Aerobic and anaerobic microbial activity in deep subsurface sediments from the savannah river plant

Methanogenesis, sulfate reduction, and rates of carbon mineralization were determined for samples derived at different depths from four cores drilled at the Savannah River Plant, Aiken South Carolina. Three‐gram subsamples of the sediments were dispensed to 10‐mL serum bottles under 5% H₂/95% N₂ and amended with 0.5 mL degassed distilled water with or without the following solutes: formate plus acetate, bicarbonate, lactate, and radiolabeled sulfate, glucose, and Índole. After incubating 1 to 5 days, the sediments were assayed for methane, H₂, ³⁵S, and ^I4CO₂. No methanogenesis was detected at any depth in any core and sulfate was rarely reduced. Evolution of ¹⁴CO₂ from glucose and indole was detected in sediments as deep as 262 and 259 m, respectively. At some depths the ¹⁴CO₂ evolution rate was comparable to that of surface soils; however, at other depths no ¹⁴CO₂ evolution could be detected. Injection of sterile air into anaerobic incubations increased rates of carbon mineralization at all depths that had demonstrated anaerobic activity and stimulated mineralization activity in sediments that were inactive anaerobically, suggesting a predominance of aerobic metabolism. Increasing the concentration of added glucose and indole often increased the resulting rates of ¹⁴CO₂ evolved from these substrates. Our data indicate that both aerobic and anaerobic microorganisms are present and metabolically active in samples from deep subsurface environments.     

Characterization of the microbiology within a 21 m3section of rock from the deep subsurface

The distribution of aerobic chemoheterotrophic microorganisms within a 21 m3 section of deep subsurface rock was determined. Nineteen samples for microbiological analysis were aseptically taken by hand from the walls of a 400 m deep subsurface tunnel after an alpine miner created fresh rock faces 0.76, 1.52, 2.28, and 3.04 m into the tunnel wall. The direct counts were several orders of magnitude greater than viable counts in all samples. One of each morphologically distinct bacterial type from each sample was purified and analyzed for fatty acid methyl esters (FAME) using the Microbial Identification System (MIDI). Numbers of bacterial types, diversity, and equitability of recoverable microbial communities were the same or similar using either morphotype or FAME analyses as the basis for distinguishing between bacterial types. Twenty-nine genera (Euclidean distance of 25) were found within the rock section, while 28 of the 210 bacterial types isolated were nonculturable under the growth regime required for cluster analysis. Most isolates clustered at the genus level with Arthrobacter, Gordona, and Acinetobacter. Two genera, containing 16 isolates, were unmatched to known organisms within the MIDI data base and clustered with other isolates at a Euclidean distance greater than 50. While some species (Euclidean distance 10) were recovered from multiple sites within the rock section, most were found at 1–3 sites and usually without a definitive pattern of distribution. Offprint requests to: P. S. Amy.     

Evaluation of deep subsurface sampling procedures using serendipitous microbial contaminants as tracer organisms

Surface microbiological investigations are critically dependent on the procedures used to collect samples for study. It can be difficult to distinguish between indigenous organisms and those encountered as contaminants during the drilling process. We found that coliform bacteria contaminated drilling mud slurries. These bacteria proved useful as tracer organisms in evaluating the degree of microbial contamination accidentally encountered while drilling for subterranean samples. While these organisms were found in high numbers in both the circulating muds and in the mud reservoir, few subsurface samples harbored conforms. Subsurface slurries did not inhibit the growth of a known coliform inoculum. These results indicate that the methods used to collect and field‐process cores from Atlantic coastal plain sediments were sufficient to prevent a large degree of bacterial contamination in most samples. The microflora in drilling fluids did not quantitatively or qualitatively account for the number and diversity of bacteria in subsurface samples. We conclude that a large and viable bacterial community is present in deep regions of the terrestrial subsurface.     

Isolation and characterization of cellulose-degrading bacteria from the deep subsurface of the Homestake gold mine,Lead, South Dakota,USA

