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Microbes Deep S ingle-celled organismsbacteria, fun-

gi and protozoathrive on all parts


of the earths surface. Their habitats

inside the Earth range from the boiling hot waters of ther-
mal springs to the pleasantly cool soils of
backyard gardens. Microorganisms pro-
vide essential services to other creatures by
decomposing waste products and forming
Recently discovered microorganisms nutrients. Some microbes also inflict harm
by infecting higher organisms and causing
that dwell within the earths crust could disease. Fortunately, scientists have learned
to control many of those damaging effects
reveal clues to the origin of life and to expand on the ways microorgan-
isms benefit humankind.
Although people have used the metabolic
by James K. Fredrickson and Tullis C. Onstott activities of microorganisms for thousands
of years to produce cheese, wine and bread,
it was not until the mid-20th century that
scientists harnessed microbes to create an-
tibiotics and other pharmaceuticals. Today
people also employ microorganisms for
such diverse tasks as controlling pests,
treating sewage and degrading oil spills.
With countless novel uses still awaiting
discovery, biologists continue to scour the

BEARING

FLUID

LID

TRACER
FLUID

CORE
SAMPLE
GEORGE RETSECK

DRILL BIT

68 Scientific American October 1996 Copyright 1996 Scientific American, Inc. Microbes Deep inside the Earth
surface of the earth in search of mi- hundreds of meters below the surface. ers. These scientists routinely assumed
crobes that might prove valuable in for- Bastin and Greer speculated that these that any microbes found in groundwa-
mulating new drugs or improving microbes might have been descendants ter samples taken from great depths
industrial processes. But until recently, of organisms buried more than 300 mil- were surface contaminants.
few such bio-prospectors thought to lion years ago when the sediments that Then, during the late 1970s and early
look deep inside the earth. Long-stand- constituted the oil reservoir were depos- 1980s, concerns about the quality of
ing scientific dogma held that this realm ited. But they had no way to test this in- groundwater stimulated some investi-
was essentially sterile. But that belief, as triguing hypothesis. At the time, many gators at the U.S. Geological Survey and
it turns out, was wrong. scientists viewed with skepticism the the Environmental Protection Agency to
very idea of microorganisms living deep reevaluate their understanding of
Its Alive! underground, noting that oil-drilling groundwater chemistry. This work
techniques were not designed to obtain spurred them to reconsider the possibil-

T he first hints that microorganisms


lived in the deep subsurfacehun-
dreds to thousands of meters below
samples uncontaminated by microor-
ganisms from the surface. With little ac-
ceptance or support in the scientific
ity that microorganisms could inhabit
water-yielding rock formations. At the
same time, the U.S. Department of En-
groundemerged in the 1920s from the community, the views of Bastin and ergy (DOE) faced the daunting task of
studies of Edson S. Bastin, a geologist at Greer languished. cleaning up the industrial facilities where
the University of Chicago. Bastin ques- Interest in the microbiology of petro- nuclear materials had been produced.
tioned why water extracted from oil leum deposits temporarily revived dur- (As a cold war expedient, the DOE had
fields contained hydrogen sulfide and ing the late 1940s and 1950s, when dumped vast quantities of wastein-
bicarbonate. After puzzling for some Claude E. Zobell of the Scripps Institu- cluding organic-rich solutions, metals
time, Bastin ventured an explanation. tion of Oceanography and his colleagues and radioactive materialsinto the sub-
He knew that so-called sulfate-reducing investigated microbial processes in sed- surface at these sites.) DOE scientists
bacteria can exploit sulfate for respira- iments buried far below the seabed. But were also studying how to build under-
tion in places on the surface where no research into subsurface microbiology ground repositories that could isolate
oxygen is present. So Bastin reasoned again fell into dormancy during the high-level radioactive wastes for thou-
that such bacteria must also live in un- 1960s and 1970s. Despite the impor- sands of years.
derground oil reservoirs and produce tance of rock formations as reservoirs During this period, Frank J. Wobber,
hydrogen sulfide and bicarbonate when and conduits for water supplies, few a geologist and manager at the DOE,
they degrade organic components in oil. considered the possibility of microbial reasoned that if microorganisms were
By 1926 Bastin and Frank E. Greer, a activity deep underground. Most present well below the earths surface,
colleague at the University of Chicago researchers believed that water under- they might helpfully degrade buried or-
who specialized in microbiology, had went predominantly inorganic chemical ganic pollutants or dangerously disrupt
succeeded in culturing sulfate-reducing alterations as it passed through the the integrity of closed chambers con-
bacteria from groundwater samples ex- earth and that biological influences taining radioactive waste. But a great
tracted from an oil deposit that was were restricted to near-surface soil lay- deal of fundamental research needed to
be done before such practical concerns
could be addressed. And so he began a
special effort, called the Subsurface Sci-
ence Program, within the DOE. His idea
was to sponsor a diverse group of biolo-
gists, geologists and chemists to search
DAVID BOONE Oregon Graduate Institute

