Metagenome based analysis of groundwater from arsenic contaminated sites of West Bengal revealed community diversity and their metabolic potential.

The study of microbial community in groundwater systems is considered to be essential to improve our understanding of arsenic (As) biogeochemical cycling in aquifers, mainly as it relates to the fate and transport of As. The present study was conducted to determine the microbial community composition and its functional potential using As-contaminated groundwater from part of the Bengal Delta Plain (BDP) in West Bengal, India. Geochemical analyses indicated low to moderate dissolved oxygen (0.42-3.02 mg/L), varying As (2.5-311 µg/L) and Fe (0.19-1.2 mg/L) content, while low concentrations of total organic carbon (TOC), total inorganic carbon (TIC), nitrate, and sulfate were detected. Proteobacteria was the most abundant phylum, while the indiscriminate presence of an array of archaeal phyla, Euryarchaeota, Crenarchaeota, Nanoarchaeota, etc., was noteworthy. The core community members were affiliated to Sideroxydans, Acidovorax, Pseudoxanthomonas, Brevundimonas, etc. However, diversity assessed over multiple seasons indicated a shift from Sideroxydans to Pseudomonas or Brevundimonas dominant community, suggestive of microbial response to seasonally fluctuating geochemical stimuli. Taxonomy-based functional potential showed prospects for As biotransformation, methanogenesis, sulfate respiration, denitrification, etc. Thus, this study strengthened existing reports from this region by capturing the less abundant or difficult-to-culture taxa collectively forming a major fraction of the microbial community.

Microbial diversity and function in crystalline basement beneath the Deccan Traps explored in a 3 km borehole at Koyna, western India.

Deep terrestrial subsurface represents a huge repository of global prokaryotic biomass. Given its vastness and importance, microbial life within the deep subsurface continental crust remains under-represented in global studies. We characterize the microbial communities of deep, extreme and oligotrophic realm hosted by crystalline Archaean granitic rocks underneath the Deccan Traps, through sampling via 3000 m deep scientific borehole at Koyna, India through metagenomics, amplicon sequencing and cultivation-based analyses. Gene sequences 16S rRNA (7.37 × 106 ) show considerable bacterial diversity and the existence of a core microbiome (5724 operational taxonomic units conserved out of a total 118,064 OTUs) across the depths. Relative abundance of different taxa of core microbiome varies with depth in response to prevailing lithology and geochemistry. Co-occurrence network analysis and cultivation attempt to elucidate close interactions among autotrophic and organotrophic bacteria. Shotgun metagenomics reveals a major role of autotrophic carbon fixation via the Wood-Ljungdahl pathway and genes responsible for energy and carbon metabolism. Deeper analysis suggests the existence of an 'acetate switch', coordinating biosynthesis and cellular homeostasis. We conclude that the microbial life in the nutrient- and energy-limited deep granitic crust is constrained by the depth and managed by a few core members via a close interplay between autotrophy and organotrophy.

Characterization of the rare microbiome of rice paddy soil from arsenic contaminated hotspot of West Bengal and their interrelation with arsenic and other geochemical parameters.

Rare microbial taxa [bacterial and archaeal operational taxonomic units (OTUs) with mean relative abundance ≤ 0.001%] were critical for ecosystem function, yet, their identity and function remained incompletely understood, particularly in arsenic (As) contaminated rice soils. In the present study we have characterized the rare populations of the As-contaminated rice soil microbiomes from West Bengal (India) in terms of their identity, interaction and potential function. Major proportion of the OTUs (73% of total 38,289 OTUs) was represented by rare microbial taxa (henceforth mentioned as rare taxa), which covered 4.5-15.7% of the different communities. Taxonomic assignment of the rare taxa showed their affiliation to members of Gamma-, Alpha-, Delta- Proteobacteria, Actinobacteria, and Acidobacteria. SO42-, NO3-, NH4+and pH significantly impacted the distribution of rare taxa. Rare taxa positively correlated with As were found to be more frequent in relatively high As soil while the rare taxa negatively correlated with As were found to be more frequent in relatively low As soil. Co-occurrence-network analysis indicated that rare taxa whose abundance were correlated strongly (R > 0.8) with As also had strong association (R > 0.8) with PO42-, NO3-, and NH4+. Correlation analysis indicated that the rare taxa were likely to involved in two major guilds one, involved in N-metabolism and the second involved in As/Fe as well as other metabolisms. Role of the rare taxa in denitrification and dissimilatory NO3- reduction (DNRA), As biotransformation, S-, H-, C- and Fe-, metabolism was highlighted from 16S rRNA gene-based predictive analysis. Phylogenetic analysis of rare taxa indicated signatures of inhabitant rice soil microorganisms having significant roles in nitrogen (N) cycle and As-Fe metabolism. This study provided critical insights into the taxonomic identity, metabolic potentials and importance of the rare taxa in As biotransformation and biogeochemical cycling of essential nutrients in As-impacted rice soils.

