Retrieval of Concentrated and Undecompressed Microbial Populations from the Deep Sea

A device for sampling at depths of up to 6,000 m is described in which 3 liters of seawater is concentrated over a Nucleopore filter to about 13 ml and retrieved under in situ pressure and temperature. Subsamples can be withdrawn into transfer units that are equipped with individual gas accumulators for preventing loss of pressure during prolonged periods of storage. Transfer of samples or sample portions into sterile medium contained in pre-pressurized incubation vessels and continued subsampling therefrom permit time course experiments for the study of natural populations of deep-sea microorganisms in the absence of decompression. A test experiment with a water sample from a depth of 2,600 m supplemented with radioactively labeled Casamino Acids showed reduced rates of substrate incorporation and respiration as compared with data from a decompressed control. The barotolerance observed in this study was characterized by reduced, rather than equal, activities recorded at elevated pressures as compared with 1-atm controls.     

Methanogenic and Sulphate-Reducing Microbial Communities in Deep Groundwater of Crystalline Rock Fractures in Olkiluoto,Finland

The long-term safety of final disposal of spent nuclear fuel in the deep geosphere is dependent on stability of biogeochemical conditions at the disposal site. Microbial processes, such as sulphate reduction and methanogenesis, may have profound effects on site biogeochemistry. In this study, sulphate-reducing bacteria and methane-producing archaea were investigated at depths ranging from 68 to 545 m in crystalline rock fractures at an intended spent nuclear fuel disposal site in Olkiluoto, Finland. Denaturing gradient gel electrophoresis detected diverse sulphate-reducing bacterial communities in all samples. Although the number of dsrB gene copies was below 10³ copies ml^?1 in all analyzed samples according to real-time quantitative PCR, their abundance was highest in samples that had the highest sulphate concentrations. Several distinct mcrA gene fragments were also recovered from most of the analyzed samples by cloning, although the number of methanogens was lower than that of sulphate-reducing bacteria when measured by mcrA-targeted quantitative PCR. The detected gene fragments were most closely related to sequences obtained from aquatic and deep subsurface environments. Results imply that sulphate reduction, methanogenesis, and anaerobic methane oxidation may all take place in the Olkiluoto deep geobiosphere.     

Removing Hexazinone from Groundwater with Microbial Bioreactors

Hexazinone, a triazine herbicide that is often detected as a ground and surface water contaminant, inhibits electron transport in photosynthetic organisms and is toxic to primary producers that serve as the base of the food chain. This laboratory study evaluated the ability of two types of microbial reactors, i.e., a vegetable oil-based nitrogen-limiting biobarrier and an aerobic slow sand filter, as methods for removing hexazinone from simulated groundwater. The N-limiting biobarriers degraded hexazinone, but did so with a 52 week incubation period and a removal efficiency that varied greatly among replicates, with one biobarrier showing a removal efficiency of ~95% and the other an efficiency of ~50%. More consistent degradation was obtained with the aerobic sand biobarriers. Four aerobic biobarriers were evaluated and all behaved in a similar manner degrading hexazinone with removal efficiencies of ~97%; challenging two of the aerobic biobarriers with large amounts of influent hexazinone showed that these barriers are capable of efficiently remediating large amounts (>100 mg L⁻¹) of hexazinone at high efficiency. The remediation process was due to biological degradation rather than abiotic processes. The long lag phase observed in both types of reactors suggests that an acclimation process, where microorganisms capable of degrading hexazinone increased in numbers, was required. Also, the isolation of bacteria that show a positive growth response to the presence of hexazinone in their growth media suggests biological degradation.     

