the genus Pseudomonas are found to be closely related to Pseudomonas putida and other Pseudomonas strains reported to have siderophore mediated Fe acquisition ability and other As resistant species obtained from As contaminated sites (Matthijs et al., 2009; Davolos and Pietrangeli, 2013). Other minor members of a-, b- and g-Proteobacteria such as Ralstonia, Enterobacter have been shown to bear lineages with their type strains. Members of Firmicutes and Actinobacteria form minor component of the community bearing close lineages with formerly reported isolates from other As contaminated site (Tiago et al., 2004; Liao et al., 2011). Previous reports on bacterial diversity revealed presence of diverse genera (up to 22) at various As-contaminated estuaries and soils (Jackson et al., 2003; Achour-Rokbani et al., 2007). In contrast, As contaminated groundwater, including those
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Fig. 1 e Distribution of bacterial genera as detected in the As contaminated ground water samples (a) and overall abundance of bacterial genera (b).
collected from Bengal basin, presented relatively narrow depth of diversity with only few genera affiliated to a-, g-, b-Proteobacteria, Bacteroidetes, Acidobacteria, Verrucomicrobia, etc. (Sutton et al., 2009; Liao et al., 2011). Abundance of aProteobacteria, known for their ability to grow under low organic carbon containing subsurface environment as observed in several samples studied here is in line with earlier reports including one from West Bengal (Sarkar et al., 2013). Bacterial genera detected in our samples count species that are known for their ability to reduce As(V), siderophore production and chemolithotrophic growth under various nutrient conditions. Among these genera the predominance of Brevundimonas seems most notable. Although there are reports indicating presence of this genus in As contaminated habitats (including the groundwater from Bengal delta plain) their role in As transformation and mobilization is yet not clear (Drewniak et al., 2008; Cavalca et al., 2010; Sarkar et al., 2013). So, the high abundance of Brevundimonas could possibly be a
subject of further investigation. The b proteobacterial member Acidovorax covering a significant percentage of abundance is supported by the studies of Huang et al. (2012) where it has been reported to be a chemoorganotrophic, nitrate reducing bacterium, capable of mobilizing iron in the subsurface environment. Pseudomonas and Acinetobacter of g Proteobacteria have previously been detected in As contaminated sites (Sultana et al., 2011; Cai et al., 2009). The role of Pseudomonas has also been linked with subsurface As release (Islam et al., 2004). In this study, we report the presence of b-proteobacterial members Herbaspirillum and Undibacterium for the first time in As contaminated sites with considerable As tolerance.
3.3.
As sensitivity of isolated bacteria
The study on As tolerance of the isolated strains revealed that 94% of the total strains, (37 obtained from control plates, 29 from As(V) enrichment and 23 from As(III) amended plates)
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Fig. 2 e 16S rRNA gene based Neighbour-Joining phylogenetic tree of isolates obtained from As-contaminated groundwater [the numbers at the branches depict bootstrap values].
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irrespective of their enrichment condition (during isolation) and taxonomic affiliation could withstand 100 mM of As(V) and all except 25% could resist 10 mM of As(III) (Fig. 3a). Resistance of microbes to these threshold concentrations of As can be regarded as As tolerant (Jackson et al., 2003). Out of the 13 genera found, except one, all the Acidovorax strains collected from the groundwater samples of northern part of Brahmaputra are found to be hypertolerant to As(V) as well as As(III) (isolates named with A). In contrast, the isolates of the same taxon obtained from southern part (isolates named with J) showed hypertolerance to As(V) but no resistance to As(III) at all. Within the genus Brevundimonas, 64% of the isolates were hypertolerant to both the forms of As, two third of which could withstand even higher concentrations of As(V) i.e. 550 mM and only 3 among them grew at 50 mM of As(III). Hypertolerance to As(V) was seen in all the Acinetobacter
strains representing the samples from Jorhat (J). Nevertheless, As(III) resistance was found to be only upto the threshold level (10 mM) except three of these isolates. Other minor representatives such as Herbaspirillum and Rhizobiales members could also withstand both the forms of As well above the threshold value. It may be noted that there is a general distribution of resistance property among the bacterial isolates independently of the level of As contamination of sampling sites as few of the highly As resistant bacteria have been obtained from the groundwater sample having least As contamination. The UPGMA for the tolerance values of these isolates also support this observation (Fig. 3b). It is distinctly observed that isolates obtained from As enrichment posses higher resistance to As. In this analysis the total population has been clustered broadly into two clades where the effect of
Fig. 3 e UPGMA clustering of As resistance properties (a) As(III) and As(V) resistance of bacterial isolates (b) [Isolates belonging to subclades 1e8 have been named in Supplementary information].
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enrichment in isolating As sensitive and resistant bacteria has become prominent (See Supplementary table). The upper clade (subclades 1, 2, and 3) represented all those isolates with either no As resistance or those that have just the tolerance at threshold values. Another clade (subclades 4, 5, 6, 7 and 8), on the other hand represented the strains with extremely high resistance to both form of As. These are mainly seen to be isolated from enriched condition. Thus, it can be concluded
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that the highly tolerant isolates prefer to grow in As enriched conditions. Microbial resistance to concentration greater than 10 mM As(III) and 100 mM As(V) is regarded as very high, while resistance to 300e500 mM As(V) or 30 mM As(III) is a hyper tolerance property (Jackson et al., 2003). Except six, most of the isolates, irrespective of their site of isolation showed As resistance. Resistance to As have been reported in previous
Fig. 4 e Metabolic profiling of bacterial strains affiliated to a Proteobacteria (a) b Proteobacteria (b) and g Proteobacteria (c). Response towards various test parameters are scored as positive or negative and marked as red and yellow respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4 e (continued).
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studies for Sinorhizobium, Phyllobacterium, Variovorax, Pseudomonas and Acinetobacter along with Actinobacteria and Firmicutes up to 150e350 mM As(V) and 1.5e14 mM As(III) (AchourRokbani et al., 2007). Recently, Sarkar et al. (2013) showed a minor population of As resistant Brevundimonas from a high As rich groundwater of West Bengal. Our study confirmed the presence of highly resistant Brevundimonas sp. and Acidovorax sp. The study suggested that As resistance property is not limited to the As contaminated sites only, rather the most highly resistant isolates belonging to the genus Brevundimonas have been isolated from the sample A18 having very low or negligible As content. But these highly resistant isolates IIIA210, VA18-4 and VA18-5 could be isolated only from the As amended plates. Therefore, enrichment effect is prominent in isolating highly As resistant bacteria. From their growth preference on As enrichment plates, it may be suggested that these isolates are not only resistant to As by employing some detoxifying mechanism to pump out the toxic species from cell interior, but also may use As in their energy transduction (Oremland and Stolz, 2003).
3.4.
