EPA/600/2-91/027
July 1991
MICROBIAL DEGRADATION OF ALKYLBENZENES UNDER
SULFATE-REDUCING AND METHANOGENIC CONDITIONS
by
Harry R. Beller, Elizabeth A. Edwards, Dunja Grbic-Galic, Martin Reinhard
Department of Civil Engineering
Stanford University
Stanford, California 94305-4020
CR-815721
Project Officer
Stephen R. Hutchins
Processes and Systems Research Division
Robert S. Kerr Environmental Research Laboratory
Ada, Oklahoma 74820
ROBERT S. KERR ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
ADA, OKLAHOMA 74820
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TECHNICAL REPORT DATA
(Please read tnslrucuons on the reverse before com/ilel
1. REPORT NO.
EPA/600/2-91/027
2.
3
y
4. TITLE and subtitle
MICROBIAL DEGRADATION OF ALKYLBTSNZENES UNDER
REDUCING AND METHANOGENIC CONDITIONS •
SULFATE-
5. REPORT DATE
July 1991
6 PERFORMING ORGANIZATION CODE
7. AUTHoms)
IF. BELLI®, E. EDWARDS, D. GRBIC-GALIC, AND M.
RE1NHARD
a. 'EflFCflM'NG ORGANISATION REPORT NO
9. PE RFORM1NG ORGANIZATION NAME AND ADDRESS
DEPARTMENT OF CIVIL ENGINEERING
10. PROGRAM ELEMENT NO.
CA2H1.A
STANFORD UNIVERSITY
STANFORD CA 94305-4020
11. CONTRACT.'GRANT NO.
CR-8L5721
¦
12. SPONSORING AGENCY NAME AND ADDRESS
ROBERT S. KERR ]£NVIROEKENTAL RE,SEARCH LAB. -
ADA, OK
13. 7VPE OF REPORT AND PERIOD COVERED
FINAL REPORT 05/89 - 04/91
U.S. ENVIRONMENTAL PR0TECTI0
P.O. BOX 1198
ADA, OK 74820
M AGENCY
11 SPONSORING AGENCY CODE
EPA/600/15
15. SUPPLEMENTARY NOTES
PROJECT OFFICER: Stephen R.
Hutchins, FTS: 743-2327
16. ABSTRACT
Aquifer solids and soils obtained from various hydrocarbon-contaminated sites were used to Investigate the
ability of indigenous microorganisms to degrade monoaromalic hydrocarbons under strictly anaerobic conditions.
In anaerobic microcosms inoculated with fuel-contaminated soil from the Patuxent River site, toluene degradation
occurred concomitantly with sulfate reduction and ferric iron reduction. Similar results were obtained with
suspended enrichments derived from the microcosms. Stoichiometric data and other observations suggested that
sulfate reduction Was closely linked to toluene degradation, whereas iron reduction was a secondary, potentially
abiotic, reaction between ferric Iron and biogenic hydrogen sulfide. The presence of mlllimolar concentrations
of amorphous Fe(OH)3 in Patuxent River microcosms and enrichments either greatly facilitated the onset of
toluene degradation or accelerated the rate once degradation had begun. Fermentative/methanogenic microcosms
and enrichments that degraded toluene and o-xylene without added exogenous electron acceptors (except C02)
were developed from creosote-contaminated Pensacola samples. The microcosms initially underwent an
acclimation lag of several months; however, once the degradation of aromatic hydrocarbons was Initiated, it
proceeded at a relatively rapid rate, and it was complete (resulting in mineralization to C02 and CH4). Benzene,
ethylbenzene, and p-xylene were not degraded.
1 7
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DISCLAIMER
Although the information in this document has been funded wholly or in part by the United
States Environmental Protection Agency under CR-81572] to Stanford University, it does not
necessarily reflect the views of the Agency and no official endorsement should be inferred.
Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
All research projects making conclusions or recommendations based on environmentally
related measurements and funded by the Environmental Protection Agency are required to
participate in the Agency Quality Assurance Program. This project was conducted under an
approved Quality Assurance Project Plan. The procedures specified in this plan were used without
exception. Information on the plan and documentation of the quality assurance activities and
results are available from the Principal Investigator.
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FOREWORD
EPA is charged by Congress to protect the Nation's land, air, and water systems. UndeT a
mandate of national environmental laws focused on air and water quality, solid waste management
and the control of toxic substances, pesticides, noise and radiation, the Agency strives to formulate
and implement actions which lead to a compatible balance between human activities and the ability
of natural systems to support and nurture life.
The Robert S. Kerr Environmental Research Laboratory is the Agency's center of expertise
for investigation of the soil and subsurface environment. Personnel at the Laboratory are
responsible for management of research programs to: (a) determine the fate, transport, and
transformation rates of pollutants in the soil, the unsaturated and the saturated zones of the
subsurface environment; (b) define the processes to be used in characterizing the soil and
subsurface environment as a receptor of pollutants; (c) develop techniques for predicting the effect
of pollutants on ground water, soil, and indigenous organisms; and (d) define and demonstrate the
applicability and limitations of using natural processes indigenous to the soil and subsurface
environment, for the protection of this resource.
This report describes research conducted to evaluate the ability of indigenous aquifer- and
soil-derived microorganisms to degrade simple aromatic hydrocarbons, such as toluene, under
anaerobic conditions. Monoaromatic hydrocarbons are pervasive groundwater contaminants in
areas near leaking underground fuel storage tanks and other uncontrolled releases of gasoline and
aviation fuel. The research specifically assesses degradation under sulfate-Teducing and
methanogenic conditions, and evaluates the effects of certain geochemical factors, such as the
presence of iron minerals, on degradation rates.
Clinton W. Hall
Director
Robert S. Kerr Environmental
Research Laboratory
iii
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ABSTRACT
Aquifer solids and soils obtained from various hydrocarbon-contaminated sites were used
to investigate the ability of indigenous microorganisms to degrade monoaromatic hydrocarbons
under strictly anaerobic conditions. Hydrocarbon-degrading microflora from two sites, an aviation
fuel storage facility located near the Patuxent River (MD) and a creosote-contaminated aquifer near
Pensacola (FL), were studied most extensively.
Anaerobic microcosms containing fuel-contaminated soil from the Patuxent River site
displayed evidence of toluene degradation, sulfate reduction, and ferric iron reduction; all three
processes appeared to be strongly linked and proceeded concomitantly. Stoichiometry and other
considerations indicated that toluene degradation was closely linked to the activity of sulfate-
reducing bacteria. To our knowledge, this is one of the first reports of the degradation of
alkylbenzenes under sulfate-reducing conditions. Stoichiometric data were also used to evaluate
whether ferric iron reduction was more closely linked with toluene oxidation (where ferric iron
could serve as a terminal electron acceptor for toluene) or with sulfate reduction (where ferric iron
could serve as the electron acceptor for the oxidation of biogenic hydrogen sulfide). Although the
data do not yield a definitive conclusion, they indicate that toluene was oxidized to carbon dioxide
by sulfate-reducing bacteria and that the resulting hydrogen sulfide was oxidized in a secondary,
potentially abiotic, reaction with ferric iron.
The presence of millimolar concentrations of amorphous Fe(OH)3 in Patuxent River
microcosms either greatly facilitated the onset of toluene degradation (observed in one experiment)
or accelerated the rate once degradation had begun (observed in another experiment). Several
hypotheses are proposed in this report to explain the effect of added iron on toluene degradation.
Active enrichments were successfully generated from Patuxent River microcosms. These
enrichments behaved similarly to the microcosms in that (1) strongly linked toluene degradation,
sulfate reduction, and iron reduction were observed, and (2) the presence of amorphous Fe(OH)3
markedly enhanced toluene degradation rates.
iv
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Fermentative/methanogenic microcosms that degraded toluene and o-xylene without added
exogenous electron acceptors (except CO2) were developed from creosote-contaminated aquifer
solids derived from the Pensacola site. The microcosms initially underwent an acclimation lag of
several months; however, once the degradation of aromatic hydrocarbons was initiated, it occurred
at a relatively rapid rate, and it was complete (resulting in mineralization to CO2 and CH4).
