Figure 1. Sample processing laboratory inside trailer at drill site. Ar-filled glove bag with sample processing tools is located in center of room. Torpedo tube at front is airlock for moving core into glove bag. Onstott (left), Jim Fredrickson (right), Tim Griffin (near rear) and Rick Colwell (far rear) prepare to process core during the midnight shift at Parachute Creek.
Research into subsurface life remained largely dormant until emerging ground-water quality issues forced scientists to reevaluate the possibility that microorganisms inhabited water-yielding rock formations (aquifers). In 1990, scientists from the U.S. Department of Energy (DOE) were able to prove that a diverse community of microorganisms were living at depths up to 400 meters below the surface of South Carolina and were actively modifying the chemical composition of 35,000 year old ground water. Spurred on by this result, Associate Professor Tullis Onstott *81 joined a team of microbiologists and molecular biologists assembled from various universities and federal laboratories in 1992, to search for microbial life at still greater depths.
Recognizing that the integrity of subsurface samples had been a crucial issue with the findings of Bastin and Greer, Onstott helped develop strategies to detect microbial contamination (Fredrickson and Onstott, 1996). For example, coring tools were designed to expedite core retrieval and minimize contact of the core with drilling fluids (Figure 1). Chemical and particulate tracers were developed that would indicate penetration of the cores by foreign microorganisms during drilling. The microbial populations in the drilling mud were compared to those found in the cores. Finally, the physiology of the microorganisms in the cores were compared to the geochemical environment at depth.
The first opportunity to utilize this new technology occurred in May, 1992, when Texaco was exploring for gas in a deeply-buried, Triassic age, 230-200 million years (Ma), sedimentary basin named Taylorsville Basin, Virginia, located near Washington, D.C. Within six weeks, a field team and processing laboratory were mobilized. Once Texaco penetrated organic-rich, sedimentary rock at 2.8 kilometers depth, microbial coring began. Limitations in funding permitted the collection of only a few small cores during one run with a side-wall coring tool. When that tool was returned to the surface, the cores were removed and immediately placed in a sterile glove bag for processing The glove bag was filled with inert gas because the environment was expected to contain anaerobic microorganisms, those bacteria that may be poisoned by oxygen.
The outermost portions of each core were pared away with sterile tools, leaving only the innermost material that was least likely to be exposed to drilling fluids and other sources of contamination. These inner cores were then shipped to research laboratories across North America. Within 72 hours after their removal from the subsurface, the inner cores were placed in a variety of growth media at temperatures, salinities, and pH designed to reproduce the environment at depth as determined by Onstott from analyses of geophysical logs. Although vast quantities of oxygen-utilizing, moderate temperature, bacterial colonies rapidly grew from the drilling mud, little growth appeared in the uncontaminated rock samples. But as months passed, several strains of thermophilic (heat-loving), saline tolerant, anaerobic bacteria slowly began to emerge. Most of these bacteria used oxidized forms of manganese, iron and sulfur to oxidize organic matter for energy and were quite distinct from the bacteria present in the drilling mud. Only 1 to 10 of these bacteria existed in one gram of rock, compared to 109 bacteria/gram typical for soils.
As isolates were prepared for genetic characterization, Onstott's research group focused on the chemical and physical characteristics of the core samples, and the geological history of the Taylorsville Basin. The first question to address was that of Bastin and Greer. Did these microbial communities represent the descendants of microorganisms trapped during deposition of the sediments in an ancient Triassic lake? This would be true only if the present-day environment at depth had not changed radically over the geological history of the basin. By studying microscopic pockets of fluid trapped within minerals, fluid inclusions, Hsin-Yi Tseng, a graduate student working with Onstott, was able to show that at one time the temperature in the rock strata was 160°C. This temperature exceeds not only the maximum growth temperature of the bacteria recovered from the rocks, but also that of any known bacteria grown in a laboratory (Tseng et al., 1995). Upon exhaustive analyses of the thermal/fluid flow history of Taylorsville Basin, she discovered that the microbially-sampled strata cooled to the present-day temperatures no earlier than 160 million years ago.
Did the bacteria infiltrate the rock strata subsequent to that time as ground water migrated through the formation? When Onstott analyzed the pore structure in the rock samples, he discovered that the maximum pore throat diameter was only 0.03 microns. This is too small for even the tiniest bacteria to squeeze through (Figure 2). But when did this pore structure form? Fortunately, the pores were filled with clay minerals, including a Potassium-rich clay known as illite. Since Onstott had just developed a technique for determining the age of microgram quantities of illite using his laser microprobe, he proceeded to date the clay from these cores with the help of scientists at Exxon and Harvey Cohen*92, a postdoctoral associate in Onstott's lab. The analyses indicated that the illite clay crystallized 80 millions years ago. This age is not only consistent with Tseng's constraint on the timing of microbial colonization, but it also indicates that the bacteria have been entombed within their mineral cage since the Mesozoic. The results of DNA analyses of the microbial isolates, which were completed within the same week as the illite age, revealed that all the bacteria appear to be new species, never identified before on the surface. One strain has been named Bacillus Infernus, or bacteria from hell. Does this mean that during at least 80 million years of isolation from the surface, these bacteria slowly evolved into new species?
