United States
Environmental Protection
Agency
Robert S. Kerr Environmental
Research Laboratory
Ada, OK 74820
Research and Development
EPA/600/S2-91/027 Aug. 1991
vxEPA Project Summary
Microbial Degradation of
Alkylbenzenes under Sulfate-
Reducing and Methanogenic
Conditions
Harry R. Belter, Elizabeth A. Edwards, Dunja Grbi'c-Galic, Stephen R. Hutchins,
and Martin Reinhard
Aquifer solids and soils obtained
from vsrlous hydrocarbon-contami-
nated sites were used to Investigate
ths ability of indigenous microorgan-
isms to degrade monoaromatlc hydro-
carbons under strictly anaerobic con-
ditions. Hydrocarbon-degrading micro-
flora from two sites, an aviation fuel
storage facility near the Patuxent River
(MD) and a creosote-contaminated aqui-
fer near Pensacola (FL), were studied
most extensively.
In anaerobic microcosms Inoculated
with fuel-contaminated soil from the
Patuxent River site, toluene degrada-
tion occurred concomltantly with sul-
fate reduction and ferric iron reduc-
tion. Similar results were obtained with
suspended enrichments derived from
the microcosms. Stoichlometrlc date
and other observations suggested that
sulfate reduction was closely linked to
toluene degradation, whereas iron re-
duction was a secondary, potentially
abiotic, reaction between ferric Iron and
biogenic hydrogen sulfide. To our
knowledge, this Is one of the first re-
ports of the degradation of alkylben-
zenes under sulfate-reducing condi-
tions. The presence of milllmolar con-
centrations of amorphous Fe(OH) In
Patuxent River microcosms and enrich-
ments either greatly facilitated the on-
set of toluene degradation or acceler-
ated the rate once degradation had be-
gun.
Fermentative/methanogenic micro-
cosms and enrichments that degraded
toluene and o-xylene without added ex-
ogenous electron acceptors (except
CO,) were developed from creosote-
contaminated Pensacola samples. The
microcosms initially underwent an ac-
climation lag of several months; how-
ever, once the degradation of aromatic
hydrocarbons was Initiated, It pro-
ceeded at a relatively rapid rate, and It
was complete (resulting in mineraliza-
tion to CO, and CH4). Benzene,
ethylbenzene, and p-xylene were not
degraded.
This Project Summary was developed
by EPA'3 Robert 5. Kerr Environmental
Research Laboratory, Ada, OK, to an-
nounce key findings of the research
project that Is fully documented In a
separate report of the same title (see
Project Report ordering Information at
back).
Introduction
In 1986, the U.S. EPA reported that up
to 35 percent of the nation's underground
fuel storage tanks may be leaking. To-
gether with surface spill accidents and
landfill leachate intrusion, such leaks
greatly contribute to groundwater contami-
nation by gasoline and other petroleum
derivatives. Although most gasoline con-
stituents are readily degraded in aerobic
surface water and soil systems, similar
processes in the subsurface are signifi-
cantly retarded because of insufficient con-
centrations of oxygen and/or nutrients, and
consequently tow numbers of active aero-
bic microorganisms. In recognition of the
fact that it is not always feasible to intro-
duce oxygen into the subsurface to main-
tain aerobic microorganisms, and that con-
sumption of oxygen by indigenous micro-
-------
organisms often results in the develop-
ment of anaerobic conditions, this study
was undertaken to assess microbial deg-
radation of fuel-derived aromatic hydro-
carbons under anaerobic conditions.
In the absence of oxygen, degradation
of gasoline constituents can take place
only with the use of alternative electron
acceptors, such as nitrate, sulfate, or fer-
ric iron, or fermentatively in combination
with methanogenesis. To date, complete
degradation of benzene, toluene, and/or
xylene isomers by aquifer-, sediment-, or
sewage-derived microorganisms has been
reported by various investigators for deni-
trifying conditions, methanogenic condi-
tions, and ferric iron-reducing conditions.
Very recently (since 1990), pure cultures
of organisms that can degrade
alkylbenzenes under nitrate-reducing con-
ditions (Doffing et al., 1990)2 and ferric
iron-reducing conditions (Lovley and
Lonergan, 1990)3 were isolated. Despite
this research activity, anaerobic degrada-
tion of monoaromatic hydrocarbons is not
as well understood as aerobic degrada-
tion; further study of anaerobic degrada-
tion of these compounds for the range of
potential electron-accepting conditions is
warranted.