Benzene, ethylbenzene, and p-xylene were not degraded.
Active methanogenic enrichments were obtained from the Pensacola microcosms. These
enrichments, upon stabilization, mineralized 50 fiM of toluene and o-xylene to CO2 and CH4 in
one to two weeks; and slowly degraded w-xylene. The estimated doubling time of the consortium
was nine days. The presence of a small amount of aquifer solids supported the degradation of
aromatic hydrocarbons and increased the degradation rate. Exogenous electron acceptors (nitrate
and sulfate) slowed down the transformation.
This report was submitted in fulfillment of the grant EPA CR-815721-01-0 by the
researchers in the Environmental Engineering and Science program (Department of Civil
Engineering, Stanford University), under the sponsorship of the U.S. Environmental Protection
Agency. This report covers a period from 5/1/1989 to 4/30/1991, and work was completed as of
4/30/1991.
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CONTENTS
Foreword iii
Abstract iv
Figures vii
Tables ix
Acknowledgments x
1. Introduction 1
2. Conclusions 3
3. Recommendations 6
4. Materials and Methods 7
Construction and maintenance of microcosms and enrichments 7
Experimental design 12
Analytical methods 18
5. Results and Discussion 22
Sulfate reduction and ferric iron reduction in Patuxent River soils 22
Discussion of the role of iron in toluene degradation (Patuxent River
experiments) 38
Nitrate reduction in Patuxent River microcosms 43
Attempts to enrich iron-reducers in Seal Beach and Traverse City
microcosms 45
Methanogenic degradation of toluene and o-xylene by microorganisms
from the Pensacola aquifer 48
References 59
vi
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FIGURES
Number Page
1 Average toluene concentrations vs. time in Experiment PR1 microcosms. 23
2 Cumulative toluene disappearance vs. time in Experiment PR1
microcosms 25
3 Toluene and sulfate vs. time in Experiment PR2 microcosms 26
4 Sulfate vs. toluene consumed in Microcosm C8 28
5 Cumulative Fe(II) appearance and sulfate and toluene disappearance in
Microcosm C8 29
6 Cumulative toluene disappearance vs. time in Microcosms C6 and C8 .. . 33
7 Cumulative toluene disappearance vs. time in two enrichments from
Experiment PR2 34
8 Cumulative Fe(IT) appearance and sulfate and toluene disappearance in
Enrichment E7 36
9 Test of mutual dependence of toluene degradation and sulfate reduction for
a Patuxent River enrichment 37
10 Test of the effect of iron oxidation state on toluene degradation using
Patuxent River enrichments 42
11 Toluene and nitrate, and toluene and sulfate vs. time in Microcosm C3 . .. 44
12 Toluene vs. time in Seal Beach microcosms 46
13 Toluene and ethylbenzene vs. time in two Traverse City microcosms .... 47
14 14c mass balance in suspended cultures fed 14C-labeled toluene 49
15 Degradation of toluene and o-xylene in suspended mixed culture 51
16 Effect of initial cell count on the rate of toluene degradation in a mixed
methanogenic culture 53
17 Cell count vs. time during toluene degradation by a mixed methanogenic
culture 53
vii
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Number Page
18 Effect of temperature on toluene degradation in suspended culture 55,
19 Effect of pH on toluene degradation in suspended culture 55
20 Effects of nitrate (1 mM), sulfate (5 mM), and sulfide (2 mM), all as
sodium salts, on toluene degradation in suspended culture 56
21 The Length of acclimation Lag in toluene-degrading, methanogenic
microcosms under different environmental conditions 57
viii
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TABLES
Number
Page
1
Medium 1 composition
8
2
Medium 2 composition
9
3
Initial conditions for Seal Beach microcosms
13
4
Initial conditions for Traverse City microcosms
13
5
Initial conditions for Experiment PR1 microcosms
14
6
Initial conditions for enrichments of Experiment PR1
14
7
Initial conditions for Experiment PR2 microcosms
15
8
Initial conditions for enrichments (Day 88) of Experiment PR2
15
9
Initial conditions for enrichments (Day 235) of Experiment PR2
15
10
Initial conditions for Experiment PR3 .