systematically for deep-seated life-


forms and examine their activities.
Because water brought up from deep
drill holes is easily contaminated with
organisms living near the surface, the
team assembled by Wobber decided to
study pieces of rock instead. But first the
group needed a way to collect clean, in-
tact samples of rock (cores) from deep
SUBSURFACE EXPLORATION (far left) requires a great length of rotating steel pipe to in the crust.
snake downward from a drilling derrick to an underground target. As the pipe rotates, a di- Tommy J. Phelps of Oak Ridge Na-
amond-studded drill bit at the bottom of the borehole (detail, bottom left) cuts away at the tional Laboratory and W. Timothy Grif-
underlying rock and surrounds a cylindrical sample that is later extracted when the pipe is fin of Golder Associates rose to the chal-
withdrawn. Lubricating fluid with a special tracer substance is pumped down the center of lenge by designing a special drilling ap-
the pipe (detail, top left) and out through holes in the bit (arrows). The cylindrical rock sam- paratus that minimized contact of the
ple remains in place as the pipe and bit rotate because it sits within a stationary inner barrel
core samples with the drilling fluid need-
that is supported by a bearing. As a core of rock fills the inner barrel, a bag of concentrated
tracer material above it breaks open and coats the outer surface of the sample (yellow). ed to provide lubrication in a borehole.
Cores recovered in this way are cut into short segments from which the outer rind marked by And James P. McKinley of Battelle, Pa-
the tracer is removed to avoid contamination (above, left). Within pristine inner core sam- cific Northwest National Laboratory,
ples, deep-living bacteria (above, right) can be found. along with F. S. (Rick) Colwell of Idaho

Microbes Deep inside the Earth Copyright 1996 Scientific American, Inc. Scientific American October 1996 69
National Engineering Lab- least likely to have been ex-
oratory, formulated special posed to bacterial contami-
tracersadditives that nants in the drilling fluid. If
could be mixed with the seepage of the tracer chemi-

PHIL LONG Battelle, Pacific Northwest National Laboratory


drilling fluid to indicate cal indicated that a particu-
whether this liquid (and any lar specimen might have
microorganisms carried in- been tainted, the scientist
side it) could have penetrat- dissecting it noted that the
ed the core samples. core from which it came was
very possibly contaminated.
Striking It Rich Pristine inner core samples
recovered in this way were

T he search for subsurface


microbes began in 1987,
when the DOE arranged to
then placed in sterile contain-
ers filled with nitrogen,
which were packed in ice
drill several deep boreholes and shipped to research lab-
in South Carolina near the GLOVE BOX, with its rubber gloves protruding inward, allows oratories across North
Savannah River nuclear ma- scientists working near the drill sites to manipulate solid samples America. Within 72 hours af-
terials processing facility. extracted from the subsurface. These plastic enclosures are filled ter the removal of the rocks
With the operators of the with an unreactive gas to prevent oxygen from damaging deli- from the subsurface, other
drilling rig there, a field cate microbes within the recovered cores of rock. members of the research
team of scientists labored to group based at many differ-
avoid microbial contamination. Re- with an unreactive gas (nitrogen) as a ent institutions were subjecting the
searchers diligently added tracers and precaution to protect any so-called oblig- samples to a battery of tests designed to
monitored procedures around the clock atory anaerobesbacteria that would evaluate the rocks and the microorgan-
as drilling proceeded. When the drillers be quickly poisoned by the oxygen in isms they harbored. After these initial
brought a core to the surface, a member the air. experiments, researchers sent the mi-
of the team quickly encapsulated the Using surgical rubber gloves attached crobes they had extracted from the sub-
sample and placed it in a glove bag to the interior of these bags, members surface samples to special repositories
for processing. Those plastic containers of the team used sterile tools to pare in Florida and Oregon to be stored in
provided a sterile environment filled away the outermost rind of each core liquid nitrogen at -96 degrees Celsius.
sample, leaving only the part that was The first results of this quest for deep-
seated life-forms were extraordinary.
GEORGE RETSECK

