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.

Exploring the diversity and hydrocarbon bioremediation potential of microbial community in the waste sludge of Duliajan oil field, Assam, India.

Microbial community analysis of crude oil containing sludge collected from Duliajan oil field, Assam, India, showed the predominance of hydrocarbon-degrading bacteria such as Pseudomonas (20.1%), Pseudoxanthomonas (15.8%), Brevundimonas (1.6%), and Bacillus (0.8%) alongwith anaerobic, fermentative, nitrogen-fixing, nitrate-, sulfate-, and metal-reducing, syntrophic bacteria, and methanogenic archaea. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis indicated gene collection for potential hydrocarbon degradation, lipid, nitrogen, sulfur, and methane metabolism. The culturable microbial community was predominated by Pseudomonas and Bacillus with the metabolic potential for utilizing diverse hydrocarbons, crude oil, and actual petroleum sludge as sole carbon source during growth and tolerating various environmental stresses prevailing in such contaminated sites. More than 90% of the isolated strains could produce biosurfactant and exhibit catechol 2,3-dioxygenase activity. Nearly 30% of the isolates showed alkane hydroxylase activity with the maximum specific activity of 0.54 µmol min-1 mg-1. The study provided better insights into the microbial diversity and functional potential within the crude oil containing sludge which could be exploited for in situ bioremediation of contaminated sites.

Geochemical, metagenomic, and physiological characterization of the multifaceted interaction between microbiome of an arsenic contaminated groundwater and aquifer sediment.

Geomicrobiological details of the interactions between groundwater microbiome (GWM) and arsenic (As)-rich aquifer sediment of Bengal basin was investigated through microcosm incubations. Role of key microorganisms and their specific interactions with As-bearing minerals was demarcated under organic carbon- amended and -unamended conditions. Acinetobacter (50.8 %), Brevundimonas (7.9 %), Sideroxydans (3.4 %), Alkanindiges (3.0 %) dominated the GWM. The microbiome catalysed considerable alterations in As-bearing mineral [Fe-(hydr)oxide and aluminosilicate] phases resulting in substantial changes in overall geochemistry and release of As (65 µg/L) and Fe (118 µg/L). Synergistic roles of autotrophic, NH4+-oxidizing Archaea (Thaumarchaeota) and chemoheterotrophic bacteria (Stenotrophomonas, Pseudomonas, Geobacter) of diverse metabolic abilities (NH4+-oxidizing, NO3-, As/Fe-reducing) were noted for observed changes. Organic carbon supported enhanced microbial growth and As mobilization (upto 403.2 µg As/L) from multiple mineral phases (hematite, magnetite, maghemite, biotite, etc.). In presence of high organic carbon, concerted actions of anaerobic, hydrocarbon-utilizing, As-, Fe-reducing Rhizobium, fermentative Escherichia, anaerobic Bacillales, metal-reducing and organic acid-utilizing Pseudomonas and Achromobacter were implicated in altering sediment mineralogy and biogeochemistry. Increase in abundance of arrA, arsC, bssA genes, and dissolution of Fe, Ca, Mg, Mn confirmed that dissimilatory-, cytosolic-As reduction, and mineral weathering fuelled by anaerobic (hydro)carbon metabolism are the predominant mechanisms of As release in aquifers of Bengal basin.

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.

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.

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.

Biostimulation and bioaugmentation of native microbial community accelerated bioremediation of oil refinery sludge.

Scope for developing an engineered bioremediation strategy for the treatment of hydrocarbon-rich petroleum refinery waste was investigated through biostimulation and bioaugmentation approaches. Enhanced (46-55%) total petroleum hydrocarbon (TPH) attenuation was achieved through phosphate, nitrate or nitrate+phosphate amendment in the sludge with increased (upto 12%) abundance of fermentative, hydrocarbon degrading, sulfate-reducing, CO2-assimilating and methanogenic microorganisms (Bacillus, Coprothermobacter, Rhodobacter, Pseudomonas, Achromobacter, Desulfitobacter, Desulfosporosinus, T78, Methanobacterium, Methanosaeta, etc). Together with nutrients, bioaugmentation with biosurfactant producing and hydrocarbon utilizing indigenous Bacillus strains resulted in 57-75% TPH reduction. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis revealed enhanced gene allocation for transporters (0.45-3.07%), ABC transporters (0.38-2.07%), methane (0.16-1.06%), fatty acid (0.018-0.15%), nitrogen (0.07-0.17%), butanoate (0.06-0.35%), propanoate (0.004-0.26%) metabolism and some xenobiotics (0.007-0.13%) degradation. This study indicated that nutrient-induced community dynamics of native microorganisms and their metabolic interplay within oil refinery sludge could be a driving force behind accelerated bioremediation.

