EPA-660/2-73-OH
September 1973
Environmental Protection Technology Series
Subsurface Biological Activity
In Relation To Ground Water Pollution
National Environmental Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Corvallis, Oregon 97330
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series ares
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
i». Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL
PROTECTION TECHNOLOGY series. This series
describes research performed to develop and
demonstrate instrumentation, equipment and
methodology to repair or prevent environmental
degradation from point and non-point sources of
pollution. This work provides the new or improved
technology required for the control and treatment
of pollution sources to meet environmental quality
standards.
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EPA-660/2-73-014
September 1973
SUBSURFACE BIOLOGICAL ACTIVITY IN RELATION
TO GROUND WATER POLLUTION
By
William J. Dunlap
James F. McNabb
Robert S. Kerr Environmental Research Laboratory
National Ground Water Research Program
P. O. Box 1198
Ada, Oklahoma 74820
Project 21 AKQ-10
Program Element 1B1024
NATIONAL ENVIRONMENTAL RESEARCH CENTER
OFFICE OF RESEARCH AND MONITORING
U. S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 - Price 90 cents
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ABSTRACT
Biological activity occurring in subsurface regions below
the soil zone may be of considerable importance in determining
the fate and effect of pollutants in ground water, but this
possibility has received little previous attention. This
paper comprises a discussion of subsurface biological activity
in regard to ground-water pollution as reflected by available
literature references. The subsurface environment is discussed
in terms of factors likely to be of greatest significance in
regard to the development of biological systems, and previous
investigations of subsurface microbial activity are reviewed.
Available information indicates the presence in the upper
continental crust of the earth of numerous regions, particu-
larly those of sedimentary origin, which are probably suitable
habitats for many microbial species. Previous investigations
of subsurface microbial activity clearly show the presence
of diverse microbial populations in many subsurface regions
below the soil zone. Hence, microbial activity appears both
possible and probable in most subsurface regions of importance
in regard to ground water. Further elucidation of the extent
and nature of microbial activity in subsurface regions is
needed in developing methods for predicting the impact on
ground-water quality of pollutants released into the earth's
crust.
11
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TABLE OF CONTENTS
Page
ABSTRACT ii
LIST OF TABLES iv
ACKNOWLEDGEMENTS V
SECTIONS
I CONCLUSIONS 1
II RECOMMENDATIONS 3
III INTRODUCTION 4
IV THE SUBSURFACE AS A BIOLOGICAL HABITAT 7
SPATIAL LIMITATIONS 7
WATER 11
TEMPERATURE 14
HYDROSTATIC PRESSURE 16
OXIDATION-REDUCTION CONDITIONS 18
NUTRIENTS 23
GENERAL ENVIRONMENTAL OBSERVATIONS 27
V INVESTIGATIONS PERTAINING TO SUBSURFACE 29
BIOLOGICAL ACTIVITY
TECHNIQUES AND PROBLEMS IN STUDYING 30
SUBSURFACE BIOLOGY
RESULTS OF INVESTIGATIONS OF SUBSURFACE 33
MICROBIAL ACTIVITY
SIGNIFICANCE OF PREVIOUS SUBSURFACE 48
MICROBIAL STUDIES AND NEEDED
ADDITIONAL INVESTIGATIONS
VI REFERENCES 51
iii
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LIST OF TABLES
No. Page
1 REPRESENTATIVE POROSITY AND PERMEABILITY 9
RANGES FOR SELECTED SEDIMENTARY ROCKS
2 CONTENT OF ORGANIC CARBON IN REPRESENTATIVE 25
GROUND WATERS
3 DEPTH OF PENETRATION OF A BACTERIAL TRACER 32
(Serratia marcescens) FROM DRILLING MUDS
INTO DRILL CORES
4 REPRESENTATIVE REPORTS OF ORGANISMS ISOLATED 34
FROM SUBSURFACE ROCKS AND WATERS
5 COMPARISON OF THE AVERAGE BACTERIAL AND 45
CHEMICAL COMPOSITION OF THREE DIFFERENT
ZONES WITHIN OIL-BEARING AQUIFERS
6 SOME RESULTS OF BACTERIOLOGICAL AND CHEMICAL 47
INVESTIGATIONS OF THE GROUND WATERS OF AN
OIL-BEARING AQUIFER IN SOUTH TEXAS
iv
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ACKNOWLEDGEMENTS
This project would not have been possible without the
professional expertise and personal interest of Lorene
Fuller, Librarian, Robert S. Kerr Environmental Research
Laboratory, and Judy Thompson, Library Technician. Their
assistance is gratefully acknowledged.
Special thanks are due to Linda Harmon for her assistance
in organizing the report and for typing and retyping the
various drafts cheerfully and efficiently.
The patience and encouragement of Jack W. Keeley throughout
the period of preparation of this paper are sincerely
appreciated.
v
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SECTION I
CONCLUSIONS
Currently available information permits the following
conclusions concerning subsurface biological activity
in relation to ground-water pollution.
1. The upper continental crust of the earth comprises
a highly structured and complex environment, char-
acterized by limited open space and a myriad of
environmental possibilities. In most subsurface
regions, particularly those of sedimentary origin,
these environmental possibilities do not appear so
severe as to preclude microbial activity until
depths are attained where temperatures exceed
microbial tolerance levels. These depths are
apparently in excess of 2,000 m in most regions.
2. Field investigations pertaining to subsurface
microbial activity are difficult due to the
inaccessability of subsurface regions and the
consequent difficulty in obtaining uncontaminated,
representative samples from such regions. Never-
theless, a number of credible investigations re-
ported in the literature clearly show the presence
of diverse microbial populations in many subsurface
regions lying below the soil zone.
3. Available information indicates strongly that
microbial activity is both possible and probable
in most subsurface regions below the soil zone
which are of importance in regard to ground water.
Hence, possible biochemical alteration of pollutants
in these regions, as well as possible effects of
pollutants on subsurface ecosystems, must be consid-
ered in attempting to control pollution of ground
water.
4. Investigations to further elucidate the extent and
nature of microbial activity in subsurface regions
below the soil zone should be included in general
studies pertaining to the movement, fate, and effect
of pollutants in subsurface waters if these studies
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are to provide an effective basis for assessing the
probable impact of human activities on the ground-water
resource and for regulating and controlling the conduct
of such activities to minimize the threat to ground-
water quality.
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SECTION II
RECOMMENDATIONS
It is recommended that investigations be instigated to further
elucidate the potential role in ground-water pollution and
pollution control of microbial activity in subsurface regions
lying below the SOil Zone. ThgfLA jjnw»g-Mgat-ions ghrm"M hf>
anjlntegral part of more extensive studies pertaining bg_the
movemen€7^fate, and effect" of pollutants' in subsurface~waters.
The extent and nature of native microbial activity in the zone
of aeration below the soil zone and in the upper regions of
the zone of saturation should be systematically investigated.
These investigations should attempt to establish relationships
between native microbial populations and subsurface environ-
mental characteristics, particularly geology and oxidation-
reduction conditions.
Microbial activity and microbe-pollutant interactions in
subsurface regions receiving pollutants should be subjected
to thorough, integrated investigations. In-situ studies
should be employed to the fullest possible extent. These
studies should seek to correlate native microbial populations,
environmental situations (geology, oxygen and nutrient avail-
ability, pressure, temperature, etc.), nature of pollutants,
and microbial populations after introduction of pollutants
with the fate and effect of pollutants.
The ultimate objective of the recommended investigations should
be the development of information needed for defining the
capacity of subsurface regions as pollutant receptors and,
hence/ for predicting the probable impact on ground water
of activities entailing the release of pollutants into the
earth's crust.
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SECTION III
INTRODUCTION
Ground water—water beneath the earth's surface—is a little-
appreciated but valuable national resource. Approximately
20 percent of the total quantity of water currently used for
all purposes in the United States is supplied by subsurface
sources. It is estimated that ground water comprises at
least 95 percent of the Nation's fresh-water reserves, with
surface water accounting for only 5 percent of the total.1
Burgeoning requirements for fresh water and limited surface
water supplies almost certainly will result in increasing
utilization of the ground-water resource to satisfy future
water needs.
Although ground waters are less easily polluted than surface
waters, the activities of a growing and affluent population
are increasingly subjecting subsurface waters in the United
States to potential pollution. About 13 million septic
systems serving approximately 50 million people are presently
in use in the United States,* and numerous lagoons or leaching
ponds are employed for disposal of domestic, industrial, and
agricultural wastes. Soil treatment systems, involving
application of high volumes of liquid wastes to large areas
of surface soil, as well as water reuse systems entailing
percolation of treated waste waters through the earth to
recharge ground water, are being utilized with increasing
frequency. Sanitary landfills are used for disposal of much
of the country's solid waste, and large quantities of
chemicals are applied to the soil in intensive agricultural
operations. Transport and storage of commercial chemicals,
both inorganic and organic, entail accidental introduction
of these substances into the ground as a result of transport
vehicle accidents or-leakage of storage vessels and pipelines.
Subsurface disposal of wastes by means of injection wells is
employed by the oil industry for disposal of brines, and is
becoming increasingly attractive to industries with problem
wastes as a means to avoid either excessive waste treatment
expenses or violation of surface water quality standards.
Also, artificial recharge of ground-water aquifers by direct
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injection of surface waters is being practiced to store and
conserve surface waters, or to maintain a hydraulic head to
prevent contamination of fresh-water aquifers with undesirable
waters such as sea water. In total, these activities entail
the potential for introduction into subsurface waters of a
vast array of pollutants, including a myriad of synthetic
organic chemicals. It is imperative that this potential for
pollution of ground waters be thoroughly evaluated in order
that procedures necessary to prevent or minimize such pol-
lution may be instigated. This need is particularly urgent
because pollution effects almost invariably persist much
longer and are more difficult to eradicate in ground waters
than in surface waters.
Examination of the major potential sources of ground-water
pollution reveals that pollutants are most likely to enter
ground waters by either of two principal pathways: perco-
lation through the unsaturated portion of the earth's crust
above the water table or discharge directly into the sub-
surface water. Both during percolation through the crust
of the earth above the water table and within saturated
ground-water zones pollutants are subject to possible
sorption, abiotic chemical alteration, and chemical alter-
ation resulting from biological activity. Of the three
possibilities, biochemical alteration may well be of greatest
potential importance in determining the ultimate effect of a
pollutant on ground-water quality.
The topmost layers of the earth's crust, comprising that
region considered by the soil scientist as true soil as
opposed to subsoil and underlying substratum, have been
rather thoroughly studied, principally because of their
importance to agriculture, and have been shown to comprise
a region of relatively intense biological activity. Hence,
pollutants percolating through the soil zone of the earth's
crust are known to be subject to potential biochemical alter-
ation as the result of the metabolic activities of a multi-
tudinous population of biochemically diverse microorganisms
within this region. However, those regions of the earth's
crust lying below the soil zone, including both unsaturated
zones above the water table and saturated ground-water zones,
have received much less attention in regard to their biolog-
ical activity than has the soil zone. Consequently, the
possibility of biochemical alteration of pollutants in these
regions, as well as possible effects of pollutants on sub-
surface ecosystems, has been given little serious consideration.
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With increasing release of pollutants into the crust of
the earth through accidents, waste disposal, agricultural
activities, subsurface disposal, and artificial recharge,
questions regarding subsurface biological activity and
attendant biochemical alteration of pollutants are assuming
great importance in regard to control of ground-water pol-
lution. This is especially true since many pollutants are
relatively intractable synthetic organic compounds which may
survive passage through the upper soil layers and since
many pollutants may be introduced directly into ground
water without ever passing through the soil zone. Knowledge
of the biological activity which occurs or can be expected
to occur in the subsurface environment is a necessary
component for predicting the probable impact of human
activities on the ground-water resource and for planning
and conducting such activities to minimize the threat to
ground-water quality.