16
11
Amendments for Experiment PR2 microcosms
17
12
Theoretical and observed stoichiometry of iron reduction and toluene
oxidation
30
13
Theoretical and observed stoichiometry of iron reduction via hydrogen
sulfide
31
14
Influence of H2 concentration in the headspace on toluene degradation and
methane production in suspended methanogenic cultures degrading
toluene as the sole substrate
52
ix
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ACKNOWLEDGMENTS
We thank the following people, who graciously provided aquifer solids and soils for study:
Michael E. Godsy of the U.S. Geological Survey (Menlo Park, CA), who provided aquifer solids
from the Pensacola, Florida creosote-contaminated site (a U.S. Geological Survey national
research demonstration area); Ron Hoeppel of the Naval Civil Engineering Laboratory (PoiT
Heuneme, CA), who provided the Patuxent River samples; Stephen R. Hutchins of U.S. EPA
(Ada, OK), who provided aquifer solids from the Traverse City, Michigan site; and Harry
Ridgway of the Orange County Water District (Fountain Valley, CA), who provided the Seal
Beach aquifer solids.
x
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SECTION 1
INTRODUCTION
The U.S. EPA has reported that up to 35 percent of the nation's underground fuel storage
tanks may be leaking (U.S. EPA, 1986). Together with surface spill accidents and landfill leachate
intrusion, such leaks greatly contribute to groundwater contamination by gasoline and other
petroleum derivatives. Although most gasoline constituents are readily degraded in aerobic surface
water and soil systems, similar processes in the subsurface are significantly retarded because of
insufficient concentrations of oxygen and/or nutrients, and consequently low numbers of active
aerobic microorganisms. If oxygen and essential microbial nutrients are added, however, the
aerobic biotransformation of major gasoline constituents in groundwater can proceed rapidly. This
was demonstrated by Raymond (1974) and was applied for in-situ treatment of a contaminated
aquifer in the early 1970's (Raymond et al., 1975). This bioreclamation process, with variations,
has been used repeatedly since then (Lee et al., 1988). Unfortunately, it is not always feasible to
introduce enough oxygen into the subsurface. Such limitations lead to the complete consumption
of oxygen by indigenous microorganisms and result in the development of 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 ferric 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 found under
denitrifying conditions (Hutchins et al., 1991; Kuhn et al., 1985, 1988; Major et al., 1988; Zeyer
et al., 1986, 1990), methanogenic conditions (Grbic-Galic and Vogel, 1987; Vogel and Grbic-
Galic, 1986; Wilson et al., 1986, 1987), and ferric iron-reducing conditions (Lovley and
Lonergan, 1990; Lovley et al., 1989). Very recently, pure cultures of organisms that can degrade
alkylbenzenes anaerobically have been isolated (Dolfing et al., 1990; Lovley and Lonergan, 1990).
Despite this research activity, anaerobic degradation of monoaromatic hydrocarbons is not as well
understood as aerobic degradation; further study of anaerobic degradation of these compounds for
the range of potential electron-accepting conditions is warranted.