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.

Enrichment and characterization of hydrocarbon-degrading bacteria from petroleum refinery waste as potent bioaugmentation agent for in situ bioremediation.

Intrinsic biodegradation potential of bacteria from petroleum refinery waste was investigated through isolation of cultivable strains and their characterization. Pseudomonas and Bacillus spp. populated the normal cultivable taxa while prolonged enrichment with hydrocarbons and crude oil yielded hydrocarbonoclastic bacteria of genera Burkholderia, Enterobacter, Kocuria, Pandoraea, etc. Strains isolated through enrichment showed assemblages of superior metabolic properties: utilization of aliphatic (C6-C22) and polyaromatic compounds, anaerobic growth with multiple terminal electron acceptors and higher biosurfactant production. Biodegradation of dodecane was studied thoroughly by GC-MS along with detection of gene encoding alkane hydroxylase (alkB). Microcosms bioaugmented with Enterobacter, Pandoraea and Burkholderia strains showed efficient biodegradation (98% TPH removal) well fitted in first order kinetic model with low rate constants and decreased half-life. This study proves that catabolically efficient bacteria resides naturally in complex petroleum refinery wastes and those can be useful for bioaugmentation based bioremediation.

Biostimulation of Indigenous Microbial Community for Bioremediation of Petroleum Refinery Sludge.

Nutrient deficiency severely impairs the catabolic activity of indigenous microorganisms in hydrocarbon rich environments (HREs) and limits the rate of intrinsic bioremediation. The present study aimed to characterize the microbial community in refinery waste and evaluate the scope for biostimulation based in situ bioremediation. Samples recovered from the wastewater lagoon of Guwahati refinery revealed a hydrocarbon enriched [high total petroleum hydrocarbon (TPH)], oxygen-, moisture-limited, reducing environment. Intrinsic biodegradation ability of the indigenous microorganisms was enhanced significantly (>80% reduction in TPH by 90 days) with nitrate amendment. Preferred utilization of both higher- (>C30) and middle- chain (C20-30) length hydrocarbons were evident from GC-MS analysis. Denaturing gradient gel electrophoresis and community level physiological profiling analyses indicated distinct shift in community's composition and metabolic abilities following nitrogen (N) amendment. High throughput deep sequencing of 16S rRNA gene showed that the native community was mainly composed of hydrocarbon degrading, syntrophic, methanogenic, nitrate/iron/sulfur reducing facultative anaerobic bacteria and archaebacteria, affiliated to γ- and δ-Proteobacteria and Euryarchaeota respectively. Genes for aerobic and anaerobic alkane metabolism (alkB and bssA), methanogenesis (mcrA), denitrification (nirS and narG) and N2 fixation (nifH) were detected. Concomitant to hydrocarbon degradation, lowering of dissolve O2 and increase in oxidation-reduction potential (ORP) marked with an enrichment of N2 fixing, nitrate reducing aerobic/facultative anaerobic members [e.g., Azovibrio, Pseudoxanthomonas and Comamonadaceae members] was evident in N amended microcosm. This study highlighted that indigenous community of refinery sludge was intrinsically diverse, yet appreciable rate of in situ bioremediation could be achieved by supplying adequate N sources.

Molecular analysis of microbial community in arsenic-rich groundwater of Kolsor, West Bengal.

Bacterial community composition within the highly arsenic (As) contaminated groundwater from Kolsur, West Bengal was analyzed over a period of 3 years using 16S rRNA gene clone library and Denaturing Gradient Gel Electrophoresis (DGGE). Molecular phylogenetic study revealed abundance of α-Proteobacteria (56%) and Firmicutes (29%) along with members of ß-Proteobacteria, Verrucomicrobia and Sphingobacteria as relatively minor groups. Along with consistent physicochemical environment, a stable microbial community structure comprising of bacterial genera Agrobacterium-Rhizobium, Ochrobactrum, Pseudomonas, Anoxybacillus and Penibacillus was recorded over the three years study period. Presence of cytosolic arsenate reductase (arsC) gene was observed within the microbial community. Phylogenetic analyses revealed that all the arsC sequences were closely related to the same gene from γ-proteobacterial members while the community was consisted of mainly α-proteobacterial groups. Such phylogenetic incongruence between 16S rRNA and arsC genes possibly indicated horizontal gene transfer (HGT) of the ars genes within the groundwater community. Overall, the study reported a nearly stable geomicrobial environment and genetic determinant related to As homeostasis gene, and provided a better insight on biogeochemistry of As contaminated aquifer of West Bengal.