Information pertaining to subsurface biological activity
is relatively scarce and widely scattered through the
literature of several scientific disciplines, notably
geohydrology, gjLQehsmjLstry^ qeomicrobiolgqy_, microbial
ecology7~and petroleum microbiology. This papelTpresents
a discussion of subsurface microbial activity in relation
to ground-water pollution as reflected by available refer-
ences in these diverse literatures. Principal attention
has been devoted to those regions of the earth's crust lying
below the soil zone, since numerous compilations of information
concerning biological activity in the soil are widely available,
The first portion of the paper is devoted to a discussion of
the subsurface environment in terms of those factors likely
to be of most significance in regard to the development of
biological systems. This is followed by a discussion of
investigations pertaining to subsurface biological activity,
including techniques and problems in studying subsurface
biology, results of field investigations of subsurface
microbial activity, and the significance of such investi-
gations in regard to ground-water pollution and need for
additional studies.
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SECTION IV
THE SUBSURFACE AS A BIOLOGICAL HABITAT
The upper continental crust of the earth may be described in
a very general way as consisting of crystalline basement rock
interspersed with and overlain by sedimentary and volcanic
rocks in deposits of varying sizes, shapes, and depths. (The
term rock is used here in the broad sense, meaning any solid
constituent of the earth's crust.) The basement rock consists
mainly of granitic rocks with masses of metamorphosed volcanic
and sedimentary rocks, while the sedimentary and volcanic
deposits are comprised of unconsolidated rocks such as clay,
silt, sand, gravel, and volcanic ash and consolidated rocks
such as shale, sandstone, limestone, dolomite, gypsum, anhy-
drite, rock salt, and lava. These rock materials, together
with the liquids and gases filling the voids or interstices
within them, comprise the subsurface environment in which
subsurface ecological communities must exist and function.
The subsurface environment is a highly structured environment
characterized by limited open space. Like the surface environ-
ment, it is not homogeneous but varies from point to point,
encompassing a wide spectrum of environmental possibilities.
In appraising the subsurface as a potential biological habitat,
the limitations imposed by these environmental possibilities
on biological systems are of principal concern. The nature
and extent of these limitations are best revealed by examining
available information concerning the subsurface environment
in regard to those factors likely to be of most significance
in controlling the development of subsurface biota, including
spatial limitations, water temperature, hydrostatic pressure,
oxidation-reduction conditions, and nutrients.
SPATIAL LIMITATIONS
Organisms which may inhabit the subsurface will be essentially
restricted to the space provided by the voids, or interstices,
of the rock comprising the upper portion of the crust of the
earth. This fact would appear to virtually eliminate sub-
surface regions below the soil zone as potential habitats
for any life forms other than microorganisms. Of principal
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concern, therefore, is the "living space" which interstices
in the rocks of the subsurface might afford to microorganisms.
The interstices of the rocks which comprise the earth's crust
reflect the diverse geological processes by which the rocks
were formed and later modified. They are of two major classes
original (or primary) interstices, which came into existence
when the rocks were formed; and, secondary interstices,
resulting from modification of the rock by processes to
which they were subjected after formation. Original inter-
stices consist of spaces between adjacent fragments of
sedimentary rocks and cavities or intercrystal spaces formed
in igneous rocks during their congealing. Secondary inter-
stices are mainly joints, fractures, and solution cavities.
Interstices differ widely in size, shape, arrangement, and
aggregate volume. They range in size from large solution
cavities through all gradations to minute pores and may be
closely interconnected or relatively isolated.
Porosity is a measure of the interstitial space of a rock
and is usually expressed as the percentage of the total
volume of that rock which is occupied by interstices. The
ability of a rock to transmit fluid, called its permeability,
provides an indication of the size and degree of intercon-
nection of the interstices. In ground-water investigations,
permeabilities are usually stated in terms of the coefficient
of permeability, which is defined as that quantity of water
in gallons per day which will flow through a cross-sectional
area of one square foot of rock under a hydraulic gradient
of one foot per foot.
In Table 1 are presented some representative porosity and
permeability ranges for selected sedimentary rocks, as given
by Walton3 and Todd.** As these data show, the aggregate
volume of the interstices in sedimentary rocks usually
comprises a significant portion of the total volume of
these rocks. However, the individual interstices may some-
times be extremely small or poorly interconnected, resulting
in very low permeability of the rock even though its porosity
is appreciable, as in the case of clay, silt, shale, and
some sandstones. Crystalline rocks are usually quite low
in both porosity and permeability, unless they contain
significant secondary interstices.
8
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Table 1. REPRESENTATIVE POROSITY AND PERMEABILITY
RANGES FOR SELECTED SEDIMENTARY ROCKS
Rocks
Clay
Silt
Sand
Gravel
Sandstone
Shale
Limestone
Porosity,
%
45-55
40-50
35-40
30-40
10-20
1-10
1-10
Permeability,
gpd/ft2
0.001-2
0.001-2
100-3000
1000-15000
0.1-50
0.00001-0.1
No data available
Porosity data indicate that the total interstitial space per
unit volume of most rocks comprising the upper crust of the
earth, with the possible exception of massive crystalline
rocks containing no joints or fractures, is adequate to
permit at least some microbial activity, provided the sizes
of the individual interstices are sufficient to accomodate
the dimensions of the microorganisms. Permeability, however,
provides only a qualitative indication of interstice size,
and the sizes of interstices in most rocks have not been
quantitatively defined. Hence, it cannot be presently
ascertained if spatial limitations definitely preclude
microbial activity in any subsurface regions above those
extreme depths where interstitial space becomes practically
nonexistent. However, Berea sandstone, a rather dense rock
with permeability in the neighborhood of 1 gpd/ft2, was
reported by Kalish, et al.,to have a mean pore diameter of
7 to 12 microns,5 which would accomodate the dimensions of
most bacteria, particularly the smaller organisms; and
bacteria (32p-labeled-Serratia marcescens) were found by
Myers and McCready to be transported by water through a
2-inch by 14.25-inch core of this rock in 84 hours.° The
latter investigators also reported that S_. marcescens
passed through Mississippian and Early Devonian limestone
and Late Mesozoic sandstone with permeabilities in the
proximity of 0.1-0.001 gpd/ft2. These data suggest that
bacteria might find adequate space to exist and function
in wide regions of the subsurface. Certainly, spatial
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limitations would not appear likely to exclude as microbial
habitats those subsurface regions of sufficient porosity
and permeability to permit transmission and storage of
appreciable quantities of water. These are, of course,
the regions of greatest interest in regard to ground-water
pollution control.
Spatial limitations of the subsurface environment are also
of concern in the transport of microorganisms into and within
subsurface regions by moving water. Obviously, physical
difficulty in negotiating small interstices as well as
possible sorption on solid surfaces are likely to greatly
impede the movement of microbes through the interstitial
space of subsurface rocks. However, as previously noted,
investigations by Myers and McCready indicated that the
bacterium S_. marcescens can, with time, pass through sand-
stones and limestones of low permeability.6 Also, several
investigations, reviewed by Mailman and Mack,7 revealed
considerable potential for transport of bacteria through
relatively permeable sands by moving water. These studies,
which pertained to the pollution of ground water by microbes
from sewage, actually indicated that coliform organisms from
sewage did not move great distances through the sands studied,
However, the principal deterrent to movement of the coliforms
was apparently not physical impediment within the interstices
of the sand, but rather the development of a mat of solids
from the sewage at the interface where the liquid entered
the sand with resulting removal of the organisms from the
infiltrating water by filtration or sorption on this mat.
Before development of the mat, coliform organisms moved
rather rapidly through sand for distances as great as 30 m
(100 ft). It seems likely that this distance was largely
a function of the life span of the coliforms in an environ-
ment which was not most conducive to their growth, rather
than an indication of physical impediment of movement of
the microbes.
These investigations suggest that bacteria which are capable
of adapting to the subsurface environment might be able to
gain access even to deep subsurface habitats by moving,
perhaps over long periods of time, from the surface with
percolating water through the interstices of the rocks
comprising the earth's crust.
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WATER
Regardless of the nature of their habitat, microbes are
fundamentally aquatic organisms which require free water
for growth. The cells must be surrounded by a layer of
water that is adequate to permit diffusion of nutrients
and toxic products and to maintain the internal water
budget in order that metabolism and reproduction may proceed.
It is necessary, therefore, in considering the subsurface as
a possible microbial habitat, to examine the distribution and
nature of subsurface water.
Subsurface water is generally distributed vertically within
the earth's crust in two major zones, the zone of aeration
(also called the zone of infiltration or vadose zone) and
the zone of saturation. The zone of aeration, in which the
interstices of the rocks comprising the crust of the earth
contain varying proportions of water and gas, overlies the
zone of saturation, in which the interstices are completely
filled with liquid, usually water. The depth to the upper
boundary of the zone of saturation at a specific geographical
location depends on geological and climatic factors and may
vary from essentially the surface to many hundreds of feet.
Since the zone of aeration lies between the land surface and
the zone of saturation, its thickness varies with and is
equivalent to the depth to the zone of saturation.
The zone of aeration may be divided into three subzones: the
soil water zone; the intermediate zone; and the capillary zone,
The soil water zone extends from the surface down through the
major root zone and varies in depth with soil type and vege-
tation. Water contained within this zone may be classified
as follows:
1. Hygroscopic water - water which is very strongly
adsorbed by soil particles, forming a thin film
around them; it is present even in extremely dry
soils in times of drought and in deserts, but is
generally unavailable to plants and soil microbes.
2. Capillary water - water which is held in the soil
interstices by surface tension, and which is avail-
able to soil organisms.
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3. Gravitational water - excess water which is
not held by surface tension or absorptive
forces, and which drains into underlying
zones under the influence of gravity; it
is normally present only during periods of
relatively high infiltration resulting from
rainfall, irrigation, or liquid waste
disposal to the soil.
The intermediate zone extends from the lower edge of the
soil water zone to the top of the capillary zone. It is
primarily a region through which water must pass to reach
the zone of saturation and may be nonexistent or several
hundred feet thick. It contains gravitational water during
periods of infiltration and otherwise holds a significant
portion of capillary water, sometimes called pellicular
water.
The capillary zone extends from the top of the zone of
saturation to the limit of capillary rise of water. Its
thickness is a function of the size of the interstices of
the rock in which it occurs. Hence, it may extend several
feet in clay and only a fraction of an inch in gravel.
The zone of saturation may extend to great depths within
the crust of the earth, particularly in deep sedimentary
basins. Kuznetsov, et al., point out that Filatov has
estimated gravitational water to be capable under natural
conditions of moving through sedimentary rock to the depth
of about 4,000 m (13,000 ft).8 Some wells have actually
obtained water at depths of more than 3,000 m (10,000 ft).9
Water contained in the zone of saturation is by definition
ground water. The vertical distribution of ground water
within the zone of saturation is not usually uniform but
reflects the varying porosity and permeability of the suc-
cessive strata of rocks comprising the earth's crust at
any given geographical location. Hence, there may be at
successively greater depths several saturated strata of
sufficient porosity and permeability to yield significant
quantities of water, separated by other strata which are,
in the vast majority of cases, also saturated but which
are relatively impermeable. Of course, regions in which
water has been displaced by petroleum liquids or gases are
often found in the zone of saturation, usually at greater
depths. These regions are, in a sense, anomalous.