Thermoplasmata and Nitrososphaeria as dominant archaeal members in acid mine drainage sediment of Malanjkhand Copper Project, India.

Acid mine drainage (AMD) harbors all three life forms in spite of its toxic and hazardous nature. In comparison to bacterial diversity, an in-depth understanding of the archaeal diversity in AMD and their ecological significance remain less explored. Archaeal populations are known to play significant roles in various biogeochemical cycles within the AMD ecosystem, and it is imperative to have a deeper understanding of archaeal diversity and their functional potential in AMD system. The present study is aimed to understand the archaeal diversity of an AMD sediment of Malanjkhand Copper Project, India through archaea specific V6 region of 16S rRNA gene amplicon sequencing. Geochemical data confirmed the acidic, toxic, heavy metal-rich nature of the sample. Archaea specific V6-16S rRNA gene amplicon data showed a predominance of Thermoplasmata (BSLdp215, uncultured Thermoplasmata, and Thermoplasmataceae) and Nitrososphaeria (Nitrosotaleaceae) members constituting ~ 95% of the archaeal community. Uncultured members of Bathyarchaeia, Group 1.1c, Hydrothermarchaeota, and Methanomassiliicoccales along with Methanobacteriaceae, Methanocellaceae, Haloferaceae, Methanosaetaceae, and Methanoregulaceae constituted the part of rare taxa. Analysis of sequence reads indicated that apart from their close ecological relevance, members of the Thermoplasmata present in Malanjkhand AMD were mostly involved in chemoheterotrophy, Fe/S redox cycling, and with heavy metal resistance, while the Nitrososphaeria members were responsible for ammonia oxidation and fixation of HCO3- through 3-hydroxypropionate/4-hydroxybutyrate cycle at low pH and oligotrophic environment which subsequently played an important role in nitrification process in AMD sediment. Overall, the present study elucidated the biogeochemical significance of archaeal populations inhabiting the toxic AMD environment.

Variable response of arsenic contaminated groundwater microbial community to electron acceptor regime revealed by microcosm based high-throughput sequencing approach.

Arsenic (As) mobilization in alluvial aquifers is facilitated by microbially catalyzed redox transformations that depend on the availability of electron acceptors (EAs). In this study, the response of an As-contaminated groundwater microbial community from West Bengal, India towards varied EAs was elucidated through microcosm based 16S rRNA gene amplicon sequencing. Acinetobacter, Deinococcus, Nocardioides, etc., and several unclassified bacteria (Ignavibacteria) and archaea (Bathyarchaeia, Micrarchaeia) previously not reported from As-contaminated groundwater of West Bengal, characterized the groundwater community. Distinct shifts in community composition were observed in response to various EAs. Enrichment of operational taxonomic units (OTUs) affiliated to Denitratisoma (NO3-), Spirochaetaceae (Mn4+), Deinococcus (As5+), Ruminiclostridium (Fe3+), Macellibacteroides (SO42-), Holophagae-Subgroup 7 (HCO3-), Dechloromonas and Geobacter (EA mixture) was noted. Alternatively, As3+ amendment as electron donor allowed predominance of Rhizobium. Taxonomy based functional profiling highlighted the role of chemoorganoheterotrophs capable of concurrent reduction of NO3-, Fe3+, SO42-, and As biotransformation in As-contaminated groundwater of West Bengal. Our analysis revealed two major aspects of the community, (a) taxa selective toward responding to the EAs, and (b) multifaceted nature of taxa appearing in abundance in response to multiple substrates. Thus, the results emphasized the potential of microbial community members to influence the biogeochemical cycling of As and other dominant anions/cations.