This report summarizes research on the
degradation of alkylbenzenes under sul-
fate-reducing and fermentative/methane—
genie conditions, with an emphasis on the
former conditions. Notably, definitive evi-
dence of the coupling of alkytoenzene deg-
radation to sulfate reduction has not yet
been published, However, some co-au-
thors of this summary and researchers at
the R.S. Kerr Environmental Research
Laboratory (U.S. EPA) have observed deg-
radation of toluene under sulfate-reducing
conditions in contaminated subsurface
materials. In addition, other researchers
(including Bak and WkJdel,11986; Szewzyk
and Pfennig, 1987)4 have shown that
sulfate-reducers are capable of degrading
a number of oxygen-containing aromatic
compounds, some of which may be inter-
mediates in ^anaerobic toluene degrada-
tion (e.g., p-cres-ol, benzoic acid, 2- and 4-
hydroxybenzoic acid, phenol, catechol, res-
orcinol, and hydroquinone).
Procedure
The initial goals of the project were to
enrich ferric iron-reducing or fermentative
microbial communities that could degrade
monoaromatic hydrocarbons. To enrich
iron-reducing bacteria, the initial experi-
mental approach consisted of screening
sediments for hydrocarbon-degrading ac-
tivity using a basal mineral medium that
was either amended with ferric iron or
was not amended with significant concen-
trations of any potential electron acceptor.
Sulfate was present in Patuxent River
materials and eventually became a sus-
pected electron acceptor. To enrich fer-
mentative/methanogenic microorganisms,
aquifer solids (Pensacola, PL) were incu-
bated with basal mineral medium and vi-
tamins; carbon dioxide was added as the
only exogenous electron acceptor.
Construction and Maintenance
of Microcosms and
Enrichments
Microcosms and enrichments were pre-
pared under strictly anaerobic conditions
in an anaerobic glove box *(Coy Labora-
tory Products, Inc., Ann Arbor, Ml). The
microcosms and enrichments were con-
tained in glass, 250-mL, screw-cap bottles
that were sealed with Mininert PTFE valves
*(Alltech Associates, Inc. Deerfield, IL);
the Mininert valves provided a tight seal
for the bottles while allowing for sampling
of headspace and culture medium via sy-
ringe. The combined volume of medium
and wet solids (in microcosms) or medium
and culture inoculum (in enrichments) was
200 mL in most experiments. The re-
maining volume of the bottles was
headspace. Thirty grams (wet wt.) of sol-
ids were used in Patuxent River micro-
cosms and 100 grams were used in
Pensacola microcosms. Five aromatic
hydrocarbons (benzene, toluene,
ethylbenzene, and o- and p -xylenes) were
initially spiked at concentrations in the
range of 40 to 100 p.M per compound.
Sterile controls were removed from the
glove box after sealing and were auto-
claved at 121°C. Incubation was carried
out at 35°C in an anaerobic glove box.
Replicate microcosms and controls were
used in all the experiments. After degra-
dation had begun, regular re-spiking with
aromatic hydrocarbons and sulfate was
performed as necessary.
Growth Media and Ferric Iron
Source
The composition of the defined mineral
medium used for all microcosms and en-
richments in the ferric iron-reduction and
sulfate reduction studies (Medium 1) was
based on medium used by Lovley and co-
workers. This medium included the fol-
lowing compounds at the concentrations
(mM) specified in parentheses: NaHCO
(30), NH4CI (28), NaHPO4- HO (4.4),
NaCI (1.7), KCI (1.3), CaCI2-2H2O (0.68),
* Mention of trademarks or commercial products does
not constitute endorsement or recommendation for
use by the U.S. Environmental Protection Agency.
MgCL- 6H2O (0.49), MgSO • 7H2O (0.41),
MnCI • 4KO (0.025), and r4a2MoO4- 2H2O
(0.004). The final pH of the medium was
approximately 7.
A medium designed to support fermen-
tative/methanogenic bacteria (Medium 2)
included the following compounds at the
concentrations (mM) specified in paren-
theses: NaHCO, (14.3), NH.CI (10),
KH2P04(2.0), K2HP04 (2.0), MgSO4-7 H2O
(0.51), CaCI • 2H2O (0.48), FeCI2- 4H2O
(0.1). In addition, Medium 2 contained
trace minerals, vitamins, resazurin (a re-
dox indicator), and amorphous ferrous sul-
fide as a reducing agent. The final pH of
the medium was approximately 7, but was
later adjusted to 6 because the particular
methanogenic communities operated bet-
ter at a lower pH.
