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Scientific deep drilling at Koyna, western India provides a unique opportunity to explore microbial life within deep biosphere hosted by ~65 Myr old Deccan basalt and Archaean granitic basement. Characteristic low organic carbon content, mafic/felsic nature but distinct trend in sulfate and nitrate concentrations demarcates the basaltic and granitic zones as distinct ecological habitats. Quantitative PCR indicates a depth independent distribution of microorganisms predominated by bacteria. Abundance of dsrB and mcrA genes are relatively higher (at least one order of magnitude) in basalt compared to granite. Bacterial communities are dominated by Alpha-, Beta-, Gammaproteobacteria, Actinobacteria and Firmicutes, whereas Euryarchaeota is the major archaeal group. Strong correlation among the abundance of autotrophic and heterotrophic taxa is noted. Bacteria known for nitrite, sulfur and hydrogen oxidation represent the autotrophs. Fermentative, nitrate/sulfate reducing and methane metabolising microorganisms represent the heterotrophs. Lack of shared operational taxonomic units and distinct clustering of major taxa indicate possible community isolation. Shotgun metagenomics corroborate that chemolithoautotrophic assimilation of carbon coupled with fermentation and anaerobic respiration drive this deep biosphere. This first report on the geomicrobiology of the subsurface of Deccan traps provides an unprecedented opportunity to understand microbial composition and function in the terrestrial, igneous rock-hosted, deep biosphere.

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.

Sulfate- and iron-reducing heterotrophic bacteria represented minor proportion of the indigenous microbial community of highly acidic, oligotrophic acid mine drainage (AMD), but they can be successfully stimulated for in situ bioremediation of an AMD impacted soil (AIS). These anaerobic microorganisms although played central role in sulfate- and metal-removal, they remained inactive in the AIS due to the paucity of organic carbon and extreme acidity of the local environment. The present study investigated the scope for increasing the abundance and activity of inhabitant sulfate- and iron-reducing bacterial populations of an AIS from Malanjkhand Copper Project. An AIS of pH 3.5, high soluble SO42− (7838 mg/l) and Fe (179 mg/l) content was amended with nutrients (cysteine and lactate). Thorough geochemical analysis, 16S rRNA gene amplicon sequencing and qPCR highlighted the intrinsic metabolic abilities of native bacteria in AMD bioremediation. Following 180 days incubation, the nutrient amended AIS showed marked increase in pH (to 6.6) and reduction in soluble -SO42− (95%), -Fe (50%) and other heavy metals. Concomitant to physicochemical changes a vivid shift in microbial community composition was observed. Members of the Firmicutes present as a minor group (1.5% of total community) in AIS emerged as the single most abundant taxon (∼56%) following nutrient amendments. Organisms affiliated to Clostridiaceae, Peptococcaceae, Veillonellaceae, Christensenellaceae, Lachnospiraceae, Bacillaceae, etc. known for their fermentative, iron and sulfate reducing abilities were prevailed in the amended samples. qPCR data corroborated with this change and further revealed an increase in abundance of dissimilatory sulfite reductase gene (dsrB) and specific bacterial taxa. Involvement of these enhanced populations in reductive processes was validated by further enrichments and growth in sulfate- and iron-reducing media. Amplicon sequencing of these enrichments confirmed growth of Firmicutes members and proved their sulfate- and iron-reduction abilities. This study provided a better insight on ecological perspective of Firmicutes members within the AMD impacted sites, particularly their involvement in sulfate- and iron-reduction processes, in situ pH management and bioremediation.

BackgroundSustainable 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.ResultsAll 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.ConclusionRefinery 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.Electronic supplementary materialThe online version of this article (10.1186/s12866-018-1275-8) contains supplementary material, which is available to authorized users.