12
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Movement of subsurface water within the zone of aeration is
essentially vertical in direction, while the movement of
ground water in the zone of saturation is mainly lateral,
towards points of discharge to the surface. The rate of
movement of ground water is quite slow, usually being
measured in feet per day or feet per year. In the permeable
formations comprising the upper part of the zone of saturation
rates of movement are usually highest, so that residence times
for water in these formations most generally do not exceed a
few years. Water movement in the deeper permeable strata of
the zone of saturation, ranging in depth from hundreds to
thousands of feet, is generally much slower, with residence
times of decades to thousands of years being common. Some
waters deep within the zone of saturation, usually several
thousand feet below the land surface, are essentially stagnant,
exhibiting no discernable movement. These waters appear to
have been trapped for geologic ages in permeable formations
which are completely isolated from the natural circulating
system of the hydrologic cycle by impermeable strata.
Because of the great solvent power of water, ground water
usually reflects to some extent the chemical composition
of the rock formations through which it moves. In general,
the mineral content of ground waters increases with depth
and may reach levels in excess of 20 percent total mineral
content in deep formations, particularly those which appear
to be hydrogeologically isolated.8/10 Ground waters of high
mineral content are also found in relatively shallow rock
formations which are partly composed of salt beds deposited
from ancient seas.
This description of the nature and distribution of subsurface
water, though brief and oversimplified, shows clearly that
water is virtually ubiquitous in the subsurface environment
but varies considerably in quantitative distribution, move-
ment, and mineral content.
The lowest relative humidity at which microbial growth is
possible depends on the species,11 but most soil microbes
cannot grow when the soil contains only hygroscopic water.
Hence, microbial growth in the soil water zone may be
greatly retarded during periods of drought, but quickly
resumes, often at increased levels, when water replenishment
occurs.*2 In the deeper regions of the zone of aeration,
however, it seems unlikely that moisture levels would ever
become so low as to effectively limit microbial activity.
13
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In those regions of the zone of saturation where ground waters
are highly mineralized the availability of water to many
microbes would, in effect, be severely restricted because of high
osmotic pressures. However, bacteria differ greatly in their
ability to withstand and adapt to high osmotic pressure, and
many species have been found to grow in highly mineralized
waters, including some which tolerate as much as 30 percent
salt.13 Therefore, considering the general abundance of water
in the zone of saturation, it seems a logical conclusion that
practically no subsurface environmental possibility would be
completely hostile to all microorganisms because of limited
availability of water.
TEMPERATURE
Temperature is one of the most important environmental factors
controlling the metabolic activity of microorganisms in any
type of habitat, and considerably information has been developed
concerning the relationship of temperature to the growth and
survival of microorganisms. In general, microbial metabolic
activity is roughly doubled for each 10°C rise in temperature
until a temperature is reached at which the rate of thermal
denaturation of enzyme proteins exceeds the rate of thermal
stimulation of enzyme reaction rates. For mesophilic organisms,
which comprise by far the greatest number of microbes, denatur-
ation becomes significant above 40°C; hence, the optimum growth
range for these organisms is 25-40°C. Thermophilic organisms
have an optimum growth temperature of 55-60°C, but some can
continue to grow at temperatures as high as 75-90°C at
atmospheric pressure, evidently because they produce enzymes
which are less prone to heat denaturation. The maximum tem-
perature that all but a few heat-resistant spore-forming
bacteria can tolerate without being killed is normally
considered to be 100°C, but most microorganisms are actually
killed by temperature much above 50°C. lk
The minimum temperature limits at which all microorganisms
cease to grow in nature are determined primarily by their
need for water in the aqueous phase and may be as low as
-15°C in waters with a high concentration of solutes. 15
Many psychrophilic bacteria are found in nature which can
grow fairly rapidly between 0° and 5°C. However, the optimum
growth temperatures for many psychrophiles actually are often
in the same range of 25°-40°C as most other bacteria. Ikf1&
14
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The temperature tolerances of microorganisms may be affected
by other environmental factors. For example, high acidity
seems to lower the maximum temperature at which growth may
occur.17 Pressure and temperature are definitely interrelated,
the maximum temperature for growth being raised 5°-20°C by
compression according to ZoBell.18
Temperatures in the earth's crust rarely fluctuate, except
in the topmost layer of approximately 10 m (33 ft) which may
be affected by seasonal temperature variations.8 The normal
ground-water temperature at depths of 9-18 m (30-60 ft)
roughly corresponds to the mean annual air temperature of
the given region and ranges from about 3°C (37°F) to 25°C
(77°F) in the United States.1 As the depth increases, the
temperature of the subsurface rocks and waters increases;
this increase is roughly estimated to be 30°C per 1,000 m
(3,281 ft).19
Based on upper temperature limits of 80°C and 100°C,
respectively, for microbial growth and survival, and
assuming a 30°C increase in temperature per 1,000 m of
depth, it would appear that high temperatures would likely
prevent most microbial activity in subsurface environments
lying below approximately 1,800 m (5,900 ft) and would
virtually preclude the existence of microbes in those
environments at depths below about 2,500 m (8,200 ft).
These values are, however, only very rough guidelines subject
to wide variation, since temperatures within the earth's
crust at any particular geographical location are likely
to reflect not only depth but also geologic structure and
geothermal activity. Hence, high temperatures may sometimes
occur at relatively shallow depths, while temperatures
considerably lower than predicted may be encountered in
deep formations. For example, on the basis of 4,500 thermo-
graphs and 700 individual measurements from more than 5,000
deep bore holes throughout the European USSR, Pokrovskii,
et al., concluded that the 100° isotherm in this region lay
at varied depths generally ranging from 2,900 to 5,500 m
(9,500 to 18,100 ft).20 In a few areas, however, temperatures
of 100°C were encountered within 300-400 m (1,000-1,300 ft)
of the surface because of thermal springs, and in other areas
the 100° isotherm was indicated to be as deep as 10,000-
15,000 m (32,800-41,200 ft) because of low geothermal activity.
It is obvious, therefore, that temperature extremes suffi-
cient to preclude microbial activity are not likely to occur
in those regions of the subsurface that are of greatest
concern in regard to ground-water pollution, with the
possible exception of a few regions of extreme geothermal
15
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activity and some very deep strata which may be employed for
subsurface disposal of waste waters. In fact, the elevated
constant temperatures of 25-40°C which are likely to occur
in many subsurface regions would be ideal for growth of
mesophilic organisms.
HYDROSTATIC PRESSURE
The pressure within the crust of the earth increases progres-
sively with depth. This increase is due principally to the
weight of the overlying rocks and of the water contained within
the rock interstices. The hydrostatic pressure within rock
interstices, in which any subsurface microbial activity must
occur, usually is not influenced to any great extent by the
overburden pressure exhibited by the rocks but approximates
the pressure which would be produced by a column of water
extending from the surface to the depth of the measurement.
Hence, interstitial hydrostatic pressure generally increases
downward at an average rate of 0.1 atin per meter or about
0.43-0.47 psi per ft, although pressures considerably higher
or lower than predicted may be encountered with some frequency.21
For example, in an extensive study of pressures in 76 oil wells
in the Gulf Coast Region of Texas and Louisiana actual pres-
sures were found to agree quite well with predicted values
down to a depth of about 2,200 m (7,200 ft), ranging from
37 atm at about 365 m to approximately 221 atm at 2,160 m.22
At greater depths positive pressure deviations were quite
common, with a pressure of 530 atm at about 3,200 m (10,200 ft)—
a deviation of 64 percent—being noted in one case. These
excessive pressures may have been due to the development of
gases and to the partial transmission of overburden pressures
to confined interstitial fluids in isolated deep formations.
;Kuznetsov,et al., concluded, on the basis of pressures observed
in oil wells in the USSR, that the hydrostatic pressure Should
not exceed 300-400 atm in the majority of subsurface regions
where temperatures are not too high for microbial activity.8
This appears a valid assumption, although there may well be
exceptions in regions of low geothermal activity.
Microorganisms differ considerably in their response to hydro-
static pressure. Some may survive for prolonged periods at
pressures greater than 2,000 atm, but none have been found that
can grow at such high pressures. Most microbes which normally
are found growing in habitats near atmospheric pressure appear
16
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capable of at least some growth at 200-300 atm at 30°C,
although some species are completely inhibited or killed
by these or even lower hydrostatic pressures.l 8,23,21* At
higher pressures the growth of organisms from surface or
near-surface habitats is usually severely to completely
retarded, and the survival of such microbes for significant
periods at pressures of 500-600 atm is doubtful.
On the other hand, many microorganisms isolated from deep
sea habitats have exhibited the ability to grow readily
at pressures above 400 atm during laboratory studies.
Sulfate-reducing bacteria from the Philippine Trench
(depth about 10,000 m) were observed by ZoBell to grow
very Slowly at pressures as high as 1,400 atm, but rela-
tively few species of microbes from all habitats have been
successfully cultivated under laboratory conditions at
pressures in excess of 800 atm.25
Pressure-tolerant microbes which have been investigated
exhibit significant variability in regard to the range
of pressures within which they are capable of growth.
Many also can grow at low hydrostatic pressures. This is
illustrated by an investigation of ZoBell and Budge, who
found that four of eight species of marine facultative
anaerobes capable of growing at 1,000 atm grew best at
1 atm, while the optimum pressures for growth of the other
four species were in the range of 100-200 atm.26 Other*
pressure-tolerant microbes, however, grow poorly or not
at all at low pressures. For example, sulfate-reducing
bacteria from the Weber Deep in the Indian Ocean were
cultivated for several years at 700 atm at temperatures
of 3-5°C by ZoBell and Morita but failed to grow at
1 atm.27
Microorganisms growing at pressures near their maximum
tolerance levels often exhibit indications of altered
metabolric processes. Filamentous growth resulting from
failure of cells to divide in the normal manner is commonly
noted, and decreased synthesis of macromolecules such as
nucleotides and proteins has been observed by several
investigators.28,29/30 Also, the ability of microbes to
grow at high pressures appears to be enhanced by the
plentiful availability of organic metabolites (amino acids,
vitamins, etc.) in the surrounding medium, which probably
reflects decreased synthetic capability at higher pressures.25
The mechanisms by which increased pressures induce alterations
in microbial metabolic processes have not been elucidated, but
17
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inhibition of enzyme systems, possibly through adverse
effects on formation of enzyme-substrate complexes, may
be involved. Such a mechanism appears plausible because
the formation of an enzyme-substrate complex entails an
increase in molecular volume, and increased hydrostatic
pressure tends to inhibit reactions requiring increases
in molecular volume.31'32 This mechanism would be consistent
with the observation that pressure tolerance levels of
microbes are usually raised by limited increases in temp-
erature,18 since increased temperature tends to increase
molecular volume and hence might be expected to attenuate
the effects of increased pressure.
In summarizing the information presented above, it seems a
reasonable conclusion that hydrostatic pressures are not
likely to exclude microbial activity in subsurface regions
which are otherwise suitable as microbial habitats. However,
pressures in deep formations employed for subsurface disposal
of liquid wastes may be sufficiently high to kill or to
severely affect the metabolic activity of microbes from
surface habitats which may be indigenous to the injected
wastes.
OXIDATION-REDUCTION CONDITIONS
Determinations of both molecular oxygen concentrations and
oxidation-reduction potentials in subsurface environments are
subject to formidable methodological problems because of the
difficult accessibility of these regions. Consequently,
definitive data concerning subsurface oxidation-reduction
conditions are scarce. However, those data which have been
reported, coupled with general observations concerning the
probable availability and consumption (reduction) of molecular
oxygen and other reducible substances in subsurface regions,
provide a limited insight regarding probable oxidation-
reduction conditions in subsurface zones.