Genome Sequence of Hydrocarbon-Degrading Cronobacter sp. Strain DJ34 Isolated from Crude Oil-Containing Sludge from the Duliajan Oil Fields, Assam, India.

We report here the 4,856,096-bp draft genome sequence of hydrocarbon-degrading Cronobacter sp. strain DJ34 isolated from crude oil-containing sludge from the Duliajan oil fields, India. DJ34 contains genes that mediate hydrocarbon degradation, metal resistance, and biosurfactant production. This is the first report of the genome sequence of Cronobacter sp. inhabiting an oil-contaminated environment.

Diversity, metabolic properties and arsenic mobilization potential of indigenous bacteria in arsenic contaminated groundwater of West Bengal, India.

Arsenic (As) mobilization in alluvial aquifers is caused by a complex interplay of hydro-geo-microbiological activities. Nevertheless, diversity and biogeochemical significance of indigenous bacteria in Bengal Delta Plain are not well documented. We have deciphered bacterial community compositions and metabolic properties in As contaminated groundwater of West Bengal to define their role in As mobilization. Groundwater samples showed characteristic high As, low organic carbon and reducing property. Culture-independent and -dependent analyses revealed presence of diverse, yet near consistent community composition mostly represented by genera Pseudomonas, Flavobacterium, Brevundimonas, Polaromonas, Rhodococcus, Methyloversatilis and Methylotenera. Along with As-resistance and -reductase activities, abilities to metabolize a wide range carbon substrates including long chain and polyaromatic hydrocarbons and HCO3, As3+ as electron donor and As5+/Fe3+ as terminal electron acceptor during anaerobic growth were frequently observed within the cultivable bacteria. Genes encoding cytosolic As5+ reductase (arsC) and As3+ efflux/transporter [arsB and acr3(2)] were found to be more abundant than the dissimilatory As5+ reductase gene arrA. The observed metabolic characteristics showed a good agreement with the same derived from phylogenetic lineages of constituent populations. Selected bacterial strains incubated anaerobically over 300 days using natural orange sand of Pleistocene aquifer showed release of soluble As mostly as As3+ along with several other elements (Al, Fe, Mn, K, etc.). Together with the production of oxalic acid within the biotic microcosms, change in sediment composition and mineralogy indicated dissolution of orange sand coupled with As/Fe reduction. Presence of arsC gene, As5+ reductase activity and oxalic acid production by the bacteria were found to be closely related to their ability to mobilize sediment bound As. Overall observations suggest that indigenous bacteria in oligotrophic groundwater possess adequate catabolic ability to mobilize As by a cascade of reactions, mostly linked to bacterial necessity for essential nutrients and detoxification.

Arsenic biotransformation and release by bacteria indigenous to arsenic contaminated groundwater.

Arsenic (As) biotransformation and release by indigenous bacteria from As rich groundwater was investigated. Metabolic landscape of 173 bacterial isolates indicated broad catabolic repertoire including abundance of As(5+) reductase activity and abilities in utilizing wide ranges of organic and inorganic respiratory substrates. Abundance of As homeostasis genes and utilization of hydrocarbon as carbon/electron donor and As(5+) as electron acceptor were noted within the isolates. Sediment microcosm study (for 300 days) showed a pivotal role of metal reducing facultative anaerobic bacteria in toxic As(3+) release in aqueous phase. Inhabitant bacteria catalyze As transformation and facilitate its release through a cascade of reactions including mineral bioweathering and As(5+) and/or Fe(3+) reduction activities. Compared to anaerobic incubation with As(5+) reducing strains, oxic state and/or incubation with As(3+) oxidizing bacteria resulted in reduced As release, thus indicating a strong role of such condition or biocatalytic mechanism in controlling in situ As contamination.

Microbial diversity, community composition and metabolic potential in hydrocarbon contaminated oily sludge: prospects for in situ bioremediation.

