'In situ' Characterization or
Microorganisms Indigenous to
Water-Table Aquifers
Florida State Univ., Tallahassee
Prepared for
Robert S. Kerr Environmental Research Lab.
Ada, OK
Sep 84
U.S, Department of Co!vwser':c
Technical lnformatk),« Setuke
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LPA-600/0-S4-232
September 19S4
IN £ITU CHARACTERIZATION OF MICROORGANISMS
INDIGENOUS TO WATER-TABLE AQUIFERS
D. L. Balv.will1 and W. C. Ciiorse2
Department of Biological Science
Florida State University
Tallahassee, Florida 32306, U.S.A.
and
^Department of Microbiology
Cornell University
Ithaca, New York 14853, U.S.A.
Cooperative Agreement
CR-806931
LPA Project Officer
James F. McNabb
ROBERT S. KERR ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PPOlECiION AGENCY
ADA, OK 74820
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TECHNICAL REPORT DATA
(Plcaie n-L J Imirucroiu on l':e rci iT5V telore f<
I. hEPOHT NO.
3. RfcCIP'ENT'S ACCESSIOffNQ.
rc.S 5 101731
•S. TITLE A\D SUBTITLE
In Situ Characterization of Microorganisms Indigenous
to Water-Table Aquifers
5. REPORT DATE
—spnTf>[nhr-r j^>J
6. PERFORMING ORGANIZATION CODE
7. AUTHOR S)
D.L. Balkwill and W.C. Ghiorse
8. PERFORMING ORGANIZATION REPORT NO
9. PERFORMING ORGANIZATION NAME AND «DDFiESS
Dept. of Biological Sci.
Florida State University
Tallahassee, FL 32306
Dept. of Microbiology
Cornell Universit>
Ithaca, NY 1435;
10. PROGRAM bl EMENT NO.
CBPC1A
11. CONTRAC1VCRANT NO.
CR-8C6931
12. SPONSORING AGENCY NAME AND ADDRESS
U.S. EPA, R.S. Kerr Environ. Research Laboratory
P.O. Box 1198
Add, OK 74820
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA 600/15
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Core material from the deeper subsurface was examined for the presence
and activity of microbes. Methods included acridine orange direct counts, of
the total number of cefls, 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-nhenyl
tetrazoliuni chloride reduction assays of the number of respiring cells,
plate counts of the number of viable cells, and examination of the ultra-
structural characteristics of any microbes by transmission electron
microscopy. The results demonstrated conclusively that appreciable
numbers (1-10 million per gram) of bacteria reside in shallow, water-table
aquifers. This observation is important because (1) it contradicts the
traditional belief that such environments are almost devoid of life and
^2) the numbers are large enough to potentially affect ground water quality.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b. IDENTIFIERS/OPF.V ENDED TERMS
COSATI l-icld/Group
Ground water quality
Ground water microbiology
Soil microbiology
AODC
INT Reduction
Tetrazolium dye
reduction
6F
6M
8. DISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (This Report/
Unclassified
21. NO OF PAGES
16
SO. SECURITY CLASS tThisfage)
Unclassified
27. PRICE
EPA Form 2220-1 O-73)
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NOTICE
This document has been reviewed in accordance with
U.S. Environmental Protection Agency policy and
approved for publication. Mention of trade names
or commercial products does not constitute endorse-
ment or recommendation for use.
ii
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INTRODUCTION .
Water-table aquifers are environmental]y significant because they contain
ground water reserves that represent .nost (95% in the United States;
Josephson, 1980) of the freshwater available r'or irrigation or consumption.
With the occurrence of organic pollu'.ants in ground waters becoming an
increasingly widespread problem (Council on Environment.il Quality, 1981), it
is important to define and develop ar, understanding of factors that affect
ground water quality.
Microorganisms could play a -najor role in maintaining ground water
quality, considering that they can profoundly affect biological and chemical
activities in surface soils ard other environments (Alexander, 1977).
However, the microorganisms in a'^uifers have been studied only rarely (Dunlap
and McNabb, 1972), perhaps because early reports of soil microbiologists (e.g.
