Depth wide distribution and metabolic potential of chemolithoautotrophic microorganisms reactivated from deep continental granitic crust underneath the Deccan Traps at Koyna, India.

Characterization of inorganic carbon (C) utilizing microorganisms from deep crystalline rocks is of major scientific interest owing to their crucial role in global carbon and other elemental cycles. In this study we investigate the microbial populations from the deep [up to 2,908 meters below surface (mbs)] granitic rocks within the Koyna seismogenic zone, reactivated (enriched) under anaerobic, high temperature (50°C), chemolithoautotrophic conditions. Subsurface rock samples from six different depths (1,679-2,908 mbs) are incubated (180 days) with CO2 (+H2) or HCO3 - as the sole C source. Estimation of total protein, ATP, utilization of NO3 - and SO4 2- and 16S rRNA gene qPCR suggests considerable microbial growth within the chemolithotrophic conditions. We note a better response of rock hosted community towards CO2 (+H2) over HCO3 -. 16S rRNA gene amplicon sequencing shows a depth-wide distribution of diverse chemolithotrophic (and a few fermentative) Bacteria and Archaea. Comamonas, Burkholderia-Caballeronia-Paraburkholderia, Ralstonia, Klebsiella, unclassified Burkholderiaceae and Enterobacteriaceae are reactivated as dominant organisms from the enrichments of the deeper rocks (2335-2,908 mbs) with both CO2 and HCO3 -. For the rock samples from shallower depths, organisms of varied taxa are enriched under CO2 (+H2) and HCO3 -. Pseudomonas, Rhodanobacter, Methyloversatilis, and Thaumarchaeota are major CO2 (+H2) utilizers, while Nocardioides, Sphingomonas, Aeromonas, respond towards HCO3 -. H2 oxidizing Cupriavidus, Hydrogenophilus, Hydrogenophaga, CO2 fixing Cyanobacteria Rhodobacter, Clostridium, Desulfovibrio and methanogenic archaea are also enriched. Enriched chemolithoautotrophic members show good correlation with CO2, CH4 and H2 concentrations of the native rock environments, while the organisms from upper horizons correlate more to NO3 -, SO4 2- , Fe and TIC levels of the rocks. Co-occurrence networks suggest close interaction between chemolithoautotrophic and chemoorganotrophic/fermentative organisms. Carbon fixing 3-HP and DC/HB cycles, hydrogen, sulfur oxidation, CH4 and acetate metabolisms are predicted in the enriched communities. Our study elucidates the presence of live, C and H2 utilizing Bacteria and Archaea in deep subsurface granitic rocks, which are enriched successfully. Significant impact of depth and geochemical controls on relative distribution of various chemolithotrophic species enriched and their C and H2 metabolism are highlighted. These endolithic microorganisms show great potential for answering the fundamental questions of deep life and their exploitation in CO2 capture and conversion to useful products.

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.

Impact of arsenic on microbial community structure and their metabolic potential from rice soils of West Bengal, India.

Paddy soil is a heterogenous ecosystem that harbours diverse microbial communities critical for maintaining ecosystem sustainability and crop yield. Considering the importance of soil in crop production and recent reports on its contamination with arsenic (As) across the South East Asia, its microbial community composition and biogeochemical functions remained inadequately studied. We have characterized the microbial communities of rice soil from eleven paddy fields of As-contaminated sites from West Bengal (India), through metagenomics and amplicon sequencing. 16S rRNA gene sequencing showed considerable bacterial diversity [over 0.2 million Operational Taxonomic Units (OTUs)] and abundance (upto 1.6 × 107 gene copies/g soil). Existence of a core-microbiome (261 OTUs conserved out of a total 141,172 OTUs) across the samples was noted. Most of the core-microbiome members were also found to represent the abundant taxa of the soil. Statistical analyses suggested that the microbial communities were highly constrained by As, Fe K, N, PO43-, SO42- and organic carbon (OC). Members of Proteobacteria, Actinobacteria, Acidobacteria, Chloroflexi, Planctomycetes and Thaumarchaeota constituted the core-microbiome. Co-occurrence network analysis displayed significant interaction among diverse anaerobic, SO42- and NO3- reducing, cellulose and other organic matter or C1 compound utilizing, fermentative and aerobic/facultative anaerobic bacteria and archaea. Correlation analysis suggested that taxa which were positively linked with soil parameters that maintain soil health and productivity (e.g., N, K, PO43- and Fe) were adversely impacted by increasing As concentration. Shotgun metagenomics highlighted major metabolic pathways controlling the C (3-hydroxypropionate bicycle), N (Denitrification, dissimilatory NO3- reduction to ammonium), and S (assimilatory SO42- reduction and sulfide oxidation) cycling, As homeostasis (methylation and reduction) and plant growth promotion (polyphosphate hydrolysis and auxin biosynthesis). All these major biogeochemical processes were found to be catalyzed by the members of most abundant/core-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.