OCEANIC CRUST

CONTINENTAL CRUST

SEDIMENTS

SUBSURFACE ENVIRONMENTS vary considerably in the composition of


the surrounding rock. Deep-living microbes pervade both oceanic and conti-
nental crust and are especially abundant in sedimentary formations. Such mi- O2 H2O (aerobic respiration)
croorganisms fail to survive only where the temperature exceeds about 110 de- MnO2 Mn 2 + (manganese reduction)
grees Celsius (orange areas). The nature of the population does, however, change
Fe 3 + Fe 2 + (iron reduction)
from place to place. For example, a porous sedimentary layer that acts as a
conduit for groundwater may contain both oxygen-rich (light blue) and oxy- SO42 H2S (sulfate reduction)
gen-poor (dark blue) zones, and the bacteria found within its different regimes
CO2 CH4 (methanogenesis)
will vary according to the chemical reactions they use for energy (bar, right).

70 Scientific American October 1996 Copyright 1996 Scientific American, Inc. Microbes Deep inside the Earth
The scientists involved quickly learned ture rises about 15 degrees C per kilo- fuel to provide the energy required for
that diverse types of microorganisms meter of depth, tolerance of 110 de- this ongoing activity.
lived beneath the Savannah River site at grees allows microbial life to extend
depths extending at least as far as 500 (on average) about seven kilometers be- From Sandstone to SLiMEs
meters beneath the surface, the deepest low the seafloor. For continental crust,
core taken. We and our many colleagues
working under the aegis of the DOEs
Subsurface Science Program have since
where the temperature is often near 20
degrees C at the surface and typically
increases by about 25 degrees per kilo-
T he types of microbes found in the
earths deep realms depend on the
particulars of the local subsurface envi-
examined many other geologic settings. meter, microscopic life should, on aver- ronment. Diverse bacterial communi-
Although we are still unsure of the ex- age, reach almost four kilometers down- ties thrive in most sedimentary rocks,
tent of fungi or protozoa, the results ward into the earth. which commonly contain a rich supply
clearly indicate that subsurface bacteria The abundance of microbes will, how- of organic compounds to nourish mi-
are ubiquitous. We have now recovered ever, vary considerably from place to croorganisms. These nutrients were
these organisms from formations with place, even at the same depth in the originally produced by plants at the
temperatures as high as 75 degrees C earth. For example, we have discovered earths surface before the loose sands,
(167 degrees Fahrenheit) and from that samples obtained from 400 meters silts or clays that constitute most sedi-
depths extending to 2.8 kilometers (1.7 below the surface of the ground can mentary formations were buried and
miles) below the surface. contain as few as 100 to as many as 10 consolidated into solid rock. As long as
What determines the maximum depth million bacteria in each gram of rock. these nutrients remain available, mi-
at which subsurface microbes can exist? John R. Parkes and his colleagues at the croorganisms living within the pores of
Mounting pressure exerts little direct University of Bristol have found some- the sediments can continue to survive
effect on microorganisms even several what higher concentrations of microor- and grow. Sedimentary rocks also sup-
kilometers below ground level. It is the ganisms living in sediments beneath the ply oxidized forms of sulfur, iron and
increasing temperature that limits the ocean floor. In comparison, agricultural manganese that can provide the energy
depth of subsurface life. The maximum topsoil typically contains more than these microbes need. The chemical
temperature that such organisms can one billion bacteria in each gram of dirt. power sources here are so-called reduc-
tolerate remains something of a mystery, It seems that the richness of life in the tion reactions (processes that involve
but biological oceanographers have deep subsurface depends not only on tol- the gain of electrons).
found bacteria that are capable of grow- erable temperatures but also on the ca- As sediments become more deeply
ing at 110 degrees C in deep-sea volcan- pacity of the local environment to sup- buried over geologic time, they are in-
ic vents, and some scientists estimate port growth and proliferation. Crucial creasingly compacted. Much of the
that subsurface microorganisms might prerequisites include the presence of wa- dwindling pore space eventually be-
be able to withstand temperatures as ter and the sheer availability of space in comes cemented with minerals that pre-
high as 140 degrees C, at least for short the pores of the rock. The region host- cipitate from fluids passing through the
periods. ing the microbes must also contain the rock. Consequently, as depth and pres-
For oceanic crust, where the tempera- nutrientssuch as carbon, nitrogen, sure increase, the opportunity for ob-
phosphorous and various trace met- taining life-sustaining materials de-
alsthat microorganisms need to syn- clines, and the overall rate of metabo-
thesize their cellular constituents, in- lism of microbial communities
cluding DNA and proteins. The envi- gradually diminishes, except in those
ronment also has to offer some form of spots that directly surround rich con-
centrations of nutrients. The distribu-
tion of microorganisms in sediments ul-
timately becomes quite patchy. Small
coloniesor even individual cellslive
well separated from one another within
the rock. Not surprisingly, then, search-
ing for microorganisms living in these
settings proves to be a hit-or-miss affair.
Todd O. Stevens of Battelle, Pacific
Northwest National Laboratory has
found, for example, that with sediment
collected near the DOEs Hanford facili-
ty in Washington State, the larger the
sample tested, the better the chances of
finding microbial activity.
SLIMES, or subsurface lithoautotrophic Although quite inhospitable, such
microbial ecosystems, exist in the pores
hardened sedimentary rock is not the
between interlocking mineral grains of
many igneous rocks. Autotrophic microbes most challenging environment for sub-
( green) derive nutrients and energy from surface microbes: some environments
inorganic chemicals in their surroundings, appear far more hostile. The bulk of the
and many other microbes (red ), in turn, continental crust is composed of igneous
feed on organics created by autotrophs. rock (that is, rock solidified from molten

