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Overview of
Advanced Diagnostics for Site Design, Management and
Closure
Stephen Koenigsberg, Environ International Corporation
Monitoring Gene Expression to Evaluate the Effectiveness
of an Oxygen Infusion System at a Petroleum Impacted
Site
Dora Ogles, Microbial Insights,
Rockford, TN
Brett Baldwin, Microbial Insights,
Rockford, TN
Joel Blair, Arctos Environmental, Long Beach, CA
Mike Purchase, Arctos Environmental, Berkeley, CA
Jeffrey M. Baker, Tesoro Companies, Inc., Auburn, WA
Greg Davis, Microbial Insights, Rockford, TN
Insights to
Dehalococcoides Biomarker Analysis and
Interpretation for Bioremediation: Opportunities
and Challenges Ahead
Kirsti Ritalahti, Georgia
Institute of Technology, Atlanta GA
Janet K. Hatt, Georgia Institute
of Technology,
Atlanta
GA
Kelly E. Fletcher, Georgia
Institute of Technology,
Atlanta
GA
Frank E. Löffler, Georgia
Institute of Technology,
Atlanta
GA
Diagnosing the Metabolic Status of Microorganisms
Involved in Subsurface Bioremediation with Antibody
Quantification of Proteins
Toshiyuki Ueki,
University of
Massachusetts
Amherst, Amherst, MA
Jiae
Yun,
University of
Massachusetts
Amherst, Amherst, MA
Derek R. Lovley,
University of
Massachusetts
Amherst, Amherst, MA
The Use of DNA
Microarrays for Bioremediation
Aaron Peacock, Haley and Aldrich, Oak Ridge, TN
Darrell Chandler, Akonni Biosystems, Inc.,
Frederick, MD
Dora Ogles, Microbial Insights, Inc.,
Rockford,
TN
Phil Long, Pacific Northwest National Laboratory,
Richland, WA
The Use of
Archaea Technology for Site Remediation
Ernest Childs, Archaea Solutions
Overview of Advanced Diagnostics for Site Design,
Management and Closure
Stephen S. Koenigsberg, ENVIRON International
Corporation, 18100 Von Karman Avenue, Ste 600,
Irvine, CA
92612,
Tel: 949-798-3604, Email: skoenigsberg@environcorp.com
Remediation strategies for site
closure have undergone a significant paradigm shift.
Once the exclusive domain of energy intensive,
mechanically driven processes we now witness replacement
by or integration with passive in-situ
strategies. In concert with these changes, legitimate
questions about site closure endpoints have been raised
as recalcitrant and asymptotic
conditions confound clean-up goals.
Consequently, monitored natural attenuation (MNA)
and other related options have become established in
their own right as remedies.
Recently, a number of validated advanced methods
have been used to generate new lines of evidence in
support of MNA.
The use of a variety of diagnostic tools has not
only been valuable in site design and management
decisions, but has now shown proven worth in structuring
the final phase of the remedy.
A comprehensive program should
include the use of molecular biological protocols,
emergent chemical analytical tools, and novel
applications of extant chemical methods such as compound
specific isotope analysis, state-of-the-art geophysical
techniques, fate and transport modeling and risk
analysis.
This array of options can be put into two categories –
those involving data collection and those involving a
more virtual component that is software driven. The
recent emergence of molecular biological tools (MBTs),
compound specific stable isotope analysis (CSIA) and
advanced geotechnical protocols “enviro-tomography”) are
rich in content and powerful in scope of application on
the data collection side.
Coupled with this are the standard risk analyses,
novel forms of fate and transport modeling and more
recently sustainable remediation principles and
practices that provide compelling arguments for MNA when
that form of resolution is appropriate.
This presentation sets forth the
elements of a comprehensive program in remedy
optimization and site exit strategies. Specific examples
will be presented that illustrate the achievement of
cost-effective and expedited closure goals using these
tools.
