Dana L. Haldeman

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.

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.

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.

Comparison of drilling and mining as methods for obtaining microbiological samples from the deep subsurface

Abstract Ashfall tuff samples for microbiological analysis were obtained by mining and drilling within a 400 m deep tunnel system at Rainier Mesa, Nevada Test Site. Comparison of microbiota revealed that bacteria recovered from the core samples were similar to those recovered from the mined samples in abundance, diversity, evenness of distribution, and the numbers of distinct colony types. Cluster analyses based on the characterization of one of each distinct bacterial type from cored and mined samples indicated morphological and physiological similarities between some of the microbiota that were recovered. Drilling fluid microbiota were more abundant, and were distinct from those recovered from cored or mined samples. Storage of both the mined and cored samples for 1 week at 4°C led to the recovery of increased numbers of culturable cells, but with decreased diversity. The presence of bromide tracer indicated penetration of drilling fluids into the cored rock samples, a problem not encountered with mining. However, abundance and diversity values, and isolate characterization indicate that valuable information can be obtained from cored rock samples.

Heterogeneity of deep subsurface microorganisms and correlations to hydrogeological and geochemical parameters

Nineteen samples were obtained from a 21‐m3 section of zeolitized volcanic ash‐fall tuff, 390 m below the surface of Rainier Mesa, Nevada. Rock mined aseptically from the walls of deep subsurface tunnels provided pristine samples for microbiological and geochemical analyses. Microbiological parameters measured on all samples included direct counts and the abundance, diversity, morphology, and metabolic traits of culturable organisms. Physical and chemical parameters measured included ionic and nutrient chemistries, mineralogy, porosity, moisture content, and permeability. The results indicate that the culturable microbiological community size and composition exhibit random spatial variability within the geologically/geochemically homogeneous rock section. The relative abundance of microorganisms testing positive for nitrate reductase demonstrated a spatial trend along the vertical and front‐to‐back axes of the rock section by gradient analysis. The porewater concentration of nitrate correlated with number...

Comparison of the microbiota recovered from surface and deep subsurface rock, water, and soil along an elevational gradient

The isolation of viable microorganisms from deep volcanic rock formations (50–450 m) within Rainier Mesa, Nevada Test Site has posed questions regarding the origin of in situ bacteria. One hypothesis under investigation is that microbial transport to depth has been facilitated via recent natural water flow from surface bacterial populations. Recoverable microbiota from surface topsail, paleosol, rock, and outflow spring water were compared to those from subsurface rock and fracture flow water to determine if relationships existed between microbial communities along an elevational gradient. Direct counts were higher than culturable counts in all samples. Top‐soil samples had higher culturable counts and numbers of nitrogen‐fixing and sulfur‐reducing bacteria than samples from the subsurface rock. Heterogeneity in microbial communities from the different samples (measured by diversity and evenness indices and by comparison of morphological and physiological tests of representative isolates) was demonstrated...

Laboratory and field evidence for long-term starvation survival of microorganisms in subsurface terrestrial environments

Biogeochemical modeling of groundwater flow and nutrient flux in subsurface environments indicates that inhabitant microorganisms experience severe nutrient limitation. Using laboratory and field methods, we have been testing starvation survival in subsurface microorganisms. In microcosm experiments, we have shown that strains of two commonly isolated subsurface genera, Arthrobacter and Pseudomonas, are able to maintain viability in low-nutrient, natural subsurface sediments for over one year. These non- spore-forming bacteria undergo rapid initial miniaturization followed by a stabilization of cell size. Membrane lipid phospholipid fatty acid (PLFA) profiles of the Pseudomonas are consistent with adaptation to nutrient stress; Arthrobacter apparently responds to nutrient deprivation without altering membrane PLFAs. To test survivability of microorganisms over a geologic time scale, we characterized microbial communities in a sequence of unsaturated sediments ranging in age from modern to > 780,000 years. Sediments were relatively uniform silts in eastern Washington State. Porewater ages at depth (measured by the chloride mass- balance approach) were as old as 3,600 years. Microbial abundance, biomass, and activities (measured by direct counts, culture counts, total PLFAs, and radiorespirometry) declined with sediment age. The pattern is consistent with laboratory microcosm studies of microbial survival: rapid short-term change followed by long-term survival of a proportion of cells. Even the oldest sediments evinced a small but viable microbial community. Microbial survival appeared to be a function of sediment age. Porewater age appeared to influence the makeup of surviving communities, as indicated by PLFA profiles. Sites with different porewater recharge rates and patterns of Pleistocene flooding had different communities. These and other studies provide evidence that microorganisms can survive nutrient limitation for geologic time periods.