Planktonic Microbial Communities Associated with Fracture-Derived Groundwater in a Deep Gold Mine of South Africa

The vertical distribution and function of terrestrial planktonic microbial communities at depths greater than 600 m remain poorly established. Culture-independent methods using 16S rRNA genes and geochemical approaches were employed to investigate the heterogeneity and potential function of microbial communities residing within fractures at 0.7 to 1.4 kilometers below land surface of Beatrix Au Mine, South Africa. The salinity (26 to 47 mM Cl^?), temperature (33 to 40°C) and age (1 to 5 Ma) of these fracture water increased with depth. The δD and δ¹⁸O values of fracture water ranged from ?44 to ?39‰ and from ?7 to ?4‰ VSMOW, respectively, and exhibited a mixing trend with fracture water collected from the same mine in a previous study where isotopic signatures were indicative of hydrothermal origin. Fracture water from Beatrix Mine was distinct from the groundwater in the overlying Karoo sedimentary strata in terms of its Cl^?, He and CH₄ concentrations, and its δD and δ¹⁸O signatures and from Vaal River (source of service water) in terms of its δD and δ¹⁸O signatures. The differences constrain the maximum amount of mixing with service water or shallow groundwater to be less than 4%. The 16S rDNA analyses revealed diverse and numerous novel 16S rRNA genes affiliated with Proteobacteria, Firmicutes, Nitrospira, Chlorobi, Thermus, Candidate Division OP3 and Euryarchaeota. The proportion of each phylum in clone libraries varied markedly among samples and suggests km-scale, spatial heterogeneity in community structures. Potential metabolisms inferred from the presence of 16S rRNA genes are generally consistent with estimates of the available free energy.     

Successive Microbial Populations in Calimyrna Figs

Smyrna-type (Calimyrna) figs have essentially sterile internal tissue until visited by the pollinating fig wasp, Blastophaga psenes, which introduces a specific microflora consisting of Candida guilliermondii var. carpophila and Serratia plymuthica. This flora persists and develops in numbers throughout the ripening period until maturity of the fruit. These organisms do not cause spoilage. The presence of C. guilliermondii var. carpophila appears to increase the attractiveness of the fruit to drosophilae. Drosophila (mainly D. melanogaster) carry spoilage yeasts and bacteria on their exterior body parts, and introduce these organisms during ovipositing in the fruit cavity. The spoilage yeasts consist almost entirely of apiculate yeasts (Hanseniaspora valbyensis, H. uvarum, and Kloeckera apiculata) and of Torulopsis stellata, which cause active fermentative spoilage. Spoilage bacteria (primarily Acetobacter melanogenus) are also introduced with the yeasts. Organic acids are produced by these yeasts as well as by the Acetobacter. A number of minor spoilage yeasts were also identified.     

CRISPR-Induced Distributed Immunity in Microbial Populations

In bacteria and archaea, viruses are the primary infectious agents, acting as virulent, often deadly pathogens. A form of adaptive immune defense known as CRISPR-Cas enables microbial cells to acquire immunity to viral pathogens by recognizing specific sequences encoded in viral genomes. The unique biology of this system results in evolutionary dynamics of host and viral diversity that cannot be fully explained by the traditional models used to describe microbe-virus coevolutionary dynamics. Here, we show how the CRISPR-mediated adaptive immune response of hosts to invading viruses facilitates the emergence of an evolutionary mode we call distributed immunity - the coexistence of multiple, equally-fit immune alleles among individuals in a microbial population. We use an eco-evolutionary modeling framework to quantify distributed immunity and demonstrate how it emerges and fluctuates in multi-strain communities of hosts and viruses as a consequence of CRISPR-induced coevolution under conditions of low viral mutation and high relative numbers of viral protospacers. We demonstrate that distributed immunity promotes sustained diversity and stability in host communities and decreased viral population density that can lead to viral extinction. We analyze sequence diversity of experimentally coevolving populations of Streptococcus thermophilus and their viruses where CRISPR-Cas is active, and find the rapid emergence of distributed immunity in the host population, demonstrating the importance of this emergent phenomenon in evolving microbial communities.     