Waksman, 1916) indicated thac the number of microorganisms in soil drops
sharply with increasing depth. More recent studies have shown that microorga-
nisms can be present at considerable depths in the subsurface (Dockins et al.,
1980; Dunlap et al., 1972; Whitelaw and Edwards, 1980: Whitelaw and Rees,
1980),' but problems of contamination by surface soil have hampered the inter-
pretation of these results. Thus, detailed data on the occurrence and numbers
of microorganisms in aquifers remains scant (Dunlap and McNabb, 1973). Even
less is known about the in situ metabolic activities cf such organisms or
about how these activities may affect organic contaminants of ground water.
In 1979, we initiated efforts to obtain more detailed information on the
aquifer rr.icroflora by direct observation of ir\ situ microorganisms in subsur-
face samples. Traditional cultural methods were deemed mostly unsuitable for
these studies because they were not likely to select for many of the signifi-
cant organisms in subsurface samples (Ghiorse and Balkwill, 1981). Instead,
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light and electron microscopical methods for direct observation of microbial
cells in surface soils were modified for application to aquifer and otr._-r
subsurface materials. The present report reviews the information regarding
the characterization of microorganisms in water-table aquifers and other
subsurface materials that we have obtained with this approach (Ghiorse and
Balkwill, 1981, 1983; Wilson et al., 1983).
.MATERIALS AND METHODS
Description of Samples
Samples were collected from a total of four sites in Louisiana, Oklahoma,
and Texas, from'above and below the water table at each site. Subsurface
regions situated above aquifers were sampled because they are likely to affect
water that travels from the surface to aquifers below. The samples and their
origins are listed in Table 1; for more detailed information, see the original
references cited in the table. Aquifer and subsurface samples were collected
aseptically by using a modification (Wilson et al., 1983) of the procedures
developed by Dunlap et al. (1977).
Acridine Orange Direct Counts (AODC)
Epifluorescence light microscopy (LM) of acridine orange (AO)-stained
samples was used to determine the morphological characteristics and the total
numbers of cells by direct counts (AODC). A modification of Trolldenier's
(1973) method was used to determine the AODC as described by Ghiorse and
Balkwill (1983).
Respiring Bacteria
The proportion of AODC bacteria capable of reducing 2-(p-iodophenyl)-3-
(p-nitrophenyl)-S-phenyl tetrazolium chloride (INT), i.e., the proportion of
respiring bacteria (Zimmermann et al. 1978), was determined by mixing 2.5 g of
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subsurface material with 20 ml of filter-sterilized 0.1 % sodium pyrophosphate
(SPP) in a 125-.T.1 Erlenmeyer flask. The mixture was shaken at room tempera-
ture for 15 min at 160 rpm, 2.5 ml of 0.'2 % aqueous INT was added, and shaking
was resumed for an additional 15 min. Excess INT was removed by decanting and
centrifuging the entire contents of the flask at 10,OOO rpm for 10 min. The
supernatant fluid was decanted and the pellet was washed by centrifugation in
10 ml of 0.1 % SPP. The final pellet was resuspended in 22.5 ml of O.I % SPP
and the AODC procedure described by Ghiorse and Balkwill (1983) was followed.
To count bacteria containing INT-formazan deposits, green fluorescent
cells were first identified under epi-illumination. These were then inspected
for the presence of INT-formazan employing a 100 X bright field objective
lens. Care was taken to use briyht field illumination conditions that opti-
mized recognition of the red fonr.azan deposits in the cells. This included
adjusting the substage 5ris diaphragm and the illuminator rheostat to the same
setting each time, as well as the use of neutral density filters to reduce
brightness of the field.
INT-containing bacteria of two types were counted. One type was charac-
terized by diffuse but distinctly reddish cells with no apparent granules.
The second type was characterized by the presence of distinct red granules
inside the cell.