Iron was added to microcosms in the
form of amorphous Fe(OH)3. This iron
phase was prepared by neutralizing a 0.1
M ferric chloride solution with sodium hy-
droxide. The amorphous Fe(OH)3 was
prepared with sterilized glassware and re-
agents that were prepared in sterilized
Milli-Q water, but the iron phase itself could
not be autoclaved because the elevated
heat and pressure would have facilitated
crystallization, which was not desired.
Soil and Sediment Inocula
Patuxent River soil was collected at the
Naval Air Station, Patuxent River (MD) in
September, 1987 and was provided by
Ron Hoeppel of the Naval Civil Engineer-
ing Laboratory (Port Heuneme, CA). The
Patuxent River site was extensively con-
taminated with aviation fuel (e.g., JP-5).
The sample was collected near a hydro-
carbon seep in a marshy area and was
received in water-saturated form in a plas-
tic, screw-capped container, h was stored
at 4°C until its use in microcosms roughly
two years after collection. The soil was
fine-grained and appeared to be rich in
organic matter (including some plant detri-
tus).
Aquifer solids from Pensacola, FL, were
provided by E.M. Godsy (U.S. Geological
Survey, Menlo Park, CA). The Pensacola
aquifer (which is a U.S. Geological Survey
national research demonstration area),
consists of fine-to-coarse sand deposits,
interrupted by discontinuous silts and clays.
The upper 30 m of the aquifer are con-
taminated by creosote and pentachloro-
phenol. The samples were obtained from
an actively methanogenic, sandy zone of
the aquifer, downgradient from the con-
tamination source, at a depth of approxi-
mately 6 m. The groundwater at this depth
contained tens of mg/L of nitrogen hetero-
cycles, simple polynuclear aromatic hy-
-------
drocarbons, and phenols. The sampling
was performed with a hollow-stem auger
and a split-spoon core sampling device.
After the sampler was withdrawn from the
borehole and split lengthwise, a portion of
the core was removed with a sterile
spatula. The center of the core was then
subsampled by pushing a sterile brass
tube into the core, extruded with a sterile
syringe plunger, and stored (at 4°C) in
sterile, sealed containers previously
flushed with argon.
Experimental Design
Many experimental trials for this project
were attempts to screen for activity of
iron-reducing bacteria. Thus, experimen-
tal design typically consisted of setting up
parallel series of microcosms, one with
and one without added iron. Two micro-
cosm experiments with this general de-
sign were performed with Patuxent River
material (Experiments PR1 and PR2). En-
richments of certciin iron-amended micro-
cosms were prepared in an anaerobic
glove box by shaking the microcosm, re-
moving 20 percent (by volume) of the
combined liquid and solids, adding this
inoculum to a new bottle, and diluting to
200 ml with fresh medium. Series of
enrichments were prepared after 88 and
235 days of incubation of selected micro-
cosms from Experiment PR2.
In the experiments with fermentative/
methanogenic bacteria, two types of mix-
tures of aromatic compounds were spiked
to the microcosms. In the first case, the
mixture consisted only of aromatic hydro-
carbons (toluene, ethylbenzene, o-xylene,
and p-xylene), each at a concentration of
ca. 40 u.M. In the second case, p-cresol
(0.46 mM), a putative intermediate of tolu-
ene transformation under anaerobic con-
ditions, was added to the hydrocarbon
mixture. No other organic amendments
were made and no exogenous electron
acceptors were added, except CO2.
Analytical Methods
Aromatic substrates were measured by
a static headspace technique using an
HP Model 5890A gas chromatograph
'(Hewlett-Packard Company, Palo Alto,
CA) with a HNU Model PI 52-02A photo-
ionization detector (10.2 eV lamp; HNU
Systems, Inc., Newton, MA) and a 30-m
DB-624 megabore fused silica capillary
column (3.0 u.m film thickness; J & W
Scientific, Folsom, CA). Analyses were
isothermal (65°C) and splitless. Sampling
and analysis of headspace from micro-
cosms, enrichments, and standards was
performed identically: 300 \iL of
headspace was sampled through a
Mininert valve with a 500 p.L gas-tight
syringe that included a PTFE plunger tip
and a side-port needle.
HCI-extractable Fe(ll) was measured as
described by Lovley and Lonegran. An
aliquot of 0.1 - 0.2 g of culture medium
was removed via syringe and was weighed
into a glass vial containing 5.0 mL of 0.5
M HCI. After approximately 15 minutes of
acid extraction, 0.2 ml of the mixture was
added to a glass vial containing 5.0 mL of
ferrozine (1 g/L) in 50 mM HEPES buffer
(adjusted to pH 7). After being mixed for
15 seconds, the mixture was filtered
through a 0.2 ^im, Nylon 66 syringe filter
and the absorbance at 562 nm was mea-
sured with an HP Model 8451A diode
array spectrophotometer (Hewlett-Packard
Company, Palo Arto, CA).
