Penny S. Amy

The microbiology of the terrestrial deep subsurface

Denizens of the Deep. Considerations for Sampling. Geohydrologic and Geochemical Characterization. Methods for Obtaining Deep Subsurface Microbiological Samples by Drilling. The Collection of Subsurface Samples by Mining. The Storage-Related Phenomenon: Implications for Handling and Analysis of Subsurface Samples. Microbial Heterogeneity in the Terrestrial Subsurface and Approaches for Its Description. Microbial Ecology and Related Methods. Identity and Diversity of Microorganisms Cultured from Subsurface Environments. Utility of the Signature Lipid Biomarker Analysis in Determining the In Situ Viable Biomass, Community Structure, and Nutritional/Physiological Status of Deep Subsurface Microbiota. Life in the Slow Lane: Activities of Microorganisms in the Subsurface. Phylogenetic Analysis and Implications for Subsurface Microbiology. Microbial Dormancy and Survival in the Subsurface. Subsurface Microbiology and the Evolution of the Biosphere. Movement of Bacteria in the Subsurface. Applications. Bioremediation. European Microbiology Related to the Subsurface Disposal of Nuclear Waste. Subsurface Microbiology: Effects on the Transport of Radioactive Wastes in the Vadose Zone. Biofilm Processes in Porous Media-Practical Applications. NTI Copy: Already written for June 1996 sales meeting.

Microbial Abundance and Activities in Relation to Water Potential in the Vadose Zones of Arid and Semiarid Sites

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.

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.

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.

Boundaries for Biofilm Formation: Humidity and Temperature

ABSTRACT Environmental conditions which define boundaries for biofilm production could provide useful ecological information for biofilm models. A practical use of defined conditions could be applied to the high-level nuclear waste repository at Yucca Mountain. Data for temperature and humidity conditions indicate that decreases in relative humidity or increased temperature severely affect biofilm formation on three candidate canister metals.

Effect of Gamma Radiation on Native Endolithic Microorganisms from a Radioactive Waste Deposit Site

A time-course experiment was conducted to evaluate the effects of gamma radiation on the indigenous microbiota present in rock obtained from Yucca Mountain, Nevada Test Site. Microcosms were constructed by placing pulverized Yucca Mountain rock in polystyrene cylinders. Continuous exposure (96 h) at a dose rate of 1.63 Gy/min was used to mimic the near-field environment surrounding waste canisters. The expected maximum surface dose rate from one unbreached canister designed to contain spent nuclear fuels is 0.06 Gy/min. Considering the current repository packing design, multiple canisters within one vault, the cumulative dose rate may well approach that used in this experiment. The microbial communities were characterized after receiving cumulative doses of 0, 0.098, 0. 58, 2.33, 4.67, 7.01 and 9.34 kGy. Radiation-resistant microorganisms in the pulverized rock became viable but nonculturable (VBNC) after a cumulative dose of 2.33 kGy. VBNC microorganisms lose the ability to grow on media on which they have routinely been cultured in response to the environmental stress imposed (i.e. radiation) but can be detected throughout the time course using direct fluorescence microscopy techniques. Two representative exopolysaccharide-producing isolates from Yucca Mountain were exposed to the same radiation regimen in sand microcosms. One isolate was much more radiation-resistant than the other, but both had greater resistance than the general microbial community based on culturable counts. However, when respiring cell counts (VBNC) were compared after irradiation, the results would indicate much more radiation resistance of the individual isolates and the microbial community in general. These results have significant implications for underground storage of nuclear waste as they indicate that indigenous microorganisms are capable of surviving gamma irradiation in a VBNC state.