In the zone of aeration, gas in the rock interstices is for
the most part in direct communication with the atmosphere
and, hence, is potentially subject to constant interchange
with atmospheric gases. Interchange results from molecular
diffusion of gases through the rock interstices and from
actual displacement of interstitial gas due to diurnal heating
and cooling of the earth's surface, changes in barometric
pressure, and intermittent filling of interstices by gravi-
tational water with subsequent displacement of this water by
18
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fresh atmospheric gas. It has been widely assumed, therefore,
that high oxygen tensions generally prevail within the zone of
aeration. However, the situation is probably not so simple as
this assumption implies.
Regions of oxygen deficiency are known to occur with some
frequency in the soil water subzone of the zone of aeration,
even though oxygen replenishment by gas interchange should be
most effective in this region by virtue of its close proximity
to the atmosphere. These oxygen-deficient regions probably
occur because of an unfavorable balance between oxygen replen-
ishment by gas interchange, which appears to be restricted by
interstitial spatial limitations,and oxygen utilization by
soil microorganisms. For example, it is known that soils
which are rich in organic matter are likely to become deficient
in oxygen even near the soil surface; 33 also, soils of small
particle size, and hence low permeability, have been found to
exhibit oxygen deficiency at shallow depths, while coarser
soils of greater permeability maintained a high oxygen tension
under similar conditions.34 In addition, anaerobic micro-
environments are believed to occur commonly in the soil water
zone, even in well-aerated soils.n These microenvironments
are thought to consist of soil aggregates which have developed
oxygen-deficient regions within their interiors because of
inadequate diffusion of oxygen through water surrounding and
within the aggregates and microbial depletion of oxygen on
their peripheries.35
Microbial activity and, hence, oxygen consumption is almost
certainly significantly less in the intermediate and capil-
lary sub zones of the zone of aeration than in the soil sub-
zone in most situations. However, the rate of gas inter-
change with the atmosphere must surely be related to depth
and to permeability of the rock comprising the zone of
aeration and is therefore likely to be low in these deeper
regions, particularly in formations of low permeability.
Also, the probability of survival of molecular oxygen
during movement through the rock interstices towards the
deeper regions of the zone of aeration could be relatively
low if microbial activity in the soil zone were high. It
would appear, therefore, that regions of oxygen deficiency
may be at least as likely to occur in the deeper subzones
of the zon§ of aeration as in the soil water subzone.
In summary, it appears logical to suspect that the oxidation-
reduction conditions at any specific point in the zone of
aeration will reflect the geology of the zone of aeration, the
19
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depth of this point within the zone of aeration, and the
microbial activity in the vicinity of the point and in
the overlying strata and, hence, may well vary over a
wide range of possibilities.
Oxidation-reduction conditions in the zone of saturation
likely will reflect the balance between: (1) the rates
of processes which result in the reduction of molecular
oxygen and other reducible substances in ground water;
and, (2) the rates of entry into ground water of recharge
water containing oxygen and other reducible substances
and of diffusion into ground water of oxygen from the
zone of aeration. Hence, redox conditions at any specific
point in the zone of saturation will likely depend on the
several factors presented below.
1. Redox conditions in the overlying zone of aeration. —
Obviously, the zone of saturation can receive little
or no oxygen replenishment from the zone of aeration
if the gas at the interface between the two zones is
deficient in oxygen.
2. Distance from the interface between the zones of
aeration and saturation. — Replenishment of oxygen
and other electron acceptors becomes more difficult
as the distance from the air-water interface in a
geological formation increases, since diffusion of
oxygen through water and vertical mixing of ground
water are usually very slow processes.
3. Extent of microbial activity in the ground water. —
Although abiotic reductive processes may be of some
importance in specified ground waters, particularly
in regions of high temperature and pressure, those
processes which are most likely to result in reduction
of molecular oxygen and other reducible substances in
ground water are probably associated with microbial
activity. Hence, the ultimate level of reduction
which is attained in a particular region of the zone
of saturation will likely depend to a large extent on
the level of microbial activity in that region.
4. Rate of movement of ground water. — The longer the
residence time for ground water in a specific subsurface
region, the longer it will be subject to any reductive
processes which may be operating there; hence, a reduced
environment could develop in a saturated subsurface
20
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region where watet movement is very slow, even though
the rates o£ processes resulting in reduction of mole-
cular oxygen and other reducible substances may also
be slow in this region.
5. Rate of ground-water recharge. — Recharge of ground
water ordinarily results in replenishment of oxygen
and other reducible substances in the zone of saturation
by virtue of the dissolved, oxygen and other easily
reducible substances such as ferric iron, nitrate,
sulfate, and certain organic compounds which may be
present in the recharge water. Rate of ground-water
movement also is usually related to rate of recharge.
Since the deeper regions of the zone of saturation are far
removed from the zone of aeration and the rates of ground-
water movement within these regions and the rates at which
they are recharged range from very low to essentially zero,
it appears unlikely that appreciable oxygen and other easily
reduced substances could be present in them. This supposi-
tion is supported by data obtained in the USSR during studies
related to the genesis of and exploration for petroleum. For
example, Kuznetsova found waters in deep formations in the •
oil fields of the Kuybyshev region to have redox potentials
(Eh) ranging from +20 to -250 mv.36 Also, Al'tovskii detected
no oxygen in water from 18 wells in the Groznyy oil regions
ranging in depth from 225 to 2,097 m (approximately 740 to
6,900 ft).37 The average redox potential, expressed as rH2,
for these waters was 11.0 (rH2 = Eh/0.029 + 2pH; hence at
pH = 7.0, rH2 = 11.0 would be equivalent to Eh = -87 mv) .
There is little doubt,therefore, that the deeper regions
of the zone of saturation generally comprise a reducing
environment.
On the other hand, considerable variations in rates of
reductive processes and replenishment of oxidized substances
appear possible in the upper regions of the zone of saturation,
Hence, it seems likely that oxidation-reduction conditions in
these regions may vary over a significant range of possibil-
ities, although somewhat reducing conditions would appear to
be generally favored. Data obtained by Al'tovskii from two
wells tapping an artesian aquifer in the USSR are of interest
in regard to this hypothesis.37 One well which was in the
recharge area of the aquifer was approximately 75 m (^45 ft)
deep and produced water with an rH2 of 27.2 (Eh = 382 mv at
pH 7) and an oxygen content of 4.8 ppm. The second well,
21
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which was in the discharge region of the aquifer, was about
20 m (65 ft) deep and produced water with an rH2 of 14.3
(En = 10 mv at pH 7) and which contained no oxygen. The
presence of oxygen and a high redox potential in water from
the deeper well apparently reflected the replenishment of
oxygen in the zone of saturation by oxygenated recharge
water and also possibly by diffusion from the nearby zone
of aeration. The water in the second well was separated
from the immediate overlying zone of aeration by a relatively
impermeable formation and from the recharge region by a long
distance. Hence, there was little chance for replenishment
of the oxygen which was consumed during its movement through
the aquifer.
The observation of redox potentials ranging from +540 mv
to -380 mv in water samples obtained just below a water
table ranging from 10 to 70 ft (3-21 m) beneath the surface
in the valley of the South Platte River of Colorado may
provide a further indication of the range of oxidation-
reduction conditions which can be expected in the upper
regions of the zone of aeration.38
The. oxidation-reduction conditions in which microorganisms
can grow have been cited by Vallentyne as ranging from an
upper E- limit of +850 mv at pH 3 for iron bacteria to a
lower limit of -450 mv at pH 9.5 for sulfate bacteria.15
The total exclusion of microbial activity in subsurface
environments by unfavorable oxidation-reduction conditions
therefore seems unlikely, although the possibility exists
that some deep regions of the zone of saturation might become
so reduced as to preclude microbial growth. However, since
different species of microorganisms usually are able to
grow well only within a relatively narrow range of redox
values, the oxidation-reduction conditions that prevail
within a subsurface region will limit the species of
microbes which may successfully inhabit that region and
will often control the metabolic pathways and products of
these microbes. For example, sulfate-reducing bacteria
require an environmental En of -200 mv or less for initiation
of growth,39 and reduction of nitrate by facultative microbes
in soil has been found not to proceed unless the redox poten-
tial is less than +338 mv at pH 5.1.1*0 Also, metabolic activ-
ity in soils apparently changes from aerobic to anaerobic,
with concomitant accumulation of partially oxidized organic
compounds, when the concentration of oxygen declines below
3 x 10~6 a.1*1 The implications of oxidation-reduction
conditions in regard to the fate of pollutants in subsurface
waters are obvious.
22
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NUTRIENTS
A microbial habitat must provide to resident microorganisms
the nutrients which they require for synthesis of protoplasmic
constituents and for generation of energy needed for conduct
of life processes. For synthesis of protoplasm a carbon
source, either carbon dioxide or organic matter, must be
available, along with smaller quantities of nitrogen,
phosphorus, and sulfur in either organic or inorganic form,
and low levels of various minerals. Generation of energy
by chemotrophic organisms (the absence of solar radiation
obviously excludes phototrophs from subsurface environments)
requires the availability of: (1) suitable electron donors,
such as oxidizable organic compounds, or, for growth of
chemolithotrophs, oxidizable inorganic substances such as
molecular hydrogen, reduced sulfur compounds, ammonia,
nitrite, and ferrous iron; and, (2) suitable electron
acceptors including molecular oxygen, nitrate, sulfate,
carbon dioxide, and simple organic compounds.
Potential sources of carbon for microbial utilization are
relatively plentiful in many subsurface environments, since
the upper continental crust of the earth is variously esti-
mated to contain in the neighborhood of lO^-lO^O Kg of
carbon. lf2,1*3 Most of this carbon is present as inorganic
compounds, principally carbonates, but an appreciable portion
occurs as organic matter, much of which was incorporated into
sedimentary deposits at the time of their formation and has
not been completely mineralized over geological ages* Large
quantities of organic compounds are found concentrated in
petroleum deposits but vastly greater amounts are dispersed
throughout sedimentary rocks in a finely disseminated state.
For example, analysis of 35,000 samples of ancient sediments
obtained from sedimentary formations in the United States
revealed these materials to contain an average of 1.5 percent
organic matter.4'* Similar study of 2,000 samples of recent
sediments, mostly of marine origin, showed organic matter
ranging from 0.5-10 percent.^ Other geochemical studies
have generally confirmed the apparently ubiquitous occurrence
of significant quantities of organic matter in sedimentary
deposits, although recent terrigenous sediments have received
relatively little investigation in this regard.
Organic matter in sedimentary rocks may be grossly classified
as bitumen, comprising organic substances which are extracted
by neutral organic solvents, and kerogen, consisting of organic
materials which are not readily soluble in such solvents. The
23
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bitumen fraction usually contains numerous hydrocarbon
materials, fatty acids, porphyrins and many other substances;
its composition is likely dependent to some extent on the
organic solvent employed for extraction. The kerogen fraction
constitutes the bulk of the organic matter in subsurface
environments, usually comprising 90 percent or more of the
total organic content of sedimentary materials. lt6 The compo-
sition of this fraction is very complex and subject to
considerable variation? but, in general, it appears to
consist in large part of humus-like materials.
Available information also indicates that essentially all
subsurface waters probably contain significant quantities
of dissolved organic matter, as illustrated by Table 2.
This table presents representative data for organic carbon
content for both fresh and highly mineralized ground waters
obtained at depths ranging from 6 m (19.7 ft) to 3,049 ra
(10,003 ft) at a number of different geographical locations
and, hence, provides some indication of the levels of
dissolved organic matter occurring in subsurface waters.