Sulfate in filtered culture medium was
determined by ion chromatography (Dionex
Series 4000i with a Nelson Analytical Chro-
matography Software system) equipped
with a HPIC-AS4A column (Dionex, Sunny-
vale, CA), an anion micro membrane sup-
pressor, and a conductivity detector.
Analyses were isocratic, with a sodium
bicarbonate (0.75 mM) / sodium carbon-
ate (2.2 mM) eluant. Ions were identified
and quantified by comparing retention
times and peak areas to those of external
standards.
Results and Discussions
Iron's Relationship to Toluene
Degradation In Patuxent River
Microcosms
Results of the first microcosm experi-
ment with Patuxent River material (Ex-
periment PR1) are shown in Figure 1 in
terms of toluene concentration versus time
over the first two months of incubation.
The data in Figure 1 represent averages
of three groups of microcosms: (1) two
autoclaved controls (one with added iron
and one without), (2) three microcosms
without added amorphous Fe(OH)3, and
(3) four microcosms with added amorphous
Fe(OH) [ca. 12 mM Fe(lll) on Day 0].
Although rates of toluene degradation for
microcosms with and without added iron
were very similar for the first 3 to 4 weeks
of incubation, the microcosms with added
iron clearly had faster toluene degrada-
tion rates starting at Day 30. These dif-
ferences in rates continued throughout the
next 30 days of incubation. Note that the
microcosms with added iron received ad-
ditional toluene on Day 44, whereas
those without added iron did not. The
effect of the presence/absence of iron on
toluene degradation rate was reproduc-
ible in this study. As an indication of the
variability among replicates, the average
amount of toluene that had been degraded
by Day 60 in microcosms with added iron
(0.35 ± 0.02 mM; mean ± s.d.) was signifi-
cantly greater (P < 0.001) than the aver-
age among replicates without added iron
(0.20 ± 0.02 mM).
In the second microcosm experiment
(Experiment PR2), as in the first, the pres-
ence of amorphous Fe(OH)3 had an effect
on toluene degradation, but the effect was
qualitatively different in the two experi-
ments. Toluene concentration versus time
is shown in Figure 2 for the first 47 days
of incubation of five microcosms and two
controls. Toluene was not degraded in
any microcosms during the first month of
incubation. However, the addition of ca.
10 mM amorphous Fe(OH)3 to the three
microcosms that initially contained added
iron (Microcosms C8, C9, and C10) initi-
ated toluene degradation within a few days
in each of those microcosms. Two micro-
cosms that did not receive amorphous
Fe(OH), over the period shown (Micro-
cosms C5 and C6) did not degrade tolu-
ene until roughly 40 days later than the
iron-amended microcosms.
Hypotheses were developed to explain
the apparent stimulation of toluene degra-
dation by iron, including the possible need
for iron as a micronutrient required for the
synthesis of enzymes involved in toluene
degradation, the possible role of iron in
reducing sulfide toxicrty, and the possible
presence of ferric iron-reducing bacteria
that were syntrophically associated with
sulfate-reducing bacteria. A preliminary
experiment with Patuxent River enrich-
ments was performed in an attempt to
narrow down the range of plausible hy-
potheses. This experiment, which focused
on the importance of iron oxidation state
(ferric vs. ferrous) in stimulating toluene
degradation, was not conclusive; however,
the results suggested that the hypotheses
proposing iron as a limiting micronutrient,
or as an agent to reduce sulfide toxicity,
were most plausible.
Interrelationships Among
Toluene Degradation, Sulfate
Reduction, and Iron Reduction
In Patuxent River microcosms and en-
richments, toluene degradation, sulfate re-
duction, and ferric iron reduction appeared
to be strongly linked. An example of these
relationships in a Patuxent River micro-
cosm is shown in Figure 3, in which cu-
mulative appearance of Fe(ll) (an indica-
tion of ferric iron reduction) is shown on
the far left y axis, and cumulative sulfate
reduction and cumulative toluene degra-
dation are shown on the right hand side of
the figure. As shown in the figure, not
only do these processes appear to pro-
ceed simultaneously, but their relative rates
-------
0.20
0.15-
* - - Controls (n=2)
--••-- w/0 Fe(OH)3 (n=3)
•— with Fe(OH)3 (n=4)
0.00
0 10 20 30 40 50
Time (days)
Flgun 1. Average toluene concentrations vs. time in Patuxent River (Experiment PR1) microcosms. Arrows indicate amendments of toluene.
