United States
Environmental Protection
Agency
Solid Waste and
Emergency Response
(5102G)
EPA 542-N-01-002
June 2001
Issue No. 41
-EPA TECH TRENDS
CONTENTS
Land Treatment Used at
Wood-Treating Sites page 1
Biological Treatment
of Amine Wastes from
the Gas Industry page 3
Integrated Analytical
Approach for Determining
Bioremediation
Effectiveness page 4
The Applied Technologies
Newsletter for Superfund
Removals
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[continued from page 1]
season in Montanalasts approximately
90-120 days between June and Sep-
tember.) Earlier studies had indicated
that the amount of soil capable of being
treated to aremediation level in a
specified time period is dependent on
the size of the LTU and the depth of soil
treated with each lift of a tiller. Jb_
maximize the depth of each Hftat these
sites, an 8-wheel drive, 200-horse-
power tractor was used to pull a
subsoiler plow with 3-foot tines. Asa
result, potential treatment depths were
extended to 2.5 feet.
Nitrogen and phosphorous in the form
of agricultural fertilizer were added as
nutrients to each LTU to stimulate
microorganismpeiformance. Calcula-
tions of the amounts of nitrogen and
phosphorous to be applied at each LTU
were based on soil carbon contents
measured through analytical sampling.
AttheMontanaPoleandTreatingPlant
site, liquid nutrients can be applied easily
through the irrigation system, but other
sites require granular fertilizer applica-
tion through a spreader and tilling
system. Since remediation levels for
each soil lift are normally reached
before the nutrients are depleted,
nutrients typically are added only once
to a lift of soil.
The time required to achieve
vary with the type of wood-treating fluid
used by the operating facility, as well as
site-specific factors such as soil type,
weather, and operational characteristics
of the LTU system. Table 1 provides
LTU performance information for each
study site.
It is anticipated that complete
remediation of the 45,000 cubic yards
of CPAH- andPCP-contaminated soil
at the Libby Ground Water site will be
achieved by 2003, following 14 years
of system operation. This estimate
reflects installation of a second LTU in
1998 that reduced remediation time by
16-18 years. At the BN Somers site,
where 67,000 cubic yards of~
CPAH-contaminated soil was treated
through three lifts, remediationlevels
were achieved in 2000 after six years of
LTU operation.
Soil remediation levels for PCP were
reached at the Idaho Pole Company in
2000 following five years of treatment.
This time reflects an additional 3,000
cubicfSas'dfednfaiiiiiiaesd soil ftiat." '"*"
were treated through a single lift as
follow-up to closure of the site's wood-
treating facility in 1999. AttheMontana
Pole and Treating site, approximately
52,000 cubic yards of contaminated soil
have been treated since 1997, with the
first 30,000 cubic yards reaching PCP-
target concentrations in 1998 after a
single lift treatment
Based on information gained from this
study, it is estimated that the average
cost of soil treatment in an LTU varies
from $16 to $25 per cubic yard. For
more information, contact Jim Harris
(EPA/Region 8) at 406-441-1150 or
harris.jim@epa.gov.
Site
Ufoby Ground Water
BN Somers
Idaho Pole Company
Montana Pole and
Treating Plant
Table 1. LTU Performance Summary
CPAH
Initial
Concentration
(mg/kg)
40-150
100-200
(below ctean-up
level)
(below clean-up
level)
Target
Concentration
(mg/kg)
59
57
15
4.2
Duration of
Treatment for
Each Lift
90 days to 2
seasons
2 seasons
N/A
N/A
PCP
Initial
Concentration
(mg/kg)
90-230
(not present)
50-220
30-70
Target
Concentration
(mg/kg)
36
'N/A
48
34
Duration of
Treatment for
Each Lift
90 days to 2
seasons
N/A
1 to 2 seasons
1 season
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Biological Treatment
of Amine Wastes from
the Gas Industry
by J.R. Gallagher and J.A.
Sorensen, University of North
Dakota/Energy &
Environmental Research Center
The fate and remediation options for; ?
treating subsurface contaminationfrom
the natural gas industry have not been
examined to great extent in the past. To
address these concerns, a research
project funded by the Canadian Asso-
ciation of Petroleum Producers,
Canadian Occidental Petroleum Ltd,
the U.S. Department of Energy, Gas
Researchlnstitute, Environment
Canada, and the National Energy
Board of Canada was initiated in 1995.
Researchers at the University of North
Dakota's Energy & Environmental
Research Center began studying related
site-specific issues andremediation
options for soil contaminated with
amines, amine byproducts, and salts at
a decommissioned gas plantnear
Calgary, Alberta. Preliminary studies on
the biodegradability of amines, coupled
with the relatively low mobility of
amines, indicated that bioremediation
was aremedial option at this site.
Based on these findings, a 500 cubic-
meter biopile including aeration,
irrigation, andleachate collection
systems was designed and constructed.