Microbes Deep inside the Earth Copyright 1996 Scientific American, Inc. Scientific American October 1996 71
magma), which contains little organic microbes found within such rock must ganic sources. Many types of autotrophic
carbon. Nevertheless, Stevens and Mc- have been carried there by the flow of bacteria capture energy from inorganic
Kinley discovered bacteria living within groundwater sometime after the parent chemical reactions involving iron or sul-
igneous formations that are composed magma cooled and solidified. fur. The autotrophs living in these
of layers of basalt (a dark, fine-grained Little buried organic matter is avail- basalts use hydrogen gas for energy and
type of rock). able within igneous formations, and so derive carbon from inorganic carbon
Microorganisms thrive in other igne- Stevens and McKinley were surprised dioxide. These acetogens then excrete
ous rock as well. Karsten Pedersen of the to find that microbes could flourish in simple organic compounds that other
University of Gteborg in Sweden de- basalt. They eventually discovered the bacteria can in turn consume. In these
tected bacteria in water flowing through secret. The bacterial communities living basalts the hydrogen gas is produced by
deep fractures in granitea light-col- there include so-called autotrophs, or- the reaction of oxygen-poor water with
ored, coarse-grained variety of igneous ganisms that synthesize organic com- iron-bearing minerals. Many of us call
rock. Because igneous rock is too hot to pounds (proteins, fats and other biolog- such environments SLiMEs, for sub-
support life when it is first formed, the ical molecules rich in carbon) from inor- surface lithoautotrophic microbial eco-
systems. Amazingly, SLiME microor-
ganisms can persist indefinitely without
any supply of carbon from the surface.
Biodiversity in the Subsurface
Old as the Hills?