The present study investigated the cultivable mesophilic (37°C) and thermophilic (60°C) cellulose-degrading bacterial diversity in a weathered soil-like sample collected from the deep subsurface (1.5 km depth) of the Homestake gold mine in Lead, South Dakota, USA. Chemical characterization of the sample by X-ray fluorescence spectroscopy revealed a high amount of toxic heavy metals such as Cu, Cr, Pb, Ni, and Zn. Molecular community structures were determined by phylogenetic analysis of 16S rRNA gene sequences retrieved from enrichment cultures growing in presence of microcrystalline cellulose as the sole source of carbon. All phylotypes retrieved from enrichment cultures were affiliated to Firmicutes. Cellulose-degrading mesophilic and thermophilic pure cultures belonging to the genera Brevibacillus, Paenibacillus, Bacillus, and Geobacillus were isolated from enrichment cultures, and selected cultures were studied for enzyme activities. For a mesophilic isolate (DUSELG12), the optimum pH and temperature for carboxymethyl cellulase (CMCase) were 5.5 and 55°C, while for a thermophilic isolate (DUSELR7) they were 5.0 and 75°C, respectively. Furthermore, DUSELG12 retained about 40% CMCase activity after incubation at 60°C for 8 h. Most remarkably, thermophilic isolate, DUSELR7 retained 26% CMCase activity at 60°C up to a period of 300 h. Overall, the present work revealed the presence of different cellulose-degrading bacterial lineages in the unique deep subsurface environment of the mine. The results also have strong implications for biological conversion of cellulosic agricultural and forestry wastes to commodity chemicals including sugars.     

The effect of cleaning and disinfecting the sampling well on the microbial communities of deep subsurface water samples

Our knowledge of the microbial characteristics of deep subsurface waters is currently very limited, mainly because of the methods used to collect representative microbial samples from such environments. In order to improve this procedure, a protocol designed to remove the unspecific, contaminant biofilm present on the walls of an approximately 800 m deep well is proposed. This procedure included extensive purges of the well, a mechanical cleaning of its wall, and three successive chlorine injections to disinfect the whole line before sampling. Total bacterial counts in water samples decreased from 2.5 x 10(5) to 1.0 x 10(4) per millilitre during the cleaning procedure. Culture experiments showed that the first samples were dominated by sulfate-reducers and heterotrophs, whereas the final sample was dominated by oligotrophic and hydrogenotrophic bacteria. Community structures established on the diversity of the 16S rRNA genes and data analysis revealed that the water sample collected, after a purge without removal of the biofilm, was characterized by numerous phyla which are not representative of the deep subsurface water. On the other hand, several bacterial phyla were only detected after the full cleaning of the well, and were considered as important components of the subsurface ecosystem which would have been missed in the absence of well cleaning.     

Molecular analysis of prokaryotic diversity in the deep subsurface of the former Homestake gold mine,South Dakota,USA

A culture-independent molecular phylogenetic analysis was carried out to study the prokaryotic diversity in two soil samples collected from the subsurface (1.34 km depth) of the former Homestake gold mine, Lead, South Dakota, USA at two sites, the Ross shaft and number 6 Winze. Microbial community analyses were performed by cloning and sequencing of 16S rRNA genes retrieved directly from soil samples. Geochemical characterization of soils revealed high amount of toxic metals such as As, Cd, Co, Cr, Cu, Ni, Pb, Zn, and U at both the sites. Phylogenetic analyses showed that soil samples were predominantly composed of phylotypes related to phylum Proteobacteria. Other phyla detected in libraries were Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Chlorobi, Firmicutes, Gemmatimonadetes, Nitrospirae, Planctomycetes, Verrucomicrobia, and candidate divisions OP10 and TM7. The majority (>95%) of the phylotypes retrieved in the libraries were most closely related to environmental sequences from yet-uncultured bacteria representing a hitherto unidentified diversity. The archaeal communities at both the sites exhibited lower diversity and were most closely affiliated to uncultivated species within the Crenarchaeota. Results showed the existence of diverse microbial populations in deep subsurface environment of the Homestake gold mine. Statistical analyses demonstrated that each site harbored phylogenetically distinct microbial populations that were more diverse at Ross site compare to winze site.     

Bacterial heterogeneity in deep subsurface tunnels at Rainier Mesa,Nevada test site

To characterize the deep subsurface environment of Rainier Mesa, Nevada Test Site, rock samples were taken from tunnels U 12b, U12g, U12p, and U 12n, which varied in depth from 50 m to 450 m and in gravimetric moisture content from 4% to 27%. Values for total count, viable count, biomass, Simpson diversity, equitability, similarity coefficient, and number of distinct colony types indicated microbiological variability between samples. Viable counts ranged from less than 1 × 10¹ to 2.4 × 10⁵ CFU g dry wt^–1 of rock. Direct counts and enumeration based on phospholipid determination indicated larger numbers of cells g dry wt-1 of rock than viable counts. Simpson diversity indices, equitability, and numbers of distinct colony types varied from 3.00 to 8.05, 0.21 to 0.89, and 7 to 19, respectively, and indicated heterogeneity between samples. Each distinct morphotype was purified and characterized. Gram reaction, morphology, metal and antibiotic resistances, and metabolic activities of each isolate confirmed spatial variability among microbiota isolated from different locations. Most probable numbers of nitrifying, sulfur oxidizing, and sulfur-reducing bacteria were below the limit of detection in all samples, while the numbers of nitrogen fixing bacteria ranged from below the level of detection to 7.8 × 10² cells g dry wt^–1 of rock sample, and the numbers of dentrifying bacteria ranged from below the level of detection to greater than 1.6 × 10³ cells g dry wt^–1 of rock sample. Offprint requests to: P. S. Amy.     