Microbial community composition and metabolic potential have been explored in petroleum-hydrocarbon-contaminated sludge of an oil storage facility. Culture-independent clone library-based 16S rRNA gene analyses revealed that the bacterial community within the sludge was dominated by the members of ß-Proteobacteria (35%), followed by Firmicutes (13%), δ-Proteobacteria (11%), Bacteroidetes (10%), Acidobacteria (6%), α-Proteobacteria (3%), Lentisphaerae (2%), Spirochaetes (2%), and unclassified bacteria (5%), whereas the archaeal community was composed of Thermoprotei (54%), Methanocellales (33%), Methanosarcinales/Methanosaeta (8%) and Methanoculleus (1%) members. Methyl coenzyme M reductase A (mcrA) gene (a functional biomarker) analyses also revealed predominance of hydrogenotrophic, methanogenic Archaea (Methanocellales, Methanobacteriales and Methanoculleus members) over acetoclastic methanogens (Methanosarcinales members). In order to explore the cultivable bacterial population, a total of 28 resident strains were identified and characterized in terms of their physiological and metabolic capabilities. Most of these could be taxonomically affiliated to the members of the genera Bacillus, Paenibacillus, Micrococcus, Brachybacterium, Aerococcus, and Zimmermannella, while two strains were identified as Pseudomonas and Pseudoxanthomonas. Metabolic profiling exhibited that majority of these isolates were capable of growing in presence of a variety of petroleum hydrocarbons as sole source of carbon, tolerating different heavy metals at higher concentrations (≥1 mM) and producing biosurfactant during growth. Many strains could grow under a wide range of pH, temperature, or salinity as well as under anaerobic conditions in the presence of different electron acceptors and donors in the growth medium. Correlation between the isolates and their metabolic properties was estimated by the unweighted pair group method with arithmetic mean (UPGMA) analysis. Overall observation indicated the presence of diverse groups of microorganisms including hydrocarbonoclastic, nitrate reducing, sulphate reducing, fermentative, syntrophic, methanogenic and methane-oxidizing bacteria and Archaea within the sludge community, which can be exploited for in situ bioremediation of the oily sludge.

Studies on arsenic transforming groundwater bacteria and their role in arsenic release from subsurface sediment.

Ten different Gram-negative arsenic (As)-resistant and As-transforming bacteria isolated from As-rich groundwater of West Bengal were characterized to assess their role in As mobilization. 16S rRNA gene analysis confirmed the affiliation of these bacteria to genera Achromobacter, Brevundimonas, Rhizobium, Ochrobactrum, and Pseudoxanthomonas. Along with superior As-resistance and As-transformation abilities, the isolates showed broad metabolic capacity in terms of utilizing a variety of electron donors and acceptors (including As) under aerobic and anaerobic conditions, respectively. Arsenic transformation studies performed under various conditions indicated highly efficient As(3+) oxidation or As(5+) reduction kinetics. Genes encoding As(3+) oxidase (aioA), cytosolic As(5+) reductase (arsC), and As(3+) efflux pump (arsB and acr3) were detected within the test isolates. Sequence analyses suggested that As homeostasis genes (particularly arsC, arsB, and acr3) were acquired by most of the bacteria through horizontal gene transfer. A strong correlation between As resistance phenotype and the presence of As(3+) transporter genes was observed. Microcosm study showed that bacterial strain having cytosolic As(5+) reductase property could play important role in mobilizing As (as As(3+)) from subsurface sediment.

Characterization of arsenic resistant bacteria from arsenic rich groundwater of West Bengal, India.

Sixty-four arsenic (As) resistant bacteria isolated from an arsenic rich groundwater sample of West Bengal were characterized to investigate their potential role in subsurface arsenic mobilization. Among the isolated strains predominance of genera Agrobacterium/Rhizobium, Ochrobactrum and Achromobacter which could grow chemolitrophically and utilize arsenic as electron donor were detected. Higher tolerance to As(3+) [maximum tolerable concentration (MTC): ≥10 mM], As(5+) (MTC: ≥100 mM) and other heavy metals like Cu(2+), Cr(2+), Ni(2+) etc. (MTC: ≥10 mM), presence of arsenate reductase and siderophore was frequently observed among the isolates. Ability to produce arsenite oxidase and phosphatase enzyme was detected in 50 and 34 % of the isolates, respectively. Although no direct correlation among taxonomic identity of bacterial strains and their metabolic abilities as mentioned above was apparent, several isolates affiliated to genera Ochrobactrum, Achromobacter and unclassified Rhizobiaceae members were found to be highly resistant to As(3+) and As(5+) and positive for all the test properties. Arsenate reductase activity was found to be conferred by arsC gene, which in many strains was coupled with arsenite efflux gene arsB as well. Phylogenetic incongruence between the 16S rRNA and ars genes lineages indicated possible incidence of horizontal gene transfer for ars genes. Based on the results we propose that under the prevailing low nutrient condition inhabitant bacteria capable of using inorganic electron donors play a synergistic role wherein siderophores and phosphatase activities facilitate the release of sediment bound As(5+), which is subsequently reduced by arsenate reductase resulting into the mobilization of As(3+) in groundwater.

Uranium and other heavy metal resistance and accumulation in bacteria isolated from uranium mine wastes.

