Deep Terrestrial Subsurface Research Articles

陸上深部地下生物圏の研究<br>―人間社会との関わり―

The deep subsurface biosphere has been regarded as an emerging topic in geo-bioscience and industry for the past few decades, and has been approached by terrestrial and seafloor drillings. Terrestrial sites have better proximity and greater relevance to the anthroposphere and technosphere, i.e., human habitats and societies, than do seafloor sites. Therefore, understanding the subterranean biosphere has more direct importance to issues related to a sustainable civilization, and issues such as formation/maturation of hydrocarbon reservoirs and ore deposits, disposal of radioactive wastes and carbon dioxide, and postulated association between seismogenic and microbial activities. Microbiological studies in the terrestrial deep subsurface have been prompted to respond to such human-related issues, and microbial life in sedimentary and crystalline rocks as well as pore-filling fluids has been studied to evaluate rock stability and (im) mobilization of redox-sensitive elements/nuclides, for instance. This is in contrast to subseafloor microbiology, which focuses more on microbial interactions with hydrothermal circulation, relevant biogeochemical processes including gas hydrate formation, associated diversity of life, and modern analogs of origin-of-life. Avoiding man-induced contamination of cored samples and pumped fluids has been a microbiological issue. Technical (both instrumental and operational) measures to minimize contamination were first developed in subterranean microbiology, because of easier accesses to test sites for repetition, evaluation, improvement, etc. of attempted measures on land. Then, anti-contamination expertise was introduced into subseafloor practices, and anti-contamination protocols and facilities are now better developed by the Integrated Ocean Drilling Program (IODP) than the International Continental Scientific Drilling Program (ICDP). Newly developed techniques are also applied to measure/monitor geological and geochemical parameters that are used to characterize microbial habitats and processes occurring there. Lessons from subterranean microbiology are directly applicable to subglacial microbiology that may retrieve microbial life from sub-million-year-old ice cores, although additional measures are needed for glacier drilling. Because land and icy surfaces are common in Earth-like planets or potentially life-bearing satellites, lessons (experiences and expertise) from subterranean microbiology should be applicable to astrobiological searches for extraterrestrial life.

Shrinking majority of the deep biosphere

Living microorganisms were first discovered in sediment cores from scientific ocean drilling in the late 1980s when microbiologists observed and counted DNA-stained microbial cells under the microscope. During the following decade, new observations were made from drill sites in the Atlantic and Pacific Oceans and the Mediterranean Sea (1, 2). The combined data on cell numbers from these sites showed a systematic, log-log linear decrease with depth, from approximately 109 cells⋅cm−3 near the sediment surface to 106 cells⋅cm−3 at several hundred meters below seafloor, where the sediment had been deposited many million years ago (2) (Fig. 1). In their 1998 study, Whitman et al. (3) used these data to make a grand estimate of the total number of prokaryotic cells on Earth. Their astonishing conclusion was that 55% of all microbial cells occurred in the deep seabed whereas most of the rest were found in the deep terrestrial subsurface. Even more staggering, the combined deep biospheres appeared to harbor one third of the total living biomass on Earth. The publication of Whitman et al. (3) sparked a rapidly growing interest in the deep biosphere, and the data from this study were repeatedly cited in the literature in the following years. Their biomass estimate was later supported by data on intact polar membrane lipids as indicators of living cells (4). Now, these numbers are being challenged. In PNAS, Kallmeyer et al. (5) conclude that the total cell number for the deep subseafloor biosphere is probably 12-fold lower than the earlier estimate whereas the total biomass may be 74-fold lower. Is the deep biosphere losing significance?