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.

Biodegradation of Unpretreated Low-Density Polyethylene (LDPE) by Stenotrophomonas sp. and Achromobacter sp., Isolated From Waste Dumpsite and Drilling Fluid.

Exploring the catabolic repertoire of natural bacteria for biodegradation of plastics is one of the priority areas of biotechnology research. Low Density Polyethylene (LDPE) is recalcitrant and poses serious threats to our environment. The present study explored the LDPE biodegradation potential of aerobic bacteria enriched from municipal waste dumpsite and bentonite based drilling fluids from a deep subsurface drilling operation. Considerable bacterial growth coupled with significant weight loss of the LDPE beads (∼8%), change in pH to acidic condition and biofilm cell growth around the beads (CFU count 105-106/cm2) were noted for two samples (P and DF2). The enriched microbial consortia thus obtained displayed high (65-90%) cell surface hydrophobicity, confirming their potential toward LDPE adhesion as well as biofilm formation. Two LDPE degrading bacterial strains affiliated to Stenotrophomonas sp. and Achromobacter sp. were isolated as pure culture from P and DF2 enrichments. 16S rRNA gene sequences of these isolates indicated their taxonomic novelty. Further biodegradation studies provided strong evidence toward the LDPE metabolizing ability of these two organisms. Atomic Fore Microscopy (AFM) and Scanning Electron Microscopy (SEM) revealed considerable damage (in terms of formation of cracks, grooves, etc.) on the micrometric surface of the LDPE film. Analysis of the average roughness (Ra), root mean square roughness (Rq), average height (Rz), maximum peak height (Rp), and maximum valley depth (Rv) (nano-roughness parameters) through AFM indicated 2-3 fold increase in nano-roughness of the LDPE film. FTIR analysis suggested incorporation of alkoxy (1000-1090 cm-1), acyl (1220 cm-1), nitro (1500-1600 cm-1), carbonyl (1720 cm-1) groups into the carbon backbone, formation of N-O stretching (1360 cm-1) and chain scission (905 cm-1) in the microbially treated LDPEs. Increase in carbonyl index (15-20 fold), double bond index (1.5-2 fold) and terminal double bond index (30-40 fold) confirmed that biodegraded LDPEs had undergone oxidation, vinylene formation and chain scission. The data suggested that oxidation and dehydrogenation could be the key steps allowing formation of low molecular weight products suitable for their further mineralization by the test bacteria. The study highlighted LDPE degrading ability of natural bacteria and provided the opportunity for their development in plastic remediation process.

Archaeal Communities in Deep Terrestrial Subsurface Underneath the Deccan Traps, India.

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 103-104 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 Fe2O3-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, Fe2O3 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.

Microbial Carbonic Anhydrases in Biomimetic Carbon Sequestration for Mitigating Global Warming: Prospects and Perspectives.

All the leading cities in the world are slowly becoming inhospitable for human life with global warming playing havoc with the living conditions. Biomineralization of carbon dioxide using carbonic anhydrase (CA) is one of the most economical methods for mitigating global warming. The burning of fossil fuels results in the emission of large quantities of flue gas. The temperature of flue gas is quite high. Alkaline conditions are necessary for CaCO3 precipitation in the mineralization process. In order to use CAs for biomimetic carbon sequestration, thermo-alkali-stable CAs are, therefore, essential. CAs must be stable in the presence of various flue gas contaminants too. The extreme environments on earth harbor a variety of polyextremophilic microbes that are rich sources of thermo-alkali-stable CAs. CAs are the fastest among the known enzymes, which are of six basic types with no apparent sequence homology, thus represent an elegant example of convergent evolution. The current review focuses on the utility of thermo-alkali-stable CAs in biomineralization based strategies. A variety of roles that CAs play in various living organisms, the use of CA inhibitors as drug targets and strategies for overproduction of CAs to meet the demand are also briefly discussed.

