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.     

Assessing the correlation between anaerobic toluene degradation activity and <Emphasis Type="Italic">bssA</Emphasis> concentrations in hydrocarbon-contaminated aquifer material

The assessment of biodegradation activity in contaminated aquifers is critical to demonstrate the performance of bioremediation and natural attenuation and to parameterize models of contaminant plume dynamics. Real time quantitative PCR (qPCR) was used to target the catabolic bssA gene (coding for benzylsuccinate synthase) and a 16S rDNA phylogenetic gene (for total Bacteria) as potential biomarkers to infer on anaerobic toluene degradation rates. A significant correlation (P = 0.0003) was found over a wide range of initial toluene concentrations (1–100 mg/l) between toluene degradation rates and bssA concentrations in anaerobic microcosms prepared with aquifer material from a hydrocarbon contaminated site. In contrast, the correlation between toluene degradation activity and total Bacteria concentrations was not significant (P = 0.1125). This suggests that qPCR targeting of functional genes might offer a simple approach to estimate in situ biodegradation activity, which would enhance site investigation and modeling of natural attenuation at hydrocarbon-contaminated sites.     

Intracellular nickel accumulation by Pseudomonas aeruginosa and its chemical nature

AIMS: To investigate intracellular localization of nickel and its chemical nature in Pseudomonas aeruginosa. METHODS AND RESULTS: Transmission electron micrographs of Ni-loaded bacteria exhibited a darkened electron opaque zone throughout the cell periphery. Energy dispersive X-ray analysis confirmed the deposition of metallic nickel. Cell fractionation revealed that 88% of the accumulated nickel was restricted to the periplasm and membrane. X-ray diffraction patterns ascertained the chemical nature of cellular Ni as phosphide (Ni5P4, NiP2 and Ni12P5) and carbide (Ni3C) crystals. CONCLUSION: Pseudomonas aeruginosa accumulated nickel as its phosphide and carbide crystal mostly in the cell envelope region, indicating the predominant role of phosphoryl and carboxyl/carbonyl groups of cell wall/membrane components in cation sequestration. SIGNIFICANCE AND IMPACT OF THE STUDY: The data contribute significantly to a better understanding of bacteria-metal interaction and will be useful in developing biotechnological strategies for toxic metal bioremediation.     

Nickel Uptake by Pseudomonas aeruginosa: Role of Modifying Factors

Pseudomonas aeruginosa cells growing in minimal medium were 40-fold more sensitive to Ni²⁺ than cells growing in enriched medium, suggesting a possible protective role of medium ingredients. Likewise, cells pre-grown in enriched medium showed a high K _m (6.15 mM) and increased Ni²⁺ uptake (950 nmol mg⁻¹ protein, 1h) over cells pre-sown in minimal medium (K _m , 0.48 mM; 146 nmol mg⁻¹ protein, 1 h). The overall pattern indicates that cells pre-grown in enriched medium were characterized by having lowered affinity towards Ni²⁺ than those with minimal medium background. The enhanced Ni²⁺ uptake by enriched medium-grown cells can be correlated with the improved metabolic state of the cells. Ni²⁺ uptake was optimum at neutrality (pH 7.0). A major Ni²⁺ transport system was competitively inhibited by Mg²⁺, Zn²⁺, Cd²⁺, or Co²⁺ (400 μM each). Noticeably, a minor Ni²⁺ transport pathway was still operative even in the higher concentration range of Mg²⁺ (4 mM and 40 mM). The stimulation of Ni²⁺ uptake monitored in the presence of different carbon sources (0.5% wt/vol, each) showed the sequence: glucose (1.6-fold) > phenol = gallic acid (1.5-fold). Succinate, in comparison, reduced Ni²⁺ uptake (0.5-fold) possibly because of its acting as a metal chelator as well. Sensitivity of Ni²⁺ transport towards methyl viologen, azide, 2-4 DNP, and DCCD suggested that transport was energy-linked. Received: 13 January 1998 / Accepted: 21 May 1998     

Extracellular polysaccharides of a copper-sensitive and a copper-resistant Pseudomonas aeruginosa strain: synthesis,chemical nature and copper binding

Extracellular polysaccharides (EPS) of a copper-sensitive (Cu^s) and a copper-resistant (Cu^r) Pseudomonas aeruginosa strain were investigated in terms of their production, chemical nature and response towards copper exposure. The extent of EPS synthesis by the resistant strain (4.78 mg mg^–1 cell dry wt.) was considerably higher over its sensitive counterpart (2.78 mg mg^–1 dry wt.). FTIR-spectroscopy and gas chromatography revealed that both the polymers were acidic in nature, containing alginate as the major component along with various neutral- and amino-sugars. Acid content in the Cu^r EPS (480.54 mg g^–1) was greater than that in the Cu^s EPS (442.0 mg g^–1). Presence of Cu²⁺ in the growth medium caused a dramatic stimulation (approximately 4-fold) in EPS synthesis by the Cu^r strain, while in a similar condition, the Cu^s failed to exhibit such response. The polymer of the resistant strain showed elevated Cu²⁺ binding (320 mg g^–1 EPS) compared to that of the sensitive type (270 mg g^–1). The overall observations show the potential of the Cu^r EPS for its deployment in metal bioremediation.     