Nematoda from the terrestrial deep subsurface of South Africa

Since its discovery over two decades ago, the deep subsurface biosphere has been considered to be the realm of single-cell organisms, extending over three kilometres into the Earth's crust and comprising a significant fraction of the global biosphere. The constraints of temperature, energy, dioxygen and space seemed to preclude the possibility of more-complex, multicellular organisms from surviving at these depths. Here we report species of the phylum Nematoda that have been detected in or recovered from 0.9-3.6-kilometre-deep fracture water in the deep mines of South Africa but have not been detected in the mining water. These subsurface nematodes, including a new species, Halicephalobus mephisto, tolerate high temperature, reproduce asexually and preferentially feed upon subsurface bacteria. Carbon-14 data indicate that the fracture water in which the nematodes reside is 3,000-12,000-year-old palaeometeoric water. Our data suggest that nematodes should be found in other deep hypoxic settings where temperature permits, and that they may control the microbial population density by grazing on fracture surface biofilm patches. Our results expand the known metazoan biosphere and demonstrate that deep ecosystems are more complex than previously accepted. The discovery of multicellular life in the deep subsurface of the Earth also has important implications for the search for subsurface life on other planets in our Solar System.

Biodegradation of Kupferschiefer black shale organic matter (Fore-Sudetic Monocline, Poland) by indigenous microorganisms

This study provides the first evidence for the direct biodegradation of persistent organic matter extracted from the organic-rich polymetallic black shale ore Kupferschiefer, one of the most important sources of metals in the world. It was demonstrated that an enriched community of indigenous heterotrophic microorganisms isolated from black shale grown under aerobic conditions could utilize shale organic matter as the sole carbon and energy source. Colonization of shale organic matter was observed. The main biodegradation intermediates and products such as phosphonic acid dioctadecyl ester and isoindole-1,3 were detected in the aqueous phase of cultures. The bacterial community showed the ability to PAH biodegradation, assimilation of organic acids and esters as well as lipase activity. The intracellular accumulation of phosphorus by bacteria during growth on organic matter was confirmed. Strains within the genus Pseudomonas were found to dominate the bacterial population at the end of the experiment. The results of this study confirm that indigenous bacteria are likely to play a role in the biotransformation of black shale and can influence the geochemical cycles of ancient organic carbon in the deep terrestrial subsurface. This process may also occur in tailings ponds containing black shale, and cause the mobilization of potentially toxic compounds to the soil and groundwater.

Potential for Nitrogen Fixation and Nitrification in the Granite-Hosted Subsurface at Henderson Mine, CO.

The existence of life in the deep terrestrial subsurface is established, yet few studies have investigated the origin of nitrogen that supports deep life. Previously, 16S rRNA gene surveys cataloged a diverse microbial community in subsurface fluids draining from boreholes 3000 feet deep at Henderson Mine, CO, USA (Sahl et al., 2008). The prior characterization of the fluid chemistry and microbial community forms the basis for the further investigation here of the source of NH4+. The reported fluid chemistry included N2, NH4+ (5–112 μM), NO2− (27–48 μM), and NO3− (17–72 μM). In this study, the correlation between low NH4+ concentrations in dominantly meteoric fluids and higher NH4+ in rock-reacted fluids is used to hypothesize that NH4+ is sourced from NH4+-bearing biotite. However, biotite samples from the host rocks and ore-body minerals were analyzed by Fourier transform infrared (FTIR) microscopy and none-contained NH4+. However, the nitrogenase-encoding gene nifH was successfully amplified from DNA of the fluid sample with high NH4+, suggesting that subsurface microbes have the capability to fix N2. If so, unregulated nitrogen fixation may account for the relatively high NH4+ concentrations in the fluids. Additionally, the amoA and nxrB genes for archaeal ammonium monooxygenase and nitrite oxidoreductase, respectively, were amplified from the high NH4+ fluid DNA, while bacterial amoA genes were not. Putative nitrifying organisms are closely related to ammonium-oxidizing Crenarchaeota and nitrite-oxidizing Nitrospira detected in other subsurface sites based upon 16S rRNA sequence analysis. Thermodynamic calculations underscore the importance of NH4+ as an energy source in a subsurface nitrification pathway. These results suggest that the subsurface microbial community at Henderson is adapted to the low nutrient and energy environment by their capability of fixing nitrogen, and that fixed nitrogen may support subsurface biomass via nitrification.