This organic matter probably results both from leaching
of organics from sedimentary rocks into ground„water and
transport of fresh organic matter from the surface by
recharge water. Humic substances, naphthenic acids, fatty
acids, and phenols are known to comprise part of the dis-
solved organic matter in ground waters near petroleum
deposits, but the specific composition of the naturally
occurring organic matter of ground waters generally remains
largely unknown.
The growth of heterotrophic (chemoorganotrophic) bacteria
may often proceed when very low levels of organic matter
are available, ZoBell, et al., having observed marine bacteria
growing readily at concentrations of organic substances
lower than 0.1 rag/1.1*9'50 It would appear likely, therefore,
that the organic substances present in most subsurface environ-
ments of sedimentary origin are sufficient in quantity to
support microbial activity, both as sources of carbon for
synthesis of protoplasm and as electron donors for generation
of energy. However, microbial activity in the subsurface
may be limited by the quality, rather than the quantity,
of subsurface organic matter. For example, much of this
material may, as noted above, consist of substances resembling
soil humus components which yield to microbial degradative
processes only very slowly, even under the most favorable
conditions. Also, some compounds may not be utilized by
microbes in the absence of oxygen per se,51 and some may even
be microbial inhibitors.
24
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Table 2. CONTENT OF ORGANIC CARBON IN
REPRESENTATIVE GROUND WATERS
Location
Moscow Basin
(USSR)
Moscow Basin
(USSR)
South Platte
River, Valley,
Colorado
Western
Turkmenia
(USSR)
Ob ' irtysh
Basin (USSR)
Ob 'irtysh
Basin (USSR)
Orenburg
Region (USSR)
Orenburg
Region (USSR)
Orenburg
Region (USSR)
Kuibyshev
Region (USSR)
Kuibyshev
Region (USSR)
Kuibyshev
Region (USSR)
Depth
6 m (19.7 ft)
80-260 m
(262-853 ft)
6.4-10.3 m
(21-34 ft)
512 m (1,679 ft)
664-672 m
(2,178-2,205 ft)
2,314-2,317 m
(7,559-7,602 ft)
448-452 m
(1,470-1,483 ft)
1,633-1,639 m
(5,358-5,377 ft)
1,735-1,741 m
(5,692-5,712 ft)
854-860 m
C, 802-2, 821 ft)
1,360-1,390 m
(4,462-4,560 ft)
3,036-3,049 m
(9,961-10,003 ft)
Organic Carbon,
mg/1
0.3-0.6
0.64
7-51
4.2
1.23
5.28
5.0
1.77
28.4
1.45
1.92
1.35
Reference
Al'tovskii,
et al., 1961 37
Al ' tovskii ,
et al., 1961 37
Stewart, et al.
196738
Al'tovskii,
et al., 1961 37
Bars and
Nosova, 1964 k7
Bars and
Nosova, 1964 **7
Bars and
Kogan, 1964 **
Bars and
Kogan, 1964 If8
Bars and
Nosova, 1964 **7
Bars and
Kogan, 1964 **8
Bars and
Kogan, 1964 **8
Bars and
Kogan, 1964 ^8
25
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Carbon dioxide is potentially available as a carbon source
for chemolithotrophic (autotrophic) microorganisms in many
subsurface regions. Inorganic electron donors which might
be oxidized by such microbes for generation of energy are
also encountered with some frequency in subsurface environ-
ments. For example, molecular hydrogen is often present in
subsurface gases, 8 and elemental sulfur and hydrogen sulfide
are not uncommon in the earth's crust.
Nitrogen, phosphorus, and sulfur are probably present in
essentially all sedimentary formations as constituents of
organic and/or mineral matter, albeit the concentration of
one or more of these elements, most likely nitrogen and
phosphorus, may be extremely low in specific locations.
However, since the amount of carbon in microbial protoplasm
exceeds the quantity of nitrogen, phosphorus, and sulfur,
respectively, by approximately five-, twenty-, and one
hundredfold, and the latter elements are likely to be
recycled in-place in subsurface environments, the levels
of these elements required to maintain a minimum level of
microbial activity in such environments would appear very
low. Hence, it seems unlikely that nitrogen, phosphorus,
and sulfur concentrations would often be so low in subsur-
face environments as to completely preclude all microbial
activity, although the probability appears high that limited
availability of one or more of these elements in a readily
utilized form will restrict the level of microbial activity
which can possibly occur in many subsurface regions.
Inhibition of microbial activity in subsurface environments
solely because of unavailability of mineral elements would
also likely be exceptional, since all subsurface waters are
mineralized to some extent due to leaching of the rocks with
which they come in contact, and the concentrations of inorganic
ions required for microbial growth are usually extremely low.
In this connection, Kartsev states that the presence of
mineral compounds necessary for growth of hydrocarbon-utilizing
microbes has been established in a wide variety of subsurface
formations, although clay interbeds deficient in the minerals
required for development of these bacteria have been observed
in rare cases.52
As noted in the preceding discussion of subsurface redox
conditions, molecular oxygen, the electron acceptor required
by obligate aerobes and usually preferred by facultative
microbes, is undoubtedly absent from the deeper regions of
the zone of saturation and is likely available in variable
26
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and often limited quantities in the upper regions of the zone
of saturation and in the zone of aeration. However, other
substances which serve as terminal electron acceptors for
anaerobic organisms or which may be utilized as alternate
electron acceptors by facultative organisms are widely dis-
tributed in subsurface regions. Sulfate, the required ter-
minal electron acceptor of the obligately anaerobic sulfate-
reducing bacteria, is probably present to some extent in most
subsurface waters, often in relatively high quantity. Carbon
dioxide, as previously noted, is present in many subsurface
environments and may be utilized as terminal electron acceptor
by several species of methane bacteria. Nitrate, the alternate
electron acceptor for many facultative microbes, occurs often
iri subsurface waters, particularly at relatively shallow depths,
Simple organic compounds which may serve a number of microbes
as electron acceptors via fermentative metabolic pathways are
also likely present in many subsurface environments, although
such substances probably become progressively more scarce with
increasing depth.53 In total, the availability of electron
acceptors is probably sufficient in many subsurface regions
to support at least a minimum level of microbial activity.
However, the rates at which electron acceptors can be replen-
ished in various subsurface environments appear likely to
limit the levels of microbial proliferation which may be
sustained therein. Such replenishment rates are undoubtedly
very low in many subsurface regions, particularly in deeper
strata of the zone of saturation. Insufficient availability
of suitable electron acceptors to sustain significant microbial
activity may well be a principal factor in the survival for
geological ages of organic matter, particularly petroleum
deposits, in the earth's crust.
GENERAL ENVIRONMENTAL OBSERVATIONS
Environmental factors other than those discussed above may
also affect microorganisms in subsurface regions but do not
seem likely to preclude microbial activity in such regions
except in rare cases. For example, pH may limit the types
of organisms which find various subsurface regions to be
suitable habitats, and large solid surface areas character-
istic of the highly structured subsurface environment may
influence microbial activity through sorptive effects on
extracellular enzymes, nutrients, and the microbes themselves.
27
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It should also be noted that the various environmental factors,
while discussed individually above, are interacting, as illus-
trated by the interrelation between temperature and pressure
noted previously. Definitive experimental data concerning
subsurface environmental factors are relatively scarce and
incomplete and are not sufficient to define completely the
total potential effect of these interacting factors on sub-
surface microbial activity. Nevertheless, available evidence
indicates strongly that the upper continental crust of the
earth is generally not a hostile environment for microorganisms,
and probably encompasses many regions, particularly those of
sedimentary origin, which are quite suitable habitats for many
microbial species. The discovery of diverse microbial popu-
lations in a number of subsurface regions, as described in
the next section of this paper, lends credence to this
hypothesis..
The observations presented in this section concern mostly
subsurface environmental factors in natural environments
unaffected by human activities. Unquestionably such
activities could profoundly alter the environmental condi-
tions in many subsurface regions. For example, low levels
of readily utiliiable carbon sources, electron acceptors,
and possibly phosphorus and nitrogen appear most likely to
limit microbial activity in many subsurface environments.
Disposal or ground-water recharge activities which result
in the entry of liquid or solid wastes into regions below the
•oil zone would be likely to considerably alter this situation
xin the surrounding subsurface environment.
28
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SECTION V
INVESTIGATIONS PERTAINING TO
SUBSURFACE BIOLOGICAL ACTIVITY
Subsurface regions of the earth's crust have never been
studied extensively by microbiologists. Early investi-
gations by soil microbiologists on the distribution of
microbes at different soil depths indicated the presence
of relatively large populations near the surface with
numbers decreasing with depth and attaining extremely low
levels at the lower boundaries of the soil zone.5^,55,56
From these studies, many microbiologists formed the
assumption that biological life below the upper soil zone
was either nonexistent or extremely limited. This assump-
tion, when coupled with the immense technical problems and
expense involved in subsurface investigations, resulted in
many subsequent workers ignoring the subsurface as an area
of investigation. Limited numbers of scientists associated
with petroleum technology, however, have had a continuing
interest in the microbiology of subsurface regions since
the sterility of petroleum deposits came under serious
question in the 1920s. Their investigations concerning
the origin of oil and the development of bacterial methods
to aid in the exploration and production of oil are primarily
responsible for currently available knowledge concerning
microbial life in subsurface regions. Such studies have
proved beyond a reasonable doubt that many subsurface
regions are not hostile to microbial life and, in fact,
contain a varied and apparently active microflora.
Investigations of microbial activity in subsurface regions
are discussed below. The techniques and somewhat unique
technical problems of the study of subsurface biology are
first described; this is followed by a discussion of results
obtained in field investigations of subsurface microbial
activity; finally, observations regarding the significance
of previous microbial studies and the need for additional
investigations are presented.
29
-------
TECHNIQUES AND PROBLEMS IN STUDYING SUBSURFACE BIOLOGY
Obviously, terrestrial environments far below the earth's
surface cannot be directly examined because of their inac-
cessability. Instead, biological activity in subsurface
environment must be characterized by examining natural spring
waters or by drilling wells and examining the resultant
cores and well waters. Spring waters give an indication of
the chemical nature of the producing aquifer, but ate not
necessarily biologically or chemically representative of
deep stratal waters.1'6/5' In essence, they may provide
meaningful information concerning the biological situation
in the vicinity of the outcrop where the spring occurs, but
are unlikely to accurately reflect microbial activity deeper
within the aquifer formation.
Well waters are considered to provide more representative
information concerning subsurface ecosystems than spring
waters, but they are likely to reflect contamination
resulting from drilling operations, and they may not be
quantitatively indicative of the microbial population in
an aquifer since the movement of water from the surrounding
aquifer rocks into a well during pumping is likely to be
much more rapid than the movement of microbes. Samples
obtained aseptically from undisturbed cores undoubtedly
provide the most representative information concerning
subsurface microbial activity, but the acquisition of such
samples is fraught with difficulties, as discussed in more
detail below. Also, since subsurface strata are not homo-
geneous, both cores and water from a single well may not
accurately reflect the microbial population of the adjacent
rock formation.
Well drilling operations alter and contaminate the subsurface
environment, especially with wells of any depth because of
the need for circulating drilling fluids or muds to remove
the cuttings and keep the walls of the borehole from collapsing
Drilling fluids often contain various sorts of additives and
ate a definite source of bacterial contamination.1*6*58
The possibility that surface microbes can be introduced during
drilling and sampling operations has caused many to question
whether microbes isolated from subsurface samples were actually
native to that environment. This has often been a valid
question, particularly where samples were Obtained from old
boreholes or producing wells, since such samples are hardly
representative of nondisturbed, uncontaminated formations.