Metagenomics of two gnotobiotically grown aromatic rice cultivars reveals genotype-dependent and tissue-specific colonization of endophytic bacterial communities attributing multiple plant growth promoting traits.

Exploration of community structures, habitations, and potential plant growth promoting (PGP) attributes of endophytic bacteria through next generation sequencing (NGS) is a prerequisite to culturing PGP endophytic bacteria for their application in sustainable agriculture. The present study unravels the taxonomic abundance and diversity of endophytic bacteria inhabiting in vitro grown root, shoot and callus tissues of two aromatic rice cultivars through 16S rRNA gene-based Illumina NGS. Wide variability in the number of bacterial operational taxonomic units (OTUs) and genera was observed between the two samples of the root (55, 14 vs. 310, 76) and shoot (26, 12 vs. 276, 73) but not between the two callus samples (251, 61 vs. 259, 51), indicating tissue-specific and genotype-dependent bacterial community distribution in rice plant, even under similar gnotobiotic growth conditions. Sizes of core bacteriomes of the selected two rice genotypes varied from 1 to 15 genera, with Sphingomonas being the only genus detected in all six samples. Functional annotation, based upon the abundance of bacterial OTUs, revealed the presence of several PGP trait-related genes having variable relative abundance in tissue-specific and genotype-dependent manners. In silico study also documented a higher abundance of certain genes in the same biochemical pathway, such as nitrogen fixation, phosphate solubilization and indole acetic acid production; implying their crucial roles in the biosynthesis of metabolites leading to PGP. New insights on utilizing callus cultures for isolation of PGP endophytes aiming to improve rice crop productivity are presented, owing to constancy in bacterial OTUs and genera in callus tissues of both the rice genotypes.

Molecular and taxonomic characterization of arsenic (As) transforming Bacillus sp. strain IIIJ3-1 isolated from As-contaminated groundwater of Brahmaputra river basin, India.

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 reduction of As 5+ (1 mM) within 15 h of growth designated its physiological novelty. Besides O2, cells were found to reduce As5+, Fe3+, SO42-, NO3-, and Se6+ 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. 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.

Comparative genome analysis of arsenic reducing, hydrocarbon metabolizing groundwater bacterium Achromobacter sp. KAs 3-5T explains its competitive edge for survival in aquifer environment.

Whole genome sequence of arsenic (As) reducing, hydrocarbon metabolizing groundwater bacterium Achromobacter sp. KAs 3-5T was explored to understand the genomic basis of its As-ecophysiology and niche adaptation in aquifer environment. The genome (5.6 Mbp, 65.5 G + C mol %) encodes 4840 proteins, 1138 enzymes, 53 tRNAs, 11 rRNAs, 608 signal peptides, and 1.13% horizontally transferred genes. Presence of genes encoding cytosolic As5+-reduction (arsRCBH, ACR3), aromatics utilization (bph, naph, catABC, boxABCD, genACB), Fe-transformation (tonB, achromobactin, FUR, FeR), and denitrification (nar, nap) processes were observed and validated through proteomics. Phylogenomic analysis (< 90% ANI, < 50% DDH) confirmed strain KAs 3-5T to be a novel representative of the genus Achromobacter. An asymptotic open pan-genome (20,855 genes) and high correlation between genomic and ecological diversity suggested niche preference ability of this genus. Assemblage of species specific genes affiliated to transcription-regulation, membrane transport, and redox-transformation explained the strain's competitive survival strategies in As-rich oligotrophic groundwater.

Characterization and application of an anaerobic, iron and sulfate reducing bacterial culture in enhanced bioremediation of acid mine drainage impacted soil.

Development of an appropriate bioremediation strategy for acid mine drainage (AMD) impacted environment is imperative for sustainable mining but remained critically challenged due to the paucity of knowledge on desired microbiological factors and their nutrient requirements. The present study was conducted to utilize the potential of an anaerobic, acid-tolerant, Fe3+ and SO42- reducing microbial consortium for in situ remediation of highly acidic (pH 3.21), SO42- rich (6285 mg/L) mine drainage impacted soil (AIS). A microbial consortium enriched from AMD system and composed of Clostridiales and Bacillales members was characterized and tested for in situ application through microcosms. A combination of bioaugmentation (enriched consortium) and biostimulation (cellulose) allowed 97% reduction in dissolved sulfate and rise in pH up to 7.5. 16S rRNA gene-based amplicon sequencing confirmed that although the bioaugmented community could survive in AIS, availability of carbon source was necessary for superior iron- and sulfate- reduction. Quantitative PCR of dsrB gene confirmed the role of carbon source in boosting the SO42- reduction activities of sulfate reducers. This study demonstrated that native AIS harbored limited catabolic activities required for the remediation but addition of catabolically active microbial populations along with necessary carbon and energy source facilitate the bioremediation of AIS.