Metabolic characterization of isolated bacteria
The study on metabolic profile was performed based on the capacity of the isolates to utilize varying carbon sources, different electron acceptors and to produce siderophore as well as their As(V) reductase activity. The result showed that the most dominant and overlapping genus Brevundimonas and other a-proteobacterial members Rhizobium were capable of utilizing the different types of organic carbon sources on the same time being particular about using As(V) and sulphate as their electron acceptors under anaerobic growth (Fig. 4a). The next abundant genus Acidovorax is seen to be equally capable of using various carbon sources but they are less capable of growing under anaerobic conditions with alternate electron acceptors. The genera Herbaspirillum and Pseudomonas showed better capability to reduce different inorganic electron acceptors in anaerobic condition. Acinetobacter is found to be highly versatile in utilization of different carbon sources but is only capable of using As(V) as terminal electron acceptor when grown anoxically. Pseudomonas, on the other hand, is relatively able to use alternate electron acceptors. It was observed that more than 50% of the total isolates could grow anaerobically with alternate (more than three) electron acceptors. These observations were further established by a principle component analysis (PCA) (Fig. 5a). The Euclidean biplot showed that the metabolic properties of isolates had a correlation among them with an Eigen value of 0.896 and 0.445 for axis 1 and 2, respectively. This PCA could explain a cumulative percentage metabolic diversity of 65.8%. Based on their metabolic profile, the isolates are found to cluster in 4 quadrants (Fig. 5a). It is evident that the As(V) reductase activity along with siderophore production is highly related to their capability to use As(V) and Se(VI) as electron acceptors in combination with their ability to use acetate, pyruvate, arabinose, glucose, starch, raffinose and inositol. The analysis further indicates that the capability of siderophore production by the isolates is more associated with As(V) reductase activity. Another PCA analysis with the tolerance values of the isolates for As(III) as well as As(V), their As(V)reductase
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activity and siderophore production capability shows that the tolerance to As(III) and reductase activity is strongly correlated (Fig. 5b). The broad metabolic profiles were obtained for each isolates obtained from individual sampling sites. Fig. 4c represented the cumulative metabolic characteristics of the isolates obtained from each sample. UPGMA analysis reveals that based on metabolic traits of the indigenous bacteria the sampling sites can be divided into two clades mostly representing the sites at either bank of the river Brahmaputra (Fig. 5c). In the upper clade the nearest correlation is seen within the isolates of A2 (As ¼ 161 ppb) and A21 (As ¼ 2 ppb) depicting that groundwater As had no apparent influence in the metabolic adaptation of the isolates. Rather the subsurface geochemistry might have conferred some common characteristics to the resident microbes. The culturable community is also seen to be overlapping in these two samples. All these data significantly indicates that the geochemical factors and not the level of As contamination might affect the microbial diversity and their metabolic characters. Their contribution to mobility of As from sediment to the groundwater is prominent from the fact that most of the isolates are reductase positive and many of them are capable of utilizing As(V) as their alternate electron acceptor. A number of bacterial genera retrieved from As rich sites and those affiliated to genera Hydrogenophaga, members of AgrobacteriumeRhizobium group etc. or Desulfitobacterium spp., Bacillus arsenicoselenatis and Bacillus selenitireducens have been previously shown to utilize multiple electron donors and/or acceptors (Laverman et al., 1995; Gihring et al., 2001). Although it is of rare occurrence to find bacteria capable of utilizing three or more carbon sources as well as various electron acceptors, nevertheless, significant number (60%) of our isolates may be considered truly versatile with respect to versatile C source utilization and relatively less (32%) among them, are capable of utilizing alternate electron acceptors. Reports are available for pre- and post monsoon seasonal and diurnal change in level of water table creating dynamic oxiceanoxic states and hence availability of electron acceptors as well (Macur et al., 2004). This fact and our supporting results therefore suggest that the inhabiting microorganisms might have equipped themselves with ability to utilize diverse electron acceptors and donors to survive and flourish under such changing environments. In previous studies, it has been seen that As(V) reduction by the microbes is mainly performed through cytoplasmic reductases (ars) that encode As(III) extruding pump for detoxification mechanism under oxic condition or, through periplasmic respiratory reductase that dissimilates As(V) as terminal electron acceptor in anoxic condition (Oremland and Stolz, 2003). At significant depth where dissolved oxygen is negligible and anoxic condition is stable, these isolates may respire As(V) bound to the sediments along with iron and sulphur compounds. Siderophore production in various genera has been found to be the way of nutrient acquisition by chelating Fe compounds and making Fe bioavailable. Such an activity in Pseudomonas, Rhodococcus, and Rhizobium including As mobilization has been reported in various studies previously (Sriyosachati and Cox, 1986; Vala et al., 2006; Nair et al., 2007). However, such studies in Brahmaputra delta were undone upto now. In this work we found
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Fig. 5 e (continued).
siderophore activity in the dominant genera of a, b and g proteobacterial classes. Total Fe content of the groundwater being significantly low in these groundwater samples, the production of siderophore by most of the reductase positive strains further suggests the microbial role in releasing As(III) while chelating iron from the solid phase. In the meantime, As(V) is being reduced to As(III) by the reductase activity of the microbes solubilizing As(III) in the aqueous phase. This hypothesis is supported by the recent studies of Drewniak et al. (2008) and Sarkar et al. (2013). This may explain the observation by the second PCA plot (Fig. 5b) showing correlation between reductase activity and As(III) tolerance of the isolates. The more they reduce As(V) to As(III), more they are resistant to the reduced As(III).
3.5.
Fig. 5 e Plot of PCA scores on utilization of different C sources and alternate electron acceptors, As(V) reductase activity and siderophore production of the isolates (a). Each bacterial strain is represented by numbers referred to their names in Supplementary Table 2. The arrows indicate the corresponding parameters for which the isolates with higher eigen values have clustered [Q1: isolates versatile in different C source utilization, Q2: isolates relatively less versatile in terms of C source utilization but capable of siderophore production, Q3: isolates incapable of utilizing most of the carbon sources yet, well equipped to metabolize various electron acceptors, Q4: most versatile isolates capable of metabolizing different C sources as well can utilize nitrate, Fe(III), thiosulphate and sulphate as their terminal electron acceptor]. PCA analysis showing relation among As(V) and As(III) tolerance, reductase activity and ability to produce siderophore by the isolates (b). Cumulative metabolic profile represented by UPGMA for the isolates obtained from individual samples (c).
Chemolithoautotrophic growth of isolated bacteria
It has been established in various studies that organic carbon plays an important role in the microbial mobilization of As. Yet, in the last decade a few studies reported well known chemolithoautotrophic strains such as NT-26 (Santini et al., 2000) capable of utilizing As(III) as sole electron donor in autotrophic condition. During this study we were interested to see if these heterotrophically isolated bacteria could grow autotrophically and whether they could utilize As(III) as their electron donor. Incubation in chemolithotrophic medium (with bicarbonate and As(III) as sole C and e source, respectively) showed positive growth upto an O.D of 0.2e0.3 at 600 nm for 8 isolates among all in 7 days. These were subcultured in the same media under the same conditions twice. These chemolithotrophic genera were mostly Brevundimonas, followed by few Acidovorax and Acinetobacter isolated from enrichment culture (Fig. 6). Chemolithoautotrophy has been found in diverse genera in various ecosystems where phototrophy or heterotrophy is not supported due to natural constrains. Studies reveal chemolithoautotrophic growth of members of b- and g-Proteobacteria upon oxidation of sulphur or ammonia or nitrite (Chen et al., 2009). Chemolithoautotrophy using As(III) as electron donor in Pseudomonas arsenitoxidans had first been reported by Ilyaletdinov and Abdrashitova (1981). In recent studies, member of a-Proteobacteria Rhizobium NT-26 and g proteobacterial member Ectothiorhodospira MLHE-1 has been reported to oxidize As(III)
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unexplored. The present paper identifies and characterizes the metabolic diversity of bacteria from several As rich groundwater of Assam, India, thus illuminating their intrinsic capability in controlling subsurface arsenic geochemistry and mobilization. Strong correlation could be established between the property to tolerate As(III) and siderophore production with reductase activity of the isolates. The abundance of chemolithotrophic Brevundimonas, facultative anaerobic Acidovorax, etc. is observed.
Acknowledgement The authors gratefully acknowledge the support from Department of Biotechnology Govt. of India as NER Twinning Programme (Ref No. BT/19/NE/TBP/2010). Fig. 6 e Growth of chemolithoautotrophic bacteria [O.D at 600 nm taken after an incubation of 7 days at 30 C].
chemoautotrophically (Oremland et al., 2002; Santini et al., 2000). The growth of various members of proteobacterial subgroups chemolithoautotrophically oxidizing As(III) is coherent with our study. Our results indicate different genera of these proteobacterial groups to grow as facultative chemolithoautotrophs utilizing As(III) as electron donor.