Ecology, physiology, and phylogeny of deep subsurface Sphingomonas sp

Several new species of the genus Sphingomonas including S. aromaticivorans, S. stygia, and S. subterranea that have the capacity for degrading a broad range of aromatic compounds including toluene, naphthalene, xylenes, p-cresol, fluorene, biphenyl, and dibenzothiophene, were isolated from deeply-buried (>200 m) sediments of the US Atlantic coastal plain (ACP). In S. aromaticivorans F199, many of the genes involved in the catabolism of these aromatic compounds are encoded on a 184-kb conjugative plasmid; some of the genes involved in aromatic catabolism are plasmid-encoded in the other strains as well. Members of the genus Sphingomonas were common among aerobic heterotrophic bacteria cultured from ACP sediments and have been detected in deep subsurface environments elsewhere. The major source of organic carbon for heterotrophic metabolism in ACP deep aquifers is lignite that originated from plant material buried with the sediments. We speculate that the ability of the subsurface Sphingomonas strains to degrade a wide array of aromatic compounds represents an adaptation for utilization of sedimentary lignite. These and related subsurface Sphingomonas spp may play an important role in the transformation of sedimentary organic carbon in the aerobic and microaerobic regions of the deep aquifers of the ACP. Received 12 May 1999/ Accepted in revised form 25 July 1999     

Numbers,diversity, and morphological characteristics of aerobic,chemoheterotrophic bacteria in deep subsurface sediments from a site in South Carolina

The aerobic, chemoheterotrophic bacteria indigenous to deep aquifers and other subsurface sediments (depths to 265 m) at a site in South Carolina were characterized by direct microscopy, enumeration of viable cells, analysis of colony morphologies on plates, and analysis of cell morphologies of isolated strains. Substantial numbers of viable bacteria (10^5‐10⁸/g) were present in all transmissive, aquifer sediments, and their numbers did not decrease with depth. Fewer bacteria (<10³/g) were detected in nontransmissive, confining layers. The highest viable counts were obtained on dilute media, but 10–50% of the bacteria in most aquifer sediments also grew rapidly on concentrated, nutrient‐rich media (indicating a high degree of metabolic flexibility). Most of the bacteria were mesophilic; relatively few psychrophiles or thermophiles were detected (<10³/g; in many cases, none). The bacterial flora was diverse (11–62 distinct colony types on enumeration plates of most aquifer sediments). Diversity did not decrease with depth, but the composition of the microflora (based on colony analysis) varied extensively from one geological formation to another. Almost 95% of the platable colonies that grew on enumeration plates contained nonstreptomycete bacteria, more than 80% of which were gram‐negative rods. Light microscopy of films released from aquifer sediments by flotation revealed the presence of dividing cells and microcolonies, thus implying that the in situ deep aquifer microflora was more metabolically active than that seen previously in shallow aquifers.     

Arthrobacter subterraneus sp. nov., isolated from deep subsurface water of the South Coast of Korea

Strain CH7T, a pale yellow-pigmented bacterium and new isolate from deep subsurface water of the South Coast of Korea, was subjected to a polyphasic taxonomic study. CH7T grew between 5 and 37 degrees C, pH 5.3-10.5, and tolerated up to 13% NaCl. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain CH7T was associated with the genus Arthrobacter and phylogenetically closely related to the type strains Arthrobacter tumbae (99.4%) and Arthrobacter parietis (99.1%). However, DNA-DNA hybridization experiments revealed 2.1% and 12% between strain CH7T and Arthrobacter tumbae and Arthrobacter parietis, respectively. Thus, the phenotypic and phylogenetic differences suggested that CH7T should be placed in the genus Arthrobacter as a novel species, for which the name Arthrobacter subterraneus sp. nov. is proposed. In addition, the type strain for the new species is CH7T (=KCTC 9997T=DSM 17585T).     