Microcosm based analysis of arsenic release potential of Bacillus sp. strain IIIJ3-1 under varying redox conditions.

The role of indigenous bacteria in mobilization of sediment bound arsenic (As) into groundwater is investigated using subsurface sediment from Brahmaputra River Basin (BRB) and the Bacillus sp. strain IIIJ3-1, an indigenous species to BRB. Anaerobic sediment microcosms with varying organic carbon sources and terminal electron acceptors (TEAs) are used to illustrate the role of the test bacterium in As mobilization. The aquifer sediment shows an asymmetric distribution of As and Fe in its different phases. Among the TEAs added, NO3 amendment promotes higher cell growth, oxalic acid production and maximum release of sediment bound As. X-ray diffraction analysis further suggests that weathering of As bearing secondary minerals through bacterial action enhances As bioavailability, followed by dissimilatory reduction and thus promotes its mobilization into aqueous phase. Co-release pattern of other elements from the sediment indicates that release of As is decoupled from that of Fe. This study confirms that microbe-mediated mineral weathering followed by respiratory reduction of As facilitates mobilization of sediment hosted As into aqueous phase, and provides a better insight into the catabolic ability of groundwater bacteria in mobilization of sediment hosted As in BRB region.

Development of nitrate stimulated hydrocarbon degrading microbial consortia from refinery sludge as potent bioaugmenting agent for enhanced bioremediation of petroleum contaminated waste.

Stable and efficient hydrocarbon degrading microbial consortia were developed from a refinery sludge through nitrate amendment for their application in enhanced bioremediation of petroleum contaminated waste. Nitrate induced biostimulation of refinery sludge resulted in increased abundance of hydrocarbon degrading Rhodocyclaceae, Xanthomonadaceae, Syntrophaceae and Comamonadaceae members. Repeated subculturing of nitrate stimulated communities in crude oil supplemented basal medium was done under aerobic and anaerobic conditions. Aerobically enriched consortia (composed of Pseudomonadaceae, Pseudoxanthomonadaceae and unclassified Comamonadaceae) showed their ability to utilize alkanes, aromatics and crude oil as growth substrates. Anaerobically enriched consortium was predominated by Bacillaceae, Pseudomonadaceae, Xanthomonadaceae, Porphyromonadaceae and Comamonadaceae members. Anaerobic consortium was found to be relatively less efficient in terms of TPH (total petroleum hydrocarbons) degradation compared to its aerobic counterpart. These enriched microbial consortia were finally tested for their biodegradation performance and stability during bioremediation of highly contaminated refinery sludge using different strategies. A 30 days microcosm based bioremediation trial showed that bioaugmentation of aerobic cultures with refinery sludge was more effective in TPH degradation (~ 65% degradation) compared to the anaerobic consortium (only 36% TPH degradation) and a combination of bioaugmentation and nitrate amendment with sludge resulted in enhanced hydrocarbon attenuation (up to 86% TPH degradation). Subsequent community analysis at the end of bioremediation trial confirmed the stability of the added microbial populations. Thus, the strategy of bioaugmentation of specially enriched native microbial populations in combination with nitrate amendment was successfully used for the enhanced bioremediation of petroleum hydrocarbon contaminated refinery waste.

Exploring the piezotolerant/piezophilic microbial community and genomic basis of piezotolerance within the deep subsurface Deccan traps.