Monitoring Gene Expression to Evaluate the Effectiveness
of Oxygen Infusion at a Gasoline-Impacted Site
Dora Ogles,
Microbial Insights, Inc., 2340 Stock Creek Blvd.,
Rockford, TN 37853-3044, USA, Tel: 865-573-8188, Fax:
865-573-8133, Email: dogles@microbe.com
Brett Baldwin, Microbial Insights, Inc., 2340 Stock
Creek Blvd., Rockford, TN 37853-3044, USA, Tel:
865-573-8188, Fax: 865-573-8133, Email:
bbaldwin@microbe.com
Joel Blair, Arctos Environmental, 3450 E. Spring St,
Suite 212, Long Beach, CA 90806, USA, Tel: 562-988-2755,
Email: jblair@orionenv.com
Mike Purchase, Arctos Environmental,
1332 Peralta Ave,
Berkeley,
CA
94702,
USA, Tel:
510- 525-2180, mpurchase@arctosenv.com
Jeffrey M. Baker, Tesoro Companies, Inc., 3450 South 344th
Way, Ste 201, Auburn, WA 98001, USA, Tel: 253-896-8708,
Email: Jeffrey.m.baker@tsocorp.com
Greg Davis, Microbial Insights, Inc., 2340 Stock Creek
Blvd., Rockford, TN 37853-3044, USA, Tel: 865-573-8188,
Fax: 865-573-8133, Email: gdavis@microbe.com
Evaluation of corrective actions
designed to enhance biodegradation of petroleum
hydrocarbons and fuel oxygenates should include
chemical, geochemical, and microbiological lines of
evidence.
At gasoline-impacted sites, temporal monitoring and
analysis of trends in dissolved benzene, toluene,
ethylbenzene, xylene (BTEX) and methyl
tert-butyl
ether (MTBE) concentrations can be used to document
contaminant loss and provide the first line of evidence.
Likewise, temporal monitoring of geochemical
parameters can reveal changes in redox status resulting
from site activities, changes in electron acceptor
availability, and can provide a second indicator of
enhanced biodegradation.
The third and potentially most direct line of
evidence to evaluate the ability of a remediation
technology to stimulate biodegradation is to quantify
expression of the genes and activity of the organisms
responsible for contaminant biodegradation.
In the current study, quantitative polymerase
chain reaction (qPCR) and reverse-transcription qPCR
(RT-qPCR) were used to monitor microbial populations and
gene expression to evaluate the effectiveness of an
oxygen infusion system to promote aerobic biodegradation
of BTEX and MTBE.
During system startup and continuous operation,
dissolved oxygen (DO) levels at the injection points
were greater than 30 mg/L, contaminant concentrations
decreased, and transcription of aromatic oxygenase genes
(toluene dioxygenase and phenol hydroxylase) and
activity of MTBE-utilizing strain
Methylibium
petroleiphilum PM1 increased by as many as four
orders of magnitude in response to system operation.
Moreover, aromatic oxygenase gene transcription
and PM1 activity increased at downgradient locations
despite the fact that DO levels in downgradient wells
did not appreciably increase during system operation.
Conversely, BTEX- and MTBE-utilizing populations,
aromatic oxygenase gene transcription, and PM1 activity
substantially decreased when the system was temporarily
deactivated.
Overall, traditional groundwater analyses
combined with monitoring gene expression provided the
three lines of evidence needed to conclusively
demonstrate that the oxygen infusion system effectively
promoted BTEX and MTBE biodegradation at the site.
Insights to
Dehalococcoides Biomarker Analysis
and Interpretation for Bioremediation:
Opportunities and Challenges Ahead
Kirsti
M. Ritalahti,
Georgia Institute of Technology,
311 Ferst Dr.
Atlanta GA
30332,
USA, Tel:
404-385-4558, Fax: 404-894-8266, Email:
krita@ce.gatech.edu
Janet K. Hatt, Georgia Institute of
Technology, 311 Ferst Dr.
Atlanta GA 30332,
USA, Tel:
404-385-4552, Fax: 404-894-8266, Email:
janet.hatt@ce.gatech.edu
Kelly E. Fletcher, Georgia Institute of Technology,
311 Ferst Dr. Atlanta GA 30332,
USA, Tel:
404-385-4552, Fax: 404-894-8266, Email:
kelly.fletcher@gatech.edu
Frank E. Löffler, Georgia Institute of
Technology, 311 Ferst Dr.
Atlanta GA 30332,
USA, Tel: 404-894-0279,
Fax: 404-894-8266, Email: frank.Loeffler@ce.gatech.edu
Practical experience and laboratory
research has established a firm link between the
detoxification of chlorinated ethenes and the presence
and abundance of Dehalococcoides (Dhc)
bacteria in anaerobic groundwaters. Bioaugmentation
consortia containing
Dhc are
commercially available as an effective remedy at many
sites contaminated with chlorinated ethenes. While
Dhc often
occur in low abundance within natural microbial aquifer
communities, some sites require bioaugmentation (i.e.,
inoculation) with
Dhc-enriched cultures to achieve faster
detoxification rates or to initiate reductive
dechlorination as not all
Dhc possess
the repertoire of reductive dehalogenase (RDase) genes
required for chlorinated ethene detoxification. Crucial
for technology implementation and measuring the success
of bioremediation applications are unbiased assessment
and monitoring tools that establish links between the
presence and activity of
Dhc and contaminant detoxification.
Nucleic acids provide an appropriate target
molecule that can be obtained from most environmental
sample material.
Site monitoring tools targeting
Dhc biomarker
genes, including the 16S rRNA gene and the known RDase
genes tceA,
vcrA and bvcA, are used to
monitor Dhc
presence and abundance. To compensate for different
extraction efficiencies from diverse samples, internal
controls are being incorporated.
Although DNA-based tools provide valuable
information about
Dhc presence, distribution and abundance, the
DNA-targeted approaches fall short of specifically
distinguishing viable, active
Dhc cells from
irreversibly inhibited
Dhc cells.
To increase the knowledge surrounding
Dhc viability
and activity, the nucleic acid-based approach was
expanded to quantify biomarker gene transcripts (i.e.,
RNA). During
reductive dechlorination to ethene, transcript abundance
has been shown to correlate with dechlorination
activity. In
laboratory experiments for
Dhc-containing
mixed consortia, environmental challenges impact the
viability and growth of
Dhc
populations and, transcript numbers in inhibited cells
may be as high as or even higher than those in active
cells.
Although promising, transcript monitoring in groundwater
faces numerous challenges, including interpreting
Dhc DNA and
RNA biomarker data in context for making site management
decisions.
Diagnosing the Metabolic Status of Microorganisms
Involved in Subsurface Bioremediation with Antibody
Quantification of Proteins
Toshiyuki Ueki,
Department of Microbiology, University of Massachusetts
Amherst, 639 North Pleasant Street,
Amherst, MA 01003, USA, Tel: +1-413-577-4666, Fax:
+1-413-577-4660, Email: tueki@microbio.umass.edu
Jiae
Yun, Department of Microbiology, University of
Massachusetts Amherst, 639 North
Pleasant Street, Amherst, MA 01003, USA, Tel:
+1-413-577-4666, Fax: +1-413-577-4660, Email:
yunjiae@microbio.umass.edu
Derek R.
Lovley, Department of Microbiology, University of
Massachusetts Amherst, 639 North
Pleasant Street, Amherst, MA 01003, USA, Tel:
+1-413-545-9651, Fax: +1-413-577-4660, Email:
dlovley@microbio.umass.edu
Design of an optimal bioremediation
strategy requires information about the physiological
status of microorganisms involved in the bioremediation.
Quantifying environmental transcript abundance is an
effective approach for diagnosing physiological status.
However, environmental transcript analysis can be
technically challenging, which may limit its widespread
application, and it does not account for the impact of
translational modifications on protein abundance. An
alternative approach is to directly quantify the
abundance of key proteins that might be diagnostic of
physiological status. To evaluate this approach, initial
studies focused on
Geobacter
species, which play an important role in bioremediation
of groundwater contaminated with organics and/or metals
and are among the most effective organisms for
extracting electricity from organic matter in
sedimentary environments. Previous studies have
demonstrated that the transcript abundance of the
citrate synthase gene is correlated with metabolic rates
of Geobacter
species in subsurface environments.
Citrate synthase
is a key enzyme in the
TCA cycle for
the generation of energy. Levels of the citrate
synthase protein were quantified with an antibody
designed to target the unique citrate synthase of
Geobacter
species. When
Geobacter bemidjiensis, a representative of the
Geobacter
species that predominate in subsurface environments, was
grown at different dilution rates in chemostats, there
was a direct correlation between the amount of the
citrate synthase and metabolic rate. Furthermore,
analysis of the abundance of
Geobacter
citrate synthases in the groundwater from the
in situ
uranium bioremediation
site in Rifle, CO demonstrated that acetate injection,
which stimulated growth of
Geobacter
species, resulted in increase of
Geobacter citrate synthases in
the groundwater, suggesting that the amount of
Geobacter citrate synthases reflects the
metabolic activity of
Geobacter species involved in the
bioremediation.
These studies show promise for quantifying
abundance of key metabolic proteins with antibodies as a
new molecular tool for diagnosing the physiological
status of microorganisms.
The Use of DNA Microarrays for Bioremediation
Aaron D. Peacock, Haley &
Aldrich, Inc., 103 Newhaven Rd, Oak Ridge, Tennessee,
37830, USA, Tel: 913-787-4172, Fax: 913-599-5822, Email:
apeacock@haleyaldrich.com
Darrell Chandler, Akonni Biosystems, Inc., 400 Sanger Ave, Suite 300, Frederick, Maryland, 21701, USA,
Tel: 301-698-0101, Fax: 301-698-0202, Email:
dchandler@akonni.com
Dora Ogles, Microbial Insights, Inc., 2340 Stock Creek Blvd., Rockford,
Tennessee, 37853,
USA, Tel:
865-573-8188. Fax: 865-573-8133, Email:
dogles@microbe.com
Phil Long, Pacific Northwest National Laboratory, Mail
Stop K9-33
Richland, Washington, 99354,
USA, Tel:
509-372-6090, Fax: 509-372-6089
Microorganisms in the subsurface
have a direct impact on the nature, extent, and fate of
many contaminants.
Microorganisms can create conditions that
decrease contaminant mobility or directly transform
contaminants into innocuous or immobile forms.
However, there are presently very few readily
available methods for assessing
in situ
microbial community structure, activity or remediation
potential within a time frame that impacts treatment or
remediation decisions. The objective of this effort was
to develop and validate a simple-to-use, field-portable,
microarray-based system for monitoring microbial
community structure and dynamics in groundwater and
subsurface environments. The system performance and
efficacy was verified on 50 groundwater samples from an
in situ Uranium bioremediation field experiment conducted at the
Rifle, Colorado Integrated Field Research Center (IFRC).
Samples were collected over a 4 month period,
representing site status before acetate injection,
during the Fe-reduction phase; during the transition
from Fe- to SO42- reduction, and
during the SO42- reduction phase.
Sample-to-answer results for the field deployment were
obtained in 4 hours and showed an expected onset of
metal-reducer signatures within four days of acetate
addition to the subsurface.
Retrospective analysis of all samples with the
field portable system likewise showed the expected
progression of microarray and microbial signatures from
Fe- to SO42- -reducers with
changes in acetate amendment and
in situ field conditions. Microarray results and S/N ratios were in
concordance with quantitative PCR data sets and lipid
profiles, indicating that the field-portable array is a
reasonable and useful indicator of microbial presence
and response to in situ remediation of a
uranium-contaminated site.
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