Ten bacterial strains isolated from uranium mine wastes were characterized in terms of their uranium and other metal resistance and accumulation. 16S rRNA gene sequence analysis identified the strains as members of genera Bacillus, Serratia, and Arthrobacter. Strains were able to utilize various carbon sources, particularly aromatic hydrocarbons, grow at broad pH and temperature ranges and produce non specific acid phosphatase relevant for metal phosphate precipitation in contaminated environment. The isolates exhibited high uranium and other heavy metals (Ni, Co, Cu and Cd) resistance and accumulation capacities. Particularly, Arthrobacter sp. J001 and Bacillus sp. J003 were superior in terms of U resistance at low pH (pH 4.0) along with metals and actinides (U and Th) removal with maximum cell loading of 1088 µmol U, 1293 µmol Th, 425 µmol Cu, 305 µmol Cd, 377 µmol Zn, 250 µmol Ni g(-1) cell dry wt. Genes encoding P(1B)-type ATPases (Cu-CPx and Zn-CPx) and ABC transporters (nik) as catalytic tools for maintaining cellular metal homeostasis were detected within several Bacillus spp., with possible incidence of horizontal gene transfer for the later gene showing phylogenetic lineage to α Proteobacteria members. The study provides evidence on intrinsic abilities of indigenous bacteria from U-mine suitable for survival and cleaning up of contaminated mine sites.

Molecular analysis of bacterial communities in uranium ores and surrounding soils from Banduhurang open cast uranium mine, India: A comparative study.

Bacterial community structure of heavy metal rich- uranium ores and surrounding soils was explored using 16S rRNA gene based clone library analysis and denaturing gradient gel electrophoresis (DGGE) to provide baseline microbial diversity data on autochthonous communities. Sequence analysis of major ribotypes and/or DGGE bands revealed Proteobacteria and Acidobacteria as the two most frequently present bacterial phyla across the samples, although relative abundance of each phyla and identity of their members at lower taxonomic level showed marked difference. Gammaproteobacteria (Pseudomonas and Escherichia) was most abundant in U-ore samples along with the lineages of ß-Proteobacteria (Burkholderia and Janthinobacterium), α-Proteobacteria (Brevundimonas), Bacteroidetes (Spingobacterium), Firmicutes (Peptoniphilus), Actinobacteria (Corynebacterium), uncultured -Acidobacteria, -Chloroflexi and -Cyanobacterium. In contrast to this soil communities were represented by mixed populations predominated by uncultured Acidobacteria along with Gammaproteobacteria (Succinivibrio, Cellovibrio and Legionella), ß-Proteobacteria (Rhodocyclus), α-Proteobacteria (Methylocystis and Phenylobacterium), δ-Proteobacteria, unclassified bacteria, uncultured Bacteroidetes, Firmicutes (Bacillus), Cyanobacteria (Scytonema), Actinobacteria (Actinomadura) and candidate division TM7. Principle Component Analyis (PCA) of geochemical data and UPGMA cluster analysis of DGGE profiles were in close agreement showing characteristic relatedness of samples obtained from either ores or soils. Our analysis indicated that soils surrounding the ore deposit bear specific geochemical as well as microbiologial characteristics distinct from the ore deposit and therefore these data obtained at the onset of mining could serve as a baseline of information to gauge the subsequent environmnetal impact of U-mining.

Culture-independent molecular analysis of bacterial diversity in uranium-ore/-mine waste-contaminated and non-contaminated sites from uranium mines.

Soil, water and sediment samples collected from in and around Jaduguda, Bagjata and Turamdih mines were analyzed for physicochemical parameters and cultured, and yet to be cultured microbial diversity. Culturable fraction of microbial community measured as Colony Forming Unit (CFU) on R2A medium revealed microbes between 10(4) and 10(9) CFU/g sample. Community DNA was extracted from all the samples; 16S rRNA gene amplified, cloned and subject to Amplified Ribosomal DNA Restriction Analysis. Clones representing each OTU were selected and sequenced. Sequence analyses revealed that non-contaminated samples were mostly represented by Acidobacteria, Bacteroidetes, Firmicutes and Proteobacteria (ß-, γ-, and/or δ-subdivisions) along with less frequent phyla Nitrospira, Deferribacteres, Chloroflexi. In contrast, samples obtained from highly contaminated samples showed distinct abundance of ß-,γ- and α-Proteobacteria along with Acidobacteria,Bacteroidetes and members of Firmicutes, Chloroflexi, Candidate division, Planctomycete, Cyanobacteria and Actinobacteria as minor groups. Our data represented the baseline information on bacterial community composition within non-contaminated samples which could potentially be useful for assessing the impact of metal and radionuclides contamination due to uranium mine activities.