The deep biosphere is often characterized by multiple extreme physical-chemical conditions, of which pressure is an important parameter that influences life but remains less studied. This geomicrobiology study was designed to understand the response of a subterranean microbial community of the Deccan traps to high-pressure conditions and to elucidate their genomic properties. Groundwater from a deep basaltic aquifer of the Deccan traps was used to ascertain the community response to 25 MPa and 50 MPa pressure following enrichment in high-salt and low-salt organic media. Quantitative PCR data indicated a decrease in bacterial and archaeal cell numbers with increasing pressure. 16S rRNA gene sequencing displayed substantial changes in the microbial community in which Acidovorax appeared to be the most dominant genus in the low-salt medium and Microbacteriaceae emerged as the major family in the high-salt medium under both pressure conditions. Genes present in metagenome-associated genomes which have previously been associated with piezotolerance include those related to nutrient uptake and extracytoplasmic stress (omp, rseC), protein folding and unfolding (dnaK, groEL and others), and DNA repair mechanisms (mutT, uvr and others). We hypothesize that these genes facilitate tolerance to high pressure by certain groups of microbes residing in subsurface Deccan traps.

Enrichment of indigenous arsenate reducing anaerobic bacteria from arsenic rich aquifer sediment of Brahmaputra river basin and their potential role in as mobilization.

Anaerobic enrichment of As5+ reducing bacteria in the presence and/or absence of organic carbon (OC) and As5+ from As contaminated soil of Brahmaputra river basin (BRB) (Jorhat, Assam) was performed. Denaturing gradient gel electrophoresis of the 16SrRNA gene sequences amplified from the enriched microbial community indicated occurrence of maximum diversity under conditions receiving no OC (MSM) followed by moderate OC (LB). However, higher OC or As showed antagonistic effect on bacterial enrichment whereas together (BB + As) they showed a synergistic effect. Phylogenetic analysis of the prominent bands revealed an overall abundance of Lachnoanaerobaculum (39%), Clostridium (39%), Bacillus, Peptostreptococcaceae, Anaerostipes (13%), and Desulfotomaculum (8.7%). Moderate OC (LB) led to maximum As mobilization i.e. 27.42 µg/L, whereas presence of added As together with high OC (BB + As) enhanced the mobilization process. Mineralogical analyses of the sediments after incubation showed prominent weathering and loss of crystallinity in MSM and LB. Appearance of a new peak corresponding to arsenolamprite (As) in LB and LB + As indicated opening up of secondary phases of the minerals harboring As due to microbial leaching under moderate OC. This is the first study reporting Lachnoanaerobaculumas a potent As5+ dissimilating bacterium isolated from As contaminated subsurface sediment of BRB.

Petroleum hydrocarbon rich oil refinery sludge of North-East India harbours anaerobic, fermentative, sulfate-reducing, syntrophic and methanogenic microbial populations.

BACKGROUND: Sustainable management of voluminous and hazardous oily sludge produced by petroleum refineries remains a challenging problem worldwide. Characterization of microbial communities of petroleum contaminated sites has been considered as the essential prerequisite for implementation of suitable bioremediation strategies. Three petroleum refinery sludge samples from North Eastern India were analyzed using next-generation sequencing technology to explore the diversity and functional potential of inhabitant microorganisms and scope for their on-site bioremediation. RESULTS: All sludge samples were hydrocarbon rich, anaerobic and reduced with sulfate as major anion and several heavy metals. High throughput sequencing of V3-16S rRNA genes from sludge metagenomes revealed dominance of strictly anaerobic, fermentative, thermophilic, sulfate-reducing bacteria affiliated to Coprothermobacter, Fervidobacterium, Treponema, Syntrophus, Thermodesulfovibrio, Anaerolinea, Syntrophobacter, Anaerostipes, Anaerobaculum, etc., which have been well known for hydrocarbon degradation. Relatively higher proportions of archaea were detected by qPCR. Archaeal 16S rRNA gene sequences showed presence of methanogenic Methanobacterium, Methanosaeta, Thermoplasmatales, etc. Detection of known hydrocarbon utilizing aerobic/facultative anaerobic (Mycobacterium, Pseudomonas, Longilinea, Geobacter, etc.), nitrate reducing (Gordonia, Novosphigobium, etc.) and nitrogen fixing (Azovibrio, Rhodobacter, etc.) bacteria suggested niche specific guilds with aerobic, facultative anaerobic and strict anaerobic populations. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) predicted putative genetic repertoire of sludge microbiomes and their potential for hydrocarbon degradation; lipid-, nitrogen-, sulfur- and methane- metabolism. Methyl coenzyme M reductase A (mcrA) and dissimilatory sulfite reductase beta-subunit (dsrB) genes phylogeny confirmed methanogenic and sulfate-reducing activities within sludge environment endowed by hydrogenotrophic methanogens and sulfate-reducing Deltaproteobacteria and Firmicutes members. CONCLUSION: Refinery sludge microbiomes were comprised of hydrocarbon degrading, fermentative, sulfate-reducing, syntrophic, nitrogen fixing and methanogenic microorganisms, which were in accordance with the prevailing physicochemical nature of the samples. Analysis of functional biomarker genes ascertained the activities of methanogenic and sulfate-reducing organisms within sludge environment. Overall data provided better insights on microbial diversity and activity in oil contaminated environment, which could be exploited suitably for in situ bioremediation of refinery sludge.

