v>EPA
United States
Environmental Protection
Agency
National Risk Management
Research Laboratory
Ada, OK 74820
Research and Development
EPA/600/R-09/103
September 2009
ENVIRONMENTAL
RESEARCH BRIEF
The Use of Molecular and Genomic Techniques Applied to Microbial Diversity,
Community Structure, and Activities at DNAPL and Metal Contaminated Sites
Ann Azadpour-Keeley1*, Michael J. Barcelona2, Kathleen Duncan3, and Joseph M. Suflita4
Abstract
The myriad of in situ subsurface remediation tech-
nologies currently in practice result in responses by
indigenous or introduced microbial communities that
can be measured with respect to alterations in biomass,
structure, diversity, enzymatic activity, or consequent
stress. Providing a highly developed understanding
of subsurface ecologies has shown great promise,
through the use of molecular and genomictechniques,
in providing new approaches to soil and ground-water
investigations by reducing the inherent parameter
variability of more traditional approaches in bench
and pilot scale investigations as well as full scale
applications. In addition to providing a background on
classic molecular and genomic sciences, the results
and interpretation of their application to field-scale
subsurface remediation activities is discussed.
Background
A wide variety of in situ subsurface remediation strategies
have been developed to mitigate contamination by chlori-
nated solventdense non-aqueous phase liquids (DNAPLS)
and metals. Geochemical methods include: zerovalent
iron emplacement, various electrolytic applications, elec-
trosmotic mobilization, as well as the addition of various
oxidants, orreductants, chelating agents, and surfactants.
Physical methods (primarily for the chlorinated solvents)
include in situ heating by various methods, steam injec-
tion/vacuum extraction, and gas sparging. Bioremediation
1' Corresponding Author: U.S. EPA, Office of Research and
Development, National Risk Management Research Laboratory,
Subsurface Protection and Remediation Division, Ada, Oklahoma,
74820, USA. Phone: 580-436-8890; Fax: 580-436-8703;
E-mail: keeley.ann&epa.gov
2 National Research Council & Department of Chemistry, Western
Michigan University, Kalamazoo, Ml 49002
3'4 Department of Botany and Microbiology, University of Oklahoma,
Norman OK 73019
methods applied to these classes of contaminants can
be broadly categorized as active (enhanced) or passive
(monitored natural attenuation; MNA) approaches. In the
former, additions of carbon substrates including organic
esters, acids, mulch, emulsified oils, orfats are intended to
stimulate the growth of microorganisms capable of degrad-
ing chlorinated solvents orcausing immobilization of metals,
via reduction or removal as insoluble precipitates. Also,
active approaches may be supplemented by the addition
of cultured microorganisms capable of carrying out deg-
radation or immobilization under selected conditions. This
approach is called bioaugmentation.
Regardless of the basis forthe remedial action, it is critical
to recognize that subsurface microbial communities will
respond to both the presence of the contaminants or
the engineered manipulation of subsurface conditions.
Responses of the microbiota may include stress orchanges
in diversity, community structure, biomass, and activity
among others. Their response may have profound effects on
remedial progress, effective time-frames to reach regulatory
decision points, and consequences forthe environment.
Therefore, it is quite important for subsurface scientists,
engineers, regulatory officials, policy makers and the
general public to recognize the roles that microorgan-
isms play during in situ remedial efforts and the value of
subsurface ecology to soil and water resources quality. It
is also important that we employ and improve on modern
methods to measure microbial responses to contamina-
tion and remediation strategies so that subsurface ecology
may be better understood. The molecular and genomic
techniques to identify and track the response of microbial
ecosystems to human influences hold great promise in our
efforts to protect the environment.
One should keep in mind that the inherent variability in sub-
surface hydrogeology, flow phenomena and geochemical
parameters are of the order of ±20% (combined sampling
and analytical error). Measured contaminant distributions
can easily vary as much as ±140% over the time frame of
initial site characterization or tracer release (Keeley and
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Barcelona, 2006). Therefore we should carefully consider
how the measures of microbial responses to the presence
of contaminants might be expected to vary considerably
overtime and space.
The purpose of this document is to provide an overview of
the molecularand genetictechniques applicable in ground-
water investigations and to present examples of the use of
these techniques to draw site-specific inferences.
Overview of Molecular and Genomic Analysis
Methods
Classical techniques to identify, measure, or estimate
aspects of soil and subsurface microbial ecology (i.e., char-
acterizing strains, active biomass, diversity, etc.) normally
involved isolation followed by culturing the organisms. These
methods are inadequate for complex soil orsediment envi-
ronments, particularly when they are contaminated. Viable
amounts of bacteria in environmental samples determined
with classical culturing methods represent only a small
fraction (0.1 to 10%) of the active microbial community
(Amannetal., 1995; White etal., 1997). In the last twenty
years, improvements in instrumentation, more selective
and specific methods, as well as advanced statistical and
mathematical interpretational tools have been developed.
This progress has given subsurface scientists exciting
opportunities to improve our understanding of indigenous
microorganisms and their impact on the environment.
It should be clear that a major challenge in creating or
utilizing an environmental diagnostic tool is to target
and identify relevant biomarkers to provide the desired
resolution (i.e., sensitivity and specificity). A biomarker
however can be defined as any molecule that is specific
to the microorganism(s) or the process of interest. A list of
molecular and genomic tools is provided in Table 1. Most
techniques target nucleicacids; howeverlipids and proteins
are othertargets with promising expansion in environmental
settings (Table 1).
Table 1. Selected Molecular and Genomic Techniques Applied at Hazardous Waste Sites
Molecular
Lipids PLFA
Contaminants CSIA
Protpins Dehydrogenase
Proteins Enzyme Assay
Sensitive and quantitative approach with available
database support; knowledge of process-specific
biomarkers and sophisticated and expensive equipment
required. * (y), ** (y), ***(y)
In situ monitoring tool, analyzes naturally occurring
isotope ratios (C, N, H, and O) of dissolved contaminants,
sophisticated and expensive equipment required. * (y),
** ^ ***(y)
Quantitative and inexpensive approach, time-consuming,
useful for anaerobic soil and sediment metabolic
monitoring. * (y), ** (y), ***(y)
Chang etal. (2001)
Appl. Environ. Microbiol.
67:3149-3160.
Meckenstock et al.
(2004) J. Contam. Hydrol.
75:215-255.
Skujins & McLaren
(1 967) Science
22(1 58):1 569-1 570.
Genomic
16S/fg Direct PCR
16S/fg Nested PCR
16S/fg DGGE
Qualitative conventional approach, provide consistent
information. * (n), ** (y), ***(y)
To enhance sensitivity, two successive PCR
amplifications are made; an internal primer set is
required. * (n), ** (y), ***(y)
High resolution with group-specific primers, separates
PCR amplicons due to their melting behavior in
polyacrylamide gels, only useful for targeting a small
region within a target gene. * (n), ** (y), ***(y)
Major et al. (2002) Environ.
Sci. Technol. 36:5106-5116.
Lendvay et al. (2003)
Environ. Sci. Technol.
37:1422-1431.
Muyzer & Smalla (1998)
Antonie van Leeuwenhoek
73:127-141.
-------
16S/fg
PCR/Cloning/
RFLP
Lengthy and time-consuming because of cloning
(Transfer of a gene of interest into E. coli as a foreign
host. * (n), ** (n), ***(y)
Macbeth et al. (2004)
Appl. Environ. Microbiol.
70:7329-7341.
16S/fg
PCR/Cloning/
Sequencing
Most widely used technique, lengthy and time-consuming Chandler et al. (1997)
because of cloning (Transfer of a gene of interest into E. FEMS Microbiol. Rev.
coli as a foreign host. * (n), ** (n), ***(y) 20:217-230.
16S
T-RFLP
Easy to perform, widely used, low resolution community
fingerprints (16S rRNA gene) tool that only analyzes the
terminal fragments generated in the restriction digests of
PCR-amplified target gene(s). * (n), ** (n), ***(y)
Tiedje et al. (1999) Appl.
SoilEcol. 13(2): 109-122.
