Journal

of
Microbiological
Journal of Microbiological Methods 32 (1998) 165–178
Methods

Nucleic acid analytical approaches in bioremediation: site

assessment and characterization
Raymond D. Stapleton, Steven Ripp, Luis Jimenez, Sung Cheol-Koh, James T. Fleming,
Igrid R. Gregory, Gary S. Sayler*
Center for Environmental Biotechnology, University of Tennessee, Knoxville, 676 Dabney Hall, Knoxville, TN 37996, USA

Abstract

Bioremediation, the removal of environmental pollutants by living organisms, has become a viable and promising means
of restoring contaminated sites. Gene probing techniques have enhanced our ability to assess the efficacy of microbial-based
remediation efforts. DNA probes targeting specific genetic sequences, i.e. those genes responsible for the degradative ability
of the microorganism, can be used to characterize a contaminated site throughout the bioremediation program to determine
overall community structure and catabolic activity. To do so, however, requires efficient techniques for recovering nucleic
acids from environmental sites as well as methods for generating probes to the specific genetic sequences desired. This
review discusses procedures for isolating DNA, messenger RNA, and ribosomal RNA from environmental samples, the
utilization of polymerase chain reactions to construct gene probes, and hybridization methods to genetically match the probe
to the environmental sample. The use of these methods and advancement of techniques at several bioremediation sites is also
presented along with typical problems and limitations encountered. The first case study involves monitoring the effects of
nutrient addition to stimulate microbial degradation of chlorinated solvents at the DOE Westinghouse Savannah River Site.
The next case study describes the bioremediation of chlorinated solvents and low levels of BTEX at Dover Air Force Base,
Delaware. The final study is a field-scale natural attenuation project currently underway at Columbus Air Force Base,
Mississippi.  1998 Elsevier Science B.V.

Keywords: Bioremediation; DNA hybridization; Microbiology; Molecular diagnostics

1. Introduction bioremediation, where discrete microorganisms and

The union of molecular biology and microbial
ecology has led to the development of new, nucleic-
acid-based analytical techniques for identifying and
quantifying specific genetic sequences present and
their expression within natural microbial populations.
These techniques are especially useful in the field of
*Corresponding author. Tel.: 11 423 9748080; fax: 11 423
9748086; e-mail: sayler@utk.edu
microbial processes are typically employed to safely
remove environmental pollutants, either through
direct destruction or indirectly through a transforma-
tion of the contaminant to a safer intermediate. For
bioremediation to be effective, however, it is impera-
tive that scientists possess a means of quantifying
those populations of organisms responsible for de-
struction of the released contaminants as well as the
overall degradative activities associated with these
organisms — both achievable through the utilization
of molecular gene probes (Sayler and Layton, 1990).
Early environmental gene probing techniques typi-

0167-7012 / 98 / $19.00  1998 Elsevier Science B.V. All rights reserved.

PII: S0167-7012( 98 )00021-9
166 R.D. Stapleton et al. / Journal of Microbiological Methods 32 (1998) 165 – 178

cally involved DNA:DNA colony hybridizations 2. Nucleic acid methodology

(Grunstein and Hogness, 1975; Sayler et al., 1985),
which were limited since the population of cells
exposed to the probe included only those cells
capable of growth on laboratory media, estimated at
only 10% or less of the total population (Torsvik et
al., 1990). The development of methods for directly
extracting and isolating DNA from environmental
samples bypassed the need to culture organisms, thus
providing a more representative sampling of micro-
bial constituents within any given community. This
technology was augmented by the incorporation of
polymerase chain reaction methodologies along with
the more recent developments in messenger RNA
and ribosomal RNA extraction procedures, allowing
for the in situ measurement of biological activities
and further, more precise definition of total com-
munity structure. These techniques, their advantages
and limitations, and their associated use with gene
probes at various environmental sites are presented
in this review. A representative sample of gene
probes useful in site characterization and monitoring
is listed in Table 1.
The use of molecular diagnostics has greatly
enhanced our working knowledge concerning the
functional microbial community structure of soil,
sediment, sludge, and groundwater. A multitude of
nucleic acid extraction procedures exist for the
recovery of DNA, messenger RNA (mRNA), or
ribosomal RNA (rRNA) (Akkermans et al., 1996).
Direct extraction of nucleic acids from environmen-
tal samples eliminates any culture biases by pro-
viding a more representative sample of the entire
microbial community at the molecular level. DNA
extracted from the environment can be amplified by
the polymerase chain reaction (PCR), digested with
restriction enzymes, or hybridized with probes
targeting specific genes. RNA recovered from en-
vironmental samples can also be hybridized with
probes targeting specific genes or used as a template
for reverse transcription reactions, followed by PCR
amplification. Gene probes are usually generated
based on site conditions or contamination as well as
available physiological characteristics and nucleic