Genome analysis of crude oil degrading Franconibacter pulveris strain DJ34 revealed its genetic basis for hydrocarbon degradation and survival in oil contaminated environment.

Franconibacter pulveris strain DJ34, isolated from Duliajan oil fields, Assam, was characterized in terms of its taxonomic, metabolic and genomic properties. The bacterium showed utilization of diverse petroleum hydrocarbons and electron acceptors, metal resistance, and biosurfactant production. The genome (4,856,096bp) of this strain contained different genes related to the degradation of various petroleum hydrocarbons, metal transport and resistance, dissimilatory nitrate, nitrite and sulfite reduction, chemotaxy, biosurfactant synthesis, etc. Genomic comparison with other Franconibacter spp. revealed higher abundance of genes for cell motility, lipid transport and metabolism, transcription and translation in DJ34 genome. Detailed COG analysis provides deeper insights into the genomic potential of this organism for degradation and survival in oil-contaminated complex habitat. This is the first report on ecophysiology and genomic inventory of Franconibacter sp. inhabiting crude oil rich environment, which might be useful for designing the strategy for bioremediation of oil contaminated environment.

Molecular and eco-physiological characterization of arsenic (As)-transforming Achromobacter sp. KAs 3-5T from As-contaminated groundwater of West Bengal, India.

Molecular and eco-physiological characterization of arsenic (As)-transforming and hydrocarbon-utilizing Achromobacter type strain KAs 3-5T has been investigated in order to gain an insight into As-geomicrobiology in the contaminated groundwater. The bacterium is isolated from As-rich groundwater of West Bengal, India. Comparative 16S rRNA gene sequence phylogenetic analysis confirmed that the strain KAs 3-5T is closely related to Achromobacter mucicolens LMG 26685T (99.17%) and Achromobacter animicus LMG 26690T (99.17%), thus affiliated to the genus Achromobacter. Strain KAs 3-5T is nonflagellated, mesophilic, facultative anaerobe, having a broad metabolic repertoire of using various sugars, sugar-/fatty acids, hydrocarbons as principal carbon substrates, and O2, NO3-, NO2-, and Fe3+ as terminal electron acceptors. Growth with hydrocarbons led to cellular aggregation and adherence of the cells to the hydrocarbon particles confirmed through electron microscopic observations. The strain KAs 3-5T showed high As resistance (MIC of 5 mM for As3+, 25 mM for As5+) and reductive transformation of As5+ under aerobic conditions while utilizing both sugars and hydrocarbons. Molecular taxonomy specified a high genomic GC content (65.5 mol %), ubiquinone 8 (UQ-8) as respiratory quinone, spermidine as predominant polyamine in the bacterium. The differential presence of C12:0, C14:0 2-OH, C18:1 ω7c, and C 14:0 iso 3-OH/ C16:1 iso fatty acids, phosphatidylglycerol (PG), phosphatidylcholine (PC), two unknown phospholipid (PL1, PL2) as polar lipids, low DNA-DNA relatedness (33.0-41.0%) with the Achromobacter members, and unique metabolic capacities clearly indicated the distinct genomic and physiological properties of strain KAs 3-5T among known species of the genus Achromobacter. These findings lead to improve our understanding on metabolic flexibility of bacteria residing in As-contaminated groundwater and As-bacteria interactions within oligotrophic aquifer system.

An arsenate-reducing and alkane-metabolizing novel bacterium, Rhizobium arsenicireducens sp. nov., isolated from arsenic-rich groundwater.