16S/fg
RTmPCR
Quantitative technique for bioremediation monitoring
in real time based on various detection chemistries
including Taqman probes and SYBR Green. * (y), ** (y),
***(y)
Lendvay et al. (2003)
Environ. Sci. Technol.
37:1422-1431.
Quantitative fingerprinting of all microbial ribotypes in a
C0mmunity. * (Y), ** (Y), ***(n)
Yu et al. (2005) Appl.
Environ. Microbiol.
71:1433-1444
Provides sensitive detection of transcripts since RT-PCR
fg mRNA RT-RTm PCR quantifies the initial amount of starting template (specific
mRNA) used in a PCR reaction. * (y), ** (y), ***(n)
Johnson et al. (2005)
Appl. Environ. Microbiol.
71:3866-3871.
16S
FISH
Targets 16S rRNA gene(s); excellent tool but perhaps
difficult to apply in bioremediation. Variations of this
tool: microautoradiography (MAR-), substrate tracking
autoradiography (STAR), Microautoradiography
(MICRO)-FISH combine 14C autoradiography to identify
active cells. Catalyzed Reporter Deposition (CARD)-
FISH can increase the sensitivity and allow the detection
of functional genes and mRNA. * (y), ** (y), ***(n)
(1)Del_ongetal.(1999)
Appl. Environ. Microbiol.
65(12): 5554-5563.
(2) Ouverney & Fuhrman
(1999) Appl. Environ.
Microbiol. 65:1746-1752.
(3) Bakermans & Madsen
(2002) J. Microbiol. Meth.
50:75-84.
16S/fg
In situ PCR
The target gene is PCR-amplified inside the cell followed
by FISH detection; technique is technically challenging
and must be optimized for different organisms. * (y),
** (y), ***(n)
Tani et al. (2002) Appl.
Environ. Microbiol.
68:412-416.
16S/fg
This excellent research tool is a high throughput
technology which allows the parallel screening of large
Microarrays numbers of genes. Results are non-quantitative and
(PhyloChip its use for environmental samples presents challenges
Functional in terms of specificity, sensitivity, and quantification.
Gene Array) Sensitivity can be satisfactory if combined with PCR but
results require cautious interpretation. * (y/n), ** (y/n),
***(n)
Wagner et al. (2006)
Microbial Ecol. 53:498-506.
Note: Quantitative: *
Potential Application in Bioremediation: **
Applied in Bioremediation: ***
(y):Yes; (n):No
16S: 16S rRNA; fg: Functional Gene
Abbreviations: CSIA, Compound-Specific Stable Isotope Analysis; DGGE, Denaturing Gradient Gel Electrophoresis; Direct PCR, Direct
Polymerase Chain Reaction; FISH, Fluorescent In Situ Hybridization; MAR-FISH, Microautoradiography Fluorescent In Situ Hybridization;
PhyloChip, DNA microarrays; PLFA, Phospholipids Fatty Acid; RFLP, Restriction Length Polymorphism; RTm PCR, Real-Time PCR; RTm T-RFLP,
Real-Time PCR Terminal Restriction Fragment Length Polymorphism; T-RFLP, Terminal Restriction Fragment Length Polymorphism.
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Molecular Methods to Characterize Subsurface
Microbial Communities
Lipids
Lipidsmake up ~ 5 to 9% (drywt.) and the highest number
of molecules in bacterial cells (Fritsche, 1999; Madigan
et al., 2000). The phospholipids are the most abundant,
providing structure to the lipid bilayer of microbial cell
membranes (Ratledge and Wilkinson, 1988). The simplest
intact phospholipid (IPP) is phosphatidic acid:
iJiFTIfii fit
CH,OCOR1
R2COO-CH
CH2O-P-OH(orX)
Figure 1. Phosphatidic Acids
Where R1 and R2 are often fatty acid esters, or ethers (i.e.,
in Archaea) on the three-carbon glycerol backbone. The
X group can be a proton (as it is above), an alcohol, or vari-
ous alkyl, alkylamine, amino acid, (e.g., serine), or other
functional groups (Ratledge and Wilkinson, 1988). This part
of the molecule is often referred to as the polar head group.
Bacterial fatty acids are normally 12 to 24 carbons long.
They are called phospholipid-linked fatty acids or PLFA.
Often the phospholipids have one saturated fatty acid and
one unsaturated acid. Though the degree of unsaturation
may range up to six double bonds, polyunsaturated fatty
acids are found in only a few bacterial groups (e.g., cyano-
bacteria) as well as eukaryotes, fungi, plants, and animals.
The fatty acylchain may also contain branching as well as
cyclopropane rings. A brief example of the nomenclature
of PLFA compounds should prove helpful. Ex. 18:1w9cis
a PLFA with eighteen carbons, 1 double bond between the
9 and 10 carbons from the methyl end of the molecule in
the cis configuration. Branching and the incorporation of
hydroxyl groups into the fatty acid chain call for a somewhat
more complicated notation system.
It should also be noted that enzymatic hydrolysis of mem-
brane phospholipids to diglycerides may not be immediate
upon cell death particularly for those with alkyl ether side
chains (Harvey et al., 1986). PLFA or IPP determinations
require extraction of the lipid fractions from the bacteria
(i.e., neutral lipids, glycolipids, and phospholipids) with
organic solvents and further separation into three fractions
including neutral or polar lipids and glycolipids.
For IPP, the fractions may be separated by liquid chromatog-
raphyfollowed by electrospray ionization massspectrometry
LC/ESI/MS (Fang and Barcelona, 1998); or matrix assisted
laserdesorption/ionization time of flight massspectrometry,
MALDI-TOFMS (Ishida et al., 2002). There is significantly
more useful information for microbial characterization in
the intact phospholipids, but the tradeoffs are increased
analysis time and more complex instrumentation.
In the case of PLFA, the lipid fractions are treated by
saponification to separate the fatty acids and the backbone,
methyl ester formation, extraction of the fatty acid methyl
esters (FAMES), followed by analysis by gas chromatog-
raphy (GC) or gas chromatography/mass spectrometry
(GC-MS). Commercial kits are available to expedite FAME
determinations (Instant FAME, www.midi-inc.com).
Interpretations of IPP or FAME data may vary as a number
of variables may affect the total PLFA and the nature of the
fatty acids. Among the variables are: culture conditions,
carbon substrate, physiological or nutritional stress and
growth phase.
With these potential complicating factors in mind, it is pos-
sible to estimate non-viable vs. viable biomass by deter-
mination of the diglyceride fatty acids and phospholipid
fatty acids, respectively (Piotrowska-Seget and Mrozik,
2003). Forviable biomass we can convert the phospholipid
(ug/g sediment) to cells/ug PLFA using conversion factors
5.47 x 107 cells/ug PLFA from Balkwill et al. (1988), but see
White et al. (1997) for an overview of some of the difficulties
with making such conversions.
PLFA or IPP have been employed as biomarkers for dif-
ferent genera, species or microbial groups for over twenty
years. For example, if ethanolamine and glycerol are
predominant head groups and unsaturated this indicates
that the IPP were from bacteria. Signature lipid biomarker
analysis may be summarized (modified from Piotrowska
Seget and Miozik (2003)) in Figure 2.
Alkyletherlipids
(Archaea)
Neutral Lipids
Diglycerides
(non-viable cells)
Lipid
Glycolipid
Polyhydroxy Alkanoates
(Stress or unbalanced)
Growth
Lipopolysaccharides
Hydroxy Fatty Acids
(Gram negative bacteria)
Polar Lipids
(viable biomass)
Community structure
Metabolic status
Figure 2. Signature Lipid Biomarker Analysis
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Some characteristic biomarkers for microbial groups are
shown in Table 2, which would enable one to examine
community structure.
Table 2. Characteristic Biomarkers for Microbial Groups
Microeukaryotes
Aerobic prokaryote
Gram-positive bacteria
Anaerobic bacteria
Sulfate-reducing
bacteria
Gram-negative
bacteria
Actinomycetes
Cyanobacteria
Fungi
Polyunsaturated
Monounsaturated
Saturated and branched
c16-c19
Saturated and branched
c16-c19
Saturated and branched
c16-c19
Cyclopropyl
Methyl branched (C10)
Polyunsaturated
C24-C26 unsubstituted,
linoleicacid
Microbial community response to metal (i.e., Cd, Cu, Ni,
Pb, Zn) exposure in soils was tracked by Frostegard et al.