Vegetation, substrate and hydrology in floating marshes in the Mississippi river delta plain wetlands, USA

In the 1940s extensive floating marshes (locally called flotant) were reported and mapped in coastal wetlands of the Mississippi River Delta Plain. These floating marshes included large areas of Panicum hemitomon-dominated freshwater marshes, and Spartina patens/Scirpus olneyi brackish marshes. Today these marshes appear to be quite different in extent and type. We describe five floating habitats and one non-floating, quaking habitat based on differences in buoyancy dynamics (timing and degree of floating), substrate characteristics, and dominant vegetation. All floating marshes have low bulk density, organic substrates. Nearly all are fresh marshes. Panicum hemitomon floating marshes presently occur within the general regions that were reported in the 1940's by O'Neil, but are reduced in extent. Some of the former Panicum hemitomon marshes have been replaced by seasonally or variably floating marshes dominated, or co-dominated by Sagittaria lancifolia or Eleocharis baldwinii.     

Distribution of bacterial plasmids in clean and polluted sites in a South Wales river.

We isolated 400 aerobic heterotrophic bacteria from the sediment of unpolluted and polluted sites in a fast-flowing south Wales river. Isolates were subjected to taxonomic tests and screened for the presence of plasmid DNA by alkaline lysis and agarose gel techniques. There were no significant differences between sites in either the total percentage of isolates containing plasmids (unpolluted site, 9.4%; polluted site, 15%) or in the percentage of non-Pseudomonas-like isolates containing plasmids (unpolluted site, 15%; polluted site, 10%). There were significantly more Pseudomonas-like isolates with plasmids at the polluted site than at the unpolluted site (unpolluted site, 7%; polluted site, 18%). This presumably reflected a response of the nutritionally versatile Pseudomonas-like isolates to conditions at that site. The majority (86%) of the plasmids detected had molecular masses between 35 and 312 megadaltons. These plasmids were large enough to carry genes for conjugal transfer, suggesting the possibility of such transfer in this environment.     

Distribution of aerobic bacteria,protozoa, algae,and fungi in deep subsurface sediments

The distribution of microorganisms in deep subsurface profiles was determined at three sites at the Savannah River Plant, Aiken, South Carolina. Acridine orange direct counts (AODC) of bacteria were highest in surface soil samples and declined to the 10⁶ to 10⁷ per gram range in the subsurface, but then did not decline further with depth. In the subsurface, AODC values varied from layer to layer, the highest being found in samples from sandy aquifer formations and the lowest in clayey interbed layers. Sandy aquifer sediments also contained the highest numbers of viable bacteria as determined by aerobic spread plate counts (CFU) on a dilute heterotrophic medium. In some of these samples bacterial CFU values approached 100% of the AODC values. Viable protozoa (amoebae and flagellates, but no ciliates) were found in samples with high bacterial CFU values. A variety of green algae, phytoflagellates, diatoms, and a few cyanobacteria were found at low population densities in samples from two of the three boreholes. Low numbers of fungi were evenly distributed throughout the profiles at all three sites. Microbial population density estimates correlated positively with sand content and pore‐water pH, and negatively with clay content and pore‐water metal concentration. A large diversity of prokaryotic and eukaryotic microorganisms was found in samples with high population densities. A survey of bacterial strains isolated from subsurface samples revealed associations of gram‐positive bacteria with high clay sediments and gram‐negative bacteria with sandy sediments. The ability to deposit lipophilic storage material (presumably poly‐ß‐hydroxybutyrate) was found in a high proportion of isolates from sandy sediments, but only rarely in isolates from high clay sediments.     

Impact of taxon sampling on the estimation of rates of evolution at sites

The function of individual sites within a protein influences their rate of accepted point mutation. During the computation of phylogenetic likelihoods, rate heterogeneity can be modeled on a site-per-site basis with relative rates drawn from a discretized Gamma-distribution. Site-rate estimates (e.g., the rate of highest posterior probability given the data at a site) can then be used as a measure of evolutionary constraints imposed by function. However, if the sequence availability is limited, the estimation of rates is subject to sampling error. This article presents a simulation study that evaluates the robustness of evolutionary site-rate estimates for both small and phylogenetically unbalanced samples. The sampling error on rate estimates was first evaluated for alignments that included 5-45 sequences, sampled by jackknifing, from a master alignment containing 968 sequences. We observed that the potentially enhanced resolution among site rates due to the inclusion of a larger number of rate categories is negated by the difficulty in correctly estimating intermediate rates. This effect is marked for data sets with less than 30 sequences. Although the computation of likelihood theoretically accounts for phylogenetic distances through branch lengths, the introduction of a single long-branch outlier sequence had a significant negative effect on site-rate estimates. Finally, the presence of a shift in rates of evolution between related lineages can be diagnostic of a gain/loss of function within a protein family. Our analyses indicate that detecting these rate shifts is a harder problem than estimating rates. This is so, partially, because the difference in rates depends on two rate estimates, each with an intrinsic uncertainty. The performances of four methods to detect these site-rate shifts are evaluated and compared. Guidelines are suggested for preparing data sets minimally influenced by error introduced by sequence sampling.     