Studies on Uranium Removal by the Extracellular Polysaccharide of a Pseudomonas aeruginosa Strain

Extracellular polysaccharide (EPS) produced by a Pseudomonas aeruginosa strain BU2 was characterized for its ability to remove uranium from aqueous solution. The EPS was acidic in nature and found as a potent biosorbent for uranium (U), showing pH dependence and fast saturating metal sorption, being maximum (985 mg U g^{? 1} EPS) at pH 5.0. The polymer showed enhanced uranium sorption capacity and affinity with increasing solution pH, suggesting a preferential sorption of monovalent uranyl hydroxide ions over the nonhydroxylated divalent species. Pseudo-first-order and pseudo-second-order kinetic models were applied to the experimental data, assuming that the external mass transfer limitations in the system can be neglected and biosorption is sorption controlled. Equilibrium metal binding showing conformity to the Freundlich model suggested a multilayer sorption involving specific binding sites with affinity distribution. The presence of two types of metal binding sites corresponding to strong and weak binding affinity was interpreted from the Scatchard model equation. Uranium sorption by EPS was unaffected or only slightly affected in the presence of several interfering cations and anions, except iron and thorium. Fourier transform infrared (FTIR) spectroscopy ascertained the strong binding of uranium with the carboxylic groups of uronic acids of bacterial EPS at pH 5.0, whereas at lower pH, amino and hydroxyl groups played a major role in metal binding.     

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.     

Copper uptake and its compartmentalization in Pseudomonas aeruginosa strains: Chemical nature of cellular metal

Copper-sensitive (Cu^s) and copper-resistant (Cu^r) strains of Pseudomonas aeruginosa were characterized in terms of Cu²⁺ sensitivity, uptake and its compartmentalization in the possible cell sectors. Minimum inhibitory concentrations (MICs) of Cu²⁺ for the Cu^r strain (3.2 mM and 0.12 mM in enriched- and in minimal-medium, respectively) were almost 5-fold higher over that of its sensitive counterpart. While Cu^s strain accumulated Cu²⁺ to a maximum of 1.8 mol mg^–1 protein, Cu^r strain increased it to 2.37 mol mg^–1 protein. Both the strains also demonstrated energy- and pH-dependent Cu²⁺ uptake through the broad-substrate range divalent cation (Zn²⁺, Mg²⁺, Co²⁺) uptake system as well as through the system specific for Cu²⁺. Cell-fractionation study revealed that in Cu^r strain, periplasm and membrane are the main Cu²⁺ binding sites, whereas, in case of Cu^s strain, it is the cytoplasm. The overall observations indicate that the Cu^r strain restricted Cu²⁺ sequestration exterior to the cytoplasm as the possible strategy for Cu-resistance. The chemical nature of Cu²⁺ deposition in the respective strains was also ascertained by X-ray powder diffraction analysis.     

Lanthanum biosorption by a Pseudomonas sp.: equilibrium studies and chemical characterization

Lanthanum biosorption by a Pseudomonas sp. was characterized in terms of equilibrium metal loading, model fitting, kinetics, effect of solution pH, lanthanum–bacteria interaction mechanism and recovery of sorbed metal. Lanthanum sorption by the bacterium was rapid and optimum at pH 5.0 with equilibrium metal loading as high as 950 mg g⁻¹ biomass dry wt. Scatchard model and potentiometric titration suggested the presence of at least two types of metal-binding sites, corresponding to a strong and a weak binding affinity. The chemical nature of metal–microbe interaction has been elucidated employing FTIR spectroscopy, energy dispersive X-ray analysis (EDX) and X-ray diffraction analysis (XRD). FTIR spectroscopy and XRD analysis revealed strong involvement of cellular carboxyl and phosphate groups in lanthanum binding by the bacterial biomass. EDX and the elemental analysis of the sorption solution ascertained the binding of lanthanum with the bacterial biomass via displacement of cellular potassium and calcium. Transmission electron microscopy exhibited La accumulation throughout the bacterial cell with some granular deposits in cell periphery and in cytoplasm. XRD confirmed the presence of LaPO₄ crystals onto the bacterial biomass after La accumulation for a long period. A combined ion-exchange–complexation–microprecipitation mechanism could be involved in lanthanum accumulation by the biomass. Almost 98% of biomass-bound La could be recovered using CaCO₃ as the desorbing agent.