Plate Counts
Standard plate counts in triplicate were used to estimate the number of
viable microorganisms in subsurface environments. Both nutritionally rich
(PYG and/or 1-5% PYG agar; Ghiorse and Balkwill, 1983) and low-nutrient (SEA;
Wilson et al., 1983) media were used in all cases. All plates w'ere incubated
aerobically at 25 C, and colonies were counted after 1-2 weeks.
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Transmission Electron Microscopy (TEM)
Transmission electron microscopy (TEM) was used to determine the ultra-
structural characteristics of subsurface microorganisms. Microbial cells were
released and concentrated from subsurface materials prior to TEM examination
with the centrifugal washing method described by Ghiorse and Balkwill (1983).
RESULTS
Morphological Characteristics of Aquifer Microorganisms
Epifluorescence light microscopy (LM) of AO-stained preparations readily
detected microbial cells in all of the aquifer and other types of subsurface
samples examined (see Table 1). Objects that fluoresced bright green, thereby
indicating that they contained double-stranded DMA (Daley and Kobbie, 1975),
and that possessed appropriate morpnolcglcal characteristics were considered
to be microbial cells. These cells stood out clearly against a dull orange
background of fluorescing abiotic material.
Epifluorescence LM was useful for assessing the range of morphological
diversity in each sample and for detecting the occurrence of microcolonies
(for illustrations of these results, see Fig. 1 in Ghiorse and Balkwill, 1983
and Fig. 2 in Wilson et al., 1983). Microcolonies (groups of cells with
similar morphological characteristics) were present in all samples, but the
range of morphological diversity varied considerably in samples from one
location to another. Texas and Oklahoma (both Lula and Pickett) samples
contained mostly small, coccoid bacterial cells that were similar in shape to
those found in surface soils vBae et al., 1972; Balkwill and Casida, 1973;
.Balkwill et al., 1975, 1977). Few, if any, eukaryotic forms were detected.
In contrast, the samples from Louisiana contained a gn .ter variety of
bacterial forms, including: small coccoid cells, rod-shaped cells of varying
dimensions, and actinomycetes or other fila.nentous type's. Some of these
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samples contained small numbers of microeukaryotic forms. Very recently, a
cyst-forming amoeba and a fungus have been detected in Oklahoma samples by
special cultural methods (J. Sinclair, personal communication). These
microeukaryotes were present in low numbers in comparison to bacteria and,
therefore, they do not appear to account for a significant portion of the
biomass in the sample.
Ultrastructural Characteristics of Aquifer Microorganisms
Transmission electron microscopy (TEM) of aquifer and other subsurface
materials confirmed the presence of microorganisms in all samples by revealing
objects that possessed ultrastructural features (such as cell walls, mem-
branes, and intracytoplasmic inclusions) unequivocally characteristic of
microbial cells (for illustrations, see Figs. 2 and 3 in Ghiorse and Balkwill,
1983 and Fig. 3 in Wilson.et al., 1983). The dimensions and shapes of these
cells corresponded to those of the green-fluorescing objects considered to be
cells in AO-stained preparations for LM (above).
TEM of thin-sectioned microbial cells that were released and concentrated
from aquifer or other subsurface samples by blending and centrifugal washing
(see Materials and Methods) provided important information on these organisms
that could not be obtained readily with other approaches. For example, it was
possible to determine the relative proportions of Gram-positive and Gram-
negative bacteria in aquifer environments because thin sectioning revealed the
architectural details of their cell walls. Both Gram-positive and Gram-
negative forms were present in all samples, but the former were always clearly
predominant (two-thirds or more of the bacteria observed werr Gram-positive).
The cytoplasm of many subsurface bacterial cells was 'martially depleted
of the intracellular constituents (ribosomes and nuclear material) commonly
found in laboratory-cultured cells. Control experiments involving addition of
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laboratory-cultured cells to subsurface samples established that these cyto-
plasmic constituents were not lost during preparation of the samples for TEM.