Archaeal community structure and potential functions within the deep, aphotic, oligotrophic, hot, igneous provinces of ∼65 Myr old basalt and its Archean granitic basement was explored through archaeal 16S rRNA gene amplicon sequencing from extracted environmental DNA of rocks. Rock core samples from three distinct horizons, basaltic (BS), transition (weathered granites) (TZ) and granitic (GR) showed limited organic carbon (4–48 mg/kg) and varied concentrations (<1.0–5000 mg/kg) of sulfate, nitrate, nitrite, iron and metal oxides. Quantitative PCR estimated the presence of nearly 10 3 –10 4 archaeal cells per gram of rock. Archaeal communities within BS and GR horizons were distinct. The absence of any common OTU across the samples indicated restricted dispersal of archaeal cells. Younger, relatively organic carbon- and Fe 2 O 3 -rich BS rocks harbor Euryarchaeota , along with varied proportions of Thaumarchaeota and Crenarchaeota . Extreme acid loving, thermotolerant sulfur respiring Thermoplasmataceae , heterotrophic, ferrous-/H-sulfide oxidizing Ferroplasmaceae and Halobacteriaceae were more abundant and closely interrelated within BS rocks. Samples from the GR horizon represent a unique composition with higher proportions of Thaumarchaeota and uneven distribution of Euryarchaeota and Bathyarchaeota affiliated to Methanomicrobia , SAGMCG-1, FHMa11 terrestrial group, AK59 and unclassified taxa. Acetoclastic methanogenic Methanomicrobia , autotrophic SAGMCG-1 and MCG of Thaumarcheaota could be identified as the signature groups within the organic carbon lean GR horizon. Sulfur-oxidizing Sulfolobaceae was relatively more abundant in sulfate-rich amygdaloidal basalt and migmatitic gneiss samples. Methane-oxidizing ANME-3 populations were found to be ubiquitous, but their abundance varied greatly between the analyzed samples. Changes in diversity pattern among the BS and GR horizons highlighted the significance of local rock geochemistry, particularly the availability of organic carbon, Fe 2 O 3 and other nutrients as well as physical constraints (temperature and pressure) in a niche-specific colonization of extremophilic archaeal communities. The study provided the first deep sequencing-based illustration of an intricate association between diverse extremophilic groups (acidophile-halophile-methanogenic), capable of sulfur/iron/methane metabolism and thus shed new light on their potential role in biogeochemical cycles and energy flow in deep biosphere hosted by hot, oligotrophic igneous crust.

Summary 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.

Reductive transformation of toxic arsenic (As) species by As reducing bacteria (AsRB) is a key process in As-biogeochemical-cycling within the subsurface aquifer environment. In this study, we have characterized a Gram-stain-negative, non-spore-forming, rod-shaped As reducing bacterium designated KAs 5-3T, isolated from highly As-contaminated groundwater of India. Strain KAs 5-3T displayed high 16S rRNA gene sequence similarity to the members of the genus Pseudoxanthomonas, with P. mexicana AMX 26BT (99.25% similarity), P. japonensis 12-3T (98.9 0%), P. putridarboris WD-12T (98.02%), and P. indica P15T (97.27%) as closest phylogenetic neighbours. DNA-DNA hybridization study unambiguously indicated that strain KAs 5-3T represented a novel species that was separate from reference strains of P. mexicana AMX 26BT (35.7%), P. japonensis 12-3T (35.5%), P. suwonensis 4M1T (35.5%), P. wuyuanensis XC21-2T (35.0%), P. indica P15T (32.5%), P. daejeonensis TR6-08T (32.0%), and P. putridarboris WD12T (22.1%). The DNA G+C content of strain KAs 5-3T was 64.9 mol %. The predominant fatty acids were C15:0 (37.4%), C16:0 iso (12.6%), C17:1 iso ω9c (10.5%), C15:0 anteiso (9.5%), C11:0 iso 3-OH (8.5%), and C16:1 ω7c/ C16:1 ω6c (7.5%). The major polar lipids were diphosphatidylglycerol, phosphatidyldimethylethanolamine, phosphatidylcholine, and two unknown phospholipids (PL1, PL2). Ubiquinone 8 (Q8) was the predominant respiratory quinone and spermidine was the major polyamine of the strain KAs 5-3T. Cells of strain KAs 5-3T showed the ability to use O2, As5+, NO3-, NO2-, and Fe3+ as terminal electron acceptors as well as to reduce As5+ through the cytosolic process under aerobic incubations. Genes encoding arsenate reductase (arsC) for As-detoxification, nitrate- and nitrite reductase (narG and nirS) for denitrification were detected in the strain KAs 5-3T. Based on taxonomic and physiological data, strain KAs 5-3T is described as a new representative member of the genus Pseudoxanthomonas, for which the name Pseudoxanthomonas arseniciresistens sp. nov. is proposed. The type strain is KAs 5-3T (= LMG 29169T = MTCC 12116T = MCC 3121T).