Novel Microbial Populations in Ambient and Mesophilic Biogas-Producing and Phenol-Degrading Consortia Unraveled by High-Throughput Sequencing

Methanogenesis from wastewater-borne organics and organic solid wastes (e.g., food residues) can be severely suppressed by the presence of toxic phenols. In this work, ambient (20 °C) and mesophilic (37 °C) methane-producing and phenol-degrading consortia were enriched and characterized using high-throughput sequencing (HTS). 454 Pyrosequencing indicated novel W22 (25.0 % of bacterial sequences) in the WWE1 and Sulfurovum-resembled species (32.0 %) in the family Campylobacterales were the most abundant in mesophilic and ambient reactors, respectively, which challenges previous knowledge that Syntrophorhabdus was the most predominant. Previous findings may underestimate bacterial diversity and low-abundance bacteria, but overestimate abundance of Syntrophorhabdus. Illumina HTS revealed that archaeal populations were doubled in ambient reactor and tripled in mesophilic reactor, respectively, compared to the ～4.9 % (of the bacteria and archaea sequences) in the seed sludge. Moreover, unlike the dominance of Methanosarcina in seed sludge, acetotrophic Methanosaeta predominated both (71.4–76.5 % of archaeal sequences) ambient and mesophilic enrichments. Noteworthy, this study, for the first time, discovered the co-occurrence of green sulfur bacteria Chlorobia, sulfur-reducing Desulfovibrio, and Sulfurovum-resembling species under ambient condition, which could presumably establish mutualistic relationships to compete with syntrophic bacteria and methanogens, leading to the deterioration of methanogenic activity. Taken together, this HTS-based study unravels the high microbial diversity and complicated bacterial interactions within the biogas-producing and phenol-degrading bioreactors, and the identification of novel bacterial species and dominant methanogens involved in the phenol degradation provides novel insights into the operation of full-scale bioreactors for maximizing biogas generation.     

Vadose Zone Microbial Biobarriers Remove Nitrate from Percolating Groundwater

Microbial biobarriers are an established technique for cleansing contaminants from aquifers. This study evaluated their use under well-drained conditions within the vadose or unsaturated zone. Three sets of sand filled columns, the positive control, field-capacity, and sub-field-capacity groups, contained biobarriers formed by mixing sand with sawdust and soybean oil. The biobarriers were positioned 1 m from the top of the 145 cm columns. A fourth set of column, the negative control, contained no biobarrier. The positive control group’s biobarriers were saturated while biobarriers in the other groups were allowed to drain. At intervals water containing 20 mg l⁻¹ NO₃⁻–N was applied to the columns, the water was allowed to percolate through the columns, and the effluents were collected and analyzed. The biobarriers were highly effective at removing NO₃⁻. NO₃⁻–N in the effluents from the field-capacity, sub-field-capacity, and positive control groups averaged 0.4 ± 0.1, 0.6 ± 0.1, and 0.8 ± 0.1 mg l⁻¹, respectively, during the final weeks of the study while effluents from the negative control group averaged 17.9 ± 0.4 mg l⁻¹. The barriers removed NO₃⁻ even when the water content was in the 20–40% pore filled space range. During the 12-week study the field-capacity barriers lost 5.6% of their organic content while those in the sub-field-capacity group lost no detectable organic matter indicating that the barriers contained sufficient substrate to last for several years. Vadose zone biobarriers could provide a useful means of protecting surface waters and aquifers from NO₃⁻.     