Some of thft rn situ bacterial cells with a depleted cytoplasm contained
mesosome-like internal membranes and intracellular storage bodies such as
polyphosphate granules (Jensen, 1968), or more frequently, poly- -hydroxy-
butyrate (PHB) granules (Dunlop and Robards, 1973). In contrast, other
subsurface bacteria lacked such inclusions and contained the "normal" or
"healthy-looking" cytoplasm that is characteristic of laboratory-cultured
cells. A few of these bacteria possessed cross-walls or division septa,
implying that they were in the process of dividing when the samples were
fixed. This was observed in both coccoid and filamentous forms. A small
number of bacterial cells in the samples from Louisiana also contained
internal membrane systems that were reminiscent of those found in nitrifying
or methane-oxidizing bacteria. Ruthenium red staining indicated that many
subsurface bacteria were surrounded by polysaccharide-based capsules and gly-
cocalyx layers. The polysaccharide strands of these structures often extended
from the cell surface to surrounding pieces of abiotic materials.
As was true of morphological diversity (above), the internal ultrastruc-
tural diversity of the bacteria in samples from Louisiana was greater than
that of the bacteria in Oklahoma samples. TEM also confirmed that the over-
whelming majority of subsurface microorganisms were prokaryotic.
Numbers of Microorginisms in Aquifer Environments
EJ?ifluorescence LM of AO-stained samples was an effective way to obtain
direct counts (AODC) of total microbial cells in aquifers and other subsurface
environments. The resulting counts (Table 2) were lower, sometimes by two or
three orders of magnitude, than those that have been reported for typical
surface soils (see Alexander, 1977), and they were remarkably consistent from
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one sampling site to another. Samples from Oklahoma, Louisiana, and Texas
typically contained between 1-10 million AODC-cells per gram (dry weight) of
subsurface solids. Somewhat surprisingly, the numbers of cells did not
decrease appreciably with increasing depth at any site.
Plate counts on media with differing nutrient concentrations were used to
estimate the numbers of viable cells present in subsurface samples (Table 2).
Although traditional cultural methods of this type were of limited value for
obtaining meaningful information on aquifer microorganisms (s«? Discussion),
we included plate counts in our studies to provide a basis for comparison with
other environmental investigations. Plate counts on nutritionally rich media
like PYG agar were generally lower (sometimes much lower) than on low-nutrient
media like SEA (Table 2). The highest plate counts, which were usually
obtained on SEA, were always lower than the AODCs of the same sample. The
magnitude of this discrepancy varied from one sampling site to another. Plate
counts for samples from Oklahoma were sometimes as high as 50% of the AODC,
but those for samples from Louisiana and Texas were generally much lower
(0.01% of the ADOC or less).
Metabolic Activities of Aquifer Microorganisms
Plate c%unts demonstrated that some of the microorganisms residing in
aquifer and other subsurface samples were capable of growth, but this informa-
tion provided little or no indication of their activities in situ. Similarly,
direct observation of microbial cells with LM and TEM provides only limited
and indirect information on the metabolic activities of these organisms.
Therefore, it was of interest to apply methods designed to reveal in situ
metabolic activity more directly. One such method involves the use of 2-(p-
iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) as a measure
of respiratory activity of microbial cells (Zimmermann et al., 1978).
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Preliminary results with the INT method suggest that 1-1O % of the AODC
bacterial cells in samples from Texas were capable of respiration-linked i:.T
reduction. In most cases, however, INT-formazuP-containing bacteria contained
the diffuse type of deposit. Very few cells contained distinct granules.
These results suggest a low level of respiratory activity in the subsurface
bacterial population.
An alternate approach to investigating potential metabolic activities of
aquifer microorganisms was developed and usec' by J. T. Wilson (Wilson et al.,
1983). This approach involved the use of microcosms constructed from subsur-
face materials to determine whether the organisms indigenous to those
materials could degrade selected organic pollutants. Toluene was degraded
rapidly in subsurface samples from above and below the water table at Lula,
Oklahoma. Comparison of autoclaved and non-autoclaved samples indicated that
the degradation- was a biological process. Chlorobenzene was also degraded in
these samples, but (i) its degradation rate was considerably slower than that
of toluene and (ii) degradation took place only in samples from above che
water table. Broniodichloromethane was also degraded slowly, but it was not
clear whether this was a direct or indirect result of microbial metabolism.
