Factors limiting microbial growth and activity at a proposed high-level nuclear repository, yucca mountain, nevada.

As part of the characterization of Yucca Mountain, Nev., as a potential repository for high-level nuclear waste, volcanic tuff was analyzed for microbial abundance and activity. Tuff was collected aseptically from nine sites along a tunnel in Yucca Mountain. Microbial abundance was generally low: direct microscopic cell counts were near detection limits at all sites (3.2 x 10(sup4) to 2.0 x 10(sup5) cells g(sup-1) [dry weight]); plate counts of aerobic heterotrophs ranged from 1.0 x 10(sup1) to 3.2 x 10(sup3) CFU g(sup-1) (dry weight). Phospholipid fatty acid concentrations (0.1 to 3.7 pmol g(sup-1)) also indicated low microbial biomasses; diglyceride fatty acid concentrations, indicative of dead cells, were in a similar range (0.2 to 2.3 pmol g(sup-1)). Potential microbial activity was quantified as (sup14)CO(inf2) production in microcosms containing radiolabeled substrates (glucose, acetate, and glutamic acid); amendments with water and nutrient solutions (N and P) were used to test factors potentially limiting this activity. Similarly, the potential for microbial growth and the factors limiting growth were determined by performing plate counts before and after incubating volcanic tuff samples for 24 h under various conditions: ambient moisture, water-amended, and amended with various nutrient solutions (N, P, and organic C). A high potential for microbial activity was demonstrated by high rates of substrate mineralization (as much as 70% of added organic C in 3 weeks). Water was the major limiting factor to growth and microbial activity, while amendments with N and P resulted in little further stimulation. Organic C amendments stimulated growth more than water alone.

Changes in Bacteria Recoverable from Subsurface Volcanic Rock Samples during Storage at 4 degrees C.

The abundance of viable microorganisms recovered from deep subsurface volcanic rock samples increased after rock perturbation and storage for 1 week at 4 degrees C, while the diversity and evenness of recoverable heterotrophic bacterial communities generally decreased. One sample of each morphologically distinct colony type, recovered both before and after storage of U12n rock samples, was purified and characterized by fatty acid methyl ester (MIDI) and API rapid NFT strips. As determined by MIDI cluster analysis, the composition of the recoverable microbial communities changed with storage of rock samples; some groups of organisms were recovered only before, only after, or at both sample times. In general, the isolates recovered only after storage of rock samples had a greater ability to utilize the carbohydrates included in API test strips and had faster generation times than isolates recovered only on initial plating. The nutritional versatility and faster growth rates of organisms recovered in higher proportions after sample storage provide evidence that some microbial community changes may be due to the proliferation of a few bacterial types. However, because some new genera are recovered only after storage, the possibility also exists that dormant bacterial types are resuscitated during sample perturbation and storage.

Diversity within a Colony Morphotype: Implications for Ecological Research.

Sets of bacterial isolates with the same colony morphologies were selected from spread plates of bacteria from deep subsurface rock samples; each set had a unique morphology. API-rapid-NFT analysis revealed that isolates within a set were the same. Fatty acid methyl ester analysis of one set of isolates clustered organisms within the same species, defining variation between isolates at the biotype (subspecies) and strain levels. Metal resistances consistently tracked with colony morphology, while antibiotic resistances were less reliable.

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 × 10(1) to 2.4 × 10(5) 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 × 10(2) 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 × 10(3) cells g dry wt(-1) of rock sample.

Characterization of the microbiology within a 21 m(3)section 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 [Symbol: see text]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 [Symbol: see text]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.

Comparison of Identification Systems for Classification of Bacteria Isolated from Water and Endolithic Habitats within the Deep Subsurface.

One water and three rock samples were taken from a mined tunnel system, U12n, in Rainier Mesa at the Nevada Test Site. Endolithic microorganisms were cultured from ashfall tuff, which was crushed and made into slurries with a formulation of artificial pore water, on R2A agar plates. Microbial counts ranged from 10 to 10 viable cells per g (dry weight) of rock sampled. The cultured water sample yielded 10 viable cells per ml. Many of the isolates were very small (<1 mum) when viewed in the rock matrix and remained small even when cultured. Most were gram-negative rods. Individual isolates were profiled by API-NFT strip number, antibiotic and metal resistance patterns, and colony and cellular morphologies. Three identification systems, API-NFT strips, BIOLOG, and MIDI, were compared. Each system identified only a small percentage of the total isolates, and in only seven cases were the isolates identified the same way by more than one system. The same genus was identified in three of these cases, but different species were indicated. The genus Pseudomonas was the most commonly identified. The isolate profiles and the three identification systems demonstrated that water isolates were considerably different from endolithic isolates.