Notes: Improved understanding of the spatial and temporal distribution of microbiological properties and processes is critical due to the relative difficulty and high cost of obtaining large numbers of subsurface samples. Quantification of spatial patterns in subsurface environments is important because it is well known that geologic, hydrologic and geochemical properties are not constant in space; rather, they are spatially autocorrelated, or related over certain length scales. Preliminary research indicates that subsurface microbiological properties have similar length scales, and the microbiological properties appear to be spatially correlated to geologic, hydrologic and/or geochemical properties. Temporal variability can also be important in subsurface systems that receive seasonal recharge. In order to better understand heterogeneous subsurface systems, it is critical to sample such that the spatial and temporal patterns are adequately captured, and understand what is causing the variability and spatial patterns. Improved understanding in these two areas will yield more efficient sampling schemes, assist in defining factors that control the distribution of microbiological properties at the field scale, and increase the ability to predict and ultimately model the distribution of microbiological properties and the responses of microbial communities to environmental perturbations such as subsurface contaminant transport and bioremediation.

Notes: Total community DNA was isolated from a series of low-biomass subsurface sediments immediately after coring, either maintained as intact cores or homogenized and maintained at 17°C for up to 21 weeks. Eubacterial 16S rRNA genes were recovered by PCR amplification, and full-length, cloned genes were digested with CfoI. Resulting restriction enzyme patterns were used to group clones into specific RFLP groups. The abundance and distribution of individual clones within the RFLP groups was used to assess the changes in community structure as a function of storage time and sample condition (i.e., intact versus homogenized). Ninety-eight different RFLP groups were identified in toto. Large subsets of new RFLP groups were continuously identified at increasing times post-sampling. Only 12 specific RFLP patterns were identified in both homogenized and intact samples, however, indicating that the response of these communities varied significantly between the homogenized and intact sediments. Further, a comparison of clone libraries from the multiple sample treatments provided evidence that the relative abundance of clones within specific RFLP groups reflected a change in the abundance of that specific RFLP group within the microbial community. These results support the hypothesis that both growth and resuscitational processes are responsible for post-sampling stimulation of subsurface microorganisms. These and similar approaches will further enhance our ability to more rigorously analyze the composition and structure of low-biomass (e.g., 〈105 cells g−1), subsurface microbial communities.

Notes: Culture-based techniques have traditionally been the primary tools utilized for studying the microbiology of terrestrial subsurface environments. Recently, nucleic acid-based methods have been employed to further characterize the microbial diversity in subsurface sediments and rocks, but the results have not been related to individual bacteria cultivated from the same environment. Restriction fragment length profiles of 16S rRNA genes derived from bulk community DNA or bacterial isolates were compared to determine the efficacy of PCR-based methods for studying microbial diversity and phylogeny in a deep (188 m) subsurface environment. The phylogenetic relatedness between 16S rRNA genes of enrichment cultures and individual clones was also determined through DNA sequence analysis of 16S rRNA genes. Restriction fragment length profiles from PCR clone libraries accounted for 64% of recovered isolates and 55% of the estimated culturable diversity based upon their 16S rDNA RFLP signatures. DNA sequence comparisons between the 16S rDNA of the most commonly occurring isolates and clones confirmed that similar DNA sequences were contained within the RFLP groups used to categorize the isolates and clones. For 7 of 8 RFLP groups for which DNA sequences were obtained, nearest neighbor assignments corresponded at the genus level but suggested that 16S rDNA sequences from multiple genera were contained within single RFLP profiles. Phylogenetic analysis of 16S rRNA sequences supported the nearest neighbor inferences and indicated that 16S rDNA clones derived from bulk sediment were specifically related to isolates recovered on enrichment plates. This study has shown that a majority of the cultivated aerobic heterotrophic bacteria in a subsurface sediment could be described by 16S rDNA clones obtained from directly extracted DNA, but that PCR-based methods cannot account for all organisms from a given sample. Consequently, a more comprehensive assessment of microbial diversity in subsurface (and probably other) environments can be obtained by using a combination of culture- and molecular-based techniques than by using either method alone.

Notes: Abstract Numbers and activities of microorganisms were measured in the vadose zones of three arid and semiarid areas of the western United States, and the influence of water availability was determined. These low-moisture environments have vadose zones that are commonly hundreds of meters thick. The specific sampling locations chosen were on or near U.S. Department of Energy facilities: the Nevada Test Site (NTS), the Idaho National Engineering Laboratory (INEL), and the Hanford Site (HS) in southcentral Washington State. Most of the sampling locations were uncontaminated, but geologically representative of nearby locations with storage and/or leakage of waste compounds in the vadose zone. Lithologies of samples included volcanic tuff, basalt, glaciofluvial and fluvial sediments, and paleosols (buried soils). Samples were collected aseptically, either by drilling bore-holes (INEL and HS), or by excavation within tunnels (NTS) and outcrop faces (paleosols near the HS). Total numbers of microorganisms were counted using direct microscopy, and numbers of culturable microorganisms were determined using plate-count methods. Desiccation-tolerant microorganisms were quantified by plate counts performed after 24 h desiccation of the samples. Mineralization of 14C-labeled glucose and acetate was quantified in samples at their ambient moisture contents, in dried samples, and in moistened samples, to test the hypothesis that water limits microbial activities in vadose zones. Total numbers of microorganisms ranged from log 4.5 to 7.1 cells g-1 dry wt. Culturable counts ranged from log 〈2 to 6.7 CFU g-1 dry wt, with the highest densities occurring in paleosol (buried soil) samples. Culturable cells appeared to be desiccation-tolerant in nearly all samples that had detectable viable heterotrophs. Water limited mineralization in some, but not all samples, suggesting that an inorganic nutrient or other factor may limit microbial activities in some vadose zone environments.

Notes: Abstract Three unsaturated subsurface paleosols influenced by moisture recharge, including a highly developed calcic paleosol, were studied to investigate the microbiology of paleosols. Two near-surface paleosols, one impacted by moisture recharge and the other beyond the influence of recharge, were also sampled to directly assess the effect of moisture recharge on the activity and composition of the microbial community associated with paleosols. The highly developed paleosol had a higher population of culturable heterotrophs, a greater glucose mineralization potential, a higher microbial diversity based on colony morphology, and a more than 20-fold higher concentration of ATP than the two weakly developed paleosols. The recharged near-surface paleosol, as compared to the near-surface paleosol unaffected by recharge, had a lower population of culturable heterotrophs, smaller mineralization rate constant, and lower richness based on colony morphology. The recharged paleosols contained predominantly gram-negative isolates, whereas the paleosol unaffected by recharge contained predominantly gram-positive isolates. Storage at 4°C of subsurface and near-surface paleosol samples containing high water potential increased the population of culturable aerobic heterotrophs, decreased diversity in colony morphology, and increased first-order rate constants and decreased lag times for glucose mineralization. These results indicate that aerobic heterotrophs are present in deep vadose zone paleosols and that there is potential for stimulation of their in situ growth and activity.