In contrast, there was no detectable degradation of 1,2-dichloroethane, 1,1,2-
trichloroethane, trichloroethylene, or tetrachloroethylene in any of the Lula,
Oklahoma samples.
DISCUSSION
The results reviewed here demonstrate conclusively that appreciable num-
bers (1-10 million per gram) of microbial cells reside in aquifer material.
This observation is important for two reasons: (i) it contradicts the tradi-
tional belief that such environments are almost devoid of microbial life and
(ii) the numbers of cells detected were great enough to potentially atfect
ground water quality, provided that these cells were metabolically active.
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Morphological and ultrastructural data indicated that, even though the
total number of microorganisms was quite consistent from one sampling site to
another, the identity of those, organisms may have varied considerably. This
has important implications with respect to the potential effects of microorga-
nisms on ground water quality, since different microbial types carry out
different metabolic reactions and win respond differently tc specific pollu-
tants. Equally important is the fact that che range of microbial types varied
widely from one site to another. A pollutant compound might destroy all
microbial life in an aquifer that contained only a few types of bacteria,
whereas a more diverse microbial community would be more likely to include a
species that could survive or even degrade the pollutant compound. In
defining the various factors that control ground water quality, then, it
probably will be necessary not only to consider microorganisms in general, but
also to consider the specific microbial population of each aquifer system.
Sorns of the microorganisms in aquifer and other subsurface environments
must be viable because the studies reviewed here show3d that they were capable
of growth on plates. However, most of the AODC cells in typical samples did
not grow on plates. This could mean that these organisms were not viable, but
it is more likely that the growth media used for plating simply failed to meet
their possibly complex growth requirements (see also Ghiorse and Balkwill,
1983.) There is a need, then, to characterize the growth requirements of
subsurface microorganisms so that more realistic procedures for enumerating
viable cells can be developed. Alternatively, modifications of direct LM
approaches like the INT method might also serve to solve this problem.
The fact that plate counts of aquifer and other subsurface samples
usually were higher on nutritionally rich media than on relatively dilute
media implies that the jm situ microorganisms in these samples may prefer low
levels of nutrients for growth. Morphological and ultrastructural data in tho
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studies reviewed here also point to adaption by subsurface microorganisms to
low-nutrient and/or starvation conditions. The overwhelming predominance of
prokaryotes, for example, probably occurred because oligotrophic nrokaryotes
are much better adapted than eukaryotes to live in environments with very low
levels of organic matter (Poindexter, 1981). The PliB granules seen in subsur-
face bacteria also indicated an adaptation to Ic^-nutrient conditions, since
synthesis of these and other storage materials is a common bacterial strategy
for surviving periods of nutrient shortage (Poindexter, 1981; Shively, 1974).
The depleted cytoplasm of many subsurface bacteria suggests that these cells
actually were either nutrient-limited or starving at the time of sampling and,
therefore, probably were relatively inactive members of the microbial
community. On the other hand, the bacteria with a "heoithy" cytoplasm or with
division septa must have learned both to survive and to grow actively under
low-nutrient conditions.
Although data obtained by direct observation of subsurface microbial
cells with LM end TEM allowed us to draw reasonable conclusions about the
likely physiological characteristics of subsurface microorganisms (above), we
still know very little about the specific jji situ or potential i_n situ meta-
bolic activities of these organisms. Specialized techniques like the INT
procedure may prove helpful in this regard, but there is a need to develop
more powerful and sophisticated LM and TEH methods for determining ^in situ
metabolic activities in subsurface environments. Such information will be
critical in order to understand the biology of subsurface microorganisms. It
will also be critical to understand how subsurface microorganisms may affect
ground water quality and how these microorganisms themselves may be affected
by pollutants.
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ACKNOWLEDGEMENTS
This work was supported by Subcontract No. 6931-5 under U.S.E.P.A.
Cooperative Agreement No. CR806931-02.
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