(1996), who noted an increase in methyl branched fatty acids
which reflected that increased actinomyceteswere present.
Fang et al. (2000a; 2000b) examined IPP in five strains of
Pseudomonas noting significant changes in phospholipid
content and composition as an adaptive response to toluene
exposure. The results suggested that dissimilar bacteria
utilized different mechanisms to adapt to the presence of
the compound. They also showed that IPP was superior
to FAME analysis to distinguish the individual strains using
principal component analysis. However, the identification
of all microbial species in a sediment sample with IPP or
PLFA would not be possible due to the overlap of the lipids
among species (White and Ringelberg, 1990).
Catalytic Proteins
Enzyme molecules are specialized proteins that catalyze
nearly every biological reaction. These molecules are also
highly specific in their catalytic ability in that each enzyme
catalyzes only a single or a set of closely related reactions.
Although enzymatic reactions are investigated for a variety
of reasons, molecular microbiologists are often concerned
in the detection of enzymatic activity as a function of various
biological parameters. In a search which might be use-
ful for comparison and prediction of soil microbiological
activities, where they are associated with certain horizons
and change significantly in their vertical distributions, the
dehydrogenase activity emerges as a useful criterion for
the characterization of soil biological state (Skujins and
McLaren, 1967; Skujins, 1973). The effectiveness of the
dehydrogenase measurement as a general index of meta-
bolic rate(s) in subsurface microbial communities is due
to their abundance among the sulfate-reducing bacteria
(SRB), a consortium responsible for bioattenuation of
contaminants under anaerobic conditions. The index is
particularly useful in the augmented subsurface settings
(i.e., PRBs) as shown by (Benner et al., 1999) and can
be interpreted as endogenous respiration in the intrinsic
environments (Ladd, 1978).
Nucleic Acid and Genomic Methods
to Characterize Subsurface Microbial
Communities
With the accelerated developments in sequencing capa-
bilities, database expansion, bioinformatics, environmental
genomics, transcriptomics, metabolomics, and proteomics;
there exists a broad range of genomic tools to evaluate
uncultured (i.e., not-yet-cultured) microorganisms. These
tools, which have originally been developed for use in the
medical fields, can now serve microbial ecologists with
detection of specific microorganisms or characteriza-
tion of an entire microbial community involving complex
interactions of all community members. Environmental
microbiologists are also equipped with an array of tools for
detecting process-specific biomarkers and tracing genes
responsible for degradation of contaminants (Lb'ffler and
Ritalahti, 2001). Probing the catabolic activity of the key
microorganisms responsible for detoxification pathways is
an ideal indicator for evaluating bioremediation progress
and setting time frames for cleanup goals. In fact, the util-
ity of the genomic biomarkers provides opportunities to
advance bioremediation from a largely empirical practice
into a predictable science (Lovley, 2003).
As may be observed in Table 1, the genomic tools include
RNA/ribosomal DMA detection methods, quantitative PCR
(i.e., polymerase chain reaction) amplification catalyzed
reporter deposition, fluorescence in situ hybridization
(FISH) (Cottrell and Kirchman, 2004) among other gene
probing techniques.
It is generally believed that DMA provides evidence for the
presence/absence of microorganisms and metabolic poten-
tial within the environment; mRNA provides specific activity
and expression of metabolic processes or functions; and
rRNA is an indicator of cell activity and viability (Olsen and
Tsai, 1992; Stahl, 1997). Therefore, the genetic methods
depend upon their ability to directly and representatively
isolate DMA, mRNA, and rRNA, from contaminated sedi-
ment, soil, orwatersamples. At one time, this was accom-
plished by culturing organisms in the laboratory. However,
as described earlier, the utility of cultural techniques in the
community characterization should be considered as less
than 10% of the microorganisms present in environmental
samples which grow in culture media (White et al., 1997;
Torsvik, et al., 1990) leading to a gross misrepresentation.
A detailed work plan to define sampling and sample han-
dling procedures should be in place priorto the selection of
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genomictoolsto ensure that the integrity of the biomarker(s)
of interest is maintained. These strategies should address
spatial and temporal sampling locations, sampling proce-
dures, collection of solid versus liquid samples, sample
transportation and storage, and sample processing.
As most environmental contaminants are typically located in
saturated zones, more modern methods to recover nucleic
acids are being developed to target solid matrices (i.e.,
soil, sediment, aquifer material). These techniques are
normally a succession of isolation and purification steps.
An example of such a scheme is shown in Figure 3 after
the work of Ogram et al., 1987, who developed a whole
sediment method. Previous methods utilized homogeniza-
tion and fractional centrifugations. The purification scheme
is self-explanatory.
In cases where little organic matter is available, dialysis
may be employed to remove potential interference from
inorganic material (Ogram et al., 1995). The less robust
nucleicacid groups mRNA and RNAare accordingly isolated
under less strenuous conditions (Stapleton et al., 1998).
More difficulty may be encountered in the preparation of
sufficient amounts of genomic DMA from very small, low
biomass samples as encountered in the subsurface, or
where the efficiency of DMA isolation is limited. The recent
robust tools to overcome some of these difficulties include
commercially available kits to isolate microbial DMA from
soil and other solid substrates. Once the nucleic acid
fraction has been isolated and purified, amplification by
PCR to develop gene probes and hybridization reactions
to genetically match the probes may be performed (Steffen
and Atlas, 1991; Innis and Gelfend, 1990).
xamples o.
Sediment Sample
Sediment Sample
5 g Sediment
13.5 ml DMA Extraction Buffer
100nMTris-HCI(pH8),
100 nM Sodium Phosphate,
1.5MNaCI,
1% Hexadecyltrimethyl Ammonium
Bromide (CTAB),
100 ml Proteinase K (10 mg/ml)
[30 minutes shake @ 37°C]
(Zhou etal., 1996)
1 .5 ml, 20% Sodium Dodecyl Sulfate
[Incubate 2 hrs @ 65°C]
Centrifugations
,Q, ^ Sediments
Supernatant Re-extracted 2x
24:1 (v/v) CHCI3 (Isoamylalcohol)
Centrifugation
[1 6,000 g, 20 minutes]
Crude DMA Pellet
Wash Cold
[70% Ethanol]
Purified DMA
Enzymatic Analyses
PCR or Restriction Digestion
100g Sediment
200 ml 0.12 M Sodium Phosphate,
2.5 g Sodium Dodecyl Sulfate
[1 hour @ 70°C]
(Ogram etal., 1987)
Sediment
Residue
Ballistic Disintegration in Beadmill
>^1:1 (v/w) beads to sediment
.„ ,. ._ . .. ,_ . ., ..
Alkaline Extractions/Centnfugations
Aqueous Phase
Alcohol Precipitation
Concentrated Nucleic Acids,
Protein and Cell Residue
Residue
Slot Blot
Hybridization
CHCI3,
Phenol Extractions,
Alcohol Precipitation
J>
Crude DMA Pellet
tf ^
Purified
DMA
CsCI2/ Ethidium Bromide
Ultracentrifugations or
Hydroxyapatite Chromatography
Enzymatic Analyses
PCR or Restriction Digestion
Figure 3. Example of DMA Extraction Scheme
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PCR, employing athermostable DNA polymerase, is capable
of amplification of millions of copies of a specific DNA
fragment that has been identified as the target sequence,
Amplification is guided by a primer sequence, usually 10
to 25 bases in length; which generates a 3' hydroxyl group
facing the target sequence. Successive heating and cool-
ing in the PCR reaction give the technique its amplification
power but reagent concentrations, temperatures and cycle
time are quite important (Innis and Gelfand, 1990).
Primers have been developed fora variety of genes including
the universal 16S rDNAoligonucleotide (Stahl etal., 1988)
that is useful in obtaining biomass estimates and normalizing
specific genotypes as a percentage of the total population.
Its use allows one to minimize variability in sample types or
extractions and provides a basis of comparison fordifferent
sites, contaminant mixtures, or laboratories.
Catabolic gene probe values can be normalized using
the 16S rDNA gene (total community value) to compare
samples over space and time. The underlying assump-
tions are that there are seven copies of the 16S rRNA
oligonucleotide per chromosome (Smith et al., 1987),
however, the actual number can vary from one to fifteen,
depending on the organism (Klappenbach etal., 2001). For
example, in a study of accelerated anaerobic degradation
in a mixed chlorinated and aromatic hydrocarbon plume
at Dover Air Force Base, biomass estimates ranged from
8.78 x 106 organisms/g sediment (uncontaminated) to
2.11 x 109 + 2.27 x KForganisms g/sediment (center of
anaerobic area) (Stapleton et al., 1998). In the same study,
catabolic gene probe data showed increased biomass of
organisms possessing selected degradative enzymes.
The results developed with gene probes should be inter-
preted carefully as some DNA probes are susceptible to
cross-hybridization with othergenes and one may be misled
as to the identity of the organisms responsible for degrada-
tion. Another caveat is that the "copy number" of 1 for the
gene probes and 7 for 16S rDNA may vary substantially
in different species of soil bacteria (Farelly et al., 1995).
The genomic tools that are applied after the completion of
the isolation processes are broadly classified to present
qualitative or quantitative approaches that can be used
to the target biomarker(s). The 16S rRNA gene
is the most extensively used target to qualitatively evalu-
ate the presence of a specific group of organisms (Pace
etal., 1986; Stahl, 1997). As a common practice, various
techniques such as direct and nested PCR, T-RFLP, DGGE
and others are routinely used to determine the presence
of a specific 16S rRNA gene from phylogenically defined
microbial groups. The utility of these biomarkers is particu-
larly valuable if a link between 16S rRNA gene sequences
and desired activity can be confidently established (Loftier
and Ritalahti, 2001). Although there are indications that
phylogeny and phenotype may not be representative of
the cell's metabolic potentials (Amann and Ludwig, 2000),
the application of 16S rRNA gene technologies has been
proven to be successful in bioremediation in linking the
presence of specific microbial groups with contaminant
removal (Major et al., 2002; Gu et al., 2002; Lendvay et al.,
2003; Anderson et al., 2003).
Another qualitative approach is to target the "functional
genes" by tracking the process-specific biomarkers rather
than the 16S rRNA genes. Only the functional genes for
which a direct link with the process of interest has been
recognized may be targeted by PCR amplification using
primers for the functional gene of interest. For example,
DGGE may be used to further separate DNA strands by
differences in resistance to denaturation (e.g., more closely
related to GC content) and to isolate a gene that codes
for a specific catabolic enzyme, e.g., toluene dioxygenase
to probe microbial activity at a fuel contamination site. In
practice, to date, the utility of this approach has been very
limited mainly due to the insufficient knowledge to identify
the process-specificgenesformanydetoxification pathways
(Kolker etal.,2005).
As described earlier, qualitative measurements are informa-
tive, in particular when site characterization is desired and
in bioremediation when the process of interest is carried
out by phylogenically defined bacterial groups. However,
a promising tool for quantitative monitoring of 16S rRNA
and functional gene copy numbers is quantitative real-time
(RTm) PCR. During RTm PCR, an increase in fluorescent
light emission during target gene amplification serves as
a measure of the initial target concentrations. Therefore,
a large number of the target nucleic acid in the original
sample requires fewer cycles to accumulate amplification-
associated fluorescence to a specific threshold level of
detection (Ct value). Thetarget molecules arequantified by
extrapolation using linearstandard curvesforCt values (Held
etal.,1996;Mackay,2004). BothTaqMan and SYBRGreen
approaches are most commonly used in conjunction with
RTm PCR (Walker, 2002; Mackay, 2004); however, various
amplicon detection chemistries are also becoming available
commercially. The advantage of RTm PCR overtraditional
end point PCR may include quantitation, enhanced speed
and sensitivity, and lack of post-PCR steps (e.g., agarose
gel). The utilities of RTm PCR forthe quantitation of genes
of interest in laboratory cultures (He et al., 2003a,b), or in
environmental samples (Harms et al., 2003; Lendvay et al.,
2003) have been shown. Appropriate database support
is also currently available (i.e., GenBank, Ribosomal RNA
Operon Copy Number Database). However, it should be
noted that the accurate quantitation requires data on the
target gene copy number per genome. As more bacte-
rial genomes are increasingly sequenced and more data
on gene copy numbers are becoming available in public
databases,theuseofR Tm PCR will risesignificantly.
The qualitative and quantitative genomic tools described
thus far are intended to provide information on the pres-
ence of the target population orthe gene of interest. These
tools however cannot directly inform the user whether the
target organisms are alive or metabolically active. Another
approach to obtain livelihood oractivity is by detecting mRNA
transcripts. Various endpoint PCR or RTm PCR methods
may be used for measuring the abundance of transcripts in
samples, particularly those that may serve as biomarkers of
contaminant degradation including in situ (Bagasra et al.,
1994; Hodson etal., 1995;Tani et al., 1998,2002) and in situ
reverse transcriptase (Chen et al., 1997), and RNase pro-
tection assays (Sambrookand Russell, 2001). Among these
-------
techniques, RT PCR is particularly sensitive and promising for
measuring gene expression from the field samples (Freeman
et al,, 1999; Bustin, 2002). However, routine monitoring of
mRNA in environmental samples may be challenging since
mRNA molecules are less stable [short half-lives of transcripts,
e.g., between 3-18 minutes in E. coli (Bernstein et al., 2002)]
and prone to degradation in contrast to DNA. Other factors
contributing to this challenge include the lack of protocols for
extracting mRNA from soil and sediments, and difficulties in
detecting lowabundance transcripts. Manytranscripts includ-
ing those involved in contaminant degradation are present in
1 copy or fewer per cell (Velculescu etal., 1997). Therefore,
in comparing field samples that are collected over spatial and
temporal scales, it should be noted that different mRNAs have
different half-lives and the sampling and analysis work plan
should highlight the sample handling, extraction procedures,
and standardization overtime and space.
Proteomicsisthemostdirectapproachformonitoringmicrobial
activity. It obtains information by detecting enzymes (catalytic
proteins) that are involved in a specific process. The use of
proteomic mass spectrometry for microbial detection offers
distinct advantages over traditional approaches due to the
speed of analysis. High throughput proteomic technol ogies
are rapidly expanding (Fredrickson and Romine, 2005) and
the potential for combined genome and proteome to moni-
tor gene expression and activity from a biofilm community
from an acid mine site have been demonstrated (Tyson
et al., 2004; Ram et al., 2005). However, the application of
proteomi cs technologies for the detection of a key catabolic
enzyme by peptide mass fingerprinting (PMF) and peptide
sequencing (PS) (Halden et al., 2005) is currently limited in
bioremediation since a much more detailed understanding of
protein function as well as database supports are required for
routine monitoring practices.
Molecular Genomic
We have investigated the use of recent molecular and
genomictechniquesto characterize microbial communities
and activities to their effectiveness at chlorinated
and metals hazardous waste sites. Since currently there
is no single technique that can rapidly, sensitively, specifi-
cally, and cost effectively detect and characterize the entire
microbial communities in spatial and temporal scale, a
multifaceted approach described here was selected across
the contaminated sites to examine the utility of these tools.
Our comprehensive approach is further justifiable since
it is well known that contaminant concentrations in the
subsurface are often low (i.e., mg to ug per liter range).
If the contaminant acts as an electron acceptor or donor,
it is likely to support a non-substantial biomass of active
populations. This is particularly true if it is assumed that
no other substrate is available to support the growth of
the target population and only 50% of the DNA could be
recovered. Therefore, as reported by Gu et al. (2002),
the biomass estimates based on DNA could generally be
lower by several orders of magnitude than those based on
the PLFA. In the present study, we have employed PLFA
(FAMES) as indicators of biomass and community structure,
enzyme assays for monitoring microbial activity, as well as
more phylogenetically-specific DNA analyses for the pres-
ence of specific microbial groups.
A number of investigators have utilized PLFA determina-
tions to gauge microbial community responses to either
the presence of contaminants or remedial action (Hansen
et al., 2000; Pfiffner et al., 1997). Usually the fatty acid
methylesters (FAMES) have been the focus ratherthan the
intact phospholipids (IPP), to examine changes in viable
biomass, community structure, and metabolic activity.
A - Chlorinated Solvents
Experimental Methods
The aquifer material for vertical profiling of PLFA key
biomarkers at a TCE contaminated site in Florida was col-
lected using a Cone-Penetrometer (CPT) equipped with a
Mostap™ sampler (20-inch long with a 1.5-inch diameter).
The samplercontained three sterile sleeves (brass orstain-
steel) and one spacer. Each sleeve was 6 inches long
and held approximately 250 grams of soil.
The coring was accomplished by driving the sample barrel
to four different depths (7, 15, 30, and 40 ft) at each sam-
pling location. Once the sample barrel was withdrawn, the
three sleeves were extruded from the sample barrel. The
sleeves were tightly capped on both ends with plastic end
caps, sealed as quickly as possible, and labeled with the
percentage of recovery recorded and marked to designate
the top and bottom sections. The sleeves were immediately
frozen in liquid nitrogen (-150°C) and shipped overnight
(-70°C) to the laboratory for PLFA analysis.
At the laboratory, PLFA were analyzed by extraction of the
total lipid (White et al., 1979) and then separation of the
polar lipids by column chromatography. The polar lipid fatty
acids were derivatized to fatty acid methyl esters, which
were quantified using gas chromatography (Ringelberg
et al., 1989). Fatty acid structures were verified by chro-
matography/mass spectrometry and equivalent chain
length analysis.
In orderto minimize sample contamination, a strict aseptic
sampling procedure was adopted including sterilization of
brass or stainless sleeves with isopropanol (70%) bath
dipping (15 min), air drying at ambient temperature (~ 1 h),
and aluminum foil wrapping. Each three individually foil-
wrapped sleeves were placed in an autoclave bag. The
bag was placed in a heat-resistant plastic container, and
was autoclaved (121°C, 30 min). The container was tightly
capped, packed, and shipped to the field. Polyethylene
sleeve caps were not autoclaved but were surface rinsed
with isopropanol (70%) prior to use. Sterilization of drilling
equipment involved steam cleaning between samples. After
the samples were extruded, the sample barrel used to collect
the soil sample was disassembled and decontaminated in
Alconox® detergent mixed water. The sample barrel was
then rinsed with tap water, de-ionized water, and isopropanol
(70%) priorto complete air-drying (~ 1 h) and before it was
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used again. Air exposure of the subsurface was minimized
since core sampling was continued immediately after each
extraction using two sets of the Mostap™ samplers. The
sampler containing three cored sleeves was extracted,
switched with anothersterile one, and coring continued while
the sampling crew conducted aseptic sample processing
and decontamination procedures.
Results and Discussion
The microbial response, as measured by the fatty acid
profile, to three remediation technologies applied at a TCE
(trichloroethylene) contamination site at Cape Canaveral
Air Station was investigated. The technologies included:
In situ chemical oxidation (ISCO; KMnO4); six-phase heat-
ing (SPH), and steam injection. Sediment cores were
collected at four depth intervals before, during, and after
remedial operation. The conversion factor of Balkwill et al.
(1988) of 2 x106 cells/pmol of PLFA (FAMES) was used to
determine total biomass. We observed relatively low levels
of PLFA in control plot sediments ~ 9.7 to 18 picomoles/g
dry wt. (1.9 x 105to 3.6 x 105 + 1 x 105 cells/g) and the
lowest values were observed at the shallowest depth ~ 5m
from land surface. This is counter to what most workers
have noted where biomass (or its proxy, PLFA) is generally
higher near the surface due to plant and microbial release
of organic carbon in the root zone. It should be noted that
the organiccarbon level in these sandy sediments was quite
low. The types of FAMES are shown in Table 3, along with
theirphylogenic microbial association, and response trend.
It is evident from the data in the table that ISCO had the
greatest impact on biomass relative to the controls. The
increase was from ~2.92 x 104 to 1.85 x 108 cells/g and
was sharpest in the zone of initial KMnO4 injection. Five
months later in the treatment period, biomass generally
decreased. One explanation for the increase in biomass
may be that the permanganate may have reacted with
native organic matter and released lower molecular weight
compounds that served as a carbon source for microbial
growth. The impact on community structure was evident
for SPH and SI where biomarkers forthermophilic, Gram-
negative anaerobes and Gram-positive bacteria clearly
increased. ISCO resulted in the most significant influence
on community structure evident in a number of phylogenic
groups. It was most surprising that 12 months after the
treatments were ended, the biomass and community
structure in all three treatment plots returned to that of
the control plot. Even though the biomass was low, the
overall subsurface community structure reflected a mixed
anaerobic and aerobic community similar to that reported
by Ringelberg et al. (1997).
Table 3. Types of FAMES Observed, and Response
Indicator Phylogenic Association
HUHBSESEI
Terminally
Branched
C16
Monounsaturated
C18
Monounsaturated
Mid-Chain
Branched
18:Lw9c
Polyunsaturated
C13-C18 saturated
C20-C24 saturated
Branched
unsatu rates
Anaerobic Gram (-)
and Gram (+) + +
Thermophiles
Proteobacteria +++
Proteobacteria +++
Sulfate-reducers
Actinomycetes
Eukaryote (fungi) ++
Eukaryote (fungi) ++
Anaerobes
fungi
Metal- reducers
Biomass ++
B - Metals
We have used PLFA as indicators of biomass and commu-
nity structure to investigate microbiology of ground water
undergoing treatment with permeable reactive barriers
(PRB) at three metal contaminated sites. The PRBs were
selected on the basis of the reactive media that were used
in their construction including a 100% zero valent iron (ZVI)
barrierat North Carolina, a 50% ZVI:50% compost and pea
gravel barrier at South Carolina, and a 0% ZVI: 100% cow
manure and limestone barrierat Louisiana.
Sediment core samples were collected from upgradient,
upgradient/PRB interface, in the PRB, downgradient/PRB
interface, and downgradient during quarterly or annual
sampling. Each PRB was constructed in a trench stabi-
lized during construction by a guar gum slurry. This is a
common construction technique which ends up leaving a
fair amount of organic matter in the subsurface despite
application of the proprietary enzymatic degradation solu-
tion (Gu et al., 2002).
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Experimental Methods
Core samples were collected to provide information in defin-
ing the horizontal and vertical distribution of microbes at the
PRBs. The conventional coring techniques were adopted
using a Geoprobe™ to obtain continuous coring for the
entire depth of each aquifer. To minimize contamination,
after a core was obtained using strict aseptic techniques,
care was taken not to disturb or contaminate the sample.
Processing was performed as quickly as possible under
anaerobic and aseptic conditions while at the field. The
entire core sleeve (2-feet long with a 1-inch diameter) was
placed in an anaerobic field glove box. The glove box con-
tained an atmosphere of argon and was shielded from direct
sunlight. Each sleeve was labeled with the percentage of
recovery recorded prior to opening and visual inspection
for mineralogy. Surface layers of the core were scraped
away using a sterile sampling device and discarded so that
only the center of the core was placed in sterile thin-walled
plastic sample bags (Whirl-Pak®). The portion used for
DNA/PLFA analyses was rapidly frozen with liquid nitrogen
and stored on dry ice at -70°C. The samples were trans-
ported to the laboratory at -70°C and stored at the same
temperature prior to analysis.
Results and Discussion
One site at which a zero-valent iron permeable reactive
barrier (ZVI-PRB) was used was at the U.S. Coast Guard
Support Center at Elizabeth City, NC. This site had been a
hard-chrome plating shop more than 30 years. According
to Wilkin et al. (2002), chromate levels were in excess of
10 mg/L and TCE, c-DCE, and vinyl chloride were in the
ppb ranges.
The sediment cores collected at the Elizabeth City ZVI-
PRB which has been in place since 1996 were analyzed to
assess community structure and physiological status of the
bacteria. The analyses focused on the samples collected
in the midbarrier and in the zones of low and high corro-
sion particularly those at the upgradient interface of the
wall from two consecutive sampling events 2 years apart.
In general, the abundance of biomass in sediment cores
with high corrosion was one log factor higher than the
samples with low corrosion. The lowest biomass was
observed at an upgradient sandy location within the plume
(1.03 x 105 cells/g) while the highest biomass was at a
zone with the highest corrosion (5.09 x 106 cells/g). The
increased microbial biomass at the upgradient interface
of the PRB suggests a real potential for biofouling and
reduced hydraulic performance of zero-valent iron PRBs.
The PLFA profiles of the collective samples were pre-
dominantly consistent with those of proteobacteria with the
exception of the upgradientsamples in which proteobacteria
included 42% of their community structure; they had no
biomarkers indicating metal reducers, or sulfate-reducing
8-proteobacteria. The fatty acids suggest that the com-
munity structure at the upgradient/PRB interface, within
the PRB, and the downgradient/PRB interface consisted of
sulfate and metal reducers ranging from 2.06% to 14.34%
of the total community. Among these populations, metal
reducers were present in every sample regardless of the
detection of SRBs. However, zones of high corrosion were
represented by the increased percentage SRBs than metal
reducers. This could be attributed to the increase in pH
which seemed to offset the positive influence of an abun-
dance of hydrogen produced in the barrier.
The second permeable reactive barrierwas constructed at
a pilot scale to assess the efficacy of ZVI, leaf compost, and
limestone and pea gravel as the reactive material to mitigate
the principal contaminants that were arsenic (As), lead (Pb),
and acidic pH conditions from an upgradient phosphate
fertilizer manufacturing plant that had been decommissioned
in 1972 in South Carolina. The combination of reactive
materials was designed to promote microbiological sulfate
reduction and to increase pH of the ground water from an
acid producing to acid consuming condition.
The total microbial abundance based on the PLFA
was several orders of magnitude higher in the barrier
(106~8 cells/gram) than in the background sediment samples
(104"5 cells/gram). The increase in the biomass was also
positively correlated with depth. The highest biomass
(6.63 x 108 cells/gram) was observed in a sample from
11 ft bgs suggesting that the microbial community adapted
quickly to the strongly reducing conditions.
The increase in biomass continued over the next two con-
secutive sampling events which may reflect the adaptation
of the population to degradation products of the compost.
Mid-chain branched saturated PLFA structural groups
increased significantly overtime, especially compared to the
Elizabeth City ZVI, suggesting that SRBs, and to a lesser
extent metal reducers, were increasing at the expense of
the othergroups (i.e., Firmicutes, Proteobacteria, and non-
specific populations). The increase in branched PLFA in
the PRB, and the alteration of ground-water geochemistry,
including the reduction of sulfate, resulted in Gram-positive
anaerobic bacteria being a significant fraction of the overall
community. In contrast, Polyenoic FAMES composed the
most insignificant component of the total PLFA structural
groups in the wall suggesting that eukaryotes were not sup-
ported by the reactive media. In general, the polyenegics are
considered as a remnant of the guargum (Gu et al., 2002).
The third field scale PRB comprised of pasteurized cow
manure (50% by wt.) and limestone (50% by wt.) was
constructed during 2003 in Louisiana to mitigate lead
(Pb), cadmium (Cd), arsenic (As), and a low pH ground
water. Analysis of the PLFA profiles at this site showed
that the microbial community structures of the samples
varied considerably among the samples. Estimated viable
biomass, based on total PLFA concentrations ranged from
~105-7 cells/gram dry weight among the samples collected at
4 - 6 ft bgs with the majority of samples containing biomass
of approximately 107'9 cells/gram at the deeper horizons.
The majority of the samples had relatively diverse microbial
communities in which the primary members were Firmicutes,
which was >30% of the total PLFA. Firmicutes include
Bacteroides and C/osfr/cf/a-like fermenting bacteria. High
10
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proportions of Firmicutes suggest that conditions were
anaerobic at sampling locations within the barrier. Many
of the samples also contained relatively large proportions
of biomarkers for sulfate-reducing and metal-reducing
bacteria, which further supports the presence of highly
anaerobic conditions. In two-thirds of the samples the total
"anaerobic" biomarkers accounted for >48% of the total
PLFA. Methanogenesis is common in gut-dwelling archaea.
Even though the biomarkers for methanogenic archaea
may have some overlap with common actinobacteria, a
strong correlation between these biomarkers and portions
of the wall with significant methane production (as high as
50 mg/l) is suggestive of the presence of archaea PLFA.
The results from the organic based barrier in Louisiana
suggested that there was a drastic difference in the most
abundant population as compared to the Elizabeth City
ZVI-PRB as Firmicutes were dominant in the manure wall
but Proteobacteria were the most abundant group in the
zero valent iron wall. Proteobacteria not only comprised
a smaller proportion of the total population of the manure
wall as compared to the ZVI wall, the physiological status
of the Gram-negative Proteobacteria in the manure wall is
suggestive of a slowed growth rate and/or of decreased
permeability of their cell membrane. The second distinc-
tion between the community structures of the two barriers
is that the PLFA from the manure wall contained a high
percentage of normal saturated structural groups which
are found in all organisms as compared to the ZVI wall.
Summary
The biomass estimates based on FAME were generally
lower by several orders of magnitude from upgradient to
the vicinity and within the three PRBs. FAME indicators
of both the Gram-positive and Gram-negative-anaerobic
microorganisms increased from core samples near and
within the PRBs as evidenced by terminally branched and
monoenoic fatty acids.
The FAME extracts from in or near the iron-containing
PRBs showed a similar signal of increased representa-
tion of sulfate- and metal-reducing organisms extending
well downgradient from the PRB. The presence of metal
sulfides also supported the FAME analysis suggesting the
microbial reduction of sulfates in the geochemical environ-
ment of the iron PRBs.
It is important to note that methanogens did not constitute a
significant fraction ofthetotal biomass ofthe South Carolina
PRB. A similar result was also reported by Stapleton et al.
(1998). Since there is generally a relative abundance of H2
in the vicinity of ZVI-PRB produced by cathodic corrosion
ofthe iron, it may be expected that the methanogens would
predominate over the sulfate reducers. However, due to
the high levels of SO4= it is possible that the methanogens
may have been at a competitive disadvantage (Lovley and
Goodwin, 1988). Since the anaerobic microorganisms dis-
played a higher level of biomass, a reduction in the hydraulic
performance ofthe ZVI-PRB may not result from biofouling
which could be attributed to an accumulation of H2 The role
microorganisms play in the development of "green" rust,
ferrous hydroxides, etc. in ZVI-PRBs remains unknown.
Catalytic Proteins
The activity of certain enzymes in soil and sediment samples
reflects the metabolic rate of the microbial populations.
One ofthe types of enzymes most studied under anaerobic
conditions is dehydrogenases that are abundant in sulfate-
reducing bacteria (SRB). This investigation examined over
eighty samples derived from metal and chlorinated solvent
contaminated sediments for dehydrogenases activities.
The presence of SRBs among the tested samples was
confirmed prior to the enzyme assay.
Experimental Methods
Triphenyl tetrazolium (TTC) chloride is a substrate for a
numberof non-specificdehydrogenases present in microbial
communities and can be generally correlated with respiratory
activity and used as an index of microbial activity (Ladd,
1978). In orderto determine dehydrogenase activity (DH),
5 grams of core sample were placed in an anaerobic glove
box. The samples then were buffered with CaCO3 (to a
pH >6), and 1.75 ml of 0.5% 2,3,5-triphenyl tetrazolium
chloride and distilled water were added to each sediment
sample. The samples were incubated for 24 hrs in an
anaerobic glove box and were transferred to a chemical
hood and extracted sequentially with two 10 ml aliquots
of methanol and filtered through Whatman no. 42 paper.
The aliquots were then combined in a volumetric flask and
made up to 50 ml with methanol. Optical densities ofthe
extract were measured at 485 urn. The enzyme activities
were performed in triplicate and averaged. Controls were
set up using samples with distilled water without TTC.
Since the results are interpreted in terms of enzyme
activity rather than microbial number, a standard curve
was prepared using TTF in methanol in the range of 0 to
1 mg/100 ml. The optical density was linear up to a con-
centration of 150 mMTTF/ml of methanol. Using this curve,
the amount of TTF formed in each sample was converted
into ul H and the results were expressed as umoles H/g of
core sample/hr (Johnson and Curl, 1972).
Results and Discussion
We have used dehydrogenase measurement as an index of
metabolic activity at a metal contaminated site in Louisiana
and at a chlorinated solvents site in Florida. While the
ground-water remedy at the Louisiana site included a
permeable reactive barrier constructed from cow manure
and limestone, as discussed earlier, the Cape Canaveral
project in Florida was designed to field-test and compare
three technologies forthe re mediation of ground watercon-
taminated with TCE. The technologies included Six-Phase
Heating (SPH), Steam Injection (SI), and In Situ Chemical
Oxidation (ISCO). A critical aspect ofthe project was to
determine the effects ofthe remediation activities on the
indigenous microbial populations which are relied upon for
the final polishing phase ofthe treatment train.
The results indicated that dehydrogenase activity in the
sediment samples was not always correlated to the microbial
number. However, in amended environmental samples such
as the PRB barrier samples, the enzyme activity increased
11
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with increased microbial populations. It was also deter-
mined that air drying the core samples resulted in a 30 to
50 percent loss of dehydrogenase activity while storage of
wet sediment at -80°C was found to be most satisfactory
for retaining dehydrogenase activity. Therefore, the assays
were performed on fresh core sediments that were frozen
immediately upon extraction in the field.
The Louisiana PRB was constructed in two phases; the first
portion was emplaced in the aquiferforthetreatability study
which is now termed "the matured wall" with an extension to
the wall completed one year later in May 2003, at the full-
scale which is now termed "the new wall". Data show that
the enzymatic activities were significantly higheramong the
bacterial populations of the newly installed permeable reac-
tive barrier as compared to that of the more mature wall. The
results indicated that the vertical average enzyme activity of
core samples in the old wall rose from 0.547 |jmoles H/g/hr
in December 2004, to 0.677 jjmoles H/g/hr in April 2006
(16 months). In the newer extension, the average activ-
ity rose from 0.788 umoles H/g/hr in December 2004 to
2.075 umoles H/g/h in February 2005 (3 months). These
differences in enzyme activity are also reflected by the
appearance of the cores upon extraction. While in the older
wall the more active depths rendered brown cores, those
from the more active depths in the newer wall were black
indicating more reduced conditions. In either event, these
results are indicative of the ample carbon compounds or
H2 that are made bioavailable from the reactive material in
the more recently installed wall.
Although all of the core samples suggest that the highest
enzyme activity occurred in the deeper zones, it is clear
that enzyme activity in the new construction exceeded
that of the older wall. For example, representative cores
(April, 2006) show that activity from 5-9 feet in the old wall
was 0.329 umoles H/g/hr and was 1.024 [jmoles H/g/hr
in the 10-12 foot core. In the newer wall, however, the
activity was 0.659 jjmoles H/g/hr in the 5-9 foot core and
6.240 [jmoles H/g/hr in the 11 -12 foot core.
When analyzing cores from the Cape Canaveral project
from the control site as well as the ISCO treatment plot
before initiation of the remediation, it was determined that
the enzyme activity increased linearly with depth, as in the
other treatment plots. The cores above 17 feet in depth
varied between 0.556 and 0.767 (jmoles H/g/hr while the
activity increased to 4.682 [jnnoles H/g/hr at 31 feet and
5.796 Mmoles H/g/hr at 41 feet.
Cores fromthe first post treatment sampling event(0 month
after ISCO termination) showed that in the ISCO plot
the activity had actually increased over the background
(3.178 versus 0.556 (jmoles H/g/hr) at 17 feet bgs, but was
below background (3.465 versus 4.682 jjmoles H/g/hr) at
31 feet bgs. IntheSPH plot cores fromthefirstposttreatment
sampling event demonstrated that the enzyme activity still
increased with depth (0.138 at 7 ft, 1.984 at 27 ft, and 4.230
at 41 ft) but varied between 60-70 percent of background.
One second post treatment sampling (6 months after ISCO
termination) from the SPH plot shows the enzyme activity
at 8 feet bgs was only 0.003 umoles H/g/hr.
Summary
The dehydrogenase assay used in this research for the
measurement of enzyme activity underanaerobicconditions
is based on the principle that when metabolizing cells come
in contact with an aqueous solution of TTC, it is converted
intotriphenlyformazon (TTF) which can then be measured
colorimetrically. Regardless of the remedial options used,
ISCO, SPH, or permeable reactive barriers, the results
indicated that there was an increased bacterial activity as
measured by the elevated enzyme levels particularly in
response to enhanced electron acceptors/donors present in
the aquifers. Therefore, it can be concluded that this assay
can be confidently used as a sensitive index for bacterial
activity at various contaminated sites.
Genomic
Experimental Methods
DNA Extraction
Various DNA extraction protocols were evaluated on sam-
ples from two permeable reactive sites: the North Carolina
ZVI-PRB, and the Louisiana cow manure-PRB. All proto-
cols utilized a bead-beating method to lyse bacterial cells
and release DNA (Mini Bead-Beater, BioSpec Products,
Bartlesville, OK, Borneman etal., 1996; Duncan et al., 2003).
Phenol-chloroform extraction, followed by ethanol precipita-
tion (Rios-Hernandez et al., 2003), and four commercially
available kits were compared, following the manufacturers'
protocols: FastDNASpin KitforSoil(MPBiomedicals, Irvine,
CA, Duncan etal., 2003), Power Soil DNA Isolation Kit (Mo
Bio Laboratories Inc, Carlsbad, CA), UltraClean Soil DNA
Kit (Mo Bio Laboratories), and UltraClean Mega Soil DNA
Kit (Mo Bio Laboratories). Sample weights of 0.25, 0.5,
2, and 5 g were compared. Bead-beating rates of 25,000
and 36,000 rpm and beating times of 60 seconds and 90
seconds were compared for the FastDNA Spin Kit for Soil.
DNA fragment size was examined and the concentration
verified by agarose gel electrophoresis of an aliquot of the
DNA sample and viewing the Sybr®Safe (Invitrogen Inc.,
Carlsbad, CA)-stained gel under UVtransillumination.
PCR, Cloning, and DGGE
Primers were synthesized by Invitrogen Corp. (Carlsbad,
CA). Other reagents and enzymes were purchased from
Fisher Scientific or Sigma-Aldrich.
Eubacteria
1500 bp fragment: Nearly full length eubacterial 16S rDNA
(Escherichia coll positions 8-1492) forcloning was obtained
from DNA purified from cells by amplification with "univer-
sal" eubacterial primers targeting conserved regions and
the cycling conditions described in Herrick et al. (1993).
The PCR products were cloned into the TOPO-TA vector
(Invitrogen Inc., Carlsbad, CA) following the manufacturer's
recommendations to increase the numberoftransformants
when cloning from a pool of sequences. The inserted
fragment was amplified from randomly selected single
white colonies using primers complementary to the M13
12
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sequences present in theTOPO-TA vector, and then puri-
fied forsequencing as described below (DNA sequencing).
550 bp fragment (nested): Primers GM5F and D907R
(Muyzer et a!., 1993) were used to amplify a 500-bp
region of the 1500 bp PCR template above for denaturing
gradient gel electrophoresis (DGGE) analysis of differ-
ences in sequence diversity among the samples. DGGE
was performed using a D-Code 16/16-cm gel apparatus
(BioRad, Hercules, CA), at a constant voltage of 65 V for
16 hours and a constant temperature of 60°C (Muyzer et
al., 1993). The gradient was formed of 6% polyacrylamide
in TAE buffer with between 40 and 60% denaturant (7M
urea and 40% formamide is defined as 100% denaturant).
After electrophoresis, the gel was stained in a solution
of Sybr®Safe and a permanent image was captured by
the NucleoCam Digital Image Documentation System
(NucleoTech Corp., San Mateo, CA). Selected bands were
cut from the gel and incubated in PCR-grade water, then
used as template DNA in subsequent PCR reactions, as
previously described (Rios-Hernandez et al., 2003). The
PCR product was prepared for sequencing as described
below (DNA sequencing).
Archaea
600bpfragment:16S rRNA PCR products of approximately
600 bp in size were obtained for cloning by amplification with
the archaeal primers ARC333GC and 958R (Reysenbach
and Pace, 1995; Watanabe et al., 2002). Amplification
followed the touch-down procedure described in Muyzer
et al. (1993). Cloning and subsequent preparation for
DNA sequencing followed the procedure described for the
eubacterial 1500 bp fragment.
220 bp fragment (nested): A "nested" PCR reaction was
performed using archaeal 16S primers ARC20 and 958R
forthefirstamplification, and primers ARC333 (Reysenbach
and Pace, 1995) and P2 (Muyzer etal., 1993) for the sec-
ond PCR reaction. DGGE was performed to separate the
resulting 220 bp fragments (8% polyacrylamide, 0-100%
denaturant), and bands were cut from the gel for purifica-
tion and subsequent sequencing, as described above for
the eubacterial 500 bp fragment.
DNA sequencing
Amplified DNA was purified from primers and unincorporated
nucleotides and concentrated with Millipore Ultrafree-MC
30,000 NMWL Filter Devices to 20-100 ng/|jL. Sequencing
of the purified PCR products was performed on an ABI
Model 377 automated sequencer, using Ampli-TaqFS DNA
polymerase and fluorescent-labeled dNTPs in a cycle-
sequencing kit (ABI Prizm Dye Terminator Kit, PE Applied
Biosystems, Inc., Foster City, CA). The M13 primers and
two internal primers (704f f, 907r, Johnson, 1994) were
employed to sequence 1500 bp eubacterial 16S rDNA.
The M13 primers were used to sequence the 600 bp
archaeal 16S rDNA. The amplification primers were used
to sequence the nested eubacterial (550 bp) and archaeal
(220 bp) fragments. Sequencher® (Gene Codes Corp.,
Ann Arbor, Ml) was used to assemble the fragments. The
assembled sequences were compared to those in GenBank
using BLASTN (Altschul et al., 1997, National Center for
Biotechnology Information). Sequences from a BLASTN
search, that most closely matched the sequences from the
clones and sequences of selected outgroup strains, were
aligned using CLUSTAL X (ver. 1.81) (Thompson et al.,
1997). A dendrogram was constructed from the distance
matrix using the neighbor-joining method in CLUSTALX and
1000 bootstrap replicates were performed to estimate the
support for each branch (Felsenstein, 1985).
Results and Discussion
DNA extraction
All methods gave qualitatively the same results: DNA was
obtained from five sites of sufficient quantity and quality to
be amplified using universal eubacterial primers. We were
not initially able to amplify DNA from two of the Elizabeth
City samples. Tests confirmed that PCR-inhibiting sub-
stances were not responsible for the lack of amplification,
therefore, based on the high percentage sand composition
of these samples, we assumed that biomass might be
lower in these two samples. PLFA values confirmed that
the number of microorganisms was very low in samples
taken from these as well as similar sites. Our procedures
were accordingly modified to increase the amount of sample
processed and to concentrate the extracted DNA. In brief,
four 0.5 g subsamples were processed separately until an
intermediate stage in the protocol of the MoBio Power Soil
DNA Isolation Kit, and then combined prior to filtration and
final elution of the DNA. These modifications, together with
decreasing the final elution volume, allowed us to increase
the final DNA concentration by a factor of approximately
16 (standard protocols call for single 0.25 g samples and
elution in a volume of 50 |j|_ rather than 100 |j|_). Using the
modified protocol, eubacterial and archaeal16S rRNA was
successfully amplified from two Elizabeth City samples
(eubacterial only from two additional samples) and six
samples from the permeable reactive barrier in Louisiana.
Molecular identification of eubacteria and archaea
Although DGGE profiles suggested that 16S rDNAsequence
diversity was fairly low in the Elizabeth City sites, five distinct
groups of eubacterial sequences were found. A total of 9
eubacterial and 5 archaeal sequences were obtained from
cloning.The majority of the eubacterial sequences (all from
Elizabeth City, NC) showed highest affinities to those from
Firmicutes, especially to members of the high G+C groups
(Figure 4). Two sequences had very high similarity (>99%)
to Rftocfococcosstrains, including one with 1 base difference
from Rhodococcus sp. UFZ-B528 (AF235012, sequence
of an isolate from a chlorobenzene-contaminated aquifer),
three were most closely affiliated with Desulfotomaculutn
(genus of Gram-positive sulfate-reducing bacteria), and
two were mostsimilarto uncultured Clostridium sequences
obtained from a bioreactor producing methane from landfill
leachate.Two sequences were almost identical (>99%) to
that of Pseudomonaslibaniensis, isolated from spring water.
Short sequences (122-161 bp) were obtained by cutting
bands from the DGGE gels and reamplifying-to the extent
the sequences from cut bands and clones overlapped, the
13
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former confirmed the presence of the eubacterial groups
obtained by cloning. The cloned sequences were of much
higher quality as well as longer, both factors are beneficial
for phylogenetic inference.
Lane 1
Lanes: (1) iron, zone of high corrosion; (2) down gradient sand;
(3) iron, midbarrier, low corrosion; (4) iron, midbarrier; (5) iron,
low corrosion; (6) iron, high corrosion; (7) iron, low corrosion;
(8) iron midbarrier
Conditions: 40%~60% denaturant, Eubacteria, 65v for 12hrs
running
Figure 4. Elizabeth City DGGE profile
As discussed earlier, methanogenic archaea PLFA were
found within the Louisiana wall, especially at the regions of
high methane production. Furthergenomic analysis of this
PRB has also shown sequences most closely associated
with methanogens. For example, the archaeal sequences
from cloned 16S sequences (all from the Louisiana wall)
were primarily affiliated with Methanosarcinacea (4/5); the
fifth was similar (98%) to Methanobacterium.
Summary
It is clear that typical protocols used for soil analysis would
certainly fail to adequately interrogate ground-water treat-
ment systems unless they were substantially modified.
The modifications found necessary to compensate for the
low biomass in the Elizabeth City samples were two-fold:
to increase the size of the sample processed, and to use
nested PCRforvisualization of diversity patterns by DGGE.
However, cloning longer fragments is recommended for
sequence analysis.
It should be noted that the presence of the iron orsorption
of DMA macromolecules on the minerals or Fe surfaces
was not encountered during our research. Eubacterial
diversity was limited in the Elizabeth City site, but never-
theless contained members from four different groups of
eubacteria. Some of the sequences had highest affinity to
those obtained fromsites contaminated with chlorobenzene.
Conclusions
It should be clear to the reader that the PLFA indicators of
microbial biomass and community structure complement
the use of DMA for community structure, diversity and spe-
cific microbial species in the subsurface. The presence of
contaminants superimposed on both site geochemistry and
physical/chemical interventions to remediate chlorinated
solvent and metal contamination evoke a profound change
in the abundance, diversity and activity of microbiota.
Increases in biomass alone show that microorganisms can
adapt, and in some cases thrive under otherwise adverse
dynamic conditions.
In these disturbed environments, there may be a need
for more effective DMA extraction methods to magnify the
sample size which would enable improved understanding
of the microbial ecology of the subsurface. As more spe-
cific gene probes are developed, improved DMA extraction
techniques could provide a much more in-depth picture of
microbial function, diversity, activity, and interdependence.
It must be kept in mind that nearly exponential changes in
microbial biomarkers can occur due to contamination and
remediation while the geochemical parameters remain far
more stable in time and space. There is a definite need for
the application of more accurate, reliable and statistically
responsive indicators of microbial reactions to geochemi-
cal stressors.
Notice
The U. S. Environmental Protection Agency through its
Office of Research and Development funded the research
described here. This research brief has been subjected to
the Agency's peer and administrative review and has been
approved for publication as an EPA document. Mention of
trade names or commercial products does not constitute
endorsement or recommendation for use.
Acknowledgments
We are grateful to the following individuals at the NRMRL of
the U.S. EPA: Dr. Richard Wilkin for providing the Elizabeth
Citysoilsamples;andFrankBeck(deceased),PatrickClark,
and Tony Lee for participating in field sampling.
14
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