Microbial Transport, Survival, and Succession in a Sequence of Buried Sediments

A bstractTwo chronosequences of unsaturated, buried loess sediments, ranging in age from <10,000 years to >1 million years, were investigated to reconstruct patterns of microbial ecological succession that have occurred since sediment burial. The relative importance of microbial transport and survival to succession was inferred from sediment ages, porewater ages, patterns of abundance (measured by direct counts, counts of culturable cells, and total phospholipid fatty acids), activities (measured by radiotracer and enzyme assays), and community composition (measured by phospholipid fatty acid patterns and Biolog substrate usage). Core samples were collected at two sites 40 km apart in the Palouse region of eastern Washington State, near the towns of Washtucna and Winona. The Washtucna site was flooded multiple times during the Pleistocene by glacial outburst floods; the Winona site elevation is above flood stage. Sediments at the Washtucna site were collected from near surface to 14.9 m depth, where the sediment age was ∼250 ka and the porewater age was 3700 years; sample intervals at the Winona site ranged from near surface to 38 m (sediment age: ∼1 Ma; porewater age: 1200 years). Microbial abundance and activities declined with depth at both sites; however, even the deepest, oldest sediments showed evidence of viable microorganisms. Same-age sediments had equal quantities of microorganisms, but different community types. Differences in community makeup between the two sites can be attributed to differences in groundwater recharge and paleoflooding. Estimates of the microbial community age can be constrained by porewater and sediment ages. In the shallower sediments (<9 m at Washtucna, <12 m at Winona), the microbial communities are likely similar in age to the groundwater; thus, microbial succession has been influenced by recent transport of microorganisms from the surface. In the deeper sediments, the populations may be considerably older than the porewater ages, since microbial transport is severely restricted in unsaturated sediments. This is particularly true at the Winona site, which was never flooded.

Starvation-survival of deep subsurface isolates

Six deep, subsurface endolithic isolates were subjected to starvation conditions for up to 100 days in artificial pore water, formulated to mimic in situ geochemical conditions in the nearly saturated rock. Most isolates demonstrated the typical starvation-survival curve for chemoheterotrophic bacteria, and all became miniaturized during starvation. Starvation indices were developed to compare changes in viable cell counts between isolates. Two isolates retained higher viability after 100 days of starvation-survival. High survival correlated with sustained respiration, measured by iodonitrotetrazolium-formazan production, during starvation. In all but one case, isolates plated on two nutritionally dilute media, metal-containing and antibioticcontaining media, showed similar viable counts.

Time-dependent changes in viable numbers and activities of aerobic heterotrophic bacteria in subsurface samples

Vadose and saturated zone sediment cores from depths to 212 m were obtained from the U.S. Department of Energys Hanford Site in south-central Washington by cable tool drilling, and volcanic ashfall tuff samples were obtained from tunnels 400 m beneath the surface of Rainier Mesa at DOEs Nevada Test Site (NTS) in southern Nevada. Numbers of viable aerobic heterotrophic bacteria were determined by plate counts and metabolic activities were determined by [14C]glucose radiorespirometry at t = 0 and at various post-sampling time points up to 154 d in order to assess the influence of sample storage on microbiological properties of subsurface samples. Increases in post-sampling populations of viable bacteria were observed in all samples, although the magnitude of the increase and time after sampling at which the maximum population size was reached varied with the sample type. The greatest post-sampling increases in viable counts and [14C]glucose mineralization occurred in a high-organic carbon lacustrine sediment. The population of aerobic heterotrophs increased from below detection at t = 0 to > 106 CFU g−1 after 139 d. Significant increases in culturable counts were shown to occur within 24 h for tuff samples from NTS. These results indicate that precautions are necessary in the post-drilling handling of subsurface sediments and rock for microbiological analysis. In addition, these results suggest that even low biomass subsurface environments may be readily stimulated for applications such as in situ bioremediation.

Resuscitation of microorganisms after gamma irradiation.

Microbiological analysis of rock exposed to gamma-radiation doses between 0 and 9.34 kGy indicated that some microorganisms became viable but nonculturable (VBNC) and lost metabolic capacity as measured by BIOLOG microtiter plates. To investigate this phenomenon, portions of irradiated rock were placed at 4 degrees C for 2 months in an attempt to resuscitate the microbes to a culturable state. Culturable heterotrophs were enumerated and BIOLOG plates were used to determine the metabolic capability of the microbial community. Culturable bacteria that had previously been nonculturable were found at all doses. The number of colony types decreased from 26 in the nonirradiated control rock to between 9 and 10 in rock irradiated at doses ranging from 2.34 to 9.34 kGy. BIOLOG plates indicated partial recovery of metabolic capacity in all the samples tested. Fatty acid methyl ester analysis of the recovered isolates using the MIDI system (Microbial ID, Inc.) yielded three distinct groups of related bacteria. All resuscitated isolates clustered with the original nonirradiated isolates at the genus level, and 92% of them clustered at the species level. These results indicate that microbes were likely resuscitated from a VBNC state.