0.20
0.15-
0.10-
o
H
0.05
0.00
Cl (control)
C2 (control)
C5 (w/o iron)
C6 (w/o iron)
C8 (w/iron)
C9 (wAron)
C10(w/iron)
20 30
Time (days)
40
Flgun 2. Toluenevs. time in Patuxent River (Experiment PR2) microcosms. Filled arrows represent amendment of ca. 10mMFe(IH). Theoriginal
amount of Fe(lll) added to Microcosms C8, C9, and C10 was ca. 20 mM.
-------
appear to be constant. Similar plots were
obtained for other microcosms and en-
richments.
Several lines of evidence suggest, but
do not prove, that toluene degradation
was directly linked to sulfate reduction in
microcosms and enrichments: (1) the two
processes were synchronous, (2) the ob-
served Stoichiometric ratios of sulfate con-
sumed/toluene consumed were consistent
with the theoretical ratio for the complete
oxidation of toluene to bicarbonate coupled
with the reduction of sulfate to hydrogen
sulfide, and (3) toluene degradation ceased
when sulfate was depleted, and con-
versely, sulfate reduction ceased when
toluene was depleted.
The synchronism of toluene degrada-
tion and sulfate reduction is apparent in
Figure 3 and is supported by the very
strong correlation coefficients for regres-
sions of cumulative toluene degradation
vs. cumulative sulfate reduction over time
(typically r2 > 0.95 for microcosms and
enrichments). The ratio of sulfate con-
sumedAoluene consumed for the micro-
cosm shown in Figure 3 was 4.2. In
Patuxent River enrichments, which were
less susceptible than the microcosms to
complicating effects related to the pres-
ence of soils, the ratio typically ranged
from 3.5 to 4.2 in various experiments.
These values approximate the theoretical
ratios ranging from 4.5 (toluene oxidation
to bicarbonate with no bacterial cell growth;
Equation 1) to 4.0 (toluene oxidation to
bicarbonate with estimated cell growth;
Equation 2).
The observed values of this ratio and
the consistency of the ratio over months
of monitoring provide preliminary evidence
that toluene was oxidized to bicarbonate
by sulfate-reducers. Further evidence of
the link between toluene degradation and
sulfate reduction was the apparent de-
pendence of toluene degradation on the
presence of sulfate, and conversely, the
apparent dependence of sulfate reduction
on the presence of toluene (shown for an
enrichment in Figure 4).
If toluene oxidation and sulfate reduc-
tion were directly linked, what is the ex-
planation for ferric iron reduction, which
occurred concurrently with these two pro-
cesses? Two possible explanations for
ferric iron reduction are (1) direct coupling
with toluene oxidation (where ferric iron
could have served as a terminal electron
acceptor for toluene ) and (2) coupling
with sulfate reduction (where ferric iron
could have served as the electron accep-
tor for the oxidation of biogenic hydrogen
sulfide). Stoichiometric ratios were used
to examine the likelihood of these two
explanations, although data for other vari-
ables would be required to reach more
definitive conclusions. The ratio of Fe(lll)
reduced/toluene consumed was used to
investigate the possibility that ferric iron-
reducing bacteria were oxidizing toluene.
The observed ratios were considerably
lower than the theoretical ratio of 36 ex-
pected for toluene oxidation to bicarbon-
ate coupled to ferric iron reduction (e.g.,
Equation 3).
For example, the observed ratio for the
microcosm shown in Figure 3 was 12,
which suggested that not enough ferric
iron had been reduced to account for com-
plete toluene oxidation. Thus, stoichiom-
etry suggested either that ferric iron re-
ducers were not involved in toluene oxi-
dation or that they carried out incomplete
oxidation. Indeed, the ratio of sulfate con-
sumed/toluene consumed (discussed ear-
lier) further suggests that ferric iron reduc-
ers played little, if any, role in toluene
degradation.
A different Stoichiometric ratio supported
the contention that ferric iron reduction
could have been the result of an oxida-
tion-reduction reaction between ferric iron
and biogenic hydrogen sulfide. The ob-
served ratio of Fe(lll) reduced/sulfate re-
duced was used to investigate the pos-
sible reactions between hydrogen sulfide
and amorphous Fe(OH)3. For this analy-
sis, it was assumed, based on extensive
literature about sulfate-reducing bacteria,
that all reduced sulfate was converted to
hydrogen sulfide. Potential reactions be-
tween amorphous Fe(OH)3 and hydrogen
sulfide that were considered for this analy-
sis included the oxidation of hydrogen sul-
fide to either elemental sulfur or thiosul-
fate. The reactions were chosen based
on reports in the geochemical literature of
the abiotic, anoxic or oxygen-limited reac-
tions of goethrte or amorphous Fe(OH)3
with hydrogen sulfide; in these studies,
the rapid formation of ferrous sulfide, el-
emental sulfur, and/or thiosulfate was ob-
served. For the microcosm shown in Fig-
ure 3, the ratio of Fe(lll) reduced/sulfate
reduced was consistent with the formation
of thiosulfate but not elemental sulfur.