Biogeochemical Signals from Deep Microbial Life in Terrestrial Crust

In contrast to the deep subseafloor biosphere, a volumetrically vast and stable habitat for microbial life in the terrestrial crust remains poorly explored. For the long-term sustainability of a crustal biome, high-energy fluxes derived from hydrothermal circulation and water radiolysis in uranium-enriched rocks are seemingly essential. However, the crustal habitability depending on a low supply of energy is unknown. We present multi-isotopic evidence of microbially mediated sulfate reduction in a granitic aquifer, a representative of the terrestrial crust habitat. Deep meteoric groundwater was collected from underground boreholes drilled into Cretaceous Toki granite (central Japan). A large sulfur isotopic fractionation of 20–60‰ diagnostic to microbial sulfate reduction is associated with the investigated groundwater containing sulfate below 0.2 mM. In contrast, a small carbon isotopic fractionation (<30‰) is not indicative of methanogenesis. Except for 2011, the concentrations of H₂ ranged mostly from 1 to 5 nM, which is also consistent with an aquifer where a terminal electron accepting process is dominantly controlled by ongoing sulfate reduction. High isotopic ratios of mantle-derived ³He relative to radiogenic ⁴He in groundwater and the flux of H₂ along adjacent faults suggest that, in addition to low concentrations of organic matter (<70 µM), H₂ from deeper sources might partly fuel metabolic activities. Our results demonstrate that the deep biosphere in the terrestrial crust is metabolically active and playing a crucial role in the formation of reducing groundwater even under low-energy fluxes.     

Studies on Removing Sulfachloropyridazine from Groundwater with Microbial Bioreactors

Sulfachloropyridazine (SCP), an antibiotic used in aquaculture and in animal husbandry, is a common contaminant in surface and groundwaters. Two types of microbial reactors were evaluated as methods for removing SCP from flowing water. One type of reactor evaluated was a nitrogen-limiting biobarrier; the other a slow-sand-filter. Results showed that the soybean oil-fed, nitrogen-limiting biobarrier was not very effective at removing SCP from flowing water. When supplied with flowing water containing 2.4 mg l⁻¹ SCP the nitrogen-limiting biobarrier removed ~0.6 mg l⁻¹ SCP or about 28% of that present. SCP removal by the nitrogen-limiting biobarrier may not have been biological as abiotic removal was not ruled out. More efficient biological removal was obtained with the slow-sand-filter which reduced the SCP levels from 2.35 to 0.048 mg l⁻¹, a removal efficiency of ~98%. High levels of nitrate nitrogen, 50 mg l⁻¹ N, did not interfere with the removal processes of either reactor suggesting that SCP was not being degraded as a microbial nitrogen source.     

Midgut Microbial Community of Culex quinquefasciatus Mosquito Populations from India

The mosquito Culex quinquefasciatus is a ubiquitous species that serves as a major vector for west nile virus and lymphatic filariasis. Ingestion of bloodmeal by females triggers a series of physiological processes in the midgut and also exposes them to infection by these pathogens. The bacteria normally harbored in the midgut are known to influence physiology and can also alter the response to various pathogens. The midgut bacteria in female Cx. quinquefasciatus mosquitoes collected over a large geographical area from India was studied. Examination of 16S ribosomal DNA amplicons from culturable microflora revealed the presence of 83 bacterial species belonging to 31 bacterial genera. All of these species belong to three phyla i.e. Proteobacteria, Firmicutes and Actinobacteria. Phylum Proteobacteria was the most dominant phylum (37 species), followed by Firmicutes (33 species) and Actinobacteria (13 species). Phylum Proteobacteria, was dominated by members of γ-proteobacteria class. The genus Staphylococcus was the largest genus represented by 11 species whereas Enterobacter was the most prevalent genus and recovered from all the field stations except Leh. Highest bacterial prevalence was observed from Bhuj (22 species) followed by Nagrota (18 species), Masimpur (18 species) and Hathigarh (16 species). Whereas, least species were observed from Leh (8 species). It has been observed that individual mosquito harbor extremely diverse gut bacteria and have very small overlap bacterial taxa in their gut. This variation in midgut microbiota may be one of the factors responsible for variation in disease transmission rates or vector competence within mosquito population. The present data strongly encourage further investigations to verify the potential role of the detected bacteria in mosquito for the transmission of lymphatic filariasis and west nile virus. To the best of our knowledge this is the first study on midgut microbiota of wild Cx. quinquefasciatus from over a large geographical area.     