Molecular hydrogen: An abundant energy source for bacterial activity in nuclear waste repositories

A thorough understanding of the energy sources used by microbial systems in the deep terrestrial subsurface is essential since the extreme conditions for life in deep biospheres may serve as a model for possible life in a nuclear waste repository. In this respect, H 2 is known as one of the most energetic substrates for deep terrestrial subsurface environments. This hydrogen is produced from abiotic and biotic processes but its concentration in natural systems is usually maintained at very low levels due to hydrogen-consuming bacteria. A significant amount of H 2 gas will be produced within deep nuclear waste repositories, essentially from the corrosion of metallic components. This will consequently improve the conditions for microbial activity in this specific environment. This paper discusses different study cases with experimental results to illustrate the fact that microorganisms are able to use hydrogen for redox processes (reduction of O 2, NO 3 - , Fe III) in several waste disposal conditions. Consequences of microbial activity include: alteration of groundwater chemistry and shift in geochemical equilibria, gas production or consumption, biocorrosion, and potential modifications of confinement properties. In order to quantify the impact of hydrogen bacteria, the next step will be to determine the kinetic rate of the reactions in realistic conditions.

Tet 42, a Novel Tetracycline Resistance Determinant Isolated from Deep Terrestrial Subsurface Bacteria

Tet 42, a novel tetracycline resistance determinant from deep subsurface bacteria, was characterized and found to have a 30% sequence similarity to TetA(Z). The protein is a putative efflux pump that shares characteristics with previously characterized pumps, including a divergently transcribed TetR repressor, a conserved GxxSDRxGRR motif, and transmembrane domains.

Antibiotic Resistance in Bacteria Isolated from the Deep Terrestrial Subsurface

Various natural environments have been examined for the presence of antibiotic-resistant bacteria and/or novel resistance mechanisms, but little is known about resistance in the terrestrial deep subsurface. This study examined two deep environments that differ in their known period of isolation from surface environments and the bacteria therein. One hundred fifty-four strains of bacteria were isolated from sediments located 170-259 m below land surface at the US Department of Energy Savannah River Site (SRS) in South Carolina and Hanford Site (HS) in Washington. Analyses of 16S rRNA gene sequences showed that both sets of strains were phylogenetically diverse and could be assigned to several genera in three to four phyla. All of the strains were screened for resistance to 13 antibiotics by plating on selective media and 90% were resistant to at least one antibiotic. Eighty-six percent of the SRS and 62% of the HS strains were resistant to more than one antibiotic. Resistance to nalidixic acid, mupirocin, or ampicillin was noted most frequently. The results indicate that antibiotic resistance is common among subsurface bacteria. The somewhat higher frequencies of resistance and multiple resistance at the SRS may, in part, be due to recent surface influence, such as exposure to antibiotics used in agriculture. However, the HS strains have never been exposed to anthropogenic antibiotics but still had a reasonably high frequency of resistance. Given their long period of isolation from surface influences, it is possible that they possess some novel antibiotic resistance genes and/or resistance mechanisms.

Stars of the terrestrial deep subsurface: A novel ‘star‐shaped’ bacterial morphotype from a South African platinum mine

