Carl B. Fliermans

Microbial activities in deep subsurface environments

Abstract Activities of microorganisms residing in terrestrial deep subsurface sediments were examined in 46 sediment samples from three boreholes. Radiolabeled time course experiments assessing in situ microbial activities were initiated within 30 min of core recovery. [1‐C4] Acetate incorporation into lipids, [ methyl‐3H] thymidine incorporation into DNA, [2‐14C]acetate, and [U‐14C]glucose mineralization in addition to microbial enrichment and enumeration studies were examined in surface and subsurface sediments. Surface soils contained the greatest biomass and activities, followed by the shallow aquifer zones. Water‐saturated subsurface sands exhibited three to four orders of magnitude greater activity and culturable microorganisms than the dense clay zones, which had low permeability. Regardless of depth, sediments that contained more than 20% clays exhibited the lowest activities and culturable microorganisms.

Methods for recovery of deep terrestrial subsurface sediments for microbiological studies

Methods for the aseptic recovery of sediments from the terrestrial deep subsurface for microbiological analyses are defined. Sediments were recovered from depths > 300 m by rotary drilling techniques using bentonite drilling techniques. Four sampling tools were successfully used and compared for their ability to retrieve different types of subsurface materials. Upon retrieval, sediments were pared and processed under anaerobic conditions in a glove bag. Materials were stored under N2 gas and shipped via overnight express to collaborating investigators. Six quality assurance protocols were incorporated to ensure that appropriate sediments were obtained and to monitor contamination from drilling fluid infringement. Two quality assurance protocols were field-applicable, and four were performed by independent laboratories. The quality assurance protocols provided multiple techniques for detecting 10 mg contamination from drilling fluids·kg−1 sediment. These techniques, which proved appropriate for different types of subsurface sediments, provided samples which were deemed acceptable for microbiological analyses.

Geology and hydrology of the deep subsurface microbiology sampling sites at the savannah river plant, South Carotina

Abstract This paper describes important hydrologic features of the deep subsurface microbiology sampling sites. Geologic history, depositional environments, statigraphy, and groundwater movement are discussed in relation to possible mechanisms of microbial colonization of deep aquifer formations beneath the Savannah River Plant.

Microbial life in deep terrestrial subsurfaces

In 1985 the US Department of Energy (DOE) established a research program called Microbiology of the Deep Subsurface, which focuses on detecting microorganisms at greater depths, establishing fundamental scientific information, including their ecology, and exploring their potential use in clean-up of contaminated deep terrestrial sediments and groundwater environments associated with energy and defense production activities. This article describes the initial microbiological findings associated with three boreholes established at the Savannah River Plant (SRP) in Aiken, South Carolina, to a depth of 289 m beneath the soil surface and discusses how microorganisms may have come to live at these depths. Even more recent investigations have demonstrated the presence of diverse and abundant microbiological communities as deep as 520 m beneath the soil surface.

Growth ofLegionella pneumophila in two-membered cultures with green algae and cyanobacteria

Environmental and clinical isolates ofLegionella pneumophila were grown in minimal-salts media (no organic compounds added) in associated with various green algae and cyanobacteria. Growth was observed to level off after a period of hours to days with no subsequent significant loss in the numbers of viableL. pneumophila even several days after growth had ceased. Transfer to new algal or cyanobacterial cultures resulted in a new burst of growty by theL. pneumophila.

Microbial biomass and activities associated with subsurface environments contaminated with chlorinated hydrocarbons

Abstract Soil microcosms and enrichment cultures from subsurface sediments and groundwaters contaminated with trichloroethylene (TCE) were examined. Total lipids, [I‐‘4C]acetate incorporation into lipids, and [Me‐3H]thymidine incorporation into DNA were determined in these subsurface environments. In heavily TCE‐contam‐inated zones (greater than 500 mg/L) radioisotopes were not incorporated into lipids or DNA. Radioisotope incorporation occurred in sediments both above and below the TCE plume. Phospholipid fatty acids (PLFA) were not detected, i.e., less than 0.5 pmol/L in heavily contaminated groundwater samples. In less contaminated waters, extracted PLFA concentrations were greater than 100 pmollL and microbial isolates were readily obtained. Degradation of 30–100 mg/L TCE was observed when sediments were amended with a variety of energy sources. Microorganisms in these subsurface sediments have adapted to degrade TCE at concentrations greater than 50 mg/L.

Utility of radiotracer activity measurements for subsurface microbiology studies

Radiotracer activity measurements were conducted on subsurface sediments collected from the Savannah River Plant, Aiken, SC. Sediments were aseptically extruded from stainless steel core liners into a nitrogen flushed glove bag. Subsurface materials were immediately inoculated into aerobic and/or anaerobic tubes for time course experiments. Mineralization experiments utilized 14C-2-acetate and 14C-UL-glucose, while radiotracer uptake experiments included 14C-1-acetate incorporation into lipids and 3H-methyl-thymidine incorporation into microbial DNA. Microbiological activity of subsurface sediments varied 5 orders of magnitude between dense compacted clays and water-bearing sands. Aquifers tens of meters beneath the earths surface exhibited activities greater than some of the near-surface soils. Radiotracer techniques proved to be sensitive, reproducible and applicable to field implementation. Agreement was observed between water abundance, sand content and microbial activities.

Measure ofLegionella pneumophila activity in situ

Detection ofLegionella pneumophila by serogroup-specific fluorescent antibodies was combined with a tetrazolium dye (INT) to measure electron transport activity. The biological uptake and reduction of the INT dye was studied in pure cultures and in natural water samples with respect to temperature. Uptake was complete within 60 min. Controls inhibited with formaldehyde demonstrated little activity. Both the in vitro and in situ determinations suggested that the electron transport system ofLegionella was active over a temperature range of 25 to 60°C.

Productivity and species composition of algal mat communities exposed to a fluctuating thermal regime

Algal mat communities growing in thermal effluents of production nuclear reactors at the Savannah River Plant, near Aiken, SC, are exposed to large temperature fluctuations resulting from reactor operations. Rates of primary production and species composition were monitored at 4 sites along a thermal gradient in a trough microcosm to determine how these large temperature fluctuations affected productivity and algal community structure. Blue-green algae (cyanobacteria) were the only phototrophic primary producers growing in water above 45°C. These thermophiles were able to survive and apparently adapt to ambient temperatures when the reactor was shut down. The algal mat communities exposed to <45°C were composed of blue-green and eukaryotic algae that adapted rapidly to ambient temperatures. An increase in the percentage extracellular release (PER) of14C-labeled dissolved organic compounds and a decrease in primary production were observed during periods of thermal fluctuation. The results show that the dominant phototrophs in this artificially heated aquatic habitat have been selected for their ability to survive large temperature fluctuations and are similar to those of natural hot springs.

Mineralization of trichloroethylene by heterotrophic enrichment cultures

Microbial consortia capable of aerobically degrading more than 99% of exogenous trichloroethylene (TCE) (50 mg/liter) were collected from TCE-contaminated subsurface sediments and grown in enrichment cultures. TCE at concentrations greater than 300 mg/liter was not degraded, nor was TCE used by the consortia as a sole energy source. Energy sources which permitted growth included tryptone-yeast extract, methanol, methane, and propane. The optimum temperature range for growth and subsequent TCE consumption was 22 to 37 degrees C, and the pH optimum was 7.0 to 8.1. Utilization of TCE occurred only after apparent microbial growth had ceased. The major end products recovered were hydrochloric acid and carbon dioxide. Minor products included dichloroethylene, vinylidine chloride, and, possibly, chloroform.