A novel arsenic (As)-resistant, arsenate-respiring, alkane-metabolizing bacterium KAs 5-22T, isolated from As-rich groundwater of West Bengal was characterized by physiological and genomic properties. Cells of strain KAs 5-22T were Gram-stain-negative, rod-shaped, motile, and facultative anaerobic. Growth occurred at optimum of pH 6.0-7.0, temperature 30 °C. 16S rRNA gene affiliated the strain KAs 5-22T to the genus Rhizobium showing maximum similarity (98.4 %) with the type strain of Rhizobium naphthalenivorans TSY03bT followed by (98.0 % similarity) Rhizobium selenitireducens B1T. The genomic G + C content was 59.4 mol%, and DNA-DNA relatedness with its closest phylogenetic neighbors was 50.2 %. Chemotaxonomy indicated UQ-10 as the major quinone; phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol as major polar lipids; C16:0, C17:0, 2-OH C10:0, 3-OH C16:0, and unresolved C18:1 É·7C/É·9C as predominant fatty acids. The cells were found to reduce O2, As5+, NO3-, SO42- and Fe3+ as alternate electron acceptors. The strain's ability to metabolize dodecane or other alkanes as sole carbon source using As5+ as terminal electron acceptor was supported by the presence of genes encoding benzyl succinate synthase (bssA like) and molybdopterin-binding site (mopB) of As5+ respiratory reductase (arrA). Differential phenotypic, chemotaxonomic, genotypic as well as physiological properties revealed that the strain KAs 5-22T is separated from its nearest recognized Rhizobium species. On the basis of the data presented, strain KAs 5-22T is considered to represent a novel species of the genus Rhizobium, for which the name Rhizobium arsenicireducens sp. nov. is proposed as type strain (=LMG 28795T=MTCC 12115T).

Diversity, metal resistance and uranium sequestration abilities of bacteria from uranium ore deposit in deep earth stratum.

Metal resistance and uranium (U) sequestration abilities of bacteria residing in subsurface U ore was investigated using 122 pure culture strains isolated through enrichment. The cumulative frequencies of isolates resistant to each metal tested were as follows: As(V), 74%; Zn, 58%; Ni, 53%; Cd, 47%; Cr(VI), 41%; Co, 40%; Cu, 20%; and Hg, 4%. 16S rRNA gene analysis revealed that isolated bacteria belonged to 14 genera with abundance of Arthrobacter, Microbacterium, Acinetobacter and Stenotrophomonas. Cobalt did not interfere with the growth of most of the bacterial isolates belonging to different groups while U allowed growth of four different genera of which Stenotrophomonas and Microbacterium showed high U tolerance. Interestingly, tolerance to Ni, Zn, Cu, and Hg was observed only in Microbacterium, Arthrobacter, Paenibacillus¸ and Acinetobacter, respectively. However, Microbacterium was found to be dominant when isolated from other five different metal enrichments including U. Uranium removal study showed that 84% of the test bacteria could remove more than 50mgUg(-1) dry weight from 80 or 160mgL(-1) U within 48h. In general, Microbacterium, Arthrobacter and Acinetobacter could remove a higher amount of U. High resolution transmission electron microscopy (HRTEM) study of U exposed cells revealed that accumulated U sequestered mostly around the cell periphery. The study highlights that indigenous U ore deposit bacteria have the potential to interact with U, and thus could be applied for bioremediation of U contaminated sites or wastes.

Real-time PCR based analysis of metal resistance genes in metal resistant Pseudomonas aeruginosa strain J007.

A uranium (U)-resistant and -accumulating Pseudomonas aeruginosa strain was characterized to assess the response of toxic metals toward its growth and expression of metal resistance determinants. The bacterium showed MIC (minimum inhibitory concentration) values of 6, 3, and 2 mM for Zn, Cu, and Cd, respectively; with resistance phenotype conferred by periplasmic Cu sequestering copA and RND type heavy metal efflux czcA genes. Real-time PCR-based expression analysis revealed significant upregulation of both these genes upon exposure to low concentrations of metals for short duration, whereas the global stress response gene sodA encoding superoxide dismutase enzyme was upregulated only at higher metal concentrations or longer exposure time. It could also be inferred that copA and czcA are involved in providing resistance only at low metal concentrations, whereas involvement of "global stress response" phenomenon (expression of sodA) at higher metal concentration or increased exposure was evident. This study provides significant understanding of the adaptive response of bacteria surviving in metal and radionuclide contaminated environments along with the development of real-time PCR-based quantification method of using metal resistance genes as biomarker for monitoring relevant bacteria in such habitats.