Overall, the observed Stoichiometric ratios
relating Fe(ll) appearance, toluene disap-
pearance, and sulfate disappearance for
that microcosm were consistent with Equa-
tion 4, which represents toluene oxidation
coupled with sulfate reduction, and the
reduction of ferric iron by hydrogen sulfide
to form thiosulfate.
Methanogenlc Degradation of
Toluene and o-Xylene by
Microorganisms from the
Pensacola Aquifer
Pensacola microcosms were initially fed
mixtures of benzene, toluene, ethylben-
zene, o-xylene, and p-xylene at low con-
centrations (120 u.M total hydrocarbons).
Two microcosms developed activity toward
toluene and subsequently toward o-xylene.
Toluene degradation started after approxi-
mately 100 - 120 days of incubation,
whereas o-xylene degradation started af-
ter 200 - 255 days of incubation. Ben-
zene, ethylbenzene, and p-xylene were
not degraded in any microcosms. Auto-
claved controls exhibited no degradation
of any aromatic substrates. Upon re-feed-
ing active microcosms with toluene (100
u.M) and o-xylene (100 u,M), the degrada-
tion of both compounds resumed immedi-
ately.
After the third re-feeding, the micro-
cosms were used as the source for en-
richment of suspended consortia degrad-
ing toluene and o-xylene. This was ac-
complished in two steps. In the first step,
10 g of aquifer solids and 20 ml of the
culture fluid from the microcosms were
transferred into 180 ml_ of Medium 2, and
amended with 50 u,M toluene and/or o
xylene. Transformation activity was de-
tected after one to two weeks of incuba-
tion. Upon re-feeding with the aromatic
hydrocarbons, the degradation resumed
and was completed in two to three weeks.
In the second step of enrichment, only the
liquid portion of these primary enrichments
(30 ml_) was transferred into Medium 2
(170 ml). The degradation activity was
retained.
Eq.1 C7H,+4.5 SO42 + 3 H2O » 2.25 H2S + 2.25 HS + 7 HCO3- + 0.25 H'AG01 - -49 kcal/reaction
Eq. 2 C7H, + 4.03 SO42 + 0.19 NH/ + 0 75 CO2 + 3.18 H2O -2.02 H2S + 2.02 HS + 0.19 C5H7O2N (cells) + 6.8 HCO3 + 0.94 H*
Eq. 3 C7H. + 36 Fe(OH)3 (s) + 29 HCO3 - 36 FeCO3 (s) + 58 H2O + 29 OH
Eq. 4 C7H, + 11.8 Fe(OH)3 (s) + 4.5 SO,2 +3.3 HCO3 » 1.48 S2O32 +1.54 FeS (s) + 10.3 FeCO3 (s) + 18.5 H2O + 9.34 OH
5
-------
13
E
3
u
Fe(II) (diss. and partic.)
Sulfate
Toluene
o
1.0
.8
•a
•a
0.5
0.0J
g
OH
I
3
40 60
Time (days)
Figure 3. Cumulative ferric iron reduction, sulfate reduction, and toluene degradation vs. time fora Patuxent River microcosm.
o>
§
0.30
0.25-
0.20-
0.15-
0.10-
0.05-
0.00
-1.5
I
is
3
GO
-0.5
0.0
57 59
Time (days)
61
63
Figure 4. Mutual dependence of toluene degradation and sulfate reduction for a Patuxent River enrichment. Arrows indicate amendment of toluene
or sulfate. Data points represent the averages of duplicates.
-------
The stable mixed cultures (both primary
and secondary enrichments) were main-
tained on toluene and oxylene as sole
carbon sources for over a year. They
degraded toluene and o-xylene at an ap-
proximate rate of 4 fiM/day (Figure 5).
