A culture-independent molecular analysis of archaeal communities in waters collected from deep South African gold mines was performed by performing a PCR-mediated terminal restriction fragment length polymorphism (T-RFLP) analysis of rRNA genes (rDNA) in conjunction with a sequencing analysis of archaeal rDNA clone libraries. The water samples used represented various environments, including deep fissure water, mine service water, and water from an overlying dolomite aquifer. T-RFLP analysis revealed that the ribotype distribution of archaea varied with the source of water. The archaeal communities in the deep gold mine environments exhibited great phylogenetic diversity; the majority of the members were most closely related to uncultivated species. Some archaeal rDNA clones obtained from mine service water and dolomite aquifer water samples were most closely related to environmental rDNA clones from surface soil (soil clones) and marine environments (marine group I [MGI]). Other clones exhibited intermediate phylogenetic affiliation between soil clones and MGI in the Crenarchaeota. Fissure water samples, derived from active or dormant geothermal environments, yielded archaeal sequences that exhibited novel phylogeny, including a novel lineage of Euryarchaeota. These results suggest that deep South African gold mines harbor novel archaeal communities distinct from those observed in other environments. Based on the phylogenetic analysis of archaeal strains and rDNA clones, including the newly discovered archaeal rDNA clones, the evolutionary relationship and the phylogenetic organization of the domain Archaea are reevaluated.Recent molecular phylogenetic analyses based on small-subunit (SSU) rRNA gene (rDNA) sequencing have revealed that the phylogenetic diversity of Archaea in naturally occurring microbial communities is much greater than previously assumed on the basis of the results obtained with standard cultivation and isolation methods (3,6,14,15,20,24,43,45). Initially, a small collection of isolates was referred to as archaebacteria, and now this varied assemblage is known to be both ubiquitous and cosmopolitan. Molecular phylogenetic approaches have revealed that environmental archaeal populations are both diverse and complex, often consisting of uncultivated and unidentified members. Because pure-culture phenotypic characterizations of many environmental Archaea are currently not possible, the physiological features and ecological significance of archaeal communities remain difficult to assess. The phylogenetic structure derived from archaeal rDNA clones from a given habitat, however, frequently corresponds to measurable environmental constraints (8, 42). When phylogenetic features intrinsic to archaeal communities are related to the environment, they may provide important insights into the physiological functions and ecological roles of the communities.The gold mines of South Africa are the deepest accessible excavations in the world and provide a unique opportunity for direct exploration of the deep s...

[1] H 2 is probably the most important substrate for terrestrial subsurface lithoautotrophic microbial communities. Abiotic H 2 generation is an essential component of subsurface ecosystems truly independent of surface photosynthesis. Here we report that H 2 concentrations in fracture water collected from deep siliclastic and volcanic rock units in the Witwatersrand Basin, South Africa, ranged up to two molar, a value far greater than observed in shallow aquifers or marine sediments. The high H 2 concentrations are consistent with that predicted by radiolytic dissociation of H 2 O during radioactive decay of U, Th, and K in the host rock and the observed He concentrations. None of the other known H 2 -generating mechanisms can account for such high H 2 abundance either because of the positive free energy imposed by the high H 2 concentration or pH or because of the absence of required mineral phases. The radiolytic H 2 is consumed by methanogens and abiotic hydrocarbon synthesis. Our calculations indicate that radiolytic H 2 production is a ubiquitous and virtually limitless source of energy for deep crustal chemolithoautotrophic ecosystems.

Seasonal changes in total bacterial numbers and their associated mucus coatings in surficial sediments were examined. Bacterial numbers followed the temperature cycle, with highest numbers in summer. The specific surface areas of the sediments were measured rather than inferred from other granulometric properties; bacterial numbers were proportional to surface areas only for sample suites collected at the same time. Bacteria inhabited shallow depressions on sand and silt grains; they were not found on grains smaller than about 10 µm or inside smaller pores like those on weathered feldspar grains. Mucus coatings also followed a seasonal cycle, increasing in abundance and coalescence from spring into summer. These coatings accumulated clay grains, suggesting that the relationship of bacteria to surface area may be due to bacterial control of surface area rather than the reverse. Organic carbon concentrations in grain size separates of these sediments increased with decreasing size until the fine silt fraction, and decreased in the clay fraction; it is not clear, however, whether this trend is a result or a cause of bacterial colonization patterns.

Previous bacterial transport studies have utilized fluorophores which have been shown to adversely affect the physiology of stained cells. This research was undertaken to identify alternative fluorescent stains that do not adversely affect the transport or viability of bacteria. Initial work was performed with a groundwater isolate, Comamonas sp. strain DA001. Potential compounds were first screened to determine staining efficiencies and adverse side effects. 5-(And 6-)-carboxyfluorescein diacetate, succinimidyl ester (CFDA/SE) efficiently stained DA001 without causing undesirable effects on cell adhesion or viability. Members of many other gram-negative and gram-positive bacterial genera were also effectively stained with CFDA/SE. More than 95% of CFDA/SEstained Comamonas sp. strain DA001 cells incubated in artificial groundwater (under no-growth conditions) remained fluorescent for at least 28 days as determined by epifluorescent microscopy and flow cytometry. No differences in the survival and culturability of CFDA/SE-stained and unstained DA001 cells in groundwater or saturated sediment microcosms were detected. The bright, yellow-green cells were readily distinguished from autofluorescing sediment particles by epifluorescence microscopy. A high throughput method using microplate spectrofluorometry was developed, which had a detection limit of mid-10 5 CFDA-stained cells/ml; the detection limit for flow cytometry was on the order of 1,000 cells/ml. The results of laboratory-scale bacterial transport experiments performed with intact sediment cores and nondividing DA001 cells revealed good agreement between the aqueous cell concentrations determined by the microplate assay and those determined by other enumeration methods. This research indicates that CFDA/SE is very efficient for labeling cells for bacterial transport experiments and that it may be useful for other microbial ecology research as well.

Dissolved DNA and a series of microbial biomass and activity parameters were measured in offshore, coastal, estuanne and coral reef environments of the southeast Gulf of Mexico. Oceanic concentrations of dissolved DNA ranged from 0.2 to 19 vg 1-' and decreased as a function of distance from shore and depth in the water column. Dissolved DNA concentrations were greater than half the particulate DNA content in offshore environments (Z = 63 + 45 O/O), but were a smaller percentage of particulate DNA in nearshore and estuarine environments (Z = 35 f 21 %). Dissolved DNA correlated better with bacterial parameters (i.e. bacterial direct counts, particulate DNA and thymidine incorporation) than with phytoplankton parameters (chlorophyll a, primary productivity). The molecular weight (MW) of dissolved DNA (determined by agarose gel electrophoresis) ranged from 0.12 kilobase pairs (kb; 7.75 X 104 daltons) to 35.2 kb (2.32 X 10' daltons) for estuarine samples, while an oligotrophic environment contained smaller MW DNA (range 0.24 to 14.27 kb). DNA fragments in this size range are sufficient to contain gene sequences. These results are discussed in terms of the potential for transformation by dissolved DNA.