After five months of active biotreatment
andeightmonths of leaching, treatment
had removed allidentified amines and
significant amounts of organic and
inorganic nitrogen species, salts, and
organic carbon.
Environmental assessments conducted
in 1992 as part of the gas processing
plant decommissioning process revealed
a layer of sludge-contaminated soil at a
depth of 2-3 meters in the area of the
plant's former amine sludge disposal
pits. During evaluation of potential
biodegradation technologies, the site's
climate-logic and geologic characteristics
servedas significant factors. The •..-
climate in this region is semi-arid to
moist, with an average annual precipita-
tion of 488 millimeters (mm) (19 inches)
per year. Average temperatures
typically are below 10°C during 8-9
months of the year, with a frost-free
period of 75-90 days. Other
remediation options that were consid-
ered but found to be much more costly
included landfilling of the contaminated
soil or incinerationfollowedby soil
washing.
Construction of the biopile (Figure 1)
was completed over an 8-day period in
My 1998. The completed containment
cell measured 40 meters long by 10
meters wide and 1.5 meters deep.
Above a25-mil reinforced polyethylene
bottom liner in the cell was a thin layer
of crushed gravel covered by a filter
fabric. The overlaying soillayerwas
mounded and enclosed by another liner.
Approximately 450 cubic meters of
treatment soils were housed within the
constructed cell.
Soil additives to the system included
2.58 cubic meters of calcium chloride to
increase soil permeability, as well as
[continued on page 4]
Figure 1: Biopile Construction
3^^-^Srmm^-Mfe>
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[continued from page 3]
2,036 Mlograms of 10-34-00 (percent
nitrogen-phosphorus-potassium) liquid
feriilizerforincreasingthemicrobial
population and consequent biodegrada-
tionrate. ToactasabulMngagentfor
increased porosity andpermeabilily in
the biopile, 50 cubic meters of straw
were added within the treatment cell.
One 100-mm, perforated polyvinyl
chloride air vent powered by an exter-
nal blower unit provided system
aeration, along with four equally spaced,
50-mm flow ducts running the entire
length of the cell. Microbial activity was
enhanced by the addition of water
through an irrigation system comprising
five semipermeable hoses extending the
celllength andpoweredby an external
freshwatersupply. Thefrequencyand
amount of water application were
determined by weekly soilmoisture
measurements. Excessive salt leachate
was collected in a sump located directly
below the crushed gravel layer, tempo-
rarily stored in areinforced external
tank, and ultimately disposed in an
onsite injection well.
FoUowing approximately trireemonths
oftreatmentin 1998 and two months in
1999, data indicated thatbiodegrada-
tion of the amine-related materials likely
was complete. The remaining material
was considered to be leachable but not
biodegradable. At that point, thesystem
began operating in a leaching mode and
continued in this way fortheremaining
two months oftreatmentin 1999 and
four months in 2000. Overthecourse
of leaching mode operation, approxi-
mately 85,000 Imperial gallons of
water (approximately 3 pore volumes)
were applied to the biopile.
Soil sampling was conducted at
project start-up and bimonthly
throughoutthe active treatment
periods. Key soil character param-
eters used to evaluate general activity
oftnebiop^e'iMudeatoM'KjelaM"""""
nitrogen (TKN, a measure of both
ammonia and organic nitrogen),
ammonianitrogen (NI^-N), nitrate
plus nitrite nitrogen (NOx-N) com-
pounds, and total organic carbon.
Based on the results of 20 sampling
events over the course of treatment,
data showed that TKN and total
organic nitrogen concentrations
decreased, while concentrations of
ammonia and NOx-N (the byproducts
of alkanolamines and other organic
nitrogenous compounds) increased.
During final stages of the study,
however, TKN and total organic
carbon levels remained steady while
ammonia and nitrogen compound
- levels dropped significantlyfthus ~
indicatingthatthebiodegradationof
alkanolamines andtheformation of
thermal/oxidative products were
complete. Final analysis showed that
alkanolamine concentrations were
reduced to levels below the detection
limitfollowingtreatment, fromaninitial
concentration of 15,000 mg/kg.
The estimated cost of treating con-
taminated soil at this site through use of
the biopile was $45 per cubic meter
($34.40 per cubic yard), exclusive of
engineering and analytical costs.
Researchers estimate that this cost
could be reduced further in large-scale
applications andif containment liners are
not required. For more information,
contact J.R. Gallagher (University of
North Dakota) at 701-777-5030 or
jgallagher@undeerc.org.
Integrated Analytical
Approach for
Determining
Bioremediation
Effectiveness
by D. Ringelberg, U.S. Army
Engineering Research and
Development Center, and A.