We study structure and function. Credit of course, goes to TJB, for it is from him that I inherited the habit of personifying bacteria and attempting to think like a bacterium, to better understand what they do. This work has taken us to wonderful places such as Yellowstone National Park, The Canadian Arctic, Australia, and the deep subsurface in the Republic of South Africa, the subject of this manuscript. From their perspective, why they do what they do is simple, to live. How they do it, is more challenging for us to understand, so it is something that we continue to work on. The marvel of bacteria is something that I, in turn, try to pass on to my students where I hope it will find fertile ground and provide as much enjoyment as it has given me - G. Southam. A biofilm (mine-slime) collected from the Northam Platinum mine in the Republic of South Africa contained a new bacterial morphotype. Mine-slimes are generally considered to be microbiologically compromised, subsurface samples due to the likelihood of contamination from the mining environment. However, careful examination of this biofilm demonstrated that it possessed a diverse bacterial population that included organisms that are consistent with the deep subsurface, suggesting that mine-slimes represent an underutilized, 'natural' bacterial enrichment. Using scanning and transmission electron microscopy, a novel, branching, filamentous, star-shape bacterium (in cross section) has been found, adding a new bacterial morphotype and strategy that bacteria have demonstrated to increase their surface area to volume ratio.

14C in Methane and DIC in the Deep Terrestrial Subsurface: Implications for Microbial Methanogenesis

A comparison between the 14C content of the methane and dissolved inorganic carbon (DIC) in deep, terrestrial subsurface systems was used to assess the timing of microbial methanogenesis contributing to gases in fracture water samples from three mines in the Witwatersrand Basin, South Africa. The results demonstrated that the majority of methane was produced over geologic timescales. In four of the samples, the methane contained no significant radiocarbon, indicating that the estimated 90% microbial methane in these samples was produced in the geologic past by indigenous microbial communities. In two samples from different mines, methane Δ14C levels indicated a primarily ancient origin for the microbial methane with the potential for more recent contributions from ongoing indigenous microbial activities constrained to between 0 and 40%, and 0 and 24%, respectively. Microbiological evidence for methanogenic archaea was observed in both of these samples. One sample had a Δ14C CH4 that was higher than the corresponding DIC, indicating an extreme decoupling between these species and raising concerns over the representative quality of this sample. The variations in the Δ14C of DIC and CH4 between and within mines demonstrate the need for a thorough assessment of each sample to obtain an accurate understanding of the role and timing of microbiological gas production in these complex, heterogeneous, terrestrial subsurface systems. The approach detailed here introduces timing as a new and widely applicable signature for the recognition of a major geochemical marker of indigenous life in the deep subsurface.

Structural and Chemical Characterization of a Natural Fracture Surface from 2.8 Kilometers Below Land Surface: Biofilms in the Deep Subsurface

A freshly intersected water-bearing fracture zone from the Mponeng Au mine located in the Witwatersrand Basin, Republic of South Africa was sampled, providing an opportunity to examine the natural, deep subsurface biosphere. The fracture, intersected by an advancing tunnel 2.8 kilometers below land surface, possessed a millimeter thick layer of chlorite group minerals, i.e., chamosite, at the water-mineral interface. Water flowing out from the fracture zone had a temperature of 52°C, pH of 9.16 and Eh of −263 mV. Using scanning electron microscopy, the water-mineral interface was generally found to be clean, i.e., it did not possess any secondary mineral or dominant organic coatings. Irregular patches (10's of μm 2 ) of organic material, however, resembling bacterial exopolysaccharides, occurred in the presence or absence of bacteria. The surface was colonized by highly dispersed individual bacteria or by microcolonies containing up to 5 cells, with an overall cell density of 5 × 104 bacteria cm −2 . This biofilm population, although low, was 2 orders of magnitude greater than the bacteria present within the aqueous phase and provides the first direct observation of the sessile population from the terrestrial deep subsurface. Time of Flight-Secondary Ion Mass spectrometry revealed that the fracture surface was actually coated with a thin, i.e., molecular, organic conditioning film over much of its surface that was separate from the exopolysaccharide layers associated with the mineral water interface and with some of the attached cells.

Isolation of Sulfate-Reducing Bacteria from the Terrestrial Deep Subsurface and Description of Desulfovibrio cavernae sp. nov.