Analysis of the Genetic Demographic Structure of Rural Populations Neighboring the Semipalatinsk Nuclear Test Site

Rural populations neighboring the Semipalatinsk nuclear test site were used as a model to develop and test an integrated population-genetic approach to analysis of the medical genetic situation and environmental conditions in the areas studied. The contributions of individual factors of population dynamics into the formation of the genetic load were also assessed. The informative values of some genetic markers were estimated. Based on these estimates, a mathematical model was constructed that makes it possible to calculate numerical scores for analysis of the genetic loads in populations differing in environmental exposure.     

Dominant Microbial Populations in Limestone-Corroding Stream Biofilms, Frasassi Cave System, Italy

Waters from an extensive sulfide-rich aquifer emerge in the Frasassi cave system, where they mix with oxygen-rich percolating water and cave air over a large surface area. The actively forming cave complex hosts a microbial community, including conspicuous white biofilms coating surfaces in cave streams, that is isolated from surface sources of C and N. Two distinct biofilm morphologies were observed in the streams over a 4-year period. Bacterial 16S rDNA libraries were constructed from samples of each biofilm type collected from Grotta Sulfurea in 2002. β-, γ-, δ-, and -proteobacteria in sulfur-cycling clades accounted for ≥75% of clones in both biofilms. Sulfate-reducing and sulfur-disproportionating δ-proteobacterial sequences in the clone libraries were abundant and diverse (34% of phylotypes). Biofilm samples of both types were later collected at the same location and at an additional sample site in Ramo Sulfureo and examined, using fluorescence in situ hybridization (FISH). The biomass of all six stream biofilms was dominated by filamentous γ-proteobacteria with Beggiatoa-like and/or Thiothrix-like cells containing abundant sulfur inclusions. The biomass of -proteobacteria detected using FISH was consistently small, ranging from 0 to less than 15% of the total biomass. Our results suggest that S cycling within the stream biofilms is an important feature of the cave biogeochemistry. Such cycling represents positive biological feedback to sulfuric acid speleogenesis and related processes that create subsurface porosity in carbonate rocks.     

Degradation of Ethylenediaminetetraacetic Acid by Microbial Populations from an Aerated Lagoon

The ferric chelate of ethylenediaminetetraacetic acid (EDTA) was biologically degraded by a mixed population of microorganisms present in an aerated lagoon receiving this chemical in its feed. As determined radiorespirometrically, 28% of the acetate-2-C and 30% of the ethylene position of the ammonium ferric chelate of [14C]EDTA was recovered as 14CO2 after 5 days. In a separate experiment using gas liquid chromatography and the sodium ferric chelate, as much as 89% disappearance of EDTA (0.1% wt/vol) was observed during a similar time period. Optimum 14CO2 evolution was observed at a pH value between 7 and 8 and at room temperature. Degradation of NH4Fe-[2-14C]EDTA was stimulated by the addition of either unlabeled NaFe-EDTA, nitrilotriacetic acid or ethylenediamine, and inhibited by the addition of a variety of different sugars and amino acids. Consistent with the biological nature of this degradation, little or no 14CO2 evolution was observed after heat treatment of the microorganisms at 100 C for 10 min, or after the addition of antibiotics to the incubation mixtures. Gas-liquid chromatography and mass spectral analyses were performed to demonstrate EDTA disappearance and to identify possible intermediates of EDTA degradation.     

Effects of Pharmaceutical Wastes on Microbial Populations in Surface Waters at the Puerto Rico Dump Site in the Atlantic Ocean