The substrates were continuously con-
verted to CO, and CH4. The original mi-
crocosms and the first transfers from the
microcosms, which still contained a small
amount of aquifer solids, degraded tolu-
ene and o-xylene (fed at a concentration
of 50 u.M each) completely in less than
two weeks. Secondary transfers, which
no longer contained a visible amount of
aquifer solids, degraded the substrates at
a considerably slower rate (three to four
times slower). It is suspected that the
solids were necessary for attachment of
the microorganisms, or that the solids con-
tained a trace nutrient which might have
been essential for the transformations.
The degradation of toluene and o-xy-
lene in suspended mixed cultures was
associated with cell growth. Cell counts
were determined by acridine orange stain-
ing in conjunction with epifluorescence
microscopy. Stable mixed cultures
reached an average of 10* cells/ml at the
peak of the logarithmic growth phase. The
doubling time for the stable mixed culture
utilizing toluene as the sole carbon and
energy source was about 9 days. The
initial rate of growth and the rate of tolu-
ene degradation were both highly depen-
dent on initial cell count.
The effects of certain environmental con-
ditions on the rate of toluene degradation
were investigated. The cultures degraded
toluene faster at 35° C than at 20° C, which
is to be expected for enzyme-mediated
reactions. pH 6 was more favorable than
pH 7, and pH 8 appeared to be unfavor-
able. This result was consistent with the
conditions in the contaminated aquifer,
where a groundwater pH of 6 or below
was predominant. The addition of exog-
enous electron acceptors (nitrate or sul-
fate) slowed down toluene degradation,
indicating that the active community con-
sisted of fermentative and methanogenic
bacteria that were acclimated to the con-
ditions of methanogenic fermentation.
Sulfide also slowed the transformation
down, in accordance with observations of
other investigators that sulfide is inhibitory
to methanogens.
Some of the IPensacola aquifer mate-
rial, previously used in experiments of deg-
radation of creosote constituents (cour-
tesy of E.M. Godsy, U.S. Geological Sur-
vey), was used to set up new microcosms
for studying adaptation to toluene degra-
dation under various environmental condi-
tions. The following conditions were tested:
1) toluene (80 u.M) alone, with microcosms
incubated statically as before; 2) toluene
alone, with microcosms shaken vigorously
once per day; 3) toluene with p-cresol
(0.75 mM); 4) toluene with acetate (25
mM). The lag time before the onset of
toluene degradation was approximately 50
days for both static and agitated micro-
cosms. The acclimation lag for toluene
was shorter in these microcosms than in
the first study (in which the acclimation
lag for toluene was 100 - 120 days) be-
cause the aquifer material used for these
adaptation studies had already undergone
an enrichment step during creosote-deg-
radation studies. This lag time was in-
creased to about 100 days if p-cresol was
present, indicating that p-cresol was uti-
lized as a preferential substrate. Toluene
degradation started 50 days after all the
p-cresol had been depleted. Therefore,
the addition of p-cresol, a putative inter-
mediate in toluene degradation, did not
appear to shorten the acclimation lag. The
addition of acetate prolonged the acclima-
tion lag; acetate-amended microcosms did
not adapt to toluene degradation for more
than 100 days after the depletion of ac-
etate. This finding supports the hypoth-
esis that the acetoclastic methanogens
are not the rate-limiting population in the
toluene degradation process, and suggests
that acetate prevented the enrichment of
the capable aromatic-degrading commu-
nity on the aquifer material.
Conclusions and
Recommendations
Patuxent River Soils
The work with microfbra enriched from
hydrocarbon-contaminated Patuxent River
soil resulted in two general findings: (1)
degradation of toluene under sulfate-re-
ducing conditions appears to occur de-
spite the relatively small amount of energy
that this process can yield to bacteria,
and (2) the presence of iron [in the form
of amorphous Fe(OH)J can either greatly
facilitate the onset of toluene degradation
under sulfate-reducing conditions or can
accelerate the rate once degradation has
begun. Both findings relate geochemical
site conditions (e.g., oxidation-reduction
potential, the presence of sulfate, the pres-
ence of iron-containing minerals) to the
potential for in-situ biological restoration
of aquifers contaminated with refined pe-
troleum products, such as gasoline and
aviation fuel.
Pensacola Aulfer Materials
In fermentative/methanogenic micro-
cosms containing creosote-contaminated
aquifer material (Pensacola, FL), the ini-
tial acclimation lag before the onset of
toluene and o-xylene degradation was two
to three months. Although the initial ad-
aptation period was long, the degrada-
tion, once initiated, was relatively rapid,
and the rate increased upon each subse-
quent re-feeding. Benzene, ethylbenzene,
and p-xylene were not degraded. Stable
suspended methanogenic consortia en-
riched from Pensacola aquifer microcosms
degraded 50 u.M of toluene and o-xylene
to CO2 and CH4 in one to two weeks. The
length of the adaptation lag and the rates
of degradation were strongly influenced
by environmental conditions, most notably
the presence of other more readily de-
gradable substrates.