Deep subsurface sandstones in the area of Berlin (Germany) located 600 to 1060 m below the surface were examined for the presence of viable microorganisms. The in situ temperatures at the sampling sites ranged from 37 to 45 degrees C. Investigations focussed on sulfate-reducing bacteria able to grow on methanol and triethylene glycol, which are added as chemicals to facilitate the long-term underground storage of natural gas. Seven strains were isolated from porewater brines in the porous sandstone. Three of them were obtained with methanol (strains H1M, H3M, and B1M), three strains with triethylene glycol (strains H1T, B1T, and B2T) and one strain with a mixture of lactate, acetate and butyrate (strain H1-13). Due to phenotypic properties six isolates could be identified as members of the genus Desulfovibrio, and strain B2T as a Desulfotomaculum. The salt tolerance and temperature range for growth indicated that the isolates originated from the indigenous deep subsurface sandstones. They grew in mineral media reflecting the in situ ionic composition of the different brines, which contained 1.5 to 190 g NaCl x l(-1) and high calcium and magnesium concentrations. The Desulfovibrio strains grew at temperatures between 20 and 50 degrees C, while the Desulfotomaculum strain was thermophilic and grew between 30 and 65 degrees C. The strains utilized a broad spectrum of electron donors and acceptors. They grew with carbon compounds like lactate, pyruvate, formate, n-alcohols (C1-C5), glycerol, ethylene glycol, malate, succinate, and fumarate. Some strains even utilized glucose as electron donor and carbon source. All strains were able to use sulfate, sulfite and nitrate as electron acceptors. Additionally, three Desulfovibrio strains reduced manganese oxide, the Desulfotomaculum strain reduced manganese oxide, iron oxide, and elemental sulfur. The 16S rRNA analysis revealed that the isolates belong to three different species. The strains H1T, H3M and B1M could be identified as Desulfovibrio indonesiensis, and strain B2T as Desulfotomaculum geothermicum. The other Desulfovibrio strains (H1M, H1-13, and B1T) showed identical 16S rDNA sequences and similarities as low as 93% to their closest relative, Desulfovibrio aminophilusT. Therefore, these isolates were assigned to a new species, Desulfovibrio cavernae sp. nov., with strain H1M as the type strain.

Molecular evidence for the evolution of metal homeostasis genes by lateral gene transfer in bacteria from the deep terrestrial subsurface.

Lateral gene transfer (LGT) plays a vital role in increasing the genetic diversity of microorganisms and promoting the spread of fitness-enhancing phenotypes throughout microbial communities. To date, LGT has been investigated in surface soils, natural waters, and biofilm communities but not in the deep terrestrial subsurface. Here we used a combination of molecular analyses to investigate the role of LGT in the evolution of metal homeostasis in lead-resistant subsurface bacteria. A nested PCR approach was employed to obtain DNA sequences encoding P(IB)-type ATPases, which are proteins that transport toxic or essential soft metals such as Zn(II), Cd(II), and Pb(II) through the cell wall. Phylogenetic incongruencies between a 16S rRNA gene tree and a tree based on 48 P(IB)-type ATPase amplicons and sequences available for complete bacterial genomes revealed an ancient transfer from a member of the beta subclass of the Proteobacteria (beta-proteobacterium) that may have predated the diversification of the genus Pseudomonas. Four additional phylogenetic incongruencies indicate that LGT has occurred among groups of beta- and gamma-proteobacteria. Two of these transfers appeared to be recent, as indicated by an unusual G+C content of the P(IB)-type ATPase amplicons. This finding provides evidence that LGT plays a distinct role in the evolution of metal homeostasis in deep subsurface bacteria, and it shows that molecular evolutionary approaches may be used for investigation of this process in microbial communities in specific environments.

Metal resistance among aerobic chemoheterotrophic bacteria from the deep terrestrial subsurface.