Table 1
Relevant characteristics of some common DNA probes used for assessing microbial communities
Probe Size Origin Features Reference
a
alkB from OCT ¯1.4 kb Pseudomonas oleovorans Alkane hydroxylase Kok et al. (1989)
codh ¯450 bp b Methanosarcina thermophila Carbon monoxide dehydrogenase Jablonski and Ferry (1992)
fthfs ¯1 kb Clostridium thermoaceticum Acetogenic formyltetrahydrofolate Lovell and Hui (1991)
synthetase
lux ¯300 bp Vibrio fischeri MJ-1 Luciferase Baldwin et al. (1989)
mcr ¯760 bp Methanosarcina barkeri Methyl coenzyme M reductase Hales et al. (1996)
mdh ¯1 kb Methylobacterium organophilum Methanol dehydrogenase Machlin and Hanson (1988)
nahA from NAH7 ¯1 kb Pseudomonas putida G7 Naphthalene dioxygenase Simon et al. (1993)
nahG from NAH7 ¯1 kb Pseudomonas putida G7 Salicylate hydroxylase You et al. (1991)
nahH from NAH7 ¯1 kb Pseudomonas putida G7 Catechol-2,3-dioxygenase Ghosal et al. (1987)
nahR from NAH7 ¯1.1 kb Pseudomonas putida G7 NAH7 regulatory protein You et al. (1988)
smmo ¯420 bp Methylsinus trichosporium Ob3b Soluble methane monooxygenase Wackett and Gibson (1988)
component B
todC1 C2 ¯1 kb Pseudomonas putida F1 Toluene dioxygenase Zylstra and Gibson (1989)
tomA ¯1.5 kb Burkholderia cepacia G4 Toluene monooxygenase Shields et al. (1989)
xylA from pWWO ¯2 kb Pseudomonas putida Xylene monooxygenase Suzuki et al. (1991)
23S rDNA ¯380 bp Pseudomonas fluorescens 23S large subunit ribosomal DNA Festl et al. (1986)
16S Universal rDNA ¯18 bp Escherichia coli 16S small subunit ribosomal DNA Stahl et al. (1988)
Lambda ¯500 bp Lambda phage Internal standard for DNA extractions Sanger et al. (1982)
a
kb refers to kilo base pairs.
b
bp refers to base pairs.
 R.D. Stapleton et al. / Journal of Microbiological Methods 32 (1998) 165 – 178
acid sequence databases. An array of DNA and RNA
gene probes can be used to develop specific mi-
crobiological parameters to be monitored at each site
under investigation.
Isolation of DNA from soil initially centered
around the homogenization and fractional centrifuga-
tions separating microorganisms from soil particles
(Torsvik and Goksoyr, 1978; Torsvik, 1980). The
recovered cells are then lysed and the DNA purified.
Ogram et al. (1987) developed an extraction method
that eliminates the separation of microbial cells from
soil particles and directly extracts DNA in situ. Cell
lysis occurs by dissolution of the cell envelope by
heating a soil slurry in phosphate buffer and sodium
dodecyl sulfate (SDS) followed by ballistic disinte-
gration using bead-mill homogenization. The re-
leased nucleic acids are recovered in the aqueous
phase by successive alkaline extractions and centrifu-
gations of the soil. Prior to purification, recovered
nucleic acids are concentrated via alcohol precipi-
tation. Concentrating the extracts produces a greatly
reduced sample volume that is more easily pro-
cessed. Proteins, other cellular debris generated
during cell lysis, and many co-extracted contami-
nants such as soil organic matter are removed from
the nucleic acids by solvent purification using phenol
and chloroform. After purification, the samples are
again concentrated via alcohol precipitation. DNA
recovered from this alcohol precipitation is most
often of sufficient quality for slot blot hybridization.
Enzymatic based analyses including PCR and restric-
tion digests often require further sample purification
steps such as cesium chloride / ethidium bromide
ultracentrifugation or hydroxyapatite chromatog-
raphy. Samples having low amounts of organic
material may require additional dialysis steps to
reduce salt accumulation during the extraction pro-
cedure (Johnston et al., 1996; Ogram et al., 1995).
Traditional microbiological methods previously
used in characterizing hazardous waste sites often
included an assessment of microbial activity or the
potential for biodegradation of specific contaminants
using either 14 C-radiolabeled substrate assays or
microcosms supplemented with unlabeled substrates
with degradation monitored by gas chromatography
or liquid chromatography. Extraction and manipula-
tion of mRNA from the environment, pioneered by
167
Flemming et al. (1993) and Sayler et al., provides a
direct method of monitoring the induction of en-
zymatic pathways involved in bioremediation. Anal-
ysis of the mRNA allows investigators to directly
link the more modern molecular based diagnostics
with the aforementioned traditional degradation as-
says. There are many similarities between DNA and
mRNA extraction from soil or sediment. The biggest
difference in the two extraction protocols revolves
around the stability of mRNA. Great caution and
care must be used when recovering mRNA to
eliminate alkaline hydrolysis and enzymatic degra-
dation.
Extraction and manipulation of mRNA from en-
vironmental samples allows investigators to support
the presence of particular functional populations of
microorganisms with a direct molecular activity
assessment (Flemming and Sayler, 1995). Extraction
of mRNA initiates with a more gentle cell lysis than
described for DNA. Lysis is initiated in a heated
phenol:chloroform buffer supplemented with SDS.
After heat treatment, the sample slurry is shaken on a
‘wrist-action shaker’, followed by centrifugation to
recover the aqueous phase. The aqueous phase is
extracted with chloroform, then shaken and cen-
trifuged as above. Nucleic acids are alcohol precipi-
tated overnight. Recovered precipitants are dissolved
in diethylpyrocarbonate (DEPC) treated sterile water
and treated with RNase-free DNase. Phenol:ch-
loroform extraction removes the enzyme and the
samples are again alcohol precipitated overnight. The
final pellet is resuspended in DEPC-treated water and
stored at 2808C until used. This procedure produces
mRNA of sufficient quality for reverse transcription
reactions and membrane hybridizations. In the initial
use of manipulating mRNA from environmental
samples, hybridization protection assays were used
to directly quantify extracts without use of PCR
amplification procedures.
Ribosomal RNA (rRNA) can also be extracted
from the environment and used to augment infor-
mation gained from DNA analyses with a more
comprehensive community structure. Ribosomal
RNA has gained a great deal of interest by microbial
ecologists for phylogenetic analysis of previously
unculturable microorganisms (Amann et al., 1995;
Olsen et al., 1986; Ward et al., 1992; Woese, 1987).
168 R.D. Stapleton et al. / Journal of Microbiological Methods 32 (1998) 165 – 178
As with mRNA, great care must be used when
extracting rRNA to prevent degradation. In some
cases, it may be more desirable to extract DNA from
the sample and amplify 16S or 23S ribosomal RNA
genes for sequence analysis. The sequences produced
by this method lend insight into exactly what organ-
isms are present in the sample under investigation.
Devereux et al. (1992) developed a derivation of
the DNA extraction technique of Ogram et al. (1987)
for recovery of rRNA from salt marsh sediments.
Cell lysis for recovery of rRNA was performed by
bead-mill homogenization. The aqueous phase is
recovered and extracted with phenol. The purified
aqueous phase is recovered and mixed with hexade-
cyltrimethyl ammonium bromide (CTAB) and NaCl
to remove contaminants that co-extract with the
nucleic acids. CTAB:NaCl is removed from the
aqueous phase by a phenol:chloroform treatment,
followed by a chloroform:isoamyl alcohol extraction.
The recovered aqueous phase is alcohol precipitated
overnight. After centrifugation, the samples are
washed with 80% ethanol, dried, then resuspended in
sterile TE (10 mM Tris–HCl, 1 mM EDTA) buffer
or DEPC-treated water. rRNA can be further purified
on a Sephadex G-75 spin column.
Significant insight into the presence of specific
microorganisms or groups of microorganisms has
been increased by the advent of the polymerase chain
reaction (Mullis and Faloona, 1987; Hales et al.,
1996). PCR allows for amplification of millions of
copies of a specific DNA fragment in a process that
takes advantage of a heat stable DNA polymerase
and magnesium chloride. For molecular analyses of
microbial communities as they exist in the environ-
ment, DNA extracted from environmental samples is
used as the template for the PCR reaction. Primers,
defined as short segments of single-stranded DNA
ranging from 10–25 bases in length specific for
regions flanking the area of interest, are used to
generate a free 39 hydroxyl group facing the target
sequence. The PCR amplification reaction initiates at
the free 39 hydroxyl group of the primers and
extends through the target fragment. Generally, 30–
35 PCR cycles are used to generate the desired
amount of target. Several reviews and books have
recently been published concerning the manipulation
of reagent concentrations, temperatures and cycle
lengths (Innis and Gelfand, 1990; Saiki et al., 1985).
Application of PCR to environmental microbiol-
ogy extends beyond using it for detection of organ-
isms or genes in low abundance (Steffan and Atlas,
1991). The polymerase chain reaction can be modi-
fied and used for generating either single- or double-
stranded DNA probes useful in DNA:DNA hybridi-
zation studies with nucleic acids recovered from
environmental samples. Radionucleotides labeled
with 32 P can be substituted for unlabeled nucleotides
in the basic PCR mix to generate a radioactive DNA
probe. Radionucleotides that are not incorporated
into DNA strands produced by the polymerase chain
reaction can be subsequently purified from the mix
using a Nuc-Trap Push-Column Apparatus
(Stratagene) and probe intensity can then be de-
termined using a scintillation counter. Single-
stranded DNA probes are produced by the addition
of only one primer to the reaction mix (asymmetric
amplification) and do not need to be boiled prior to
hybridization. PCR generation of probes is a simple
method to generate large quantities of highly specific
DNA fragments labeled with radionucleotides used
for hybridization with DNA extracted from the
environment. False positives resulting from template
plasmid contamination of gene probes generated
from DNA clones can easily be eliminated using
PCR to generate probes. However, for PCR gener-
ated probes to be useful, the investigator must know
the sequence of the DNA fragment targeted by the
gene probe. This is facilitated by the rapidly expand-
ing databases of catabolic gene sequences.
DNA extracted from soil and sediment samples
can be quantitatively analyzed by DNA:DNA hybrid-
izations using PCR-generated radiolabeled gene
probes. Extracted DNA can be vacuum blotted and
fixed onto a nylon membrane. A set of DNA
standards of the appropriate target sequence is
included on the membrane. The fixed DNA is then
subjected to DNA:DNA hybridization with a radio-
labeled gene probe. After washing excess probe, the
membrane is exposed to film for visualization of
positive samples. Next, a computer image of the
autoradiogram must be generated. Using a personal
computer based program, such as SigmaGel from
Jandel Scientific, for manipulation of the autoradio-
gram image, a regression curve can be generated for
the known DNA standards on the membrane. Un-
known sample values can then be inserted into the
 R.D. Stapleton et al. / Journal of Microbiological Methods 32 (1998) 165 – 178
regression equation and amount of target sequences
calculated as nanograms of DNA. Cell number
values can be estimated using previously proposed
equations (Applegate et al., 1995; Sanseverino et al.,
1993). Catabolic gene probe values can be normal-
ized on total population estimates generated using
the Universal 16S rDNA oligonucleotide (Stahl et
al., 1988) to create a percent community value that is
useful in comparing different samples recovered
from one site, as well as from various sites. This is
accomplished using an assumption of seven copies of
16S rDNA genes per chromosome (Smith et al.,
1987).
3. Site assessment and characterization
3.1. Westinghouse Savannah River Site
During the early 1990s, the DOE Westinghouse
Savannah River Site (SRS) in Aiken, South Carolina
was the location of a field Integration Demonstration
Project designed by scientists representing various
fields (microbiology, hydrology, geology, geochem-
istry, etc.). The primary contaminants at SRS are
tetrachloroethylene (PCE) and trichloroethylene
(TCE) resulting from use of metal degreasing sol-
vents since 1952 (Eddy et al., 1991). The chlorinated
solvents produced a plume with concentrations of
PCE up to 180 ppm and TCE up to 1800 ppm over
an area greater than one square mile at depths up to
40 m (Eddy et al., 1991).
The Savannah River Demonstration Site provided
a unique location for testing molecular-based meth-
ods of monitoring microorganisms in the subsurface.
The basic bioremediation hypothesis tested was that
methanotrophic co-metabolism of TCE could be
stimulated by injection of air, methane or methane
and nutrients into the contaminated area. Mi-
crobiological analyses were performed on core sam-
ples recovered from both the saturated and unsatu-
rated zones. Molecular methods chosen for moni-
toring microbial response to the injection treatments
were based on DNA:DNA hybridization with gene
probes designed for aerobic biochemical pathways
known to co-metabolize TCE (Ensley, 1991). Gene
probes used at SRS included soluble methane mono-
oxygenase (smmo) to detect Type I and Type II
169
methane oxidizing bacteria possessing methane / TCE
co-oxidation capability (Fox et al., 1990; Oldenhuis
et al., 1989; Tsien and Hanson, 1992), methanol
dehydrogenase (mdh) to detect methane and metha-
nol utilizing bacteria (Machlin and Hanson, 1988),
and toluene dioxygenase (tod) to detect bacteria
capable of aerobic toluene / TCE co-oxidation (Wac-
kett and Gibson, 1988). The toluene dioxygenase
probe was used as a negative control for the smmo
and mdh gene probes. During the final stages of the
field demonstration, initial experiments attempting to
determine microbial activity measurements based on
the extraction and manipulation of soluble methane
monooxygenase mRNA and total microbial biomass
estimates based on 16S rDNA sequences were
conducted.
DNA extraction and DNA:DNA hybridization was
performed on |350 samples recovered from SRS.
Most samples suffered from low microbial abun-
dance, poor recovery of DNA, or DNA contamina-
tion with soil organic material, thus quantitative
measurements were not possible. Samples were
scored as positive or negative for the presence of the
target gene. Gene frequency was determined by
dividing the number of positive samples by the total
number of samples probed and multiplying by 100%.
In general, smmo gene abundance never exceeded
detectable limits by more than 5% in any samples
examined. The equivalent cell density, estimated
from ng DNA (Applegate et al., 1995; Sanseverino et
al., 1993), was in the range of 10 6 organisms per
gram sediment. Consequently, gene frequency was
scored on a sample basis. While organisms con-
taining the smmo genes were likely present in many
more samples, only 107 samples of the more than
350 samples examined showed gene frequencies high
enough to be scored as positive. Table 2 summarizes
the data generated for gene frequencies of smmo,
mdh and tod for the core samples. Prior to any
treatments, each catabolic genotype was present in
greater than 20% of the samples, with 43% of the
samples probing positive for toluene dioxygenase.
Initial treatment at SRS involved injection of air
into the contaminant zone, with greatest response to
this treatment seen in samples hybridizing with the
soluble methane monooxygenase gene probe increas-
ing from 22 to 42% samples probing positive.
However, soluble methane monooxygenase geno-
170 R.D. Stapleton et al. / Journal of Microbiological Methods 32 (1998) 165 – 178
Table 2
Sediment sample gene frequency in relation to treatment campaigns for TCE bioremediation at the Savannah River Site a
Treatment No. samples analyzed
Background 44
Air 120
1% Methane 43
4% Methane 31
4% Methane1nutrients 109
a
Gene frequency (given as percent) was calculated by dividing the number of positive samples by the total number of samples extracted and
probed, then multiplying by 100%.
types steadily decreased with subsequent injections
of methane. Samples possessing the smmo gene
rebounded with the final treatment of methane and
nutrients with the gene frequency rising to 28%.
The initial treatment of air unexpectedly produced
a population reduction in organisms possessing the
mdh genotype. mdh gene frequency was 31% prior
to any treatments, then 6% after the injection of air.
Injection of methane stimulated a population re-
covery of mdh genotypes, with a major population
increase occurring after injection of both methane
and nutrients. After the final treatment, mdh gene
frequency recovered to 51%.
An anomaly exists in the apparent relationship
between smmo and mdh sample detection. Since mdh
is present in virtually all methylotrophs as well as
methanotrophs, it was hypothesized that mdh should
serve as a positive control for smmo gene occur-
rence. Table 2 does not indicate such a relationship
in terms of gross comparison among samples scoring
positive for the two genotypes. However, in an
analysis of the co-occurrence of the two genotypes in
identical samples, there was a significant correlation
(C51.7) at P50.95 and a chi value of 11.3 prior to
any treatments and a correlation of C51.5 at P50.95
and a chi value of 26 after injection of methane and
nutrients.
The tod gene frequency was 43% prior to any
treatments at SRS. After a slight increase to 46%
following the injection of air, populations possessing
the tod genotype slowly decreased in gene frequency
until there were no positive samples after the final
treatment. This could be an indication that organisms
possessing the toluene dioxygenase were unable to
utilize methane for a growth substrate or were
unsuccessful in competing for other necessary nu-
trients during the investigation.
smmo mdh tod
22 31 43
42 6 46
35 21 21
6 21 13
28 51 0
Because smmo expression is suppressed by m M
concentrations of copper ion, there was a great deal
of concern that smmo gene abundance may not be
relevant to TCE degradation. However, in applying
the mRNA extraction procedure with the hybridiza-
tion protection assay, it was possible to demonstrate
that mRNA transcripts of smmo were present under
in situ conditions for various samples at a variety of
depths within the integrated field demonstration sites.
Fig. 1 presents the results of the SRS RNA analysis.
Of the 25 soil samples extracted, nine samples
probed positive for rRNA and five for smmo. No
discernible trend was demonstrated with depth. This
may be a result of the limited number of samples
analyzed. The results from the RNA analysis repre-
sent an index of not only gene presence, but also
activity under field conditions.
The oligotrophic environment at the Savannah
River Integrated Demonstration Field Site presented
many difficulties in assessing the efficacy of moni-
toring microbial populations with specific catabolic
gene probes (discussed later). The problems associ-
ated with this particular field study rendered the
hypotheses originally developed only partially test-
able.
3.2. Dover Air Force Base
In 1995, a consortium of government agencies,
universities, and manufacturing companies including
Ciba, Dow, Dupont, General Electric, Monsanto, and
Zeneca, Inc. initiated a field scale demonstration
project concerning the bioremediation of chlorinated
solvents, primarily PCE and TCE, and petroleum
hydrocarbons, primarily BTEX (benzene, toluene,
ethyl benzene and xylenes) at Dover Air Force Base
(DAFB), Delaware. The study was designed to
 R.D. Stapleton et al. / Journal of Microbiological Methods 32 (1998) 165 – 178 171

Fig. 1. Correlation of smmo RNA with sampling depth. RNA was extracted from well soil samples and plotted in the combined depth ranges
of 0–10, 11–30, and .31 m below ground surface. smmo RNA values were normalized to cell density by dividing the pg smmo RNA / g
soil by the pg total RNA.

monitor the efficacy of intrinsic and accelerated
anaerobic bioremediation of chlorinated solvents in
groundwater, as well as co-metabolic bioventing of
TCE in unsaturated subsurface strata. The overall
objective of the study is to demonstrate accelerated
anaerobic biodegradation of chlorinated solvents in
the anoxic center of the contaminant plume and
intrinsic remediation in the low-contaminant con-
centration area. A molecular-based approach for
monitoring the response of the indigenous microbial
community was chosen based on previous experience
gained during the Savannah River Integrated Demon-
stration Study, on the increase in biochemical knowl-
edge of catabolic pathways, and advancements in
analytical methods since the SRS project. Gene
probes used in this study include soluble methane
monooxygenase (smmo), methanol dehydrogenase
(mdh), toluene dioxygenase (tod), naphthalene
dioxygenase (nahA), catechol-2,3-dioxygenase
(nahH ), carbon monoxide dehydrogenase (codh),
and a Universal 16S rDNA oligonucleotide for total
biomass. Soluble methane monooxygenase and
toluene dioxygenase are aerobic enzymes previously
shown to co-metabolize TCE (Fox et al., 1990;
Oldenhuis et al., 1989; Wackett and Gibson, 1988).
Carbon monoxide dehydrogenase is an anaerobic
enzyme shown to reductively dechlorinate TCE
(Jablonski and Ferry, 1992). A total biomass esti-
172 R.D. Stapleton et al. / Journal of Microbiological Methods 32 (1998) 165 – 178

mate can be generated using a Universal oligo-
nucleotide specifically designed for a highly con-
served region of the 16S rRNA gene (Stahl et al.,
1988). A recently developed gene probe for toluene
ortho-monooxygenase, another aerobic enzyme
shown to gratuitously degrade TCE (Shields et al.,
1989), will also be applied to DNA recovered from
DAFB sediments.
Subsurface sediment material received from
DAFB was extracted for DNA and subsequently
hybridized with the aforementioned gene probes.
Distribution of genotypes targeted by gene probe
analysis is shown in Table 3. Uncontaminated
aquifer material from the study area supports an
estimated 8.78310 6 organisms per g sediment as
determined by hybridization with the 16S Universal
rDNA oligonucleotide. DNA extracted from uncon-
taminated sediment did not produce detectable hy-
bridization signals with any other gene probe used in
this investigation. The cometabolic study area sup-
ports 2.27310 8 (62.76310 7 ) organisms per g sedi-
ment as determined by hybridization with the 16S
rDNA Universal oligonucleotide. Methanol dehydro-
genase and catechol dioxygenase genes were found
in detectable levels, but no other target sequences
were detected. Total biomass in samples recovered
from the accelerated anaerobic area was estimated at
2.11310 9 (62.27310 7 ) organisms per g sediment.
Carbon monoxide dehydrogenase and catechol-2,3-
dioxygenase sequences were not found at detectable
levels. Organisms possessing mdh, nahA, smmo, or
tod genotypes ranged from 1.51310 7 (61.00310 7 )
to 1.88310 8 (63.10310 7 ) cells per g. Aquifer
material from the intrinsic remediation study site
supports the highest total biomass of 3.64310 9
(65.49310 9 ) organisms per g. Every genotype
analyzed for was represented in the intrinsic samples,
with population levels ranging from 4.73310 7
(64.38310 7 ) to 4.86310 8 (66.62310 8 ) cells per g.
The clearest trend seen in the Dover Air Force Base
gene probe data is the increase in not only catabolic
organisms, but also in total biomass in treatment
areas as compared with the uncontaminated sample.
3.3. Columbus Air Force Base
Many contaminated sites may not require an active
and aggressive approach to remediating the environ-
ment (Lee and Ward, 1985; Lee et al., 1988; Madsen
et al., 1991; National Research Council, 1993). As
an alternative, natural attenuation can be a cost-
effective means to remediate groundwater resources
that become contaminated with organic contaminants
(Davis et al., 1994; Klecka et al., 1994). For natural
attenuation to become a universally accepted means
of site restoration, a mechanistic understanding of
the response of the indigenous subsurface microbial
community must be developed. In order for a better
mechanistic understanding of natural attenuation to
occur, interdisciplinary field scale studies are needed.
One such investigation, termed the Natural Attenua-
tion (NAT) study, is currently underway at Colum-
bus Air Force Base (CAFB), Mississippi. The CAFB
Groundwater Research Facility has been the site of

Table 3
Quantitative distribution of catabolic genotypes at Dover Air Force Base a
Treatment No. samples analyzed 16S rDNA codh mdh nahA nahH smmo tod
b
Uncontaminated 1 3.61 ng bdl bdl bdl bdl bdl bdl
8.78310 6 bdl b bdl bdl bdl bdl bdl
Co-metabolic 2 93.47 ng 8.85 ng 1.07 ng bdl 0.26 ng bdl bdl
2.27310 8 3.23310 8 1.92310 7 bdl 4.78310 6 bdl bdl
Accelerated 15 865.73 ng 5.15 ng 0.83 ng 5.89 ng bdl 2.03 ng 4.60 ng
2.11310 9 1.88310 8 1.51310 7 1.08310 8 bdl 8.21310 7 8.39310 7
Intrinsic 17 1496.29 ng 8.87 ng 2.59 ng 4.76 ng 3.02 ng 11.97 ng 2.75 ng
3.64310 9 3.24310 8 4.73310 7 8.70310 7 5.41310 7 4.86310 8 5.01310 7
Detection limits 0.01 ng 0.01 ng 0.01 ng 0.01 ng 0.01 ng 0.01 ng 0.01 ng
2.43310 6 3.65310 6 1.83310 6 1.83310 6 1.83310 6 4.06310 6 1.83310 6
a
Values given as ng target sequence recovered from each sample and a cell number estimate based on the ng DNA recovered.
b
bdl, below the listed detection limits.
 R.D. Stapleton et al. / Journal of Microbiological Methods 32 (1998) 165 – 178
several field scale natural gradient tracer studies over
the past 15 years. Groundwater research initiated at
the site during the mid-1980s was primarily
concerned with the physical and chemical interac-
tions (dispersion, diffusion, and sorption) that effect-
ed the fate and transport of certain target contami-
nants. The Groundwater Research Facility currently
supports over 300 multi-level groundwater sampling
wells providing a three-dimensional monitoring net-
work of over 6000 potential sampling ports.
A macrodispersion experiment (MADE-2) was
conducted at the CAFB Groundwater Research
Facility (Boggs et al., 1993; Stauffer et al., 1997).
This study was designed as a natural gradient tracer
experiment. The study site was previously intensely
characterized (Boggs et al., 1990, 1992; Boggs and
Adams, 1992). The compounds chosen for this study
included dissolved organic compounds (benzene,
naphthalene, 14 C-labeled p-xylene, and o-dichloro-
benzene) and tritiated water as a conservative tracer.
The dissolved contaminants were monitored in
groundwater samples over a 15-month period. All
four organic tracers showed gradual disappearance
over 440 days. Six percent of benzene, 6% of
naphthalene, and 1% of p-xylene remained in the
groundwater after 15 months (Boggs et al., 1993).
Thirteen percent of o-dichlorobenzene remained at
the end of the experiment (Boggs et al., 1993).
Disappearance of the organic compounds could not
be accounted for using advection, sorption, or disper-
sion models. It was proposed that the fate of the
organics were affected by natural biotransformation
by indigenous microorganisms. Even though no
direct microbiological evidence was collected from
the site, indirect geochemical data tends to support
this conclusion. Examination of the 14 C p-xylene
revealed greater than 80% of the radiolabel was
found as 14 CO 2 and intermediate by-products indica-
tive of aerobic biodegradation (MacIntyre et al.,
1993). The estimated biological oxygen demand
(BOD) was determined to be 1 mg / l for biodegrada-
tion of the organic tracers at the observed rates.
Aquifer heterogeneity allowing the influx of oxygen
rich groundwater eliminated the concern of oxygen
depletion during biodegradation for this concentra-
tion of contaminants (|150 ppm each).
For the NAT study higher levels (.1000 ppm
each) of the hydrocarbons benzene (6 ppm), toluene,
173
ethylbenzene, p-xylene, and naphthalene, as well as
the conservative tracer bromide, were mixed with
aquifer solids and implanted within the aquifer below
the water table, then allowed to distribute in the
subsurface by normal groundwater flow. Analysis of
indigenous microorganisms was performed using
catabolic DNA probes specific for genes encoding
degradation of the target compounds comprising the
‘mock’ jet-fuel used for this field study. Gene probes
used at CAFB included alkane hydroxylase (alkB),
carbon monoxide dehydrogenase (codh), naphthalene
dioxygenase (nahA), catechol-2,3-dioxygenase
(nahH ), toluene dioxygenase (tod), xylene mono-
oxygenase (xylA), and the previously mentioned 16S
rDNA Universal oligonucleotide. For measuring
changes in specific degradative populations over
time, data generated from catabolic gene probe
values were normalized on total population estimates
produced using the 16S rDNA Universal oligonu-
cleotide. The normalized population values are given
as percent community, and allow specific compari-
sons to be made between samples. Activity assays
were performed using the 14 C-radiolabeled substrates
benzene, toluene and naphthalene.
Hydrocarbon transport was retarded for |9 months
before a measurable hydrocarbon plume evolved
from the contaminant source. Microbial response to
pollutant perturbation was readily seen as an increase
in degradative genotypes often associated with
biodegradation of the contaminant hydrocarbons. As
can be seen in Table 4, molecular techniques can be
used to assess the impact the mock jet-fuel has on
the structure and activity of indigenous microorga-
nisms. Uncontaminated samples had standard devia-
tions ranging from 1.34 ng (1.30310 7 ) for xylA to
184.63 ng (3.22310 8 ) for total biomass estimates
based on DNA hybridization with the 16S rDNA
Universal oligonucleotide. For the plume data listed
in Table 4, standard deviations ranged from 0.78 ng
(7.15310 6 ) for xylA genotypes to 111.33 ng (1.943
10 8 ) for total biomass. Standard deviations for the
plume fringe data listed in Table 4 ranged from 1.16
ng (1.06310 7 ) for xylA to 143.84 ng (2.51310 8 ) for
total biomass. Degradative organisms are ubiquitous-
ly distributed throughout the study aquifer system.
Unlike the uncontaminated control sediment used at
Dover Air Force Base, the CAFB control sediments
recovered from upgradient the source trench support
174 R.D. Stapleton et al. / Journal of Microbiological Methods 32 (1998) 165 – 178

Table 4
Distribution of catabolic genotypes at Columbus Air Force Base
No. samples analyzed 16S rDNA alkB codh nahA nahH tod xylA
Uncontaminated 15 316.70 ng 2.97 ng n/a 3.58 ng 2.60 ng 5.58 ng 1.74 ng
5.51310 8 3.62310 7 n/a 6.51310 7 3.17310 7 9.36310 7 2.05310 7
Plume 10 381.54 ng 6.90 ng 7.32 ng 7.77 ng 5.46 ng 15.33 ng 6.52 ng
6.65310 8 7.17310 7 1.30310 8 1.41310 8 9.93310 7 2.77310 8 5.95310 7
Plume fringe 15 289.76 ng 4.02 ng n/a 5.05 ng 3.41 ng 5.84 ng 3.30 ng
5.05310 8 4.90310 7 n/a 9.18310 7 4.16310 7 1.06310 8 3.01310 7
Detection limits 0.01 ng 0.01 ng 0.01 ng 0.01 ng 0.01 ng 0.01 ng 0.01 ng
2.43310 6 2.43310 6 3.65310 6 1.83310 6 1.83310 6 1.83310 6 9.13310 5

each degradative population monitored. Radiolabel
substrate assays show these organisms remain in a
state of low catabolic activity. Time course miner-
alization analysis shows these sediments require 3–
14 days for 50% degradation of the target compound.
Each probe used in DNA:DNA hybridization studies
revealed elevated levels of degradative target genes.
The most substantial increase was seen in microbial
populations possessing toluene dioxygenase, as the
amount of tod gene targets increased from 5.58 ng to
over 15 ng / g sediment. Interestingly, degradative
organisms have not drastically increased along the
fringe of the contaminant plume. The amount of
target DNA sequences found are very similar to the
upgradient samples. However, comparative activity
assays using radiolabeled substrates show that these
sediments support a higher level of degradative
activity. Mineralization of target compounds ranges
from 5% to more than 40% after only 24 h.
Naphthalene mineralization studies show up to 60%
degradation along the plume fringe at 24 h. The
increased mineralization activity along the plume
fringe reflects the inhibition of contaminant spread-
ing in the groundwater system compared to a non-
conservative bromide tracer.
A major difference between Columbus Air Force
Base and the previously mentioned case studies is
that the NAT study was designed to monitor tran-
sient changes in the microbial community. Samples
were recovered from upgradient control areas and
experimental sites both prior to and during hydro-
carbon exposure. It is almost impossible to obtain
microbiological data a priori to contamination as
well as when the pollutants travel in the groundwater
system. The system has not had a prolonged expo-
sure to contaminants and provides the ability to
monitor dynamic changes as real-time measure-
ments. Initial data suggests there are many factors
involved in producing measurable changes in specific
microbial populations at contaminated sites such as
Columbus Air Force Base. Hydrocarbon concen-
tration and length of contaminant exposure appear to
be the main driving force in population changes.
These factors first signal an increase in catabolic
activity, then elevated population levels. After the
populations have increased, thus increasing microbial
activity, dissolved oxygen is depleted and the con-
taminants proceed further from the source. This
‘ripple’ effect occurs until the system achieves a
steady state at which contaminant destruction is
maintained at sufficient levels to inhibit plume
migration, but does not deplete dissolved oxygen.
4. Addressing problems and limitations of
molecular diagnostics
Over the past decade, many advances have been
made in overcoming limitations arising in the use of
molecular diagnostics in bioremediation. Positive /
negative detection schemes have been replaced with
analyses that provide quantitative measurements of
specific microbial populations present at hazardous
waste sites. These quantitative measurements allow
investigators to monitor any particular site over time
and provide a basis for comparing various sites with
similar contaminants or soil / water characteristics.
The potential for hazardous waste site databases
 R.D. Stapleton et al. / Journal of Microbiological Methods 32 (1998) 165 – 178
exists such that investigators can deposit mi-
crobiological information obtained through nucleic
acid analyses that relate to a particular contaminant,
soil type, geographical region, climate, etc. This
would aid government regulatory agencies in de-
termining guidelines for microbiological monitoring
of sites designated for biologically based remediation
methods.
As with any newly developed technology, molecu-
lar diagnostics has drawbacks and limitations. Sever-
al concerns arose during the course of the Savannah
River Integrated Demonstration Project. Quantitative
measurements of specific genotypes being monitored
by DNA:DNA hybridization were not available. The
catabolic genotypes under investigation were simply
recorded as being present or absent. Samples re-
corded as absent for any given gene sequence may
either not have the genotype represented or it may be
maintained within the population at levels below
detection limits. To a certain extent, low gene
abundance can be overcome using large sample sizes
for nucleic acid extraction (i.e. 50 g), increasing
probe strength using nucleotides with higher specific
activities, and supporting DNA hybridization data
with PCR-based detection methods. The question of
gene abundance also is a concern when distinguish-
ing between samples that support a detectable level
of genotypes and samples that are enriched for this
particular organism or population. This is easily
overcome with quantitative measurements, either by
DNA:DNA hybridization or PCR (Steffan and Atlas,
1991) that can be directly compared.
Development of the 16S rDNA Universal oligo-
nucleotide (Stahl et al., 1988) allows specific geno-
types to be normalized as a percentage of total
population. Normalized population values using the
16S Universal oligonucleotide also help to eliminate
variabilities associated with extractions performed on
samples high in clay or soil organic matter that make
recovery of nucleic acids problematic. This ana-
lytical tool also provides a means for comparisons
between sites, investigators, and laboratories.
Another concern associated with the efficacy of
site assessment and characterization of specific mi-
crobial populations revolves around probe specificity
and the involvement of unknown biochemical path-
ways. While most current functional gene probes are
175
designed to be highly specific, some existing DNA
probes cross-hybridize with other genes. A good
example of this can be seen in comparing hybridiza-
tion patterns of organisms possessing catechol-2,3-
dioxygenase. A catechol-2,3-dioxygenase gene probe
(nahH ) originating from the NAH7 catabolic plas-
mid also hybridizes with organisms harboring the
TOL plasmid. The TOL plasmid encodes a catechol-
2,3-dioxygenase (xylE) associated with degradation
of xylenes and toluene. Ghosal et al. (1987) showed
the two genes to be 81% homologous at the nucleo-
tide level.
While quantification of nucleic acid targets in
environmental samples is well established, estima-
tion of cell numbers is difficult. Sanseverino et al.
(1993) used the following equation for estimating
cell numbers of organisms possessing the nahA
genotype in manufactured gas plant samples:
(X nah A /v) 4 (X probe ) 3 (Vtotal /m soil )
5 nahA genotypes per g soil
where X nah A is the amount of nahA detected on a
slot blot in g, v is the volume of extract applied to
the slot blot in ml, Xprobe is the mass of the probe in
grams, Vtotal is the total volume of liquid the ex-
tracted DNA is resuspended in prior to use, and m soil
is the mass of soil extracted in g. The calculation
assumes 1 nahA gene per cell and a bacterial lysis
efficiency of 100%. Another version of this equation
has recently been published (Applegate et al., 1995).
Another useful manner of comparing nucleic acid
hybridization data from different sediment samples
or sites involves the normalization of catabolic genes
on 16S rDNA genes to derive a percent community.
Percent community can be calculated using the
following equation:
% community 5
(ng catabolic genes/copy number of the catabolic gene)
]]]]]]]]]]]
(ng 16S rDNA genes/copy number of 16S rDNA genes)
The concept of addressing particular organismal
activity as a fraction of the entire microbial com-
munity was described previously (Jones and Edin-
gton, 1968). Problems arise in applying correct copy
numbers for target genes, particularly 16S rDNA.
176 R.D. Stapleton et al. / Journal of Microbiological Methods 32 (1998) 165 – 178
For this equation, we assume a copy number of 1 for
our catabolic gene probes and 7 for 16S rDNA
(Smith et al., 1987). Farrelly et al. (1995) showed
copy number of 16S rDNA genes may range greatly
between different species of soil bacteria. Limita-
tions such as this will be readily overcome as current
databases expand the amount of attainable infor-
mation. Also additional experiments comparing pure
culture spread plate analyses of microorganisms with
DNA extraction and hybridization values will help to
calibrate cell number calculations.
5. Conclusions
Over the past decade, molecular diagnostics has
rapidly gained attention and respect in assessing the
potential for microbial degradation of hazardous
chemicals at contaminated sites. Radiolabel substrate
assays are useful in determining the catabolic activity
of a particular sample, but suffer from expense,
hazardous waste disposal, and sampling / handling
artifacts. Arrays of functional gene probes can be
applied to contaminated soils, sediments and ground-
water systems to characterize the microbial com-
munity present, as well as monitor changes in
specific populations over time. Advancement in
messenger RNA analyses from environmental sam-
ples will allow investigators to directly link these
specific population changes with a particular activity
using biologically based measurements. Ribosomal
RNA is rapidly gaining a tremendous amount of
interest in piecing together microbial community
structure from a phylogenetic standpoint. As in-
formation is gathered on various sites, whether
contaminated or uncontaminated, it becomes increas-
ingly important to develop readily utilizable and
accessible databases.
Acknowledgements
The studies presented in this manuscript were
funded by the United States Air Force, Tennessee
Valley Authority, Martin Marietta, Lockheed Martin,
and the Center for Environmental Biotechnology at
The University of Tennessee, Knoxville.
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