30
-------
Despite the possibility of contamination, however, many
workers are convinced that uncontaminated samples can be
and have been obtained from subsurface regions and, hence,
microbes isolated from such samples are indeed native to the
subsurface formations sampled. These convictions are based
on several observations and lines of reasoning. For example,
as Davis points out, organisms necessarily were present at
some time prior to drilling in some formations that have been
studied because surface microbes introduced during drilling
would not have had time to produce the biogenic products
noted in these formations immediately after drilling.46
Also, surface contamination could not have produced during
the short time of drilling and coring the large populations
of bacteria capable of utilizing hydrocarbons under sub-
surface conditions observed in some formations.8 Others
have suggested that contaminating surface microbes introduced
into subsurface regions during drilling would perish at great
depths due to a lack of adaptation to high pressures and
temperatures; but, as indicated in the preceding section on
the subsurface environment, this reasoning is not likely
to apply to studies at most depths.37'59 ZoBell, who has
devoted considerable attention to the ptoblems of sampling
and contamination of samples from subsurface regions, has
noted that many cores have been obtained in which no bacteria
were found, thus indicating that the sampling techniques
employed were capable of producing uncontaminated samples.18
A full-scale field and laboratory investigation to actually
determine whether uncontaminated cores could be obtained by
drilling operations was conducted in the Soviet Union in
.1948 and 1955.58 It was found that drilling muds were
definitely a potential source of contamination, containing
a variety of bacteria, including sulfate-reducers and hydro-
carbon utilizers. To determine the range of penetration of
bacteria from drilling muds into various types of cores, a
series of special experiments were carried out. Circulating
drilling fluids were seeded with a culture of Bacterium
prodigiosuro (Serratia marcescens), a bacterium which
produces a unique red pigment when cultured. The seeded
drilling fluid was introduced and circulated in the drill
holes for 15-20 minutes before pulling the cores. The cores
were then carefully examined layer by layer for penetration
by the test organisms. Some representative results obtained
in this study are presented in Table 3. As would be expected,
it was found that the more porous a rock the greater the
possibility that the core has been contaminated by the
drilling fluid, since test bacteria penetrated throughout
cores of sand and sand layers, but barely penetrated hard,
compact rocks such as limestone. These studies emphasize
the need for exercising great care in sampling subsurface
31
-------
Table 3. DEPTH OP PENETRATION OP A BACTERIAL TRACER
(Serratia marcescens) FROM DRILLING MUDS
INTO DRILL CORES [Prom Smirnova, 1957 58J
Depth of sample,
meters/feet
Lithologic description
of sample
Depth of penetration
(mm) by S. marcescens
75 mm diameter core
308/1011
311-314.3/1020-1031
218-223.5/715-733
18.5/61
18.0/59
377-377.5/1237-1239
35.75/117
45.5/149
4.0/13
12.9/42
208-213/682-699
8.0/26
29.15/96
32.0/105
Dolomite, dense.
Limestone, dolomitic.
Sandstone, fine-grained, very firm
with calcareous cement.
Sandstone, grayish-brown, compact dry,
Sand, argillaceous, blue, marly.
Dolomite, calcareous, strongly porous
with crumpled bedding.
Clay, dense, micaceous.
Clay, soft, very micaceous.
Clay, strongly arenaceous, friable,
micaceous, black sand in lower part.
Clay, dense, with sand fragments.
Clay, with layers of green-gray sand,
micaceous
Sand, grayish-brown, laminated, with
layers of white and reddish-brown
coarse-grained sand in lower part.
Sand, grayish-brown, argillaceous,
very friable.
Sandstone, dark brown; grayish-brown,
friable sand in lower part.
1.0
1.0-1.5
1.5-2.0
2.0-3.0
3.0
4.0
5.0
5.0
6.5
7.0
Entire thickness
of core.
Entire thickness
of core.
Entire thickness
of core.
Entire thickness
of core.
-------
environments for microbial activity, but indicate that
uncontaminated cores can be obtained from many subsurface
regions if reasonable precautions are taken.
If an uncontaminated sample can be obtained, determining
the number/ type, and activity of any organisms present
is also extremely difficult. The technical problems
involved in such determinations,have been discussed in
detail by a number of microbial ecologists who all point
out that it is presently impossible to accurately enumerate
the microbial population of any ecosystem and measure the
extent of its activity in the in-situ environment. 16»t|3'60'61'62
Originally, the basic approach in attempting to enumerate
the organisms in a sample was to count them directly with
a microscope or indirectly by inoculating different culture
media and counting the organisms which grew. Both methods
of enumeration are extremely inaccurate since it is usually
impossible to differentiate between living, dormant, and
dead cells by direct microscopic methods and because
different species of organisms vary gr6atly in their ability
to grow on selected culture media, therefore, it must be
understood that microbes found in a sample by such approaches
may not give a true indication of the total microbial community
existing in the sampled habitat and the processes occurring
there. However, the repeated presence of certain microorganisms
in samples from the same kind of habitat is highly indicative
that they are native to that habitat and their metabolic capa-
bilities provide some idea of their role there and the existing
conditions.
RESULTS OF INVESTIGATIONS OF SUBSURFACE MICROBIAL ACTIVITY
Early microbiologists generally considered the deeper portions of
the earth's crust to be void of life until the 1920s when Bastin
in the United States and Ginsburg-Karagitscheva in the Soviet
Union reported the first credible discoveries of sulfate reducers
and other bacteria in oil-field waters from depths as great as
1,000 meters (3,280 ft).63,64,65,66 Although both investigators
primarily sampled producing oil wells and, therefore, the
organisms they found could have been contaminants, their reports
apparently were the impetus to increased research in petroleum
microbiology in both countries, a development which eventually
expanded to include other phases of subsurface biology. Table 4
is a chronological listing of representative investigations
33
-------
Table 4. REPRESENTATIVE REPORTS OF ORGANISMS ISOLATED
FROM SUBSURFACE ROCKS AND WATERS
Main organisms
isolated
Source of sample
Reference
w
ith
Sulfate reducers
Sulfate reducers
Sulfate reducers
Sulfate reducers,
denitrifiers,
various other
anaerobes
Pigmented sulfur-
bacteria, sulfate
reducers, denitri-
fiers
Sulfate reducers
Oil-field waters from producing
wells.
Oil-field waters from producing
wells.
Oil-field waters from producing
wells.
Waters from oil wells and sulphu-
rous springs from depths as great
as 1,000 meters (3,280 feet).
Oil-field waters from artesian
wells.
Drill-cores of sulfur-bearing
limestone and anhydrite from
a depth of 476 meters (1,560
feet).
Bastin, 192663
Bastin, 19266U
Bastin and Greer,
193065
jGinsburg-Karagitscheva,
19336S
Issatchenko, 194067
ZoBell, 194468
-------
Table 4. REPRESENTATIVE REPORTS OF ORGANISMS ISOLATED
FROM SUBSURFACE ROCKS AND WATERS (Continued)
Main organisms
isolated
Source of sample
Reference
Anaerobes (sulfate
reducers included)
Sulfate reducers
Sulfate reducers
0-117xl06 bacteria/
gm of rock (direct
counts)
Sulfate reducers
0-366x103 bacteria/
ml oil (direct
counts)
Drill cores to a depth of 54 meters
(177 feet).
Drill cores of sulfur-producing salt
domes from depths of 213-274 meters
(700-900 feet).
Drill cores of oil-bearing rocks
from depths of 1,322-1,345 meters
(4,337-4,412 feet).
Sedimentary rocks from depths as
deep as 2,093 meters (6,887 feet).
Cores of sulfur-limestone-anhydrite
and oil-bearing rocks from depths
of nearly 3,050 meters (10,000
feet).
Oil samples from depths of 238-2,296
meters (781-7,533 feet).
ZoBell, 194969
Miller, 194970
ZoBell, 195171
Ekzertsev, V. A.,
195172
ZoBell, 195818
Meskov, 195873
-------
Table 4. REPRESENTATIVE REPORTS OF ORGANISMS ISOLATED
FROM SUBSURFACE ROCKS AND CATERS (Continued)
Main organisms
isolated
Source of sample
Reference
ot
Diplococci
Five species of
Pseudomonas and
Bacterium capable
of oxidizing
naphthalene
Members of the genera
Pseudomonas, Myco-
bacterium, and
Pseudobacterium
Eight species belong-
ing to the genera
Pseudomonas/ Bac-
terium/ Pseudobac-
terium, aid Myco-
bacterium
Saphrophytes, deni-
trifiers, sulfate
reducers, heptane
oxidizers, others
Salt crystals.
Underground waters from both oil
and non-oil bearing regions.
Reiser and Tasch,
I960™
Naumova, I96075
Underground waters and cores from
gas-bearing regions.
Underground waters from oil- and
gas-bearing regions.
Telegina, 196176
Smirnova, 1961'
77
Ground waters from recharge zones,
the vicinity of oil deposits and
at discharge zones of several
aquifers.
Al'tovskii, et
19613
-------
Table 4. REPRESENTATIVE REPORTS OF ORGANISMS ISOLATED
FROM SUBSURFACE ROCKS AND WATERS (Continued)
Main organisms
isolated
Source of sample
Reference
Sulfate reducers,
sulfur oxidizers
Sulfate reducers,
others
Sulfate reducers,
methane formers,
other anaerobes
Sulfate reducers,
sulfur oxidizers
putrefiers
(Not named)
Sulfate reducers,
denitrifiers,
methane formers,
putrefiers, sulfur
oxidizers, others
Hydrocarbon oxidizers
Sulfur deposits.
Oil, water, and oil-bearing sand
taken directly from a fractured
oil bed.
Water and oil from old and freshly-
drilled oil deposits.
Ground waters from springs and oil
and gas wells.
Rocks from drill holes.
Ground waters from various strata.
Ground waters from wells (pumped,
artesian, dug).
78
Ivanov, 1962
Andreevskii, 196279
Sazonova, 1962
60
Kuznetsova, 196281
Messineva, 196232
Al'tovskii, 196283
Telegina, 196281*
-------
Table 4. REPRESENTATIVE REPORTS OF ORGANISMS ISOLATED
FROM SUBSURFACE ROCKS AND WATERS (Continued)
Main organisms
isolated
Source of sample
Reference
Saphrophytes, denitri-
fiers, sulfate re-
ducers, methane
formers, others
A Bacillus species
Seven species of
» Pseudomonas, Myco-
bacterium, and
Chromobacterium
capable of oxi-
dizing hydro-
carbons
19 cultures of Myco-
bacterium capable
of utilizing
petroleum compounds
Hydrocarbon-oxidizing
bacteria; methane,
H2&, N2 producers
Complex of sulfate
reducers and various
Pseudomonas spp.,
others
Ground waters from various Paleozoic
strata, both oil and non-oil bearing
regions.
Paleozoic salt deposits.
Waters of oil and gas beds.
Mekhtiyeva, 1962
85
Dombrowskii, 196386
Slavnina, 1965 87
Waters underlying petroleum deposits.
Cores and waters from tertiary
deposits.
Petroleum well,
Norenkova, 1966 88
Bogdanoya, 1966 89
Kuznetsova and
Gorlenko, 196690
-------
Table 4. REPRESENTATIVE REPORTS OF ORGANISMS ISOLATED
FROM SUBSURFACE ROCKS AND WATERS (Continued)
Main organisms
isolated
Source of sample
Reference
w
Sulfate reducers/
hydrocarbon oxi-
dizers, hydrogen
producers, butyric
acid bacteria,
others
Various groups of
sulfate reducers
Thiobacillus sp.,
oxidizers
Spore formers, total
heterotrophs
Sulfate reducers,
sulfur oxidizers
Waters of oil and gas deposits from
depths as great as 2,900 meters
(9,514 feet).