The metal resistance of 350 subsurface bacterial strains from two U.S. Department of Energy facilities, the Savannah River Site (SRS), South Carolina, and the Hanford site, Washington, was determined to assess the effect of metal toxicity on microorganisms in the deep terrestrial subsurface. Resistance was measured by growth inhibition around discs containing optimized amounts of Hg(II), Pb(II), and Cr(VI). A broad range of resistance levels was observed, with some strains of Arthrobacter spp. demonstrating exceptional tolerance. A higher level of resistance to Hg(II) and Pb(II) (P < 0.05) and a higher occurrence of multiple resistances suggested that metals more effectively influenced microbial evolution in subsurface sediments of the SRS than in those of the Hanford site. Common resistance to heavy metals suggests that toxic metals are unlikely to inhibit bioremediation in deep subsurface environments that are contaminated with mixed wastes.

Reduction of humic substances and Fe(III) by hyperthermophilic microorganisms

The ability of hyperthermophilic microorganisms to transfer electrons to humic substances (humics) and other extracellular quinones was evaluated. When H 2 was provided as the electron donor, the hyperthermophile, Pyrobaculum islandicum, transferred electrons to highly purified humics and the humics analog, anthraquinone-2,6-disulfonate (AQDS). A diversity of other hyperthermophilic Archaea including: Pyrodictium abyssi, Pyrococcus furiosus, Archaeoglobus fulgidus, Thermococcus celer, Methanopyrus kandleri, as well as the thermophiles Methanococcus thermolithitrophicus and Methanobacterium thermoautotrophicum, exhibited H 2-dependent AQDS reduction as did the hyperthermophilic bacterium Thermotoga maritima. AQDS acted as an electron shuttle between P. islandicum and poorly crystalline Fe(III) oxide and greatly accelerated rates of Fe(III) reduction. Electron shuttling by AQDS also promoted the reduction of the crystalline Fe(III) oxide forms, goethite and hematite. These results have implications for the potential mechanisms of Fe(III) reduction in various hot Fe(III)-containing environments such as near hydrothermal marine vents, terrestrial hot springs, and the deep terrestrial subsurface. The finding that the ability to reduce extracellular quinones is a characteristic of all of the hyperthermophiles evaluated and the fact that these hyperthermophiles are the organisms most closely related to the last common ancestor of extant organisms suggests that the last common ancestor had the ability to reduce humics. In combination with plausible geochemical scenarios, these results suggest that electron transfer to extracellular quinones and Fe(III) were initial steps in the eventual evolution of intracellular electron transport chains that employ quinones and iron-containing proteins.

Genetic Diversity among Arthrobacter Species Collected across a Heterogeneous Series of Terrestrial Deep-Subsurface Sediments as Determined on the Basis of 16S rRNA and recA Gene Sequences

This study was undertaken in an effort to understand how the population structure of bacteria within terrestrial deep-subsurface environments correlates with the physical and chemical structure of their environment. Phylogenetic analysis was performed on strains of Arthrobacter that were collected from various depths, which included a number of different sedimentary units from the Yakima Barricade borehole at the U.S. Department of Energy's Hanford site, Washington, in August 1992. At the same time that bacteria were isolated, detailed information on the physical, chemical, and microbiological characteristics of the sediments was collected. Phylogenetic trees were prepared from the 39 deep-subsurface Arthrobacter isolates (as well as 17 related type strains) based on 16S rRNA and recA gene sequences. Analyses based on each gene independently were in general agreement. These analyses showed that, for all but one of the strata (sedimentary layers characterized by their own unifying lithologic composition), the deep-subsurface isolates from the same stratum are largely monophyletic. Notably, the layers for which this is true were composed of impermeable sediments. This suggests that the populations within each of these strata have remained isolated under constant, uniform conditions, which have selected for a particular dominant genotype in each stratum. Conversely, the few strains isolated from a gravel-rich layer appeared along several lineages. This suggests that the higher-permeability gravel decreases the degree of isolation of this population (through greater groundwater flow), creating fluctuations in environmental conditions or allowing migration, such that a dominant population has not been established. No correlation was seen between the relationship of the strains and any particular chemical or physical characteristics of the sediments. Thus, this work suggests that within sedimentary deep-subsurface environments, permeability of the deposits plays a major role in determining the genetic structure of resident bacterial populations.