1
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For this project, the initial objective was to investigate the degradation of benzene and
selected alkylbenzenes under fermentative (including methanogenic) and ferric iron-reducing
conditions. Sulfate-reducing and nitrate-reducing conditions were to be studied only as
environmental factors that could influence fermentative and iron-reducing processes. As it turned
out, active fermentative/methanogenic microcosms (and subsequently stable methanogenic
consortia) that could degrade toluene and o-xylene were indeed successfully enriched. However,
iron-reducing hydrocarbon-degrading communities could not be obtained. Instead, screening for
iron reducers led to the enrichment of a sulfate-reducing consortium that could degrade toluene,
which is the focus of this report. Notably, definitive evidence of the coupling of alkylbenzene
degradation to sulfate reduction has not yet been published. However, some co-authors of this
report have observed degradation of toluene and xylene isomers under sulfate-reducing conditions
in studies of fuel-contaminated sediments from Seal Beach, California (Haag et al., in press;
Edwards et al., 1991) and Sewell et al. (1991) recently reported toluene degradation under sulfate-
reducing conditions. In addition, sulfate-reducers are capable of degrading a number of oxygen-
containing aromatic compounds, some of which may be intermediates in anaerobic toluene
degradation (e.g.,p-cresol, benzoic acid, 2- and 4-hydroxybenzoic acid, phenol, catechol,
resorcinol, hydroquinone; Bak and Widdel, 1986; Cord-Ruwisch and Garcia, 1985; Schnell et al.,
1989; Szewzyk and Pfennig, 1987; Widdel etal., 1983).
2
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SECTION 2
CONCLUSIONS
PATUXENT RIVER SOILS
Anaerobic microcosms containing contaminated soil from an aviation fuel storage facility
located near the Patuxent River (MD) displayed evidence of toluene degradation, sulfate reduction,
and ferric iron reduction; all three processes appeared to be strongly linked and proceeded
concomitantly. Stoichiometry and other considerations strongly suggested that toluene degradation
was closely linked to the activity of sulfate-reducing bacteria. To our knowledge, this is one of the
first reports of the degradation of alkylbenzenes under sulfate-reducing conditions. Degradation of
benzene, ethylbenzene, and o- andp-xylenes was not observed in these experiments.
Stoichiometric data were used to evaluate whether ferric iron reduction was more closely
linked with toluene oxidation (where ferric iron could serve as a terminal electron acceptor for
toluene) or with sulfate reduction (where ferric iron could serve as the electron acceptor for
hydrogen sulfide oxidation). The data suggested that toluene was oxidized to carbon dioxide by
sulfate-reducing bacteria and that the resulting hydrogen sulfide was oxidized in a secondary,
potentially abiotic, reaction with ferric iron.
The presence of millimolar concentrations of amorphous Fe(OH)3 in Patuxent River
microcosms either gTeatly facilitated the onset of toluene degradation (observed in one experiment)
or accelerated the rate once degradation had begun (observed in another experiment). Several
hypotheses were proposed to explain the effect of added iron on toluene degradation, including the
assimilation of iron as a limiting micronutrient that was required for the synthesis of enzymes
involved in toluene degradation.
Active enrichments were successfully generated from Patuxent River microcosms. These
enrichments behaved similarly to the microcosms in that (1) strongly linked toluene degradation,
sulfate reduction, and iron reduction were observed, and (2) the presence of amorphous Fe(OH)3
markedly enhanced toluene degradation rates.
3
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PENSACOLA AQUIFER MATERIALS
Fermentative/methanogenic microcosms capable of degrading toluene and o-xylene were
obtained from creosote-contaminated aquifer material (Pensacola, FL). The initial acclimation lag
before the onset of degradation was two to three months. The addition of p-cresol, a putative
intermediate of toluene transformation, may have shortened the acclimation period relative to
microcosms that were not amended with p-cresol. Once the degradation was initiated, it was
relatively rapid, and the rate increased upon each subsequent re-feeding. Benzene, ethylbenzene,
and p-xylene were not degraded.
Stable suspended methanogenic consortia were enriched from the Pensacola microcosms.
These cultures degraded 50 |iM toluene and o-xylene to CO2 and CH4 in one to two weeks. The
doubling time of the cultures was nine days. The presence of a trace amount of aquifer solids
enhanced the biodegradation of aromatic hydrocarbons, indicating the importance of attachment
surfaces and/or trace nutrients for the microorganisms. Exogenous electron acceptors (nitrate or
sulfate) slowed down the degradation of toluene, suggesting that the degrading community was a
well-acclimated fermentative/methanogenic consortium that operated optimally under the conditions
of methanogenic fermentation.