Waters from wells in zones of recharge,
submerged strata, and discharge.
Shallow cores below the root zone
(from below corrals, nonirrigated
fields, sod).
Sulfur-bearing and sulfur-calcite
rocks from a sulfur deposit
.beneath the ground-water level.
Zinger, 196791
Kuznetsova, 196792
Stewart, et aL,
196736
Pomerants and
Belonitskaya,
197093
-------
made since the 1920s pertaining to microbial activity in
subsurface environments. These investigations clearly show
that a diverse microflora is found in various subsurface
regions.
Initially, investigators examined oil-field waters for the
mere presence of microorganisms with little thought being
given to the possibility that the organisms they found were
sampling contaminants. With time greater efforts were
expended to determine whether microbes found in subsurface
samples were actually native to the sampled regions or were
introduced during the drilling or sampling procedures.
Perhaps the most elegant and extensive effort was.that of
ZoBell, Sisler, and personnel of the Shell Oil Company which
involved an attempt to drill an entire oil-well aseptically
in order to determine whether the bacterial corrosion of
oil-well casings at depths of several thousand feet in a
California oil field were caused by bacteria native to those
depths.71 To minimize microbial contamination, an alkaline
CpH 11.5) drilling mud was used, equipment was sterilized
by means of germicides and heat produced by blowtorches,
and special core-handling techniques were employed. The
precautions taken were considered to be successful since
no surface-type aerobic heterotrophs were found in cores..
Viable sulfate-reducing bacteria were isolated from oil-
bearing rocks taken from depths of 1,322-1,345 meters (4,337-
4,412 feet).
Sulfate-reducing bacteria were the only microorganisms isolated
in many initial studies because they were the only microbes
which the experiments were designed to detect. Eventually,
investigators examined the subsurface for other microbial
groups and found a biochemically diverse microflora existing
not only near petroleum and sulfur deposits, but also else-
where in the subsurface. Various denitrifiers, methane formers,
sulfur oxidizers, and hydrocarbon utilizers have been isolated,
including members of the genera Thiobacillus/ Pseudomonas,
Methanomonas, Mycobacterium, Actinomyces, and Pseudobacterium.
Although determinations of air numbers are inaccurate and
undoubtedly do not reflect actual population densities in
the in-situ environment, counts of several million bacteria
per milliliter have been reported in some samples.
That sulfate reducers are not the only microbial group native
to subsurface environments is illustrated by a study in which
the pattern of distribution of hydrocarbon-oxidizing and gas-
producing microflora within Pliocene and Paleocene deposits
40
-------
of the USSR was investigated.89 Although both core and
water samples were examined, the results from cores are
most significant because the outer layer of each core was
carefully pared off and only the theoretically uncontaminated
interior of the core was examined. Hydrocarbon-oxidizing,
sulfate-reducing, and methane-producing bacteria were found
in cores of various types of sandstones, marls, and clays
mixed with limestone, siltstone, and argillite from depths
of 87-504 meters (285-1,654 feet). No apparent relationship
between the distribution of various bacteria and depth was
observed, and different types of microbes were invariably
found in the same core sample. Only one of twenty-nine
cores examined did not contain any microbes which could
be detected by the methods employed.
The presence in subsurface environments of nearly all the
organisms which have been reported as residing in such
regions is not contraindicated by available knowledge
concerning their physiological capabilities. Reviews on
the ecology of sulfate-reducing bacteria point out that
sulfate reducers, primarily the vibrio Desulfovibrio
desulfuricans, are uniquely adapted to grow in subsurface
environments, and it would not be surprising if they were
almost ubiquitious in such regions.8rr8»39,«+6,9k sulfate
reducers can tolerate an extreme range in osmotic and
hydrostatic pressure, temperature, and pH. As a class
they utilize a number of organic compounds such as various
fatty acids, amino acids, simple alcohols, carbohydrates,
and possibly som6 hydrocarbons. They may use elemental
hydrogen. Oxygen is not required as a terminal electron
acceptor since they are able to reduce a variety of sulfur
compounds (sulfates, sulfites, thiosulfates, hyposulfites,
and sulfur) to hydrogen sulfide (H2S), which they tolerate
at levels normally toxic for microorganisms. Sulfate-
reducing bacteria are often found in association with
other bacteria, a relationship which may be mutually
beneficial 8rlfl»1*6 Associated organisms evidently are able
to oxidize organic substances unusable by sulfate reducers,
producing compounds which sulfate reducers can subsequently
utilize as nutrients, with redox conditions conducive to
growth of sulfate reducers being created during the process.
Conversely, sulfate reducers may provide sulfur for associ-
ated organisms in a form more utilizable than sulfate. The
pumping of large quantities of fresh water into formations
containing organic deposits is thought by some to stimulate
eventual sulfate reduction by the development of an aerobic,
organic-oxidizing microflora and the resultant production of
41
-------
nutrients usable by sulfate reducers. 90f95 Ironically, sulfate
reducers evidently are adversely affected by conditions such
as high temperatures not directly inhibitory to them but which
are inhibitory to associated organisms.90
In general, bacteria such as Pseudomonas, Mycobacterium, and
Actinomyces species found in subsurface samples are also
commonly found in upper soil layers. They are capable of
growth in anaerobic environments by using nitrate or organic
compounds as electron acceptors instead of oxygen. As a
group they utilize a wide range of organic compounds as
nutrients. A number of different species have been isolated
from subsurface samples which in laboratory studies could
oxidize hydrocarbons ranging from napthalene to alkanes
containing from one to ten carbons.7^76'77'87'88'96 The number
of compounds that organisms such as various Pseudomonas
species are able to attack and degrade is vast, although
they may be able to attack many substances only under aerobic
conditions.
The presence in subsurface environments of some organisms
reported to have been isolated from subsurface samples has
been questioned because such organisms seemed to be unsuited
for the conditions theoretically present in those environ-
ments. For example, some early Russian investigators
reported the isolation of purple sulfur bacteria from
stratal waters of some oil deposits. 67 Since such bacteria
cannot grow in the dark without oxygen their presence in
deep subsurface waters seemed unlikely. It is now believed
that they were introduced into the oil strata via injection
wells and were able to survive by using the oxygen contained
in the air and water pumped into the well and by utilizing
the byproducts of hydrocarbon-oxidizing bacteria also present
in the strata. 97
Another seeming contradiction has been the isolation of both
sulfate reducers and sulfur oxidizers from the same samples.
However, ThiobacilluS denitrificans, one of the sulfur bacte-
ria, is a facultative anaerobe capable of using nitrate
instead of oxygen as an electron acceptor while oxidizing
HjS, sulfur, or thiosulfate to sulfate.98 It may be possible
for such organisms to live in association with sulfate
reducers, although this has not been examined under control-
led experimental conditions.
It is impossible to determine how long microorganisms have
been in subterranean formations where they are found,
especially since microorganisms isolated from such form-
ations do not differ from surface microbes to any obvious
42
-------
extent. The microorganisms found in deep stratal waters
may have been deposited with the sediments millions of
years ago or they may have migrated into such strata from
th-i surface over an undetermined period of time. 18,37,7^,82,86
Bacteria are not only carried by underground waters moving
through fractures, fissures, channels, solution cavities,
and pore spaces, but they are also often able to propel
themselves through water, perhaps to reach better conditions
or to escape unfavorable environments. Such movement is
extremely slow, ranging from a few microns to a few centi-
meters per day.14 It is possible that organisms could
migrate outward from drilled wells over a period of years,
contributing to colonization of some formations, since
proliferation of microbes in the immediate vicinity of
wells often causes severe corrosion and clogging prob-
lems. 45f99/100f101The extent to which such growth radiates
from wells is uncertain, however. Studies on the movement
and growth of microorganisms in aquifers near wells have
pertained mainly to tracing the movement of coliform
bacteria, various pathogens, or other nonnative microbes
over relatively short periods of time.'7'102'103 As noted
previously, the limited migration of such microbes in
subsurface regions is likely due in part to their limited
ability to survive and proliferate in such environments.
The outward migration over a significant period of time
of bacteria more suited to subsurface conditions has
apparently not been investigated to any extent.
The broad spectrum of environmental possibilities in
subsurface regions obviously affect in many ways the
distribution and activity of microorganisms in subsur-
face regions. There have been several studies in which
the chemical and physical conditions in various subsur-
face regions were compared with the microflora found there
in an attempt to determine the effect of various conditions.
Some of the first studies embodying this approach were begun
by Soviet workers in the 1950s who attempted to relate the
organic, inorganic, and bacterial composition of several
oil-bearing aquifers to the origin of petroleum in these
aquifers.37'81 To do so they compared the bacterial and
chemical compositions of subsurface waters from three
distinctly different zones in each oil-bearing aquifer.
These zones were (1) the zones where the aquifers were
recharged, (2) the zones where the moving waters were
associated with oil and gas deposits (anticlinal traps),
and (3) the zones whfere the waters were discharged to the
surface. They found that the microflora of various zones
43
-------
within the same aquifer cliff erect sharply, the differences
apparently being related to the temperature of the ground
waters, the oxidation-reduction potential, and the content
of organic material, oxygen, and sulfate. This is best
illustrated by Kuznetsova's data on ground waters of the
Grozny-Dagestan province in the USSR (Table 5) ,81 Waters
with low temperatures (<50°C) , high oxidation-reduction
potentials, and oxygen contained primarily "putrefying"
bacteria ranging in numbers from 59,000 to 1,000,000 per
milliliter. Waters with high temperatures (>50°C) and
lowered oxygen and oxidation-reduction values contained
greater quantities of H2S indicating the activity of sulfate
reducers. Temperatures greater than 50°C resulted in
considerably reduced counts. Sulfur-oxidizing bacteria
were abundant when the oxidation-reduction potential of
the water was between rH2 values of 11.6-21.
Mektiyeva investigated the microflora in ground waters of
Paleozoic strata and compared the distribution of microbes
in relation to the water's contact with petroleum.85 She
found that waters which were not in direct contact with
petroleum or gas deposits still contained an active micro-
flora, although not as abundant and varied as the micro-
flora of waters in contact with petroleum deposits.
Although the number of bacteria decreased with depth,
the distribution of the microorganisms was related to
the depths they were found only to the extent that the
strata at that depth affected the chemical composition
of the ground waters. For example, it was felt that the
microflora in some samples obtained from deep formations
was limited not because of the depth, but because the
waters from that strata were highly saline, lacked
sulf ates and other electron acceptors , had high Cl-Na/Mg
ratios, and contained up to 1^400 ppm bromine.
A study along the same lines as that of Russian workers
was conducted by the Socony Mobil Oil Company on a large
oil-bearing fresh-water aquifer in the Carrizo Formation
in south Texas.1*6 in this study an effort was made to
correlate the organic, inorganic, and bacteriological
characteristics of the ground waters of the aquifer with
the presence or absence of oil. Nine wells, six of which
were free-flowing, were sampled. These wells were spread
along a line roughly 36 miles long which started with
Well No. 1 above the oil field at the recharge area and
44
-------
Table 5. COMPARISON OF THE AVERAGE BACTERIAL AND
CHEMICAL COMPOSITION OF THREE DIFFERENT
ZONES WITHIN OIL-BEARING AQUIFERS
[From Kuznetsova81]
Temperature, °C
rH2
O2, mg/1
H2S, mg/1
Direct microscopic
count of bacteria
per ml (xlO3) :
@ temperatures
<50°C
@ temperatures
>50°C
Relative abundance
of bacteria:
Desulfurizing
Sulfur-Oxidizing
Putrefying
Recharge
zone
12-21
17.8-30
0.6-5.6
0-0.2
59-1036
—
+ or -
+ or -
+++
Oil deposit
zone
23-89
9.7-17.8
0
0-40
105-9600
25-72
++
++
+ or -
Discharge
zone
(springs)
12-87
1.6-15
0
4-57
144-3137
9-40
+++
++++
+
45
-------
proceeded with increasing depth in the direction of sub-
surface water movement until the oil field was passed.
