Life on our planet may have evolved first at depth, then migrated to the surface as the environment became more tolerable. If this is so, the study of deep, subsurface microbial communities may provide another key to exploring our distant past. Ironically, these tiny creatures also may carry keys to our future.
by the DOE Subsurface Science Program's Taylorsville Basin Working Group
The subsurface microbial community constitutes a large fraction of the Earth's biomass, yet only a small fraction of it has been characterized. The microbiology above 50-m depth has been examined in various environments by many groups, but similar studies of deeper strata have been few and far between despite the interesting findings they have generated. One such study at the Atlantic coastal plain aquifers of the Savannah River, S.C., turned up a surprisingly large and diverse community occupying the sandy aquifers. At a crystalline bedrock site in Sweden, a large but homogenous subsurface community constrained to shear zones was found.
The U.S. Department of Energy's (DOE) Subsurface Science Program has actively pursued research in the deep (50-500 m) subsurface biosphere since 1985 at sites in Washington, Nevada, and Idaho, and in 1992, investigation began at a site in the Taylorsville Basin, Va.
Many federally funded research institutions, as well as industrial sites and military bases, have significant groundwater pollution at depths greater than 50 m. Cleaning up these sites with current technologies would be a trillion dollar enterprise requiring several decades, so cheaper and less intrusive biotechnologies that put the indigenous subsurface microbial communities to work are being developed. An understanding of the ecology of these subsurface communities is necessary to successfully implement bio-cleanup.
A second motivation for such research is that many countries will store significant quantities of nuclear waste in deep subsurface repositories. The long-term containment of this waste may be affected by its interaction with either the indigenous microbial communities, with those transported to the site.
Subsurface microorganisms (for an example see Figure 1) also hold promise for the medical community. Under the extreme conditions of the deep subsurface environment, bacteria may have developed unique enzymes or metabolites that may prove to be of medical or biotechnological benefit. The National Cancer Institute and a private pharmaceutical firm are screening DOE's culture collection of 5,000 subsurface isolates for anticancer and anti-AIDS agents.
Controversy surrounds the origin, diversity, evolution, and extent of deep subsurface communities, and the roles they play or have played in the transformation of organic and inorganic materials at low temperatures (á120°C) are largely unresolved. The contribution of the subsurface microbial community to the early evolution of life on the Earth's surface is the subject of speculation, and the extent to which the geological and hydrological environment influences the long-term survival or evolution of subsurface microorganisms is unknown.
Fig. 1. Electron micrograph of strain TH-23 isolated by Y. Liu. D. R. Boone of the Oregon Graduate Institute of Science and Technology is proposing that TH-23 be referred to as Bacillus Infernus (bacillus from hell or deep underground). Micrograph taken at 39,200 power.
One of the most significant challenges facing investigators of deep subsurface microbiology is the acquisition of rock material uncontaminated by surface organisms. It is almost impossible to completely avoid contamination of subsurface samples. The contamination is not just biological in nature, and it occurs during drilling, coring, retrieval, and sample processing.
Consequently, DOE investigators developed an array of sampling procedures and other quality control techniques to minimize contamination. These procedures include decontamination of the drilling and coring equipment, enclosing the drilling fluid system, and the introduction of physical and chemical tracers to the drilling circulation system.
To preserve more sensitive anaerobic microorganisms, all sampling of deep subsurface environments where anaerobic conditions prevail requires sterile and non-oxidizing containment of the recovered samples followed by rapid transport to microbiology labs. For this reason, nitrogen or argon gas is used in some drilling operations, and an argon-filled glove bag encloses all sample-paring tools, sample labeling, and packaging operations, in a core-receiving lab. These extensive quality control procedures increase the confidence that the numbers and diversity of the microorganisms present are reflective of the physical and chemical environment existing at depth.
Some subsurface microorganisms may be survivors or progeny of bacteria incorporated near the time of deposition of the sedimentary strata. Persistence over millions of years would require adaptations for long-term survival in low-nutrient environments or the development of communities that are sustained through complementary interactions among members. The study of this aspect of subsurface microbial evolution and ecology requires sampling a deep subsurface site where recent transport of microorganisms is highly unlikely.
A sedimentary basin site would be characterized by the following: it was deposited quickly to great depths, it was then isolated hydrologically, it contains strata with adequate energy sources (for example, organic compounds for microbial sustenance), and it has not been recently heated to temperatures sufficient to sterilize the environment.
In spring 1992, the Taylorsville Basin was identified as a site meeting all of these criteria. The Taylorsville Basin is a buried Triassic-age, rift-related ditch in Virginia (see Figure 2). It is filled with Middle to Upper Carnian (225-230 Ma) lake- and river-derived sediments, and subsequent to its deposition and burial, it was intruded by sills and dikes, and then covered by 500 m of Cretaceous and Tertiary sediments. Today only a minor fraction of the Triassic sequence is exposed on the southern margin of the basin.
Fig. 2. Cross section of the Taylorsville basin showing projected location of Thorn Hill #1 and other drill holes.
By February 1992, drilling of the Taylorsville Basin by Texaco and the Eastern Exploration Company had begun at Thorn Hill #1, the final hole in a series of exploration wells. With the drilling nearing completion, principal investigators responded quickly to capture a window of opportunity. Within 3 weeks a team installed a core processing lab and began sampling at the site. The sampling was conducted between 8700 and 9180 feet below the surface in tightly cemented, low porosity limestone sands and organic-rich, laminated, fine-grained sedimentary rock. The team recognized that the risk of microbial contamination of the core samples by the drilling fluid was particularly acute for this hardened material at such extreme depths.
Investigators also tested circulating drilling fluids for distinct surface bacteria. Consequently, samples of the makeup water, the soil at the drill site, and drilling mud at various depths were collected and the microorganisms present were characterized with the same procedures used for those in the rock samples. Both the quantity and type of bacteria present in these samples were then compared to those observed from the rock samples.
Once Texaco and Eastern drilled the hole to 10,000 feet, sidewall cores were extracted at five depths between 8700 and 9180 feet. The cores were then pared, split, and bagged in sterile packs within the argon-filled tent. Within 48 hours both samples and blanks_which were not identified as such to off-site investigators_were received by the home labs of the sampling team and off-site teams. The teams then began inorganic, organic, and tracer analyses of these samples and culturing of bacteria.
Over the next year, about 100 isolates were cultured from a dozen cores and drilling mud samples. Characterization of these isolates revealed the presence of aerobic and anaerobic bacteria. Some bacteria were growing and thriving in a moderate environment (mesophilic), whereas others thrived in a hotter environment (thermophilic); some preferred nonsaline media, while others were saline-tolerant. Mesophilic aerobes were concentrated at higher levels in the drilling muds, while the thermophilic anaerobes appeared below 7000 feet in the drilling mud.
These thermophilic anaerobic microbes were present in the drilling muds at concentrations 100 times greater than in the rock samples. Yet, evidence indicates that the thermophilic anaerobes isolated from the pared sidewall cores are indigenous and not from the drilling mud. First, those same drilling muds still yielded 10 times as many aerobes as anaerobes, but no aerobes were detected in the pared cores. If the anaerobes extracted from the pared cores were derived from drilling mud, then a correspondingly larger number of aerobes also should have been present.
Next, the nutrient requirements of bacterial cultures extracted from drilling muds were compared to those from the pared sidewall core samples through a process that tests bacterial growth in 96 inorganic and organic media. The microorganisms from the pared cores could metabolize amino acids, whereas those in the drilling muds, soils, and waters could only metabolize simple carbohydrates.
Finally, some of the thermophilic anaerobes exhibit a tolerance to high salinity and rely upon the reduction of Fe and Mn, such as strain TH-23 (see Figure 1). This combination of physiological characteristics helps them survive in deep subsurface environments and is not a combination normally associated with soil-dwelling microorganisms.
The anaerobic thermophiles cultured from the pared cores are probably indigenous to the Triassic sediments. Some of the organisms exhibit a combination of physiological traits never before reported. The cultures also reveal unusual lipid and lipopolysaccharide components. Sequencing of their 16S rRNA is underway to ascertain whether or not some of the isolates represent new bacteria.
Are the microorganisms observed in these samples the progeny of ancestral microorganisms laid down at the time of deposition? Certainly, the Triassic lake-derived sediments would have had abundant microbial communities when deposited. Initial fluid inclusion data suggests that hydrothermal fluids with temperatures of 160°-220°C migrated through this portion of the Taylorsville basin sometime early in the basin's history. Such fluids would probably have eradicated the microorganisms originally deposited with the sediments. Analyses of phosphate minerals indicate that these strata cooled sufficiently to permit rehabitation by the present microorganisms by approximately 140 Ma.
Have the present organisms been metabolically active in the subsurface at this depth over this time interval? If so, petrographic, geochemical, and isotopic evidence of their activity should exist in the rock samples. Does such evidence elucidate the conditions under which they survived, and whether or not those conditions changed over time? Does long-term survival in the deep subsurface incur detectable variations in bacterial DNA? Further work is necessary to identify the specific physiological mechanisms and environmental conditions that enabled these microorganisms to remain metabolically active or to exist over geological timescales.
Possible sites for future sampling of the deep subsurface biosphere are being sought, but because deep drilling is quite expensive, sites that can be drilled with cooperating public or private sector partners are highly desirable. The Thorn Hill #1 operation demonstrated that microbial sampling methods can be adapted to commercial operations, paving the way for more extensive collection of deep subsurface microbial communities.
Source: Eos, Vol. 75, p. 385, August 23, 1994.
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