Peacock, University of
Tennessee/Center for Biomarker
Analysis
Researchers at the U.S. Army's
Engineering Research andDevelopment
Center and the University of
Tennessee's Center for Biomarker
Analysis have developed aholistic
_ ApproachtoevaluatingJiepqtential for
bioremediation at sites with
contaminated soil. Dredged harbor
sediment contaminated withpolycyclic
aromatic hydrocarbons (PAHs)
resultingfrompastfuelreleases was
removed from the Milwaukee Confined
Disposal Facility near the South
Milwaukee Harbor in Wisconsin and
examined for in situ biodegradative
capacity. By integrating analytic
chemistry, microbiology, andmolecular
biology techniques, the successional
characteristics of indigenous microbiota
[continued on page 5]
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[continued from page 4]
were determined during a four-month
bioslurry evaluation. Bench-scale
testing showed that an intrinsic
biodegradation potential of 52 percent
for total PAHs was possible. In
addition, soil chemistry at the facility
indicated that PAH degradation likely
biological means.
This integrated approach focuses on the
use of "whole-sample" characterization
techniques or direct assays of microbes
in their natural environments, without
impactingthem during the measurement
process. The approach eliminates the
bias introduced when using a selected
medium for the enrichment of native
organisms, and overcomes the inability
to culture a significant percentage of the
native microbiota (90-99 percent of
which are not culturable using standard
**- bacteriological techniques). In addition,
these techniques collectively provide the
activity measurements or phenotypic/
genotypic descriptions necessary for
defining in situ biodegradation poten-
tials, which no single assay can address.
Figure 2: Integrated Approach to Determining Bioremediation Potential
Characterization
Contamination
or Site
^ ~~
Plume ^|^
^^^B^^^RBSt^"
Sampling Event Source Middle
Parameters
Analysis
General
Cleanup
L _,J
Physical/Chemical Geno/Phenotypic
Contaminant PLFA
Concentration (biomass,
(TCE, PCE, etc.) community
structure,
Geo-Chemical metabolic activity)
Parameters
(suifate, sulflde,
pH, redox, etc.)
L 1
1
— ~^
.— -^
Fringe
1
Genotypic
(bacterial profiles,
identification of
prominent organisms)
Functional Genes
(detection of specific
enzymes involved in
degradation)
1
Integrated Data Assessment
1
Model Generation/Biodegradation Potential
and Endpoint Estimation
In this comprehensive process (Figure
2), unique soil chemicalmarkers such as
phospholipids and nucleic acids are
recovered quantitatively at the contami-
nated site and subjected to analysis.
Phospholipids, which constitute the
mass bulk of most microbes, are used
as an index of biomass, while microbial
membrane lipids analysis is an effective
toolfor monitoring microbial responses
to their environment. Analysis of
phospholipids fatty acids (PLFA)
profiles, whichreflect both the natureof
the intracellular components and the
extracellular environmental conditions,
indicates what types of microbes (i.e.,
bacteria, fungi, and algae) are present in
a system, and how the microbes are
reacting to environmental factors such as
pollution and physical disturbances.
Nucleic acids also are used to provide
the phylogenic specificity necessary for
precise species identifications. Once
microbial DNA is recovered, unique
subsets of organisms can be targeted to
define the diversity associated with that
particular group of microbes. The
diversity pattern that emerges from one
soil sample can be compared to that of
another to determine the co-occurrence,
novelty, or absence of particular mi-
crobes across a site.
During the South Milwaukee Harbor
bioslurry evaluation, PLFAprofiles,
multiplexpolymerasechain reaction
(PCR) of targeted genes, andradio-
respirometry techniques were used to
define in situ microbial phenotypic,
genotypic, and metabolic responses,
[continued on page 6]
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fcontinued from page 5]
respectively. MicrobialPLFAanalysis
revealedathree-foldincreasein
biomass during the slurry test, and the
resulting microbial community
composition showed a strong
correlation with observed changes in
PAH chemistry. Genes encoding PAH-
degrading enzymes increased by as
much as four orders of magnitude, and
changes in gene copy numbers showed
strong correlations with shifts in specific
subsets of the extant microbial
community. Specifically, declines in the
concentrations of the three-ring PAH
components correlated with PLFA
microbial community. This approach
gives project managers a greater
understanding of the processes involved
in the biodegradation of targeted
analytes, and the capability to build
remediation models and conduct
treatment tests in native soil without
manipulation. Additionally, these
techniques canresultin significant cost
savings when compared to traditional
methods requiring analytical results from
bacteria (i.e., Rhodoccoccus sp. and/or
actinomycetes) and genes encoding for
naphthalene, biphenyl, andcatechol
degradative enzymes.
Results of this study suggest thatthe
mtrinsicbiodegradative potential of a
contaminated site can be derived from
polyphasic characterization of the in situ
across a full site. For further
information, contactDaveRingelberg
(U.S . Army Corps of Engineers/
CRREL) at 603-643-4744 or
DavidŁ.Ringelberg@erdc.usac«.army.mil
or Aaron Peacock (University of
Tennessee/CenterforBiomarker
Analysis) at 865-974-8014 or
apeacock@utk.edu.
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EPA542-N-01-002
June 2001
Issue No. 41
TECH TRENDS
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