Thermophilic Fe(III)-Reducing Bacteria from the Deep Subsurface: The Evolutionary Implications

Thermophilic (45° to 75°C) bacteria that reduce amorphous Fe(III)-oxyhydroxide to magnetic iron oxides have been discovered in two geologically and hydrologically isolated Cretaceous- and Triassic-age sedimentary basins in the deep (&gt;860 meters below land surface) terrestrial subsurface. Molecular analyses based on 16 S ribosomal RNA (rRNA) gene sequences revealed that some of these bacteria represent an unrecognized phylogenetic group of dissimilatory Fe(III)-reducing bacteria. This discovery adds another dimension to the study of microbial Fe(III) reduction and biogenic magnetism. It also provides examples for understanding the history of Fe(III)-reducing microorganisms and for assessing possible roles of such microorganisms on hot primitive planets.

Bacillus infernus sp. nov., an Fe(III)- and Mn(IV)-reducing anaerobe from the deep terrestrial subsurface.

Bacillus infernus sp. nov. was isolated from ca. 2,700 m below the land surface in the Taylorsville Triassic Basin in Virginia. B. infernus was a strict anaerobe that grew on formate or lactate with Fe(III), MnO2, trimethylamine oxide, or nitrate (reduced to nitrite) as an electron acceptor, and it also grew fermentatively on glucose. Type strain TH-23 and five reference strains were gram-positive rods that were thermophilic (growth occurred at 61 degrees C), halotolerant (good growth occurred in the presence of Na+ concentrations up to 0.6 M), and very slightly alkaliphilic (good growth occurred at pH 7.3 to 7.8). A phylogenetic analysis of its 16S rRNA indicated that B. infernus should be classified as a new species of the genus Bacillus. B. infernus is the only strictly anaerobic species in the genus Bacillus.

Optimization of media for enumeration and isolation of aerobic heterotrophic bacteria from the deep terrestrial subsurface

The objective of this study was to develop an agar plate-count medium to maximize the number of colony-forming units (CFU) recovered from terrestrial subsurface sediment samples. A Plackett-Burman experimental design was used to test the effects of 13 possible medium variables, including various ingredients and culture conditions. These experiments used sediment samples from different surface and subsurface areas to test the resulting formulations. Microorganisms in different sediments did not respond uniformly to any variable. Variables that always had a positive effect, when significant, included addition of vitamins, addition of activated charcoal (added as a scavenger of toxic oxygen forms), use of pour plates rather than spread plates, and elevated, rather than in situ, incubation temperature. Lowered water potential always led to a negative response, where significant. Varying concentrations of most common medium components, including dilution of carbon sources, had few significant effects over the ranges tested. A medium formulation for enhanced recovery of aerobic heterotrophic bacteria from subsurface sediments was developed, based on these results.

Microbial transformation of heterocyclic molecules in deep subsurface sediments

Recently attempts have been made to establish the presence and to determine the metabolic versatility of microorganisms in the terrestrial deep subsurface at the Savannah River Plant, Aiken, SC, USA. Sediment samples obtained at 20 different depths of up to 526 m were examined to determine carbon mineralization under aerobic, sulfate-reducing, and methanogenic conditions. The evolution of(14)CO2 from radiolabelled glucose was observed under aerobic conditions in all sediments, whereas pyridine was transformed in 50% of the 20 sediments and indole was metabolized in 85% of the sediments. Glucose mineralization in certain sediments was comparable to that in the surface environment. Sulfate was reduced in only five sediments, and two were carbon limited. Methane production was detected in ten sediments amended with formate only after long-term incubations. The transformation of indole and pyridine was only rarely observed under sulfate-reducing conditions and was never detected in methanogenic incubations. This study provides information concerning the metabolic capability of both aerobic and anaerobic microorganisms in the deep subsurface and may prove useful in determining the feasibility of microbial decontamination of such environments.