J ust as countless kinds of life-forms cover the surface of the earth, many dif-
ferent types of bacteria live deep inside the crust. But because different mi-
crobes often look very much alike under the microscope, scientists have to L ike Bastin and Greer working decades
before us, we wondered whether
resort to creative methods to gauge the extent of this bacterial diversity. subsurface bacterial colonies might sur-
Certain methods allow researchers to avoid having to culture the microbes vive for as long as the rocks that host
first. Biologists can, for example, apply a procedure called epifluorescence mi- them. Such longevity is clearly not al-

SANDRA A. NIERWICKI-BAUR Rensselaer Polytechnic Institute (micrograph); SOURCE: DAVID C. WHITE AND DAVID B. RINGELBERG University of Tennessee; GEORGE RETSECK (diagram)
croscopy to visualize bacteria living within rock samples. This technique takes ways possible. The continuing burial of
advantage of the unique makeup of the ribosomal RNA found in different types sediments can ultimately raise tempera-
of bacteria (ribosomes are structures used by the cells to construct protein mol- tures sufficiently to purge an entire rock
ecules). By first fashioning short strands of DNA so that they bind to particular formation of live bacteria. More local
kinds of ribosomal RNA, one can rapidly determine the variety of bacterial fami- sterilization may also occur where fiery
lies in a sample. These DNA probes include a fluorescent dye so that when bac- hot magma impinges on sedimentary
teria accumulate this substance, they seem to glow when viewed in an epifluo- strata, leaving a body of igneous rock
rescence microscope (micrograph ). with some well-baked sediments sur-
Another way to assess bacterial communities is to analyze samples for dis-
rounding it. Once such newly solidified
tinctive organic molecules called phospholipid fatty acids. These long carbon
rock cools, or tectonic forces lift hot,
chains are the building blocks of bacterial cell membranes. Their molecular
deeply buried sedimentary layers to a
structure (which can be ascertained using modern laboratory instrumentation)
provides a fingerprint for different bacterial families. If many different types of cooler position closer to the surface,
the fatty acid chains are found within a given sample, a diverse bacterial com- bacteria carried by groundwater will
munity exists within it. In contrast, finding a small number of distinct fatty acid then colonize the formerly sterile zones.
molecules indicates a community of limited variety. At a site near the Depart- Yet that process of infiltration can be
ment of Energys Hanford facility in Washington State, drilling revealed striking exceedingly slow. Ellyn M. Murphy of
variation in the bacterial diversity of different subsurface environments. Battelle, Pacific Northwest National
Laboratory has determined, for exam-
SURFACE SOILS
ple, that the groundwater now present
42
deep beneath the Savannah River facility
has not been in contact with the surface
DRY SAND AND GRAVEL for thousands of years. In the deepest
7 sites we have examined, our measure-
ments and computer modeling indicate
that the groundwater has been isolated
ANCIENT SOILS from the surface for millions of years.
28 Because microorganisms could not have
traveled downward from the surface
faster than the groundwater descended,
LAKE SEDIMENTS some subsurface microbial communities
28 must be at least several million years old.
How do microorganisms manage to
persevere for so long? In some cases (for
MAXIMUM NUMBER example, SLiMEs), bacteria can survive
OF PHOSPHOLIPID because the essential nutrients are con-
FATTY ACID TYPES
IN ROCK SAMPLE stantly renewed; although in most oth-
er sorts of formations, food and energy
sources are relatively scarce. Neverthe-

72 Scientific American October 1996 Copyright 1996 Scientific American, Inc. Microbes Deep inside the Earth
less, the resident bacteria ap- diverse subsurface environments.
pear to have adapted to these These isolatescontaining a
rather spartan living condi- vast assortment of bacteria and
tions. Bacteria must rely on in- about 100 types of fungiare a
ternal reserves during periods source of novel microbial life
of long-term starvation (as do that have not yet been fully test-
higher organisms), and most ed for commercially applicable
types of bacteria shrink from a properties.
healthy size of a few microns to Of the small percentage of
less than a thousandth of their the collection that researchers
normal volume as they use up have examined in detail, a sur-
their stores. Thomas L. Kieft of prisingly high proportion show
the New Mexico Institute of potentially valuable capabili-
Mining and Technology has ties. Examples of such traits in-
JAMES K. FREDRICKSON