A series of cruises during 1979 and 1980 to the pharmaceutical dump site located 64 km north of Arecibo, Puerto Rico, in the Atlantic Ocean, was carried out to evaluate effects of wastes on the ecology of the microflora of surface waters of the dump site. In addition to bacteriological monitoring of the waste plume created by the release of wastes from the disposal barge, stations along a series of transects, extending north from coastal waters through and beyond the dump site, were sampled. Largest numbers of culturable bacteria on marine agar were found at stations closest to shore and in the vicinity of the dump site. Bacteria recovered on marine agar were predominantly Vibrio and Aeromonas spp., with the relative abundance of these organisms decreasing as gram-positive organisms (staphylococci, micrococci, and bacilli) became dominant in areas immediately affected by waste dumping. Total numbers of bacteria (determined by acridine orange direct counts [AODC]), which were relatively stable throughout the region, and a direct estimate of viable cells (DVC), i.e., those cells responsive to additions of yeast extract and nalidixic acid, were determined by acridine orange staining and epifluorescence microscopy. Heterotrophic bacterial activity, measured by the uptake (V_max) of ¹⁴C-labeled amino acids, declined relative to distance from land. Increases in specific activity indices (DVC/AODC and V_max/AODC) were observed near the dump site. The composite results of this study, i.e., increased specific activities (determined by two methods), increased numbers of culturable marine bacteria, and marked alteration of the taxonomic composition of the culturable bacterial community in waters within and surrounding the Puerto Rico dump site, indicate demonstrable changes in the marine microbial community in the region used for waste disposal.     

Sulphate-controlled Diversity of Subterranean Microbial Communities over Depth in Deep Groundwater with Opposing Gradients of Sulphate and Methane

The groundwater system in Olkiluoto, Finland, is stratified with a mixing layer at a depth of approximately 300 m between sulphate-rich, methane-poor and sulphate-poor, methane-rich groundwaters. New sequence library data obtained by 454 pyrotag sequencing of the v4v6 16S rDNA region indicated that sulphate-reducing bacteria (SRB) dominated the mixing layer while SRB could not be detected in the deep sulphate-poor groundwater samples. With the indispensable support of the sequence data, it could be demonstrated that sulphate was the only component needed to trigger a very large community transition in deep sulphate-poor, methane-rich groundwater from a non-sulphate-reducing community comprising Hydrogenophaga, Pseudomonas, Thiobacillus, Fusibacter, and Lutibacter to a sulphate-reducing community with Desulfobacula, Desulfovibrio, Desufobulbaceae, Desulfobacterium, Desulfosporosinus, and Desulfotignum. Experiments with biofilms and planktonic microorganisms in flow cells under in situ conditions confirmed that adding sulphate to the sulphate-poor groundwater generated growth of cultivable SRB and detectable SRB-related sequences. It was also found that the 16S rDNA diversity of the biofilms was conserved over 103 d and that there was great similarity in diversity between the microorganisms in the biofilms and in the flowing groundwater. This work demonstrates that the presence/absence of only one geochemical parameter, i.e., sulphate, in the groundwater significantly influenced the diversity of the investigated subterranean microbial community.     

Phenol- and Toluene-Degrading Microbial Populations from an Aquifer in Which Successful Trichloroethene Cometabolism Occurred

We characterized the bacterial populations that grew in a Moffett Field, Calif., aquifer following three sequential field tests of phenol- or toluene-driven cometabolism of trichloroethene (TCE). Reducing the toluene and phenol concentrations in most-probable-number (MPN) tubes from 50 to 5 ppm increased the population density measured for these degraders by 1.5 and 1 log units, respectively, suggesting that natural populations might be quite sensitive to these substrates. Phenol and toluene degraders were isolated from the terminal MPN dilution tubes; 63 genetically distinct strains were identified among the 273 phenol- and toluene-degrading isolates obtained. TCE was cometabolized by 60% of the genetically distinct strains. Most strains (57%) grew on both phenol and toluene, and 78% of these strains hybridized to the toluene ortho-monooxygenase (TOM) probe. None of the strains hybridized to probes from the four other toluene oxygenase pathways. Gram-positive strains comprised 30% of the collection; all of these grew on phenol, and 47% of them also grew on toluene, but none hybridized to the TOM probe. Among the gram-negative strains, 86% of those that grew on both toluene and phenol hybridized to the TOM probe, while only 5% of those that were TOM-positive grew on toluene alone. A larger proportion of TCE degraders was found among gram-negative than gram-positive strains and among organisms that grew on phenol than those that grew on toluene. Hybridization of strains to the TOM probe was somewhat predictive of their TCE-cometabolizing ability, especially for strains isolated on toluene, but there was also a significant number (20%) of strains that hybridized to the TOM probe but were poor TCE cooxidizers. No Moffett Field isolates were as effective as Burkholderia cepacia G4 in cooxidizing TCE. Most of the aquifer strains ranged from moderately effective to ineffective in TCE cooxidation. Such populations, however, apparently accounted for the successful phenol- and toluene-stimulated TCE removal that occurred during the field assessment of this remediation process. This suggests that naturally occurring communities of only moderate TCE-cooxidizing ability may support successful TCE bioremediation as long as the phenol or toluene present is not limiting. This activity, however, may not be sustainable for the long term, because TCE-inactive populations that consumed toluene at rates equal to that of the best TCE degraders were present and hence would be expected to eventually dominate the community.     