4.
Conclusion
This study reports diversity of culturable bacteria in the As contaminated sites of North Eastern State of India (an important part of GBM delta) for the first time. We report the abundance of chemolitho/organo trophic genera Brevundimonas, Acidovorax and Acinetobacter in the studied samples. High As resistance and broad metabolic versatility of the isolates, particularly the most abundant population is noted. Metabolic diversity indicates a correlation among the siderophore production and As(V) reductase activity simultaneous with As(V) and selenate based anaerobic metabolism. This might lead to the hypothesis that inhabitant bacteria use siderophore for acquisition of sediment bound Fe. As Fe is dislodged, As (arsenate) also gets released from the bound solid phase. This As(V) is either anaerobically respired or aerobically detoxified by the resistant microbes to release As(III) which is highly soluble in aqueous phase. The broad metabolic repertoire as detected within the groundwater bacteria could be attributed as an evolutionary outcome to provide crucial advantage to the respective microbes for their survival within such contaminated environment.
Statement of novelty Microbial role in disseminating the arsenic contamination in groundwater of GBM delta is a major problem yet, till date microbial biogeochemistry in Brahmaputra river basin remain
Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.watres.2013.08.044.
references
Achour-Rokbani, A., Bauda, P., Billard, P., 2007. Diversity of As(III) transporter genes from As-resistant soil bacteria. Res. Microbiol. 158, 128e137. Battaglia-Brunet, F., Dictor, M.C., Garrido, F., Crouzet, C., Morin, D., Dekeyser, K., Clarens, M., Baranger, P., 2002. An As(III)-oxidizing bacterial population: selection, characterization and performance in reactors. J. Appl. Microbiol. 93, 656e667. Cai, L., Liu, G., Rensing, C., Wang, G., 2009. Genes involved in As transformation and resistance associated with different levels of As-contaminated soils. BMC Microbiol. 9, 4. Cavalca, L., Zanchi, R., Corsini, A., Colombo, M., Romagnoli, C., Canzi, E., Andreoni, V., 2010. As-resistant bacteria associated with roots of the wild Cirsium arvense (L) plant from an As polluted soil, and screening of potential plant growthpromoting characteristics. Syst. Appl. Microbiol. 33, 154e164. Chatterjee, A., Das, D., Chakraborti, D., 1993. A study of ground water contamination by As in the residential area of Behala, Calcutta due to industrial pollution. Environ. Pollut. 80, 57e65. Chen, Y., Wu, L., Boden, R., Hillebrand, A., Kumaresan, D., Moussard, H., Baciu, M., Lu, Y., Murrell, C., 2009. Life without light: microbial diversity and evidence of sulfur- and ammonium-based chemolithotrophy in Movile Cave. ISME J. 3, 1093e1104. Davolos, D., Pietrangeli, B., 2013. A molecular study on bacterial resistance to arsenic-toxicity in surface and underground waters of Latium. Ecotoxicol. Environ. Saf. 96, 1e9. Drewniak, L., Styczek, A., Lopatka, M.M., Sklodowska, A., 2008. Bacteria, hypertolerant to As in the rocks of an ancient gold mine, and their potential role in dissemination of As pollution. Environ. Pollut. 156, 1069e1074. Enmark, G., Nordborg, D., 2007. Arsenic in the Groundwater of the Brahmaputra Floodplains, Assam, India e Source, Distribution and Release Mechanisms. Arbetsgruppen fo¨r Tropisk Ekologi Minor Field Study 131 Committee of Tropical Ecology. Uppsala University, Sweden.
7004
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Fan, H., Su, C., Wang, Y., Yao, J., Zhao, K., Wang, Y., Wang, G., 2008. Sedimentary arsenite-oxidizing and arsenate-reducing bacteria associated with high arsenic groundwater from Shanyin, Northwestern China. J. Appl. Microbiol. 105, 529e539. Fendorf, S., Michael, H.A., Van Geen, A., 2010. Spatial and temporal variations of groundwater As in south and south east Asia. Science 328, 1123e1127. Gault, A.G., Cooke, D.R., Townsend, A.T., Charnock, J.M., Polya, D.A., 2005. Mechanisms of arsenic attenuation in acid mine drainage from Mount Bischoff, western Tasmania. Sci. Total Environ. 345, 219e228. Gihring, T.M., Druschel, G.K., McCleskey, R.B., Hamers, R.J., Banfield, J.F., 2001. Rapid As(III)oxidation by Thermus aquaticus and Thermus thermophilus: field and laboratory investigations. Environ. Sci. Technol. 35, 3857e3862. Gorra, R., Webster, G., Martin, M., Celi, L., Mapelli, F., Weightman, A.J., 2012. Dynamic microbial community associated with ironearsenic co-precipitation products from a groundwater storage system in Bangladesh. Microb. Ecol., 64,171e64,186. Hery, M., Van Dongen, B.E., Gill, F., Mondal, D., Vaughan, D.J., Pancost, R.D., Polya, D.A., Lloyd, J.R., 2010. As release and attenuation in low organic carbon aquifer sediments from West Bengal. Geobiology 8, 155e168. Huang, Y., Li, H., Rensing, C., Zhao, K., Johnstone, L., Wang, G., 2012. Genome sequence of the facultative anaerobic arseniteoxidizing and nitrate-reducing bacterium Acidovorax sp. strain NO1. J. Bacteriol. 194, 1635e1636. Ilyaletdinov, A.N., Abdrashitova, S.A., 1981. Autotrophic oxidation of arsenic by a culture of Pseudomonas arsenitoxidans. Mikrobiologiya 50, 197e204. Islam, E., Sar, P., 2011. Culture-dependent and -independent molecular analysis of bacterial community within uranium ore. J. Basic Microbiol., 1e13. Islam, F.S., Boothman, C., Gault, A.G., Polya, D.A., Lloyd, J.R., 2005. Potential role of the Fe(III)-reducing bacteria Geobacter and Geothrix in controlling arsenic solubility in Bengal delta sediments. Mineral. Mag. 69, 865e875. Islam, F.S., Gault, A.G., Boothman, C., Polya, D.A., Charnock, J.M., Chatterjee, D., Lloyd, J.R., 2004. Role of metal-reducing bacteria in As release from Bengal delta sediments. Nature 430, 68e71. Jackson, C.R., Jackson, E.F., Dugas, S.L., Gamble, K., Williams, S.E., 2003. Microbial transformations of As(III)and As(V) in natural environments. Recent Res. Dev. Microbiol. 7, 103e118. Kar, S., Maitya, J.P., Jeana, J., Liua, C.,C., Nath, B., Yanga, H.J., Bundschuha, J., 2010. Arsenic-enriched aquifers: occurrences and mobilization of arsenic in groundwater of Ganges Delta Plain, Barasat, West Bengal, India. Appl. Geochem. 25, 1805e1814. Kazy, S.K., Sar, P., Asthana, R.K., Singh, S.P., 1999. Copper uptake and its compartmentalization in Pseudomonas aeruginosa strains: chemical nature of cellular metal. World J. Microbiol. Biotechnol. 15, 599e605. Laverman, A.M., Blum, S.J., Schaefer, J.K., Philips, E.J.P., Lovley, D.R., Oremland, R.S., 1995. Growth of strain SES-3 with As(V) and other diverse electron acceptors. Appl. Environ. Microbiol. 61, 3556e3561. Luli, G.W., Talnagi, J.W., Strohl, W.R., Pfister, R.M., 1983. Hexavalent chromium resistant bacteria isolated from river sediments. Appl. Environ. Microbiol. 46, 846e854. Li, D., Li, Z., Yu, J., Cao, N., Liu, R., Yang, M., 2010. Characterization of bacterial community structure in a drinking water distribution system during an occurrence of red water. Appl. Environ. Microbiol. 76, 7171e7180. 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 As(V)reducing bacteria associated with As-rich groundwater in Taiwan. J. Contam. Hydrol. 123, 20e29.