GENERAL CONCLUSIONS
Of the various microbial inocula used in this study, the microflora from some of the
examined sites displayed anaerobic catabolism of aromatic hydrocarbons that can be characterized
as anaerobic respiration (e.g., sulfate reduction or denitrification in Patuxent River microcosms).
These microbial communities required an exogenous electron acceptor to degrade aromatic
. hydrocarbons as electron donors/carbon sources/energy sources. The microflora from other sites,
however, exhibited fermentative/methanogenic catabolism of aromatic hydrocarbons (Pensacola
microcosms), which did not require any exogenous electron acceptors except CO2. Furthermore,
the addition of exogenous electron acceptors to some of these communities (Pensacola cultures)
even slowed down the transformation. It should be noted that the aquifer solids from the
Pensacola site were derived from actively methanogenic zones of this aquifer, which can explain
the enrichment of fermentative/ methanogenic activity.
4
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All of the inoculum sources tested showed at least some kind of anaerobic catabolism of
alkylbenzenes (toluene, xylenes, ethylbenzene), which indicates that anaerobic degradation of these
compounds might be widespread in the subsurface environment. In contrast, degradation of
benzene was not observed using any of the inocula tested in this project, and benzene has proven
to be highly recalcitrant 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 history of that site,
the presence of specific microbial groups, the availability of exogenous electron acceptors, and a
variety of environmental factors, including the geochemical characteristics of the site. Additional
efforts are warranted in order to learn more about the physical and chemical factors that influence
anaerobic catabolism of aromatic hydrocarbons.
5
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SECTION 3
RECOMMENDATIONS
The results of this project leave open several avenues of research that were not explored
because of time constraints. General areas of research that would be productive extensions of this
project include:
• Isolation and characterization of pure, toluene-degrading cultures from Patuxent
River and Pensacola enrichment cultures, or, if pure cultures cannot be obtained,
examination of syntrophic relationships that appear to be required for toluene
degradation.
Determination of the metabolic pathways of toluene degradation under
. methanogenic and sulfate-reducing conditions, and examination of the effect of iron
on the metabolic pathway in the sulfate-reducing (Patuxent River) cultures.
• Examination of the role of iron in facilitating toluene degradation in Patuxent
River enrichments and closer examination of the relevance of this process to in-situ
toluene degradation in aquifers under sulfate-reducing conditions.
The results of this study suggest that anaerobic transformation of alkylbenzenes may be
ubiquitous in the subsurface. Anaerobic microorganisms could be taken advantage of at
contaminated sites where it is not possible to introduce sufficient oxygen for aerobic degradation.
However, samples from such sites would require preliminary laboratory investigation to determine
whether (and which) exogenous electron acceptors were required, whether the addition of these
acceptors and/or nutrients was necessary at the site, and which environmental factors were most
important in controlling the anaerobic biodegradation process.
6
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SECTION 4
MATERIALS AND METHODS
The primary goals of the project were to enrich iron-reducing or fermentative microbial
communities that could degrade monoaromatic hydrocarbons. To enrich iron-reducing bacteria,
the initial experimental approach consisted of screening sediments for hydrocarbon-degrading
activity using a basal mineral medium that was either amended with ferric iron or was not amended
with significant concentrations of any potential electron acceptor. Monitoring originally focused on
aromatic hydrocarbons (the substrates) and the appearance of Fe(II) (as an indication of ferric iron
reduction). Sulfate was present in some sediment inocula (e.g., Patuxent River) and eventually
became a suspected electron acceptor; thus, subsequent monitoring included analysis for sulfate as
well. To enrich fermentative/methanogenic microorganisms, aquifer solids (Pensacola, FL) were
incubated with basal mineral medium and vitamins; carbon dioxide was added as the only
exogenous electron acceptor. Monitