Bioremediation of Relcalcitrant Compounds - Chapter 4 ppt

51

chapter four

The federal integrated
biotreatment research
consortium (flask to field)

Jeffrey W. Talley

Contents



4.1 Introduction 51
4.1.1 Chlorinated solvents 52
4.1.2 PAHs 53
4.1.3 PCBs 53
4.2 Technical approach 54
4.3 Thrust area project results 55
4.3.1 Chlorinated solvents 55
4.3.2 PAHs 55
4.3.3 PCBs 56
References 56

4.1 Introduction

The Department of Defense (DOD) has thousands of sites that have been
contaminated with organic compounds that pose a serious threat to the
environment. The remediation of these sites using existing technologies was

problematic from an economic, technical, and political point of view. The
Environmental Protection Agency (EPA) and DOD funded development and
application of innovative remediation technologies to solve these problems.
Of all of the innovative technologies, bioremediation was considered the
most promising.

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52 Bioremediation of Recalcitrant Compounds

Biotreatment processes had been successfully demonstrated for treat-
ment of a wide variety of easily degraded compounds, such as low-molec-
ular-weight fuels and phenols. Strong potential existed for development of
biotreatment processes directed toward contaminant groups traditionally
more difficult to degrade, such as explosives, chlorinated solvents, polycyclic
aromatic hydrocarbons (PAHs), and polychlorinated biphenyls (PCBs). All
of these compounds represented a major contaminant problem to the DOD.
This project’s objectives enhanced existing funded efforts in DOD pro-
grams, complemented both the EPA and DOD research strategies, and
addressed problems experienced by environmental engineers involved in
Superfund, Resource Conservation and Recovery Act (RCRA) activities, and
international technology exchange programs.
Dr. Jeffrey Talley, P.E., was the project director and was assisted by
Deborah K. Belt.

4.1.1 Chlorinated solvents

Chlorinated solvents entered the environment in massive amounts during
the 1950s, 1960s, and 1970s. These contaminants have migrated through the

subsurface and impacted groundwater at more than 1000 DOD sites. Con-
taminated aquifers can be remediated by removing the solvents in the porous
media of the subsurface. Laboratory and pilot-scale experiments have dem-
onstrated the potential of cosolvent-enhanced

in situ

extraction to remove
dense nonaqueous phase liquids (DNAPLs) in porous media. Although this
method is effective for mass removal, residual amounts of cosolvents and
contaminants are expected to remain at levels that could preclude meeting
regulatory requirements. However, with the bulk of the DNAPLs extracted

in situ

, biotreatment becomes a viable polishing procedure. This was the
emphasis of the work that was conducted by Dr. Guy Sewell, EPA.

In situ

biotreatment may transform the remaining contaminants to non-
hazardous compounds at a rate in excess of the rate of dissolution or dis-
placement. The efficacy of

in situ

bioremediation of chlorinated solvents is
usually limited by transport and mixing considerations, i.e., supplying
excess electron donors in conjunction to the chlorinated solvents at appro-
priate concentrations. The delivery-and-extraction process facilitated the

cosolvency effect and supplied electron donors (cosolvent, ethanol) and elec-
tron acceptors (chlorinated solvent, tetrachloroethylene (PCE)) to the inher-
ent bacteria. The synergism between these abiotic and biotic processes could
minimize problems associated with the individual approaches and lead to
the development of a treatment train approach, which could attenuate or
eliminate the risks posed to human health and the environment by DNAPL
sites.

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Chapter four: The federal integrated biotreatment research consortium 53

4.1.2 PAHs

PAHs include industrial wastes such as petroleum and fuel residues, tars,
and creosote that contaminate soils and sediments. Land farming is a com-
mon treatment option for PAH-contaminated soils, but the removal of the
high-molecular-weight (HMW) PAHs by this method is often problematic.
The goal of this project, coordinated by Dr. Hap Pritchard Navel Research
Laboratory (NRL), was to modify land farming by using bioaugmentation
to improve degradation of PAHs. Bioaugmentation involved the addition of
a biosurfactant-producing bacterium (strain Pa 64), a bulking agent (rice
husks), and a carbon/nitrogen source (dried-blood fertilizer) (Pritchard et
al., 1999). Microcosm studies conducted at NRL validated the method and
determined the degradation kinetics.
Lance Hansen conducted a pilot-scale study at the U.S. Army Engineer
Research and Development Center (ERDC) in Vicksburg, MS, implementing
bioaugmentation technology for PAH remediation. The study consisted of
three metal pans (10 feet long

×



3 feet wide

×

2 feet deep), each filled with
approximately 1 cubic yard of PAH-contaminated soil. One pan was
untreated, one received bulking agent and dried blood, and the third
received bulking agent, dried blood, and the bacteria. The pan study was
designed to define the sampling strategy required to measure the effective-
ness of bioaugmentation and to provide a realistic cost estimate for the
bioaugmentation treatment (U.S. Army Corps of Engineers, 1996). Methods
were developed and refined to monitor the progress and effectiveness of
bioremediation. These included molecular biological techniques to monitor
the presence of the inoculated organisms and their

in situ

activity (White
and Ringelberg, 1998; Balkwill et al., 1988), respirometric techniques that
monitor relative microbial activity based on CO

2

production, and genetic
techniques that monitor the presence or absence of enzymes involved in

nitrogen use and PAH degradation (Perkins et al., 2001). These techniques
were correlated to standard contaminant analytical chemistry methods and
were applied to the cost optimization of land-farming PAHs.

4.1.3 PCBs

Research on microbial degradation of PCBs has been ongoing for more than
25 years and has shown that bioremediation requires a more sophisticated
technology than the simplistic attempts that have been tried so far. The
project conducted by Dr. Jim Tiedje at Michigan State University addressed
key barriers to bioremediating PCBs:
• Developing microorganisms that will grow on the major congeners
produced by anaerobic dechlorination of PCBs
•Improving bioavailability of PCBs through the use of surfactants
• Optimizing field delivery of anaerobic or aerobic PCB bioremediation
technologies

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54 Bioremediation of Recalcitrant Compounds

Genetically engineered microorganisms (GEMs) were developed that
were capable of using PCB congeners as growth substrate under aerobic
conditions. GEMs were modified to exhibit dechlorination genes that
enabled the removal of chlorine before chlorocatechols were formed, avoid-
ing toxicity (Tsoi et al., 1999). This approach avoids the need to manage
cometabolism, which can be difficult

in situ

. These organisms can be used
to remove products of anaerobic reductive PCB dechlorination, predomi-
nantly ortho-chlorinated and ortho- + para-chlorinated congeners (Hrywna
et al., 1999). Verniculite, as a carrier for the bacterial inoculum, improved
survival of the GEMs in Picatinny soil.

4.2 Technical approach

Figure 4.1 illustrates the technical approach used to develop new biotreat-
ment technologies during the Flask to Field project. The technical approach
within the consortium was to develop the most promising biotreatment
processes at the bench scale and then validate the technology at the pilot
and field scales. Engineering groups worked closely with scientists in eval-
uating the potential of the resulting technologies and in the transfer of
technologies from bench scale to field. The Technical Advisory Committee
(TAC) periodically reviewed projects for technical merit. The recommenda-
tions of these biotechnology experts to the thrust area coordinators served
to further enhance the projects. This approach ensured that effective reme-
diation technologies were developed within a reasonable time frame.

Figure 4.1

Biotreatment process development in Federal Integrated Biotreatment
Research Consortium (FIBRC).
Chlorinated
Solvents
Explosives
hPAHs
PCBs

Process Engineering
CANDIDATES FOR DEMONSTRATION
AND VALIDATION

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Chapter four: The federal integrated biotreatment research consortium 55

4.3 Thrust area project results

A brief summary describing a major project from each thrust area is given
below.

4.3.1 Chlorinated solvents

The chlorinated solvents project, Solvent Extraction Residual Biotreatment
(SERB), concentrated on the remediation of tetrachloroethylene. SERB tech-
nology was validated in a field-scale study at Sage’s Dry Cleaner site, Jack-
sonville, FL (Mravik et al., 1999; Sewell et al., 2000). PCE concentration was
reduced by 70% in the aquifer using this technology. Significant levels (4
mg/l) of the dechlorination product, cis-1,2-dichloroethene (cis-DCE), were
detected in groundwater samples in the area exposed to residual ethanol
after 4 months and increased to 16 mg/l after 10 months. Maximum and
minimum observed rates of dechlorination (based on cis-DCE production)
were 43.6 and 4.2

µ

g/l/day, respectively. These results indicated that over

time, biotransformation had been enhanced.
Microbial ecology studies using site materials indicated that the site
remained biologically active. Microcosm studies indicated that anaerobic
microbial populations generated a reducing equivalent balance by oxidation
of the cosolvent (ethanol) that was linked to the reductive dechlorination of
PCE. Molecular methods indicated the presence of known groups of dechlo-
rinators. Overall, the project was successful. SERB research is still ongoing
at the Jacksonville site and represents an attractive alternative for chlorinated
solvents’ remediation.

4.3.2 PAHs

Results from the PAH pilot-scale study implementing bioaugmentation tech-
nology indicated that bioaugmentation did enhance PAH degradation
(Hansen et al., 2000). Degradation of HMW PAHs into four-ring compounds
(including BaP toxic equivalent compounds) was achieved. Low-molecu-
lar-weight PAHs were extensively degraded in the first 2 to 3 months, and
degradation of the HMW PAHs commenced in the fourth month in micro-
cosms bioaugmented and treated with dried-blood fertilizer. A reduction of
total PAHs (86 to 87%) was realized after 16 months in the pans that had
been bulked, amended with dried-blood fertilizer, and bioaugmented. Com-
parison of the two methods indicated that the time required to achieve 50%
degradation of PAHs was decreased by half through bioaugmented land
farming over traditional land farming methods. The soil used for this
research was heavily contaminated with PAHs (7200 ppm) and would tra-
ditionally not be considered a candidate for bioremediation. This research
indicates that land farming that incorporates bioaugmentation
technology may be an alternative to incineration for remediation of heavily
PAH-contaminated sites. Bioaugmentation would be more cost effective than

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56 Bioremediation of Recalcitrant Compounds

incineration because of minimal soil excavation, material handling, and
energy costs.

4.3.3 PCBs

The PCB project has been very successful in cloning genes in bacteria that
combine cometabolism of PCBs to chlorobenzoates, and the dechlorination
and mineralization of chlorobenzoates, as a growth substrate. Microbiolog-
ical, biochemical, and physiological characterization of the selected biphenyl
degraders was completed. Based on the growth on PCB mixtures, toxicity
testing, and survival in soil microcosms, a combination of two GEMs,

Rhodo-
coccus

RHA1 tfcb and

Burkholdena

K W, were the most effective for achieving
PCB degradation. The use of surfactants increased the solubility and reme-
diation rates of the contaminant. Molecular probes were developed and used
to track the bacteria in Picatinny arsenal soils and river sediments, using
both genetic and polymerase chain reaction (PCR)–based techniques. The
recombinant organisms survived in nonsterile sediment from Red Cedar

River contaminated with Aroclor 1242 and maintained degradative activity,
evidenced by reducing PCB levels by 78%.
A pilot-scale study involving three different soil loadings (low, medium,
and high solids reactors) is ongoing at ERDC. This study will evaluate the
effects of different moisture contents on PCB bioremediation and application
of GEMs, as well as determine the maximum soil loading rate optimum for
GEMs’ activity to avoid or offset the subsequent costs of disposing of the
stabilized soil.
Bioremediation of PCBs is effective and offers lower energy and opera-
tions costs than other technologies, but it may take longer to remediate the
soil, and desorption kinetics may limit degradation rates.

References

Balkwill, D.L., Leach, F.R., Wilson, J.T., McNabb, J.F., and White, D.C. 1988. Equiva-
lence or microbial biomass measures based on membrane lipid and cell wall
components adenosine triphosphate, and direct counts in subsurface aquifer
sediments.

Microb. Ecol.

16: 73–84.
Hansen, L.D., Nestler, C., and Ringelberg, D. 2000. Bioremediation of PAH/PCP
contaminated soils from POPILE wood treatment facility. In

Proceedings of the
Second International Conference on Remediation of Chlorinated and Recalcitrant
Compounds

, G.B. Wickramanayake, A.R. Gavaskar, J.T. Gibbs, and J.L. Means,

Eds. Battelle Press, Columbus, OH, pp. 145–152.
Hrywna, Y., Tsoi, T.V., Maltseva, J.F., Quensen, J.F., III, and Tiedje, J.M. 1999. Con-
struction and characterization of two recombinant bacteria that grow on
ortho- and para-substituted chlorobiphenyls.

Appl. Environ. Microbiol

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2163–2169.
Mravik, S.C., Sewell, G.W., and Wood, A.L. 1999. Field evaluation of the Solvent
Extraction Residual Biotreatment Technology in

Abstracts of the 4th Interna-
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. ISSM, Vail, CO.

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Perkins, E., Hansen, L.D., Nestler, C.C., and Bymes, J. 2001. Changes in Abundance
of

In-Situ

Aromatic Degrading Bacteria during a Pilot Scale Landfarming of
a Polycyclic Aromatic Hydrocarbon Contaminated Soil. Paper presented at
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Novel Approaches for Bioremediation of Organic
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, R. Fass, Y. Flashner, and S. Reuveny, Eds. Kluwer Academic/Plenum
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Pseudomonas aerug-
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strain 142 ohb genes coding for oxygenolytic ortho-dehalogenation of
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