General Conclusions and
Recommendations
All of the inoculum sources tested in
this project (including some materials not
discussed in this summary) showed at
least some evidence of anaerobic catabo-
lism of alkylbenzenes, which indicates that
anaerobic degradation of these com-
pounds might be widespread in the sub-
surface environment. In contrast, degra-
dation of benzene was not observed us-
ing any of the inocula tested in this project,
and benzene has proven to be highly re-
calcitrant in other studies as well. The
type of anaerobic degradation of aromatic
hydrocarbons that will occur at a specific
site will depend on the contamination his-
tory of that site, the presence of specific
microbial groups, the availability of exog-
enous electron acceptors, and a variety of
environmental factors, including the geo-
chemical characteristics of the site.
Additional efforts are warranted in order
to learn more about the physical, chemi-
cal, and biochemical factors that influence
anaerobic catabolism of aromatic hydro-
carbons. With respect to the Patuxent
River and Pensacola microflora studied
for this project, important areas for future
work include (1) isolation and character-
ization of pure, toluene-degrading cultures,
or, if pure cultures cannot be obtained,
examination of syntrophic relationships that
appear to be required for toluene degra-
dation; (2) detailed examination of the role
of iron in facilitating toluene degradation
in Patuxent River enrichments (e.g., the
importance of iron oxidation state) and
closer examination of the relevance of this
process to in-situ toluene degradation in
aquifers under sulfate-reducing conditions;
and (3) determination of the metabolic
pathways of toluene and o-xylene degra-
dation under methanogenic and sulfate-
reducing conditions.
The results of this study and the work
of other researchers suggest that anaero-
-------
bic transformation of alkylbenzenes may
be ubiquitous in the subsurface. Anaero-
bic microorganisms could be taken ad-
vantage of at contaminated sites where it
is not possible to introduce sufficient oxy-
gen for aerobic degradation. However,
samples from such sites would require
preliminary laboratory investigation to de-
termine whether (and which) exogenous
electron acceptors were required, whether
the addition of these acceptors and/or nu-
trients was necessary at the site, and
which environmental factors were most
important in controlling the anaerobic bio-
degradation process.
References
3 Bak, F. and F. Widdel, 1986. Anaerobic
degradation of phenol and phenol
derivatives by Desulfobacterium
phenolicum sp. nov. Arch. Microbiol.
146:177-80.
1 Dotting, J.J. Zeyer, P. Binder-Eicher,
and P.P. Schwarzenbach, 1990.
Isolation and characterization of a
bacterium that mineralizes toluene in
the absence of molecular oxygen. Arch.
Microbiol. 154:336-41.
2 Lovely, D.R. and D. J. Lonergan, 1990.
Anaerobic oxidation of toluene,
pheenol, and p-cresol by the
dissimilatory iron-reducing organism,
GS-15. Appl. Environ. Microbiol.
56:1858-64.
4 Szewzyk, R. and N. Pfenning, 1987.
Complete oxidation of catechol by the
strictly anaerobic sulfate-reducing
Desulfobacterium catecholium sp.nov.
Arch. Microbiol. 147:163-8
0.08
0.06-
0.04-
0.02-
0.00
Toluene sterile control
o-xylene sterile control
Toluene
o-xylene
5 10
Time (days)
Flgun 5. Simultaneous degradation of toluene and o-xylene by a mixed culture enriched from Pensacola aquifer sediment under methanogenic
conditions.
•&U.S. GOVERNMENT PRINTING OFFICE: 1992 - 648-080/40218
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Many R. Better, Elizabeth A. Edwards, Dunja Grbic-Galic, and Martin Reinhardare with
Stanford University, Stanford, CA 94305-4020; Stephen R. Hutchln* (also the
Project Officer, seebelow) is with Robert S. Kerr Environmental Research Laboratory,
U.S. Environmental Protection Agency, Ada, OK 74820
The complete report, entitled "MicrobiaJ Degradation of Akytbenzones Under Sulfate-
Redudng and Methanogenk Conditions," (Order No. PB91-212 324/AS; Cost:
$17.00, subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Robert S. Kerr Environmental Research Laboratory
U.S. Environmental Protection Agency
Ada, OK 74820
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati, OH 45268
BULK RATE
POSTAGE & FEES PAID
EPA PERMIT NO. G-35
Official Business
Penalty for Private Use $300
EPA/600/S2-91/027
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