Utility of thermo-alkali-stable γ-CA from polyextremophilic bacterium Aeribacillus pallidus TSHB1 in biomimetic sequestration of CO2 and as a virtual peroxidase.

Aeribacillus pallidus TSHB1 polyextremophilic bacterium produces a γ-carbonic anhydrase (ApCA), which is a homotrimeric biocatalyst with a subunit molecular mass of 32 ± 2 kDa. The enzyme is stable in the pH range between 8.0 and 11.0 and thus alkali-stable and moderately thermostable with T1/2 values of 40 ± 1, 15 ± 1, and 8 ± 0.5 min at 60, 70, and 80 °C, respectively. Activation energy for irreversible inactivation "E d " of carbonic anhydrase is 67.119 kJ mol-1. The enzyme is stable in the presence of various flue gas contaminants such as SO32-,SO42-, and NO3- and cations Mg2+, Mn2+, Ca2+, and Ba2+. Fluorescence studies in the presence of N-bromosuccinimide and fluorescence quenching using KI and acrylamide revealed the importance of tryptophan residues in maintaining the structural integrity of the enzyme. ApCA is more efficient than the commercially available bovine carbonic anhydrase (BCA) in CO2 sequestration. The enzyme was successfully used in biomineralization of CO2 from flue gas. Replacement of active site Zn2+ with Mn2+ enabled ApCA to function as a peroxidase which exhibited alkali-stability and moderate thermostability like ApCA.

Suitability of the alkalistable carbonic anhydrase from a polyextremophilic bacterium Aeribacillus pallidus TSHB1 in biomimetic carbon sequestration.

Carbonic anhydrase (CA) was produced from the polyextremophilic (halotolerant, moderately thermophilic and alkaliphilic) bacterium Aeribacillus pallidus TSHB1 isolated from water and sediment samples of Choti Anhoni hot spring of Pipariya, Madhya Pradesh (India), is being reported to be suitable for carbon sequestration. Growth and CA production were inhibited at higher CO2 concentration (5-10 %). Under optimized culture variables (tryptone 0.8 %, yeast extract 0.08 %, glucose 1 %, micronutrient solution 1 %, inoculums size 1.10 %, agitation 200 at pH 8, and temperature 55 °C), 3.7-fold higher CA production was attained than that under unoptimized conditions. The zymogram analysis of the partially purified CA revealed an activity band corresponding to 32 kDa. The enzyme is stable in the pH range between 8.0 and 11.0 with T 1/2 of 40, 15, and 8 min at 60, 70, and 80 °C, respectively. The CA of A. pallidus displayed a marked enhancement in the rate of CaCO3 precipitation from aqueous CO2. The CA-aided formation of CaCO3 was 42.5 mg mg(-1) protein. Scanning electron microscopy revealed the formation of rhomboid calcite crystals. This is the first report on the production and applicability of CA from the polyextremophilic A. pallidus in carbon sequestration.

Statistical media optimization and cellulase production from marine Bacillus VITRKHB.

Marine Bacillus species are potent producers of novel enzymes. Marine Bacillus VITRKHB was observed to be efficient for cellulolytic activity. It was employed for the production of extracellular cellulase. Cellulase was partially purified to 1.6-fold in a stepwise manner by ammonium sulfate precipitation, dialysis, and DEAE ion exchange chromatography. The molecular weight of purified protein was found to be about 33 kDa by SDS-PAGE. Its specific activity was recorded as 1.92 IU/mg. The effect of various carbon and nitrogen sources on cellulase production was investigated. The maximum enzyme activity was recorded in the fermentation media containing xylose as carbon source and beef extract as nitrogen source. The combined interactive effect of different variables on cellulase production was studied by response surface methodology. The optimized combination of variables for maximum enzyme activity was determined as; xylose 5.0 %, beef extract 6.9 %, pH 7.83, NaCl 1.17, and temperature 25.84 °C, after 24 h of incubation.