found that such tiny, starved clude the ability to degrade tox-
microbes (called dwarf bacteria ic organic compounds as well
or ultramicro-bacteria) com- as to produce antibiotics, heat-
monly inhabit the subsurface. stable enzymes and even novel
The metabolic rate of such pigments. Pfizer is now screen-
starved bacteria is probably ing 3,200 kinds of subsurface
PIGMENTED BACTERIA inhabit parts of the subsurface
much lower than when they near Idaho Falls, Idaho. Cultures of these microorganisms bacteria for the production of
are well fed. As a result, the av- vary in appearance from purple to red because they pro- new antimicrobial products,
erage frequency of cell division duce copious amounts of a brightly colored substance that and ZymoGenetics, a biotech-
for a subsurface microbe may shifts in hue according to the ambient acidity. nology company, is currently
be once a century, or even less, examining at least 800 isolates
whereas surface microorganisms repro- (where the flux of water is minimal) will from this archive for production of oth-
duce in a matter of minutes, hours, grow in a culture dish. er useful substances.
days or, at most, months. Microorgan- It may be that failure to culture most Perhaps many commercial products
isms living in the deep subsurface limit subsurface bacteria is a result of our in- will result from these investigations. But
their metabolism in order to endure ability to properly reproduce necessary even without such quick practical re-
starvation for geologically significant conditions in the laboratory. Or per- turns, the effort to probe the earths in-
lengths of time. These bacteria can re- haps these organisms are simply no terior for microorganisms will surely
main viable at little or no metabolic cost. longer alive. In rocks where the flux of reward scientists with a fuller under-
The sluggish pace of microbial metab- nutrients and water is low, dead cells standing of how life can exist in isola-
olism in the subsurface makes it diffi- decompose exceedingly slowly, and so tion from the surface. More study of
cult to define just how many of the bac- some of our biochemical assays would subsurface communities may, for in-
teria found entombed in these rocks are count them along with the few living stance, indicate how life functioned on
truly alive. One approach is to count cells. Alternatively, most of the organ- the early earth, before photosynthesis
only those microbes that can be grown isms could be functioning but may have evolved. It may also provide insight into
in the laboratory. More than 10 percent lost the ability to replicate. whether microbes might be living even
of the cells extracted from sandy sedi- now under the surface of Mars or be-
ments where water and nutrients can The Prospects Underground low the icy exterior of some of the larg-
generally flow freely will proliferate er moons of the outer solar system. See-
when given a supply of nutrients in the
laboratory. In contrast, less than one S
o far our colleague David L. Bulk- ing how microbes survive the rigors of
will of Florida State University has deep burial on the earth, we are more
tenth of 1 percent of the cells drawn catalogued and preserved more than inclined to believe tiny extraterrestrials
from sediments in the arid western U.S. 9,000 strains of microorganisms from might indeed be lurking out there. SA

The Authors Further Reading


JAMES K. FREDRICKSON and TULLIS C. ONSTOTT conduct re- The Deep Subterranean Biosphere. Karsten Pedersen in
search for the Department of Energys Subsurface Science Program. Earth Science Reviews, Vol. 34, No. 4, pages 243260; Au-
Fredrickson is an environmental microbiologist at Battelle, Pacific North- gust 1993.
west National Laboratory and also serves as editor in chief of the journal Ground-Water Microbiology and Geochemistry.
Microbial Ecology. He has specialized in applying molecular and isotopic Francis H. Chapelle. John Wiley and Sons, 1993.
methods to investigations of subsurface bacteria (including some obtained The Biosphere Below. Daniel Grossman and Seth Shul-
from his wine cellar). Onstott is a professor in the department of geologi- man in Earth: The Science of Our Planet, Vol. 4, No. 3,
cal and geophysical sciences at Princeton University. His expertise is in pages 3440; June 1995.
studying the history of fluid and heat flow within the earths crust. He be- Geomicrobiology. Third edition. Henry L. Ehrlich. Mar-
gan working with members of the Subsurface Science Program in 1993 to cel Dekker, 1996.
help determine the age of deeply buried microbial communities, and he Information on the U.S. Department of Energy Subsurface
quickly caught the mysterious subsurface bug that has infected this large Science Program is available on the World Wide Web at
group of scientists with a peculiar enthusiasm for their joint research. http://www.er.doe.gov/production/oher/EPR/subprog.html

Microbes Deep inside the Earth Copyright 1996 Scientific American, Inc. Scientific American October 1996 73

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