The results of sampling are discussed in detail by Davis.1*6
Some average results are shown in Table 6. The waters in
this aquifer are fresh, having a total solids content of
less than 800 ppm and a chloride content of less than
100 ppm. The water moves at a very fast rate of 50-100 feet
per year. Bacterial plate counts of well samples were
relatively low (<100,000 per liter). Aerobes were found
consistently only in Well No. 1, near the recharge area.
Sulfate reducers were found in all wells except No. 7 and 8.
Their absence in these two wells could possibly be due to
the high temperatures although sulfate reducers were found
in Well No. 9 where the temperature was also high. Sulfur
oxidizers were not found in any well. Facultative bacteria
were most numerous in Wells No. 1 and 6 where the organic
matter was the highest. It should be noted, however, that
these two wells were pumped wells while the others checked
for facultative bacteria were free-flowing; also, the
analytical methods employed would detect only a part of the
dissolved organic matter present and, hence, the data
presented undoubtedly do not accurately reflect the total
organic matter in these subsurface waters and rocks. Davis
believes that the increase of sulfide and possibly the
increase of ammonia was due to bacterial activity as the
water moved through the formation.
Obviously, most investigations of subsurface biology have
been concerned with the soil zone or with deeper regions
of the zone of saturation. Very little information has
yet been developed pertaining to biological activity in
the upper regions of the zone of saturation and those
regions of the zone of aeration lying below the soil water
zone. One study which did include microbiological investi-
gations of these regions was concerned with the distribution
of nitrates and other pollutants under fields and corrals in
the valley of the South Platte River in Colorado.38 In this
study, conducted by the United States Department of Agriculture,
cores were taken in nonirrigated pastures, cultivated fields,
and corrals at one-foot intervals down to the water table or
bedrock, and the cores and water samples were subjected to
various chemical and bacteriological analyses. Results
from microbiological analyses showned that microbial activity
continued below the root zone and that often there was a
distinct increase in total counts at or immediately above
the water table. A higher bacterial population was found
46
-------
Table 6. SOME RESULTS OF BACTERIOLOGICAL AND CHEMICAL INVESTIGATIONS
OF THE GROUND WATERS OF AN OIL-BEARING AQUIFER IN SOUTH TEXAS
[After Davis "6I
Type of well
Location to oil
accumulation
Well flow
Well depth, ft
Temperature , "C
PH
Organic matter,
Ammonium, ppm
Sulfate, ppm
Sulfide, ppm
Sulfate-reducing
bacteria/liter
Facultative
bacteria/liter
Well No. 1
Water well
Up-dip
Pumped
215
23
6.6
28.7
0.09
12.0
0
1,000
110,000
Well No. 2
Water well
Up-dip
Free-
flowing
1,137
30
6.9
2.9
0.05
36.0
0
1,200
-
Well No. 3
Water well
Up-dip
Free-
flowing
1,800+
34.5
7.2
2.6
0.1
33.0
0
7,000
360
Well No. 4
Oil well
-
Pumped
1,690
-
8.6
-
0.
2.4
0
-
-
Well No. 5
Water well
-
Free-
flowing
2,100
38.5
7.3
-
0.1
52.0
0
-
-
Well No. 6
Oil well
-
Pumped
1,846
32
8.5
4.000
0.36
3.4
0
1,000
900,000
Well No. 7
Water well
Down-dip
Free-
flowing
3,200
47
8.1
15.0
0.36
12.0
14.5
0
4,000
Well No. 8
Water well
Down-dip
Free-
flowing
4,100
58
8.3
8.3
0.5
46.0
4.8
0
0
Well No. 9
Water well
Down-dip
Free-
flowing
4,200
58
8.5
11.5
0.45
34.0
22.0
10,000
20,000
Includes only that organic matter obtained from water by carbon filter and recovered from the carbon by extraction with chloroform
-------
below corrals than beneath fields, possibly because of a
greater amount of available carbon. Nitrifying bacteria
were not found to any great extent below the upper few
feet. The investigators felt that denitrification was
probably occurring in deeper regions of the zone of aeration
under corrals since nitrate was absent and total bacteria
counts were high. It should be noted that this study
provides no information concerning the presence of obligate
anaerobes since microbiological cultures were grown under
aerobic conditions only. Also, the examined samples were
initially frozen and kept for an unstated period of time
until they were analyzed, which probably influenced the
results obtained in the microbial studies to some extent.
SIGNIFICANCE OF PREVIOUS SUBSURFACE MICROBIAL STUDIES
AND NEEDED ADDITIONAL INVESTIGATIONS
The information obtained in the previous investigations of
subsurface microbial activity described above clearly illus-
trates the presence of a diverse microbial population in
many regions of the earth's crust lying below the soil zone.
This information, in conjunction with available information
concerning the suitability of subsurface environments as
microbial habits, indicates strongly that microbial activity
is both possible and probable in most subsurface regions which
are of importance in regard to ground water. The significance
of such activity to ground-water pollution and pollution
control, of course, resides in the potential interactions of
pollutants and microorganisms in subsurface regions and the
ultimate effect of such interactions on the quality and
availability of ground water. Subsurface microbe-pollutant
interactions are likely to be mainly beneficial, producing
such desirable results as elimination of organic pollutants
from subsurface waters by mineralization or removal of
nitrate by denitrification. Such interactions might also
sometimes be detrimental, resulting in production of unde-
sirable metabolic products which enter and pollute ground
water or reduction of aquifer permeability through clogging
of interstitial space by cells and polymeric products of
metabolism.
Previous investigations of subsurface microbial activity
have been concerned primarily with delineation of the types
and numbers of native microbes in essentially undisturbed
subsurface environments, mostly in formations deep within
the zone of saturation. These investigations provide a
48
-------
limited indication of possible microbe-pollutant interactions
which might be expected to occur in some subsurface environ-
ments. However, they provide practically no information
concerning: (1) the extent and nature of native microbial
activity in the zone of aeration below the soil zone and in
the upper regions of the zone of saturation; and (2) micro-
bial activity in subsurface environments altered by the
introduction of pollutants.
Microbial activity in the zone of aeration below the soil
and in the upper portions of the zone of aeration is obvi-
ously of very great potential importance in regard to ground-
water pollution and pollution control. Systematic studies
to define the extent and nature of native microbial activity
in these regions, correlating microbial data with environ-
mental characteristics, particularly geological and oxidation-
reduction conditions, are needed. These studies would provide
data which would be useful in preliminary efforts to predict
the fate of pollutants in these regions of the earth's crust,
and which would serve both as a basis and complement to more
extensive investigations pertaining to the movement, fate,
and effect of pollutants in subsurface waters.
Microbial populations, and hence microbe-pollutant inter-
actions, in subsurface regions receiving pollutants are
likely to be profoundly affected by the nature and quantity
of pollutants introduced and their effect on the native
subsurface environment. For example, microbial species
which are very minor members of the native ecological com-
munity may become dominant in the environment created by
entry of pollutants. Also, microbes indigenous to the
pollutant-containing waste introduced into a subsurface
region may proliferate there, becoming the dominant species
and controlling the fate of pollutants entering that region.
The above observations indicate the need for thorough,
integrated investigations of microbe-pollutant interactions
in subsurface regions receiving pollutants. In-situ investi-
gations should be employed where possible, but laboratory
simulation work utilizing core samples would probably often
be required, particularly in the study of deep formations.
Such studies should seek to correlate native microbial popu-
lations, environmental situations (geology, oxygen and
nutrient availability, pressure, temperature, etc.), nature
of pollutants, and microbial populations subsequently
developing after the introduction of pollutants, with the
fate of pollutants and their effect on ground-water quality
49
-------
and availability. Investigations of this type would
necessarily be complex and expensive, but they would
provide information needed in developing methods which
would permit prediction of the probable impact on ground
water of various activities entailing release of pollu-
tants into the earth's crust, including waste disposal,
agricultural, and ground-water recharge activities, and
which, therefore, would provide the basis for logical
regulation and control of such activities. Such studies
might also indicate how microbial activity in subsurface
regions below the soil zone could best be managed and
utilized to achieve maximum degradation of pollutants in
subsurface waters. For example, improved knowledge of
the factors controlling subsurface microbial activity
might permit alteration of subsurface environmental
factors and/or microbial populations to bring about
mineralization of hydrocarbons or other organic compounds
accumulating in aquifers as the result of accidents
involving transport or storage equipment, or to achieve
beneficial alteration of wastes intentionally introduced
into deep formations.
50
-------
SECTION VI
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11. McLaren, A. D., and G. H. Peterson. Soil Biochemistry.
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52
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33. Skinner, F. A. The Anaerobic Bacteria of Soil. In:
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43. Alexander, M. Microbial Ecology. New York, John Wiley
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50. ZoBell, C. E., C. W. Grant, and H. F. Haas. Marine
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54. Waksman, S. A. Principles of Soil Microbiology.
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57. Hem, J. D. Study and Interpretation 6f the Chemical
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66. Ginsburg-Karagitscheva, T. L. Microflora of Oil
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60
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SELECTED WATER
RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
.'. Ret
w
SUBSURFACE BIOLOGICAL ACTIVITY IN RELATION TO
GROUND WATER POLLUTION,
Dunlap, W. J., and McNabb, J. F.
;,;? n:zeticn Environmental Protection Agency
Robt. S. Kerr Environmental Research Lab.
National Ground Water Research Program
Ada, Oklahoma
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U. S. Environmental Protection Agency Report
No. EPA-660/2-73-014, September 1973.
IK Abstract
Biological activity occurring in subsurface regions below the soil zone
may be of considerable importance in determining the fate and effect of
pollutants in ground water, but this possibility has received little
previous attention. This paper comprises a discussion of subsurface
biological activity in regard to ground-water pollution as reflected by
available literature references. The subsurface environment is discussed
in terms of factors likely to be of greatest significance in regard to the
development of biological systems, and previous investigations of
subsurface raicrobial activity are reviewed. Available information
indicates the presence in the upper continental crust of the earth of
numerous regions, particularly those of sedimentary origin, which are
probably suitable habitats for many microbial species. Previous
investigations of subsurface microbial activity clearly show the
presence of diverse microbial populations in many subsurface regions below
the soil zone. Hence, microbial activity appears both possible and
probable in most subsurface regions of importance in regard to ground
water. Further elucidation of the extent and nature of microbial activity
in subsurface regions is needed in developing methods for predicting tfee
impact of pollutants on ground-water quality.
17a. Descriptors
*Ground water, *Subsurface investigations, *Microbiology, *Subsurface
waters, *Habitats, Water pollution, Water pollution effects, Path of
pollutants, Zone of aeration, Zone of saturation, Biological communities,
Biodegradation, Temperature, Hydrostatic pressure. Anaerobic bacteria.
17b. Identifiers
*Subsurface microbiology,*Subsurface environment, Microbial activity
17c. COWRR Field & Group
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Send To:
WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON, D. C. 2O24O
W. J. Dunlap
RSKerr Environmental Research Lab.
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