Macur, R.E., Jackson, C.R., Botero, L.M., Mcdermott, T.R., Inskeep, W.P., 2004. Bacterial populations associated with the oxidation and reduction of As in an unsaturated soil. Environ. Sci. Technol. 38, 104e111. Mailloux, B.J., Alexandrova, E., Alison, R., 2009. Microbial mineral weathering for nutrient acquisition releases As. Appl. Environ. Microbiol. 75, 2558e2565. Matthijs, S., Laus, G., Meyer, J.M., Abbaspour-Tehrani, K., Schafer, M., Budzikiewicz, H., Cornelis, P., 2009. Siderophoremediated iron acquisition in the entomopathogenic bacterium Pseudomonas entomophila L48 and its close relative Pseudomonas putida KT2440. Biometals 22 (6), 951e964. Muyzer, G., de Waal, E.C., Uitterlinden, A.G., 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reactionamplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59, 695e700. Nair, A., Juwarkar, A.A., Singh, S.K., 2007. Production and characterization of siderophores and its application in As removal from contaminated soil. Water Air Soil Pollut. 180, 199e212. Nazina, T.N., Kosareva, I.M., Davydov, A.S., Turova, T.P., Novikova, E.V., Khafizov, R.R., Poltaraus, A.B., 2000. Physicochemical and microbiological characteristics of groundwater from observation boreholes of a deep radioactive liquid waste repository. Mikrobiologiia, 105e112. Nickson, R.T., McArthur, J.M., Ravenscroft, P., Burgess, W.G., Ahmed, K.M., 2000. Mechanism of arsenic release to groundwater, Bangladesh and West Bengal. Appl. Geochem. 15, 403e413. Oremland, R.S., Kulp, T.R., Switzer, B.J., Hoeft, S.E., Baesman, S., Miller, G., Stolz, J.,F., 2005. A microbial As cycle in a salt saturated, extreme environment. Science 308, 1305e1308. Oremland, R.S., Hoeft, S.E., Santini, J.M., Bano, N., Hollibaugh, R.A., Hollibaugh, J.T., 2002. Anaerobic oxidation of arsenite in mono lake water and by a facultative arseniteoxidizing chemoautotroph, strain MLHE-1. Appl. Environ. Microbiol. 68, 4795e4802. Oremland, R.S., Stolz, J.F., 2003. The ecology of As. Science 300, 939e944. Reasoner, D.J., Geldreich, E.E., 1985. A new medium for the enumeration and subculture of bacteria from potable water. Appl. Environ. Microbiol. 49, 1e7. 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, 92e97. Sarkar, A., Kazy, S.K., Sar, P., 2013. Characterization of As resistant bacteria from As rich groundwater of West Bengal. Ecotoxicology 22, 363e374. Shimizu, S., Akiyama, M., Ishijima, Y., Hama, K., Kunimaru, T., Naganuma, T., 2006. Molecular characterization of microbial communities in fault-bordered aquifers in the Miocene formation of northernmost Japan. Geobiology 4, 147e223. Silver, S., Phung, L.T., 2005. Genes and enzymes involved in bacterial oxidation and reduction of inorganic As. Appl. Environ. Microbiol. 71, 599e608. Singh, A.K., 2006. Chemistry of As in ground water of GangesBrahmputra river basin, India. Curr. Sci. 91, 1e7. Slyemi, D., Bonnefoy, V., 2011. How prokaryotes deal with As. Environ. Microbiol. Rep. 1, 16. Sriyosachati, S., Cox, C.D., 1986. Siderophore-mediated iron acquisition from transferrin by Pseudomonas aeruginosa. Infect. Immun. 52, 885e891. Sultana, M., Hartig, C., Planer-Friedrich, B., Seifert, J., Schlomann, M., 2011. Bacterial communities in Bangladesh aquifers differing in aqueous As concentration. Geomicrobiol. J. 28, 198e211.
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Sutton, N.B., van der Kraan, G.M., van Loosdrecht, M.C., Muyzer, G., Bruining, J., Schotting, R.J., 2009. Characterization of geochemical constituents and bacterial populations associated with As mobilization in deep and shallow tube wells in Bangladesh. Water Res. 43, 1720e1730. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596e1599.
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Tiago, I., Chung, A.P., Verissimo, A., 2004. Bacterial diversity in a nonsaline alkaline environment: heterotrophic aerobic populations. Appl. Environ. Microbiol. 70 (12), 7378e7387. Vala, A.K., Dave, B.P., Dube, H.C., 2006. Chemical characterization and quantification of siderophores produced by marine and terrestrial aspergilli. Can. J. Microbiol. 52, 603e607. Zhao, Z., Jiaa, Y., Xua, L., Zhaoc, S., 2011. Adsorption and heterogeneous oxidation of As(III) on ferrihydrite. Water Res. 45, 6496e6504.
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Identification and characterization of metabolic properties of bacterial populations recovered from arsenic contaminated ground water of North East India (Assam) Soma Ghosh, Pinaki Sar* Department of Biotechnology, Indian Institute of Technology Kharagpur, 721302 West Bengal, India
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abstract
Article history:
Diversity of culturable bacterial populations within the Arsenic (As) contaminated
Received 4 April 2013
groundwater of North Eastern state (Assam) of India is studied. From nine As contaminated
Received in revised form
samples 89 bacterial strains are isolated. 16S rRNA gene sequence analysis reveals pre-
20 July 2013
dominance of Brevundimonas (35%) and Acidovorax (23%) along with Acinetobacter (10%),
Accepted 17 August 2013
Pseudomonas (9%) and relatively less abundant (90 mg/L) in samples A1, A2, J1, J2 and J3 was noticed. As contamination in groundwater of north-eastern states of India covering a large part of GangaeBrahmaputraeMeghna Basin has been reported first by Singh (2006). The concentration of As as reported there is corroborated well with our findings.
3.2. Isolation, identification and phylogenetic study of culturable bacteria Culturable aerobic bacteria capable of tolerating toxic As were isolated using growth medium supplemented with either As(III) (2.5 mM) or As(V) (25 mM) as well as on As free control plate. A total of 89 bacterial isolates were obtained from nine ground water samples. Based on 16S rRNA gene fingerprinting 34 distinct RFLP groups or Operational Taxonomic Units (OTUs) were obtained. Taxonomic identity of isolated strains as ascertained by analysing 16S rRNA gene sequences revealed predominance of Proteobacteria (93%) within all the samples followed by minor representation of Firmicutes (2%) and Actinobacteria species (1%) and few unidentified members (4%) (Fig. 1). Samples collected from the north of Brahmaputra namely A1, A2, A18, A19, A21 and A22 showed preponderance of a-proteobacterial genera Brevundimonas (47%) followed by g, and b-proteobacterial genera Pseudomonas (12%), Acidovorax (16%). Presence of several other genera of a-, g-, b-Proteobacteria [viz. Undibacterium (4%), Herbaspirillum (4%), Rhizobium (5%), Enterobacter (2%), Rhodococcus (2%), Staphylococcus (1%), Bacillus (1%) and Caulobacter (1%)] with relatively lower abundance was observed. In contrast, samples collected from the
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south of Brahmaputra namely J1, J2 and J3 showed maximum abundance of g-Proteobacterial member Acinetobacter (40%), followed by Acidovorax (35%), Rhizobium (15%) and sparse distribution of Brevundimonas (5%). It is noteworthy that a complete reverse distribution of the bacterial (culturable) community composition was observed at the two banks of river Brahmaputra. Moreover, although Brevundimonas is seen to be present ubiquitously throughout the samples yet, they formed the major population in the highly contaminated groundwater samples from the north of the river such as A1, A2 and A19. Followed by taxonomic identification, phylogenetic lineages of our isolates to closest type strain of the same genera and other members retrieved earlier from As contaminated sites or from ground water was investigated (Fig. 2). As evident from the figure, 16 sequences representing 15 Operational Taxonomic Units (OTUs) and covering 31 isolates affiliated to Brevundimonas formed two distinct clades, and show close lineages with Brevundimonas bullata and Brevundimonas nasdae. Similarity of these isolates is also found with As resistant Brevundimonas spp. reported from As rich environments (Drewniak et al., 2008; Sarkar et al., 2013). Isolates showing similarity with N2 fixing bacteria Rhizobium have been obtained from highly contaminated samples A19, J1, J2, and J3 and represent a distant lineage with type strain Rhizobium aggregatum. Six OTUs representing 20 isolates affiliated to the genus Acidovorax show strong relatedness with previously reported As resistant Acidovorax sp. and type strain Acidovorax delafieldii (Cai et al., 2009; Fan et al., 2008). Members of this genus are found within the less contaminated sites (A21) on the north of Brahmaputra as well as a predominant group in high As contaminated areas on the south of the river. Three isolates of another N2 fixing genus Herbaspirillum bear strong relatedness with the type strain Herbaspirillum seropedicae. Interestingly, presence of this genus in As contaminated sites was not reported before. Predominance of the genus Acinetobacter is found in the southern bank of Brahmaputra. Eight isolates of a single OTU belonging to this genus revealed their relatedness with previously characterized Acinetobacter lwoffii and another Acinetobacter strain obtained from As contaminated groundwater (Nazina et al., 2000). Isolates showing 16S rRNA sequence match with Undibacterium revealed close lineage with Undibacterium pigrum and several other cultured as well as uncultured bacteria retrieved from various groundwater aquifers (Li et al., 2010; Shimizu et al., 2006). Members of the genus Pseudomonas are found to be closely related to Pseudomonas putida and other Pseudomonas strains reported to have siderophore mediated Fe acquisition ability and other As resistant species obtained from As contaminated sites (Matthijs et al., 2009; Davolos and Pietrangeli, 2013). Other minor members of a-, b- and g-Proteobacteria such as Ralstonia, Enterobacter have been shown to bear lineages with their type strains. Members of Firmicutes and Actinobacteria form minor component of the community bearing close lineages with formerly reported isolates from other As contaminated site (Tiago et al., 2004; Liao et al., 2011). Previous reports on bacterial diversity revealed presence of diverse genera (up to 22) at various As-contaminated estuaries and soils (Jackson et al., 2003; Achour-Rokbani et al., 2007). In contrast, As contaminated groundwater, including those
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Fig. 1 e Distribution of bacterial genera as detected in the As contaminated ground water samples (a) and overall abundance of bacterial genera (b).
collected from Bengal basin, presented relatively narrow depth of diversity with only few genera affiliated to a-, g-, b-Proteobacteria, Bacteroidetes, Acidobacteria, Verrucomicrobia, etc. (Sutton et al., 2009; Liao et al., 2011). Abundance of aProteobacteria, known for their ability to grow under low organic carbon containing subsurface environment as observed in several samples studied here is in line with earlier reports including one from West Bengal (Sarkar et al., 2013). Bacterial genera detected in our samples count species that are known for their ability to reduce As(V), siderophore production and chemolithotrophic growth under various nutrient conditions. Among these genera the predominance of Brevundimonas seems most notable. Although there are reports indicating presence of this genus in As contaminated habitats (including the groundwater from Bengal delta plain) their role in As transformation and mobilization is yet not clear (Drewniak et al., 2008; Cavalca et al., 2010; Sarkar et al., 2013). So, the high abundance of Brevundimonas could possibly be a
subject of further investigation. The b proteobacterial member Acidovorax covering a significant percentage of abundance is supported by the studies of Huang et al. (2012) where it has been reported to be a chemoorganotrophic, nitrate reducing bacterium, capable of mobilizing iron in the subsurface environment. Pseudomonas and Acinetobacter of g Proteobacteria have previously been detected in As contaminated sites (Sultana et al., 2011; Cai et al., 2009). The role of Pseudomonas has also been linked with subsurface As release (Islam et al., 2004). In this study, we report the presence of b-proteobacterial members Herbaspirillum and Undibacterium for the first time in As contaminated sites with considerable As tolerance.
3.3.
As sensitivity of isolated bacteria
The study on As tolerance of the isolated strains revealed that 94% of the total strains, (37 obtained from control plates, 29 from As(V) enrichment and 23 from As(III) amended plates)
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Fig. 2 e 16S rRNA gene based Neighbour-Joining phylogenetic tree of isolates obtained from As-contaminated groundwater [the numbers at the branches depict bootstrap values].
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irrespective of their enrichment condition (during isolation) and taxonomic affiliation could withstand 100 mM of As(V) and all except 25% could resist 10 mM of As(III) (Fig. 3a). Resistance of microbes to these threshold concentrations of As can be regarded as As tolerant (Jackson et al., 2003). Out of the 13 genera found, except one, all the Acidovorax strains collected from the groundwater samples of northern part of Brahmaputra are found to be hypertolerant to As(V) as well as As(III) (isolates named with A). In contrast, the isolates of the same taxon obtained from southern part (isolates named with J) showed hypertolerance to As(V) but no resistance to As(III) at all. Within the genus Brevundimonas, 64% of the isolates were hypertolerant to both the forms of As, two third of which could withstand even higher concentrations of As(V) i.e. 550 mM and only 3 among them grew at 50 mM of As(III). Hypertolerance to As(V) was seen in all the Acinetobacter
strains representing the samples from Jorhat (J). Nevertheless, As(III) resistance was found to be only upto the threshold level (10 mM) except three of these isolates. Other minor representatives such as Herbaspirillum and Rhizobiales members could also withstand both the forms of As well above the threshold value. It may be noted that there is a general distribution of resistance property among the bacterial isolates independently of the level of As contamination of sampling sites as few of the highly As resistant bacteria have been obtained from the groundwater sample having least As contamination. The UPGMA for the tolerance values of these isolates also support this observation (Fig. 3b). It is distinctly observed that isolates obtained from As enrichment posses higher resistance to As. In this analysis the total population has been clustered broadly into two clades where the effect of
Fig. 3 e UPGMA clustering of As resistance properties (a) As(III) and As(V) resistance of bacterial isolates (b) [Isolates belonging to subclades 1e8 have been named in Supplementary information].
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enrichment in isolating As sensitive and resistant bacteria has become prominent (See Supplementary table). The upper clade (subclades 1, 2, and 3) represented all those isolates with either no As resistance or those that have just the tolerance at threshold values. Another clade (subclades 4, 5, 6, 7 and 8), on the other hand represented the strains with extremely high resistance to both form of As. These are mainly seen to be isolated from enriched condition. Thus, it can be concluded
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that the highly tolerant isolates prefer to grow in As enriched conditions. Microbial resistance to concentration greater than 10 mM As(III) and 100 mM As(V) is regarded as very high, while resistance to 300e500 mM As(V) or 30 mM As(III) is a hyper tolerance property (Jackson et al., 2003). Except six, most of the isolates, irrespective of their site of isolation showed As resistance. Resistance to As have been reported in previous
Fig. 4 e Metabolic profiling of bacterial strains affiliated to a Proteobacteria (a) b Proteobacteria (b) and g Proteobacteria (c). Response towards various test parameters are scored as positive or negative and marked as red and yellow respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4 e (continued).
