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Abstract:
Defining
biodegradation rates and processes is a critical
part of assessing the feasibility of monitored
natural attenuation as a remediation method
for ground water containing organic contaminants.
During 1998–2001, the U.S. Geological
Survey conducted a microbial study at a freshwater
tidal wetland along the West Branch Canal Creek,
Aberdeen Proving Ground, Maryland, as part of
an investigation of natural attenuation of chlorinated
volatile organic compounds (VOCs) in the wetland
sediments. Geochemical analyses and molecular
biology techniques were used to investigate
factors controlling anaerobic degradation of
1,1,2,2-tetrachloroethane (TeCA), and to characterize
the microbial communities that potentially are
important in its degradation. Rapid TeCA and
daughter product degradation observed in laboratory
experiments and estimated with field data confirm
that natural attenuation is a feasible remediation
method at this site. The diverse microbial community
that seems to be involved in TeCA degradation
in the wetland sediments varies with changing
spatial and seasonal conditions, allowing continued
effective natural attenuation throughout the
year.
Rates
of TeCA degradation in anaerobic microcosm experiments
conducted with wetland sediment collected from
two different sites (WB23 and WB30) and during
three different seasons (March–April 1999,
July–August 1999, and October–November
2000) showed little spatial variability but
high seasonal variability. Initial first-order
degradation rate constants for TeCA ranged from
0.10±0.01 to 0.16±0.05 per day
(half-lives of 4.3 to 6.9 days) for March–April
1999 and October–November 2000 microcosms
incubated at 19 degrees Celsius, whereas lower
rate constants of 0 ± 0.03 and 0.06 ±
0.03 per day were obtained in July–August
1999 microcosms incubated at 19 degrees Celsius.
Microbial community profiles showed that low
microbial biomass and microbial diversity in
the summer, possibly due to competition for
nutrients by the wetland vegetation, could account
for these unexpectedly low degradation rates.
In microcosms incubated at 5 degrees Celsius,
about 50 percent of the initial TeCA in solution
was converted to daughter products within a
35-day incubation period, indicating that biodegradation
in the wetland sediments can continue during
cold winter temperatures.
Initial
pathways of TeCA degradation were the same in
the wetland sediment microcosms regardless of
the season or sediment collection site, the
reduction-oxidation conditions, and the previous
exposure of the sediment to contamination. Immediate
and simultaneous dichloroelimination and hydrogenolysis,
producing 1,2-dichloro-ethene (12DCE) and 1,1,2-trichloroethane
(112TCA), respectively, were the initial TeCA
degradation pathways in all live microcosm experiments.
The production and degradation of vinyl chloride
(VC), which is the most toxic of the TeCA daughter
compounds, was affected by spatial and seasonal
variability, reduction-oxidation condition,
and pre-exposure of the wetland sediment. TeCA-amended
microcosms constructed with WB30 sediment showed
approximately twice as much VC production as
those constructed with WB23 sediment. Results
of 112TCA-amended microcosms indicated that
the greater production of VC in the WB30 sediment
resulted from a greater predominance of the
112TCA dichloro-elimination pathway in these
sediments. VC degradation also was substantially
higher in microcosms constructed with WB30 sediment
than those constructed with WB23 sediment, resulting
in lower VC concentrations at the end of WB30
microcosms. Enrichment experiments in which
microcosm slurry was amended with high initial
VC concentrations showed that the spatial difference
in VC degradation was negligible after prolonged
incubation under methanogenic conditions. Inhibition
of methanogenic activity in microcosms by addition
of sulfate or of 2-bromoethanesulfonic acid
inhibited production and degradation of VC.
Inhibition of methanogenesis by addition of
ferric iron or of 2-bromoethanesulfonic acid
also completely inhibited VC degradation in
VC-amended enrichment experiments. Pre-exposure
to VC substantially increased degradation in
VC-amended enrichment experiments.
A
microbial consortium, rather than one microbial
species or group, likely is involved in the
degradation of TeCA, as indicated by the occurrence
of multiple degradation pathways and the variability
in VC production and degradation. A bacterial
peak at 90 base pair (bp) fragment length in
terminal-restriction fragment length polymorphism
(TRFLP) profiles was associated with TeCA hydrogenolysis
to 112TCA, and bacterial species represented
by 198 and 170 bp fragment lengths were associated
with TeCA dichloroelimination to 12DCE. Dichloroelimination
of 112TCA to VC was associated with increasing
dominance of the 198 bp bacterial peak in March–April
1999 and October–November 2000 microcosms,
whereas an 86 bp or the 170 bp bacterial peak
was associated with 112TCA dichloroelimination
in the summer experiment. Hydrogenolysis of
12DCE to VC was associated with a carbon dioxide-utilizing
methanogen at 307 bp in the March–April
1999 and October–November 2000 microcosm
experiments, whereas production of VC occurred
despite low methanogen biomass and methane production
in the July–August 1999 experiments. Production
of VC in the absence of methane production also
occurred in 12DCE-amended enrichment cultures.
The exponential production of VC in the 12DCE-amended
enrichment cultures after an initial lag indicated
growth of a microbial species or group, possibly
one of the known dehalorespiring bacteria. Molecular
analyses using specific primers targeting dehalorespiring
bacteria of the Dehalococcoides group (Dehalococcoides
ethenogenes and Dehalococcoides sp. strain FL2)
and of the acetate-oxidizing Desulfuromonas
group (Desulfuromonas sp. strain BB1 and Desulfuromonas
chloroethenica) showed the presence of these
bacteria in microcosm slurry from site WB30
but not from site WB23. Addition of hydrogen,
which is the favored substrate of Dehalococcoides,
tripled VC production in 12DCE-amended enrichment
cultures. VC degradation showed a marked association
with an increase in the relative proportion
of Methanosarcinaceae, a family of methanogens
that includes all those capable of utilizing
acetate as a substrate, in the total methanogen
community.
Half-lives
for TeCA and TCE estimated from field data were
in the range of 60 to 100 days, which agrees
well with laboratory estimates of degradation
rates considering the inherent differences in
the laboratory and field systems. Both laboratory
microcosm experiments and field data showed
that 12DCE and VC are the predominant, persistent
daughter compounds from TeCA degradation. In
addition, porewater chemistry showed higher
accumulation of VC in the wetland sediment at
site WB30 than at site WB23, as was observed
in the microcosm experiments. Molecular analyses
of grab samples of surficial wetland sediment
showed that all the microbial species or groups
linked to TeCA degradation in the microcosm
experiments were present in all sediment samples.
Microbial biomass and diversity were lowest
in an area of the wetland (transect C-C ¢)
where porewater VOC concentrations are highest,
indicating that the higher VOC concentrations
could result from lower degradation rates. The
lower microbial biomass and diversity in this
area could be caused by toxic effects of the
contaminants, or possibly from differences in
frequency and duration of tidal inundation.
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