Geomicrobiological Properties of Ultra-Deep Granitic Groundwater from the Mizunami Underground Research Laboratory (MIU), Central Japan

Although deep subterranean crystalline rocks are known to harbor microbial ecosystems, geochemical factors that constrain the biomass, diversity, and metabolic activities of microorganisms remain to be clearly defined. To better understand the geochemical and microbiological relationships, we characterized granitic groundwater collected from a 1,148- to 1,169-m-deep borehole interval at the Mizunami Underground Research Laboratory site, Japan, in 2005 and 2008. Geochemical analyses of the groundwater samples indicated that major electron acceptors, such as NO₃⁻ and SO₄²⁻, were not abundant, while dissolved organic carbon (not including organic acids), CH₄ and H₂, was moderately rich in the groundwater sample collected in 2008. The total number of acridine orange-stained cells in groundwater samples collected in 2005 and 2008 were 1.1 × 10⁴ and 5.2 × 10⁴ cells/mL, respectively. In 2005 and 2008, the most common phylotypes determined by 16S rRNA gene sequence analysis were both related to Thauera spp., the cultivated members of which can utilize minor electron donors, such as aromatic and aliphatic hydrocarbons. After a 3–5-week incubation period with potential electron donors (organic acids or CH₄ + H₂) and with/without electron acceptors (O₂ or NO₃⁻), dominant microbial populations shifted to Brevundimonas spp. These geomicrobiological results suggest that deep granitic groundwater has been stably colonized by Thauera spp. probably owing to the limitation of O₂, NO₃⁻, and organic acids.     

Geochemically Generated,Energy-Rich Substrates and Indigenous Microorganisms in Deep,Ancient Groundwater

Recent studies have shown that the biosphere extends to depths that exceed 3 km, raising questions regarding the age of the microbes in these deep ecosystems and their sources of energy for metabolism. Abiogenic energy sources that are derived from in situ, purely geochemical sources and thus independent from photosynthesis have been suggested. We sampled saline fracture water emanating from a 3.1-km deep borehole in a Au mine in the Witwatersrand Basin of South Africa and characterized the chemical constituents (including stable isotopes), groundwater age, and indigenous microorganisms. Salinity data and ratios of dissolved noble gases indicate that extremely ancient (2.0 Ga) saline fracture water has mixed with meteoric water to yield an average subsurface residence time of 20–160 Ma, the oldest age of any waters collected to date in the Witwatersrand Basin. H₂ isotope data suggest the water originated from a depth of 4 to 5 km. Sulfur isotope fractionation indicates biological sulfate reduction. Calculations of free energies and steady state energy fluxes based on water chemistry data also support sulfate reduction as the dominant terminal electron accepting process. Lipid and flow cytometry data indicate a sparse microbial community (10³ cells ml^?1), despite the presence of relatively high concentrations of energy-rich compounds (H₂, CH₄, CO, ethane, propane, butane, and acetate). The H₂ can be explained by radiolysis of water. Stable isotopic signatures of the CH₄ and short chain hydrocarbons indicate abiogenic synthesis. The persistence of energy-rich compounds suggests that other factors are limiting to microbial metabolism and growth, e.g., availability of an inorganic nutrient, such as Fe or phosphate.