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studies for Sinorhizobium, Phyllobacterium, Variovorax, Pseudomonas and Acinetobacter along with Actinobacteria and Firmicutes up to 150e350 mM As(V) and 1.5e14 mM As(III) (AchourRokbani et al., 2007). Recently, Sarkar et al. (2013) showed a minor population of As resistant Brevundimonas from a high As rich groundwater of West Bengal. Our study confirmed the presence of highly resistant Brevundimonas sp. and Acidovorax sp. The study suggested that As resistance property is not limited to the As contaminated sites only, rather the most highly resistant isolates belonging to the genus Brevundimonas have been isolated from the sample A18 having very low or negligible As content. But these highly resistant isolates IIIA210, VA18-4 and VA18-5 could be isolated only from the As amended plates. Therefore, enrichment effect is prominent in isolating highly As resistant bacteria. From their growth preference on As enrichment plates, it may be suggested that these isolates are not only resistant to As by employing some detoxifying mechanism to pump out the toxic species from cell interior, but also may use As in their energy transduction (Oremland and Stolz, 2003).
3.4.
Metabolic characterization of isolated bacteria
The study on metabolic profile was performed based on the capacity of the isolates to utilize varying carbon sources, different electron acceptors and to produce siderophore as well as their As(V) reductase activity. The result showed that the most dominant and overlapping genus Brevundimonas and other a-proteobacterial members Rhizobium were capable of utilizing the different types of organic carbon sources on the same time being particular about using As(V) and sulphate as their electron acceptors under anaerobic growth (Fig. 4a). The next abundant genus Acidovorax is seen to be equally capable of using various carbon sources but they are less capable of growing under anaerobic conditions with alternate electron acceptors. The genera Herbaspirillum and Pseudomonas showed better capability to reduce different inorganic electron acceptors in anaerobic condition. Acinetobacter is found to be highly versatile in utilization of different carbon sources but is only capable of using As(V) as terminal electron acceptor when grown anoxically. Pseudomonas, on the other hand, is relatively able to use alternate electron acceptors. It was observed that more than 50% of the total isolates could grow anaerobically with alternate (more than three) electron acceptors. These observations were further established by a principle component analysis (PCA) (Fig. 5a). The Euclidean biplot showed that the metabolic properties of isolates had a correlation among them with an Eigen value of 0.896 and 0.445 for axis 1 and 2, respectively. This PCA could explain a cumulative percentage metabolic diversity of 65.8%. Based on their metabolic profile, the isolates are found to cluster in 4 quadrants (Fig. 5a). It is evident that the As(V) reductase activity along with siderophore production is highly related to their capability to use As(V) and Se(VI) as electron acceptors in combination with their ability to use acetate, pyruvate, arabinose, glucose, starch, raffinose and inositol. The analysis further indicates that the capability of siderophore production by the isolates is more associated with As(V) reductase activity. Another PCA analysis with the tolerance values of the isolates for As(III) as well as As(V), their As(V)reductase
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activity and siderophore production capability shows that the tolerance to As(III) and reductase activity is strongly correlated (Fig. 5b). The broad metabolic profiles were obtained for each isolates obtained from individual sampling sites. Fig. 4c represented the cumulative metabolic characteristics of the isolates obtained from each sample. UPGMA analysis reveals that based on metabolic traits of the indigenous bacteria the sampling sites can be divided into two clades mostly representing the sites at either bank of the river Brahmaputra (Fig. 5c). In the upper clade the nearest correlation is seen within the isolates of A2 (As ¼ 161 ppb) and A21 (As ¼ 2 ppb) depicting that groundwater As had no apparent influence in the metabolic adaptation of the isolates. Rather the subsurface geochemistry might have conferred some common characteristics to the resident microbes. The culturable community is also seen to be overlapping in these two samples. All these data significantly indicates that the geochemical factors and not the level of As contamination might affect the microbial diversity and their metabolic characters. Their contribution to mobility of As from sediment to the groundwater is prominent from the fact that most of the isolates are reductase positive and many of them are capable of utilizing As(V) as their alternate electron acceptor. A number of bacterial genera retrieved from As rich sites and those affiliated to genera Hydrogenophaga, members of AgrobacteriumeRhizobium group etc. or Desulfitobacterium spp., Bacillus arsenicoselenatis and Bacillus selenitireducens have been previously shown to utilize multiple electron donors and/or acceptors (Laverman et al., 1995; Gihring et al., 2001). Although it is of rare occurrence to find bacteria capable of utilizing three or more carbon sources as well as various electron acceptors, nevertheless, significant number (60%) of our isolates may be considered truly versatile with respect to versatile C source utilization and relatively less (32%) among them, are capable of utilizing alternate electron acceptors. Reports are available for pre- and post monsoon seasonal and diurnal change in level of water table creating dynamic oxiceanoxic states and hence availability of electron acceptors as well (Macur et al., 2004). This fact and our supporting results therefore suggest that the inhabiting microorganisms might have equipped themselves with ability to utilize diverse electron acceptors and donors to survive and flourish under such changing environments. In previous studies, it has been seen that As(V) reduction by the microbes is mainly performed through cytoplasmic reductases (ars) that encode As(III) extruding pump for detoxification mechanism under oxic condition or, through periplasmic respiratory reductase that dissimilates As(V) as terminal electron acceptor in anoxic condition (Oremland and Stolz, 2003). At significant depth where dissolved oxygen is negligible and anoxic condition is stable, these isolates may respire As(V) bound to the sediments along with iron and sulphur compounds. Siderophore production in various genera has been found to be the way of nutrient acquisition by chelating Fe compounds and making Fe bioavailable. Such an activity in Pseudomonas, Rhodococcus, and Rhizobium including As mobilization has been reported in various studies previously (Sriyosachati and Cox, 1986; Vala et al., 2006; Nair et al., 2007). However, such studies in Brahmaputra delta were undone upto now. In this work we found
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Fig. 5 e (continued).
siderophore activity in the dominant genera of a, b and g proteobacterial classes. Total Fe content of the groundwater being significantly low in these groundwater samples, the production of siderophore by most of the reductase positive strains further suggests the microbial role in releasing As(III) while chelating iron from the solid phase. In the meantime, As(V) is being reduced to As(III) by the reductase activity of the microbes solubilizing As(III) in the aqueous phase. This hypothesis is supported by the recent studies of Drewniak et al. (2008) and Sarkar et al. (2013). This may explain the observation by the second PCA plot (Fig. 5b) showing correlation between reductase activity and As(III) tolerance of the isolates. The more they reduce As(V) to As(III), more they are resistant to the reduced As(III).
3.5.
Fig. 5 e Plot of PCA scores on utilization of different C sources and alternate electron acceptors, As(V) reductase activity and siderophore production of the isolates (a). Each bacterial strain is represented by numbers referred to their names in Supplementary Table 2. The arrows indicate the corresponding parameters for which the isolates with higher eigen values have clustered [Q1: isolates versatile in different C source utilization, Q2: isolates relatively less versatile in terms of C source utilization but capable of siderophore production, Q3: isolates incapable of utilizing most of the carbon sources yet, well equipped to metabolize various electron acceptors, Q4: most versatile isolates capable of metabolizing different C sources as well can utilize nitrate, Fe(III), thiosulphate and sulphate as their terminal electron acceptor]. PCA analysis showing relation among As(V) and As(III) tolerance, reductase activity and ability to produce siderophore by the isolates (b). Cumulative metabolic profile represented by UPGMA for the isolates obtained from individual samples (c).
Chemolithoautotrophic growth of isolated bacteria
It has been established in various studies that organic carbon plays an important role in the microbial mobilization of As. Yet, in the last decade a few studies reported well known chemolithoautotrophic strains such as NT-26 (Santini et al., 2000) capable of utilizing As(III) as sole electron donor in autotrophic condition. During this study we were interested to see if these heterotrophically isolated bacteria could grow autotrophically and whether they could utilize As(III) as their electron donor. Incubation in chemolithotrophic medium (with bicarbonate and As(III) as sole C and e source, respectively) showed positive growth upto an O.D of 0.2e0.3 at 600 nm for 8 isolates among all in 7 days. These were subcultured in the same media under the same conditions twice. These chemolithotrophic genera were mostly Brevundimonas, followed by few Acidovorax and Acinetobacter isolated from enrichment culture (Fig. 6). Chemolithoautotrophy has been found in diverse genera in various ecosystems where phototrophy or heterotrophy is not supported due to natural constrains. Studies reveal chemolithoautotrophic growth of members of b- and g-Proteobacteria upon oxidation of sulphur or ammonia or nitrite (Chen et al., 2009). Chemolithoautotrophy using As(III) as electron donor in Pseudomonas arsenitoxidans had first been reported by Ilyaletdinov and Abdrashitova (1981). In recent studies, member of a-Proteobacteria Rhizobium NT-26 and g proteobacterial member Ectothiorhodospira MLHE-1 has been reported to oxidize As(III)
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unexplored. The present paper identifies and characterizes the metabolic diversity of bacteria from several As rich groundwater of Assam, India, thus illuminating their intrinsic capability in controlling subsurface arsenic geochemistry and mobilization. Strong correlation could be established between the property to tolerate As(III) and siderophore production with reductase activity of the isolates. The abundance of chemolithotrophic Brevundimonas, facultative anaerobic Acidovorax, etc. is observed.
Acknowledgement The authors gratefully acknowledge the support from Department of Biotechnology Govt. of India as NER Twinning Programme (Ref No. BT/19/NE/TBP/2010). Fig. 6 e Growth of chemolithoautotrophic bacteria [O.D at 600 nm taken after an incubation of 7 days at 30 C].
chemoautotrophically (Oremland et al., 2002; Santini et al., 2000). The growth of various members of proteobacterial subgroups chemolithoautotrophically oxidizing As(III) is coherent with our study. Our results indicate different genera of these proteobacterial groups to grow as facultative chemolithoautotrophs utilizing As(III) as electron donor.
4.
Conclusion
This study reports diversity of culturable bacteria in the As contaminated sites of North Eastern State of India (an important part of GBM delta) for the first time. We report the abundance of chemolitho/organo trophic genera Brevundimonas, Acidovorax and Acinetobacter in the studied samples. High As resistance and broad metabolic versatility of the isolates, particularly the most abundant population is noted. Metabolic diversity indicates a correlation among the siderophore production and As(V) reductase activity simultaneous with As(V) and selenate based anaerobic metabolism. This might lead to the hypothesis that inhabitant bacteria use siderophore for acquisition of sediment bound Fe. As Fe is dislodged, As (arsenate) also gets released from the bound solid phase. This As(V) is either anaerobically respired or aerobically detoxified by the resistant microbes to release As(III) which is highly soluble in aqueous phase. The broad metabolic repertoire as detected within the groundwater bacteria could be attributed as an evolutionary outcome to provide crucial advantage to the respective microbes for their survival within such contaminated environment.
Statement of novelty Microbial role in disseminating the arsenic contamination in groundwater of GBM delta is a major problem yet, till date microbial biogeochemistry in Brahmaputra river basin remain
Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.watres.2013.08.044.
references
Achour-Rokbani, A., Bauda, P., Billard, P., 2007. Diversity of As(III) transporter genes from As-resistant soil bacteria. Res. Microbiol. 158, 128e137. Battaglia-Brunet, F., Dictor, M.C., Garrido, F., Crouzet, C., Morin, D., Dekeyser, K., Clarens, M., Baranger, P., 2002. An As(III)-oxidizing bacterial population: selection, characterization and performance in reactors. J. Appl. Microbiol. 93, 656e667. Cai, L., Liu, G., Rensing, C., Wang, G., 2009. Genes involved in As transformation and resistance associated with different levels of As-contaminated soils. BMC Microbiol. 9, 4. Cavalca, L., Zanchi, R., Corsini, A., Colombo, M., Romagnoli, C., Canzi, E., Andreoni, V., 2010. As-resistant bacteria associated with roots of the wild Cirsium arvense (L) plant from an As polluted soil, and screening of potential plant growthpromoting characteristics. Syst. Appl. Microbiol. 33, 154e164. Chatterjee, A., Das, D., Chakraborti, D., 1993. A study of ground water contamination by As in the residential area of Behala, Calcutta due to industrial pollution. Environ. Pollut. 80, 57e65. Chen, Y., Wu, L., Boden, R., Hillebrand, A., Kumaresan, D., Moussard, H., Baciu, M., Lu, Y., Murrell, C., 2009. Life without light: microbial diversity and evidence of sulfur- and ammonium-based chemolithotrophy in Movile Cave. ISME J. 3, 1093e1104. Davolos, D., Pietrangeli, B., 2013. A molecular study on bacterial resistance to arsenic-toxicity in surface and underground waters of Latium. Ecotoxicol. Environ. Saf. 96, 1e9. Drewniak, L., Styczek, A., Lopatka, M.M., Sklodowska, A., 2008. Bacteria, hypertolerant to As in the rocks of an ancient gold mine, and their potential role in dissemination of As pollution. Environ. Pollut. 156, 1069e1074. Enmark, G., Nordborg, D., 2007. Arsenic in the Groundwater of the Brahmaputra Floodplains, Assam, India e Source, Distribution and Release Mechanisms. Arbetsgruppen fo¨r Tropisk Ekologi Minor Field Study 131 Committee of Tropical Ecology. Uppsala University, Sweden.
7004
w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 6 9 9 2 e7 0 0 5
Fan, H., Su, C., Wang, Y., Yao, J., Zhao, K., Wang, Y., Wang, G., 2008. Sedimentary arsenite-oxidizing and arsenate-reducing bacteria associated with high arsenic groundwater from Shanyin, Northwestern China. J. Appl. Microbiol. 105, 529e539. Fendorf, S., Michael, H.A., Van Geen, A., 2010. Spatial and temporal variations of groundwater As in south and south east Asia. Science 328, 1123e1127. Gault, A.G., Cooke, D.R., Townsend, A.T., Charnock, J.M., Polya, D.A., 2005. Mechanisms of arsenic attenuation in acid mine drainage from Mount Bischoff, western Tasmania. Sci. Total Environ. 345, 219e228. Gihring, T.M., Druschel, G.K., McCleskey, R.B., Hamers, R.J., Banfield, J.F., 2001. Rapid As(III)oxidation by Thermus aquaticus and Thermus thermophilus: field and laboratory investigations. Environ. Sci. Technol. 35, 3857e3862. Gorra, R., Webster, G., Martin, M., Celi, L., Mapelli, F., Weightman, A.J., 2012. Dynamic microbial community associated with ironearsenic co-precipitation products from a groundwater storage system in Bangladesh. Microb. Ecol., 64,171e64,186. Hery, M., Van Dongen, B.E., Gill, F., Mondal, D., Vaughan, D.J., Pancost, R.D., Polya, D.A., Lloyd, J.R., 2010. As release and attenuation in low organic carbon aquifer sediments from West Bengal. Geobiology 8, 155e168. Huang, Y., Li, H., Rensing, C., Zhao, K., Johnstone, L., Wang, G., 2012. Genome sequence of the facultative anaerobic arseniteoxidizing and nitrate-reducing bacterium Acidovorax sp. strain NO1. J. Bacteriol. 194, 1635e1636. Ilyaletdinov, A.N., Abdrashitova, S.A., 1981. Autotrophic oxidation of arsenic by a culture of Pseudomonas arsenitoxidans. Mikrobiologiya 50, 197e204. Islam, E., Sar, P., 2011. Culture-dependent and -independent molecular analysis of bacterial community within uranium ore. J. Basic Microbiol., 1e13. Islam, F.S., Boothman, C., Gault, A.G., Polya, D.A., Lloyd, J.R., 2005. Potential role of the Fe(III)-reducing bacteria Geobacter and Geothrix in controlling arsenic solubility in Bengal delta sediments. Mineral. Mag. 69, 865e875. Islam, F.S., Gault, A.G., Boothman, C., Polya, D.A., Charnock, J.M., Chatterjee, D., Lloyd, J.R., 2004. Role of metal-reducing bacteria in As release from Bengal delta sediments. Nature 430, 68e71. Jackson, C.R., Jackson, E.F., Dugas, S.L., Gamble, K., Williams, S.E., 2003. Microbial transformations of As(III)and As(V) in natural environments. Recent Res. Dev. Microbiol. 7, 103e118. Kar, S., Maitya, J.P., Jeana, J., Liua, C.,C., Nath, B., Yanga, H.J., Bundschuha, J., 2010. Arsenic-enriched aquifers: occurrences and mobilization of arsenic in groundwater of Ganges Delta Plain, Barasat, West Bengal, India. Appl. Geochem. 25, 1805e1814. Kazy, S.K., Sar, P., Asthana, R.K., Singh, S.P., 1999. Copper uptake and its compartmentalization in Pseudomonas aeruginosa strains: chemical nature of cellular metal. World J. Microbiol. Biotechnol. 15, 599e605. Laverman, A.M., Blum, S.J., Schaefer, J.K., Philips, E.J.P., Lovley, D.R., Oremland, R.S., 1995. Growth of strain SES-3 with As(V) and other diverse electron acceptors. Appl. Environ. Microbiol. 61, 3556e3561. Luli, G.W., Talnagi, J.W., Strohl, W.R., Pfister, R.M., 1983. Hexavalent chromium resistant bacteria isolated from river sediments. Appl. Environ. Microbiol. 46, 846e854. Li, D., Li, Z., Yu, J., Cao, N., Liu, R., Yang, M., 2010. Characterization of bacterial community structure in a drinking water distribution system during an occurrence of red water. Appl. Environ. Microbiol. 76, 7171e7180. 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 As(V)reducing bacteria associated with As-rich groundwater in Taiwan. J. Contam. Hydrol. 123, 20e29.
Macur, R.E., Jackson, C.R., Botero, L.M., Mcdermott, T.R., Inskeep, W.P., 2004. Bacterial populations associated with the oxidation and reduction of As in an unsaturated soil. Environ. Sci. Technol. 38, 104e111. Mailloux, B.J., Alexandrova, E., Alison, R., 2009. Microbial mineral weathering for nutrient acquisition releases As. Appl. Environ. Microbiol. 75, 2558e2565. Matthijs, S., Laus, G., Meyer, J.M., Abbaspour-Tehrani, K., Schafer, M., Budzikiewicz, H., Cornelis, P., 2009. Siderophoremediated iron acquisition in the entomopathogenic bacterium Pseudomonas entomophila L48 and its close relative Pseudomonas putida KT2440. Biometals 22 (6), 951e964. Muyzer, G., de Waal, E.C., Uitterlinden, A.G., 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reactionamplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59, 695e700. Nair, A., Juwarkar, A.A., Singh, S.K., 2007. Production and characterization of siderophores and its application in As removal from contaminated soil. Water Air Soil Pollut. 180, 199e212. Nazina, T.N., Kosareva, I.M., Davydov, A.S., Turova, T.P., Novikova, E.V., Khafizov, R.R., Poltaraus, A.B., 2000. Physicochemical and microbiological characteristics of groundwater from observation boreholes of a deep radioactive liquid waste repository. Mikrobiologiia, 105e112. Nickson, R.T., McArthur, J.M., Ravenscroft, P., Burgess, W.G., Ahmed, K.M., 2000. Mechanism of arsenic release to groundwater, Bangladesh and West Bengal. Appl. Geochem. 15, 403e413. Oremland, R.S., Kulp, T.R., Switzer, B.J., Hoeft, S.E., Baesman, S., Miller, G., Stolz, J.,F., 2005. A microbial As cycle in a salt saturated, extreme environment. Science 308, 1305e1308. Oremland, R.S., Hoeft, S.E., Santini, J.M., Bano, N., Hollibaugh, R.A., Hollibaugh, J.T., 2002. Anaerobic oxidation of arsenite in mono lake water and by a facultative arseniteoxidizing chemoautotroph, strain MLHE-1. Appl. Environ. Microbiol. 68, 4795e4802. Oremland, R.S., Stolz, J.F., 2003. The ecology of As. Science 300, 939e944. Reasoner, D.J., Geldreich, E.E., 1985. A new medium for the enumeration and subculture of bacteria from potable water. Appl. Environ. Microbiol. 49, 1e7. 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, 92e97. Sarkar, A., Kazy, S.K., Sar, P., 2013. Characterization of As resistant bacteria from As rich groundwater of West Bengal. Ecotoxicology 22, 363e374. Shimizu, S., Akiyama, M., Ishijima, Y., Hama, K., Kunimaru, T., Naganuma, T., 2006. Molecular characterization of microbial communities in fault-bordered aquifers in the Miocene formation of northernmost Japan. Geobiology 4, 147e223. Silver, S., Phung, L.T., 2005. Genes and enzymes involved in bacterial oxidation and reduction of inorganic As. Appl. Environ. Microbiol. 71, 599e608. Singh, A.K., 2006. Chemistry of As in ground water of GangesBrahmputra river basin, India. Curr. Sci. 91, 1e7. Slyemi, D., Bonnefoy, V., 2011. How prokaryotes deal with As. Environ. Microbiol. Rep. 1, 16. Sriyosachati, S., Cox, C.D., 1986. Siderophore-mediated iron acquisition from transferrin by Pseudomonas aeruginosa. Infect. Immun. 52, 885e891. Sultana, M., Hartig, C., Planer-Friedrich, B., Seifert, J., Schlomann, M., 2011. Bacterial communities in Bangladesh aquifers differing in aqueous As concentration. Geomicrobiol. J. 28, 198e211.
w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 6 9 9 2 e7 0 0 5
Sutton, N.B., van der Kraan, G.M., van Loosdrecht, M.C., Muyzer, G., Bruining, J., Schotting, R.J., 2009. Characterization of geochemical constituents and bacterial populations associated with As mobilization in deep and shallow tube wells in Bangladesh. Water Res. 43, 1720e1730. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596e1599.
7005
Tiago, I., Chung, A.P., Verissimo, A., 2004. Bacterial diversity in a nonsaline alkaline environment: heterotrophic aerobic populations. Appl. Environ. Microbiol. 70 (12), 7378e7387. Vala, A.K., Dave, B.P., Dube, H.C., 2006. Chemical characterization and quantification of siderophores produced by marine and terrestrial aspergilli. Can. J. Microbiol. 52, 603e607. Zhao, Z., Jiaa, Y., Xua, L., Zhaoc, S., 2011. Adsorption and heterogeneous oxidation of As(III) on ferrihydrite. Water Res. 45, 6496e6504.