Figure 2. Diagram illustrating the entrapment of bacteria in the Taylorsville Basin sedimentary rocks. A. At 200 Ma the temperatures are too high for bacteria to survive. B. At 160 Ma the rocks are cool enough for bacteria to survive and the pores are large enough for bacteria to migrate into formation. The nutrient flux is high enough for significant microbial activity, perhaps even reproduction. C. At 80 Ma progressive diagenesis has decreased pore throats to the point that bacteria cannot readily migrate between pores. D. Today, nutrients can still diffuse into the pores, but at rates too slow to support colonizing population and many cells expire or become dormant.
By performing detailed electron microscopy at the Princeton Materials Institute and stable isotope analyses in Dan Schrag's (Faculty) laboratory, Onstott was able to determine how much calcite and pyrite had resulted from eons of bacterial respiration. These results indicate that the average doubling time of the observed bacterial population was on the order of thousands to million years. This growth rate is so slow, 1015 times slower than that of bacteria present in sewage sludge, that it suggests that the bacteria are surviving not reproducing. If microorganisms can only evolve by reproduction, then these data suggest that the bacteria represent species that were present in the basin at least 80 million years ago and have not changed significantly since that time. The bacteria are for all intents and purposes living microfossils!
In 1993, as the first results of the Taylorsville Basin project appeared promising, Onstott worked with Phil Long and Jim Fredrickson of Pacific Northwest Laboratories to secure DOE funding for a more ambitious program of microbial sampling of the deep subsurface at two new sites, Cerro Negro, New Mexico, and Parachute Creek, Colorado. These sites were selected, because their geological history could provide tangible constraints on the survival and the rate of migration of bacteria in the deep subsurface. At Cerro Negro, a 3 million year old basaltic intrusion had thermally altered organic-rich marine shale and sandstone of the Cretaceous period. Were the bacteria residing within the shale just outside the thermal aureole, but not within the thermal aureole itself? At Parachute Creek in the Piceance Basin, Colorado, pockets of natural gas trapped under high pressure in Cretaceous/Tertiary sandstones suggested the strata were extremely impermeable to fluid migration and hence to bacterial migration. Would these strata yield bacteria trapped since the sediment deposition?
Both sites presented new challenges to microbial sampling. At Cerro Negro, one core had to be obtained at an extremely shallow angle in order to obtain samples across the several hundred meter wide thermal aureole. At Parachute Creek, microbial samples had to be retrieved from potentially over-pressured strata at 2 kilometers depth. Field work at both sites lasted 6 months, during which 100's of meters of core were collected. Dozens of microbiologists, molecular biologists, and geologists participated in the field sampling along with Onstott, G. Gao, a postdoctoral associate with Onstott, and two undergraduates from Princeton University, Nate Fischer '94 and Witold Grzymala-busse '95.
At Cerro Negro, the microbiologists found indigenous bacterial populations ranging from 102 to 105 bacteria per gram in the sedimentary strata far away from the basaltic intrusion and within the thermal aureole. This observation indicated that bacteria had colonized low permeability shale within the thermal aureole subsequent to the time of intrusion during which the temperature exceeded that tolerated by life. Intriguingly, thermophilic bacteria appear to dominate the microbial community of the thermal aureole today even though the ambient temperature is quite moderate.
Gao endeavored to document the diagenetic/thermal history of the sandstone strata by performing detailed isotopic and electron microprobe analyses on the core samples. His analyses revealed the presence of abundant, bacterially-precipitated sulfides and minor calcite formed at low temperatures, presumably during the Cretaceous. Microbial enrichments, however, indicated that sulfate reducing bacteria dominated the present-day bacterial population in the rocks and were actively generating sulfide in the ground water. When a detailed vertical profile of the isotopic composition of the 25,000 year old ground water was obtained from the same borehole (Figure 3), however, they agreed with Gao's isotopic data on calcite cement. It is possible that the diagenetic features of sandstone did not reflect the Cretaceous paleoenvironment after all, but the recent activity of subsurface bacteria. When the rate of bacterial oxidation of organic matter was derived from the ground-water chemistry, it was found to be high enough to produce all of the observed calcite within a few hundred thousand years. This is contrary to the view of those who believe in the early diagenetic origin of calcite cementation. Actually, it is the microbiology that explains the origin of the diagenetic history rather than the other way around.
Figure 3. The vertical profile of the carbon isotope composition (d13C) of the dissolved inorganic carbon obtained by a multi-level ground- water sampler inserted into the Cerro Negro borehole. The sampler crossed the contacts between the organic-rich, Clay Mesa Shale (aquitard) and the Cubero Sandstone (aquifer), and the Oak Canyon Shale. Also shown is the carbon isotope composition of the calcite cements in the sandstone and shale (brick squares), the whole rock shale (mottled circles), and for various generations of calcite present in a concretion near the contact of the Clay Mesa Shale and the Cubero Sandstone. The unit PDB is a standard of comparison in determining isotopic composition of carbon and oxygen. Originally CO2 was prepared from belemnites collected from the Peedee Formation (Upper K) of South Carolina. The initials stand for "Peedee belemnite."
At Parachute Creek, thermophilic, metal-reducing bacteria were enriched from two sandstone bodies, one 860 meters and the other 1,996 meters below the surface. A third sandstone body, at 2,095 meters did not yield any bacterial enrichments and appeared sterile. The third sandstone body contained high concentrations of methane; whereas the other two had not. To unravel the mystery surrounding the presence of bacteria in the shallower cores and their absence in the deepest core, Qing-jun Yao, a postdoctoral associate with Onstott, determined the thermal history of the rock strata, collected water and bacterial samples from wells intercepting different horizons in the strata, and developed a fluid flow model for ground-water motion in the Piceance Basin. His results indicated that the methane formed during maximum burial of the formations, between 35 and 9 million years ago. At that time the sandstone strata were heated to temperatures of 125° to 145°C, which are high enough to eradicate known bacteria (Figure 4). During the subsequent uplift and cooling of the Colorado Plateau, the formations cooled by 60° to 80°C. The high topographic relief generated by the erosion of the Colorado River, forced meteoric water deep into the subsurface gas reservoirs flushing those with higher permeability. The same ground water probably carried bacteria, which quickly established microbial communities in the formerly sterilized environment. Those sandstone bodies still retaining high concentrations of methane gas have probably remained closed to this bacterial invasion. His model also indicates that only about one million years would be required for bacteria to migrate from the surface to the coring depths. Surprisingly, the bacteria found in the cores are very distinct from those in the recharging aquifers and thermophilic organisms still dominate the cultured communities of the shallowest core even though the present day temperatures are cool.
Although more results from the Cerro Negro and Parachute tectonically-active environments, the bacterial migration rate and microbial activity are high compared to quiescent geological terranes, such as Taylorsville Basin in Virginia. There the subsurface microbial migration has long since ceased and the bacteria are relatively dormant. Could it be that the age of deep, subsurface microbial communities corresponds to the last tectonic event when high topographic gradients and syntectonic fractures and faults produce the greatest ground water flow? In this case, then, the thermal history puts constraints on the microbial origins.
Many questions remain concerning the subsurface biosphere. For example, how big is it? Because moderate pressures exert little effect on microorganisms, the Earth's geothermal gradient controls the maximum depth for life. Assuming a maximum temperature limit for life of 120°C, typical geothermal gradients and observed biomass densities, the estimated global mass of subsurface bacteria lies somewhere between 1 x 1014 to 1 x 1018 grams of carbon. Since the surface biosphere mass is approximately 4.6 x 1017 grams of carbon, could the subsurface biosphere represent a substantial portion of the global biosphere?
How important is subsurface microbiology? Subsurface microbial ecology is clearly influenced by geological and geochemical factors, but to what extent subsurface microorganisms actively alter their environment to enhance their growth, migration and survival has yet to be ascertained. This aspect is pivotal to successful in situ bioremediation of contaminated ground water and soil. The microorganisms that have been cultured from the subsurface have yielded new antibiotics and high temperature polymers. The many, deep subsurface microorganisms that can not be cultured may potentially be characterized by the powerful tools recently developed for molecular biology and analytical chemistry. The geological story they may record and their potential for bioremediation has not been determined.
Other novel subsurface ecosystems may be discovered that provide a model for how life evolved and functioned on the early Earth, where life existed beneath the surface sheltered from intense meteorite bombardment and before photosynthesis developed. To assess this possibility, Onstott recently collected microbial samples from the deepest gold mines of South Africa. These mines have attained depths of 3.5 kilometers, as miners have pursued a 2.9 billion year old, organic-rich, gold-bearing layer. By offering direct access to the potentially thermophilic microorganisms thriving at those depths, the mines provide a unique opportunity to study subsurface microbiology without the expense of coring.
Subsurface microbiology has already provided insights on how life could conceivably exist beneath the surfaces of Mars and the larger moons within our solar system. The mere possibility of detecting subsurface life forms has dramatically changed the course of National Aeronautics and Space Administration's (NASA) planetary exploration program. Even though the microbial communities found deep beneath our planet's surface in the last few years do not compare to the grandeur of Verne's mushroom forests, their discovery has altered our perception of life in our planet, in our solar system and in the universe more than any great work of science fiction.