Patterns in vegetation, hydrology, and nutrient availability in an undisturbed river floodplain in Poland

In the undisturbed floodplain of the Biebrza river (N.E. Poland) wecompared vegetation composition, standing crop and the nutrients in standingcrop to site factors such as flood duration and inundation depth during springfloods, summer water levels and concentrations of chemical constituents inwaterand nutrient release rates from peat. Our analysis shows a number of clearspatial patterns of biotic and abiotic variables in the ca. 1 kmwide river marginal wetland. The distribution of vegetation types follows acertain pattern: Glycerietum maximae close to the river,followed by respectively Caricetum gracilis andCaricetum elatae and finally Calamagrostietumstrictae at the margin of the river plain. Species richnessincreasesand standing crop decreases from the river towards the margin. The elevation ofthe ground surface gently rises with increasing distance from the river; floodduration and flooding depths in spring decrease in the same direction.Groundwater tables in summer are less correlated to the elevation gradient buttend to be closer to the ground surface at the valley margin. These differencesalso lead to a higher amplitude close to the river and a fairly stable watertable far away from the river. Concentrations of major ions and ammoniumincrease towards the river. Nutrient release rates are also higher closer totheriver. Absence and presence of species and the variation in species compositionof the vegetation was explained best by flood variables; variables fromgroundwater explained much less of the variance. Variations in standing cropandnutrients in standing crop corresponded better to the rates of nutrient releasefrom the organic soil than to nutrient concentrations in the soil water. Weconcluded that river hydrology and nutrient release from the soil are clearlyrelated to vegetation composition, species richness and productivity of thevegetation.     

Discharge-driven flood and seasonal patterns of phytoplankton biomass and composition of an Australian tropical savannah river

Discharge is a primary determinant of river phytoplankton, but its influence can be mediated by climate, water quality and catchment development. The relationship between discharge and phytoplankton over seasonal and flood temporal scales was examined for the Daly River in the Australian tropical savannah. Phytoplankton, water quality and hydrographic data were collected over the high-discharge wet season and lower-discharge dry season. Wet season main channel river conditions were unfavourable for phytoplankton growth. Floods, however, connected the main channel to off-channel water bodies that supplied phytoplankton, predominately Cryptomonas, to the river. Wet season phytoplankton biovolume and cell concentrations were higher than in the dry season and comprised the majority of the annual phytoplankton biomass load. High discharges served to both temporally connect the river to off-channel phytoplankton sources as well as dilute these sources. In the dry season, the Daly River was groundwater-fed and disconnected from off-channel phytoplankton sources and dominated by a potamoplanktonic population of Cryptomonas and Peridinium. River phytoplankton were determined by the seasonal discharge regime which drove water quality and underpinned a shift from highly productive, spatially extensive off-channel sources in the wet season to less productive more spatially confined in-channel sources in the dry season.     

Isolation and characterization of metal-reducing thermoanaerobacter strains from deep subsurface environments of the Piceance Basin,Colorado

Five bacterial strains were isolated from anaerobic enrichment cultures that had originated from inoculations with samples collected from the deep subsurface environments of the millions-of-years-old, geologically and hydrologically isolated Piceance Basin in Colorado. Small-subunit rRNA gene-based analyses indicated that all of these bacteria were closely related to Thermoanaerobacter ethanolicus, with similarities of 99.4 to 99.5%. Three isolates (X513, X514, and X561) from the five bacterial strains were used to examine physiological characteristics. These thermophilic bacteria were able to use acetate, glucose, hydrogen, lactate, pyruvate, succinate, and xylose as electron donors while reducing Fe(III), cobalt(III), chromium(VI), manganese(IV), and uranium(VI) at 60 degrees C. One of the isolates (X514) was also able to utilize hydrogen as an electron donor for Fe(III) reduction. These bacteria exhibited diverse mineral precipitation capabilities, including the formation of magnetite (Fe(3)O(4)), siderite (FeCO(3)), rhodochrosite (MnCO(3)), and uraninite (UO(2)). The gas composition of the incubation headspace and the ionic composition of the incubation medium exerted profound influences on the types of minerals formed. The susceptibility of the thermophilic Fe(III)-reducing cultures to metabolic inhibitors specific for ferric reductase, hydrogenase, and electron transport indicated that iron reduction by these bacteria is an enzymatic process.