Ghosh et al. BMC Microbiology (2020) 20:256 https://doi.org/10.1186/s12866-020-01893-6 RESEARCH ARTICLE Open Access Molecular and taxonomic characterization of arsenic (As) transforming Bacillus sp. strain IIIJ3–1 isolated from As-contaminated groundwater of Brahmaputra river basin, India Soma Ghosh1,2, Balaram Mohapatra1,3, Tulasi Satyanarayana4,5 and Pinaki Sar1* Abstract Background: Microbe-mediated redox transformation of arsenic (As) leading to its mobilization has become a serious environmental concern in various subsurface ecosystems especially within the alluvial aquifers. However, detailed taxonomic and eco-physiological attributes of indigenous bacteria from As impacted aquifer of Brahmaputra river basin has remained under-studied. Results: A newly isolated As-resistant and -transforming facultative anaerobic bacterium IIIJ3–1 from As- contaminated groundwater of Jorhat, Assam was characterized. Near complete 16S rRNA gene sequence affiliated the strain IIIJ3–1 to the genus Bacillus and phylogenetically placed within members of B. cereus sensu lato group with B. cereus ATCC 14579(T) as its closest relative with a low DNA-DNA relatedness (49.9%). Presence of iC17:0, iC15:0 fatty acids and menaquinone 7 corroborated its affiliation with B. cereus group, but differential hydroxy-fatty acids, C18:2 and menaquinones 5 & 6 marked its distinctiveness. High As resistance [Maximum Tolerable Concentration = 10 mM As3+, 350 mM As5+], aerobic As3+ (5 mM) oxidation, and near complete dissimilatory 5+ reduction of As (1 mM) within 15 h of growth designated its physiological novelty. Besides O2, cells were found 5+ 3+ 2− − 6+ to reduce As ,Fe ,SO4 ,NO3 , and Se as alternate terminal electron acceptors (TEAs), sustaining its anaerobic growth. Lactate was the preferred carbon source for anaerobic growth of the bacterium with As5+ as TEA. Genes encoding As5+ respiratory reductase (arr A), As3+ oxidase (aioB), and As3+ efflux systems (ars B, acr3) were detected. All these As homeostasis genes showed their close phylogenetic lineages to Bacillus spp. Reduction in cell size following As exposure exhibited the strain’s morphological response to toxic As, while the formation of As-rich electron opaque dots as evident from SEM-EDX possibly indicated a sequestration based As resistance strategy of strain IIIJ3–1. (Continued on next page) * Correspondence: [email protected]; [email protected] 1Environmental Microbiology and Genomics Laboratory, Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur 721302, India Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Ghosh et al. BMC Microbiology (2020) 20:256 Page 2 of 20 (Continued from previous page) Conclusion: This is the first report on molecular, taxonomic, and ecophysiological characterization of a highly As resistant, As3+ oxidizing, and dissimilatory As5+ reducing Bacillus sp. IIIJ3–1 from As contaminated sites of Brahmaputra river basin. The strain’s ability to resist and transform As along with its capability to sequester As within the cells demonstrate its potential in designing bioremediation strategies for As contaminated groundwater and other ecosystems. Keywords: Arsenic, Bacillus cereus group, Brahmaputra river basin, Ecophysiology, Dissimilatory reduction, Groundwater, Taxonomy Background aquifers [8, 16, 30–32]. With respect to the taxonomic and Arsenic (As) contamination in groundwater of Bengal physiological characterization of DARB from diverse habi- Delta Plain (BDP) (covering large parts of Bangladesh tats, till date 32 cultivable representatives have been studied and India) has become an emergent health concern for [29, 32, 33]. Interestingly, only few members of Proteobac- millions of people over the decades [1, 2]. Consumption teria i.e. Desulfuromonas/ Pelobacter sp. WB-3 [32], Rhizo- of As contaminated drinking water and food grains has bium arsenicireducens KAs 5-22T [16], Pseudoxanthomonas been implicated with severe health crisis including arseniciresistens KAs 5-3T [34]; Achromobacter sp. KAs 3-5 arsenicosis and cancer affecting more than 100 million [35]andFirmicutes viz. B. arsenicus [36], B. indicus [37] people in BDP [3, 4]. The predominant inorganic species have been isolated from BDP (West Bengal) and character- of As in naturally (geogenic) contaminated alluvial aqui- ized thoroughly. Recently, an As tolerant siderophore pro- fers are As3+ and As5+.As3+ is more mobile in aqueous, ducing Staphylococcus sp. strain TA6 has been isolated oxic environments while As5+ tends to remain adsorbed from upper Brahmaputra River Basin (BRB) aquifer and re- to the sediments in anoxic state [5]. Relative abundance ported to have possible role in biogeochemical cycling of of these two species which eventually affects As -mobil- As therein [38]. However, except few recent documenta- ity and –toxicity in aquifers depends mainly on the pre- tions, the geomicrobiology of the vast As rich alluvial aqui- vailing redox conditions and inhabitant microbial fers of BRB remain largely unexplored [38–40]. activity [6–9]. The resident microorganisms in contami- Using anaerobic microcosms of subsurface As bearing nated aquifer involved in redox transformation of As are sediment of BRB we have recently demonstrated the considered to be the most precarious factors for As-release prominent role of Bacillus and other members of Firmi- into the groundwater [1, 10–13]. Eco-physiological, taxo- cutes in As/ Fe reduction and As mobilization [40]. Par- nomic, and molecular characterization of As transforming ticularly, strain IIIJ3-1 has been recently shown to play bacteria from As contaminated groundwater constitute an prominent role in As mobilization from sediments of - important component of subsurface geomicrobiology, par- BRB aquifer under NO3 amended anaerobic microcosm ticularly to better understand the potential of these organ- by the process of oxalate mediated mineral weathering isms in geo-cycling of As and their natural attenuation [41]. However, detailed molecular and taxonomic [13–16]. characterization of any of the As reducing taxa remained In recent years, several geomicrobiological studies have still elusive. In general, presence of Bacillus spp. in As- unanimously agreed upon the wide physiological role of contaminated groundwater of Bengal basin and other al- taxonomically diverse bacterial populations viz. Alpha-, luvial aquifers as well in various polluted environment Beta-, Gamma-proteobacteria, Firmicutes (Bacillus and rel- and their As transformation abilities have been well doc- atives), Actinobacteria, etc. influencing As bio-geochemistry umented [17, 42–45]. Dissimilatory reduction of As5+ by in alluvial groundwater [13, 17–23]. Arsenic transforming Bacillus spp. from soda lake, mono lake, mine environ- bacteria have been known to deploy an array of metabolic ment, and effluent plants (B. arsenicoselenatis, B. seleni- routes including lithotrophic to heterotrophic mechanisms tireducens, B. macyae and B. selenatarsenatis) and As3+ of As-oxidation [24], -reduction [25], -respiration [26], and oxidation (B. firmus L-148, Bacillus sp. PNKP-S2) have -methylation [27], affecting As -solubility, -speciation, and been described [36, 37, 46–49]. Metabolic versatility in- -mobilization. Heterotrophic As3+ oxidizing (HAO) and cluding the ability of dissimilatory reduction of Fe and 3+ chemoautotrophic As oxidizing (CAO) members have As or even SO4 by the members of this taxon could have been described to use As3+ as their electron source [28] strong influence of subsurface As mobility. Bacillus and dissimilatory As5+ respiring members (DARB) to use strains isolated from As contaminated sites of West Ben- As5+ as electron acceptor [29]. Among these organisms, gal (India) [36, 37], or Datong basin (China) [43] and DARB have been identified to play the crucial role in As Hetao basins (Mongolia) [50] were characterized. How- mobilization from As bearing host minerals in alluvial ever, till date, no pure culture Bacillus strain capable of Ghosh et al. BMC Microbiology (2020) 20:256 Page 3 of 20 dissimilatory As reduction has been isolated from the vast at optimal renaturation temperature (TOR) 69.95°C, and As contaminated area of North East India (covering the homoduplex of B. cereus ATCC 14579(T) was found to BRB) and studied for its taxonomic characterization, over- be 8°C (Supplementary Fig S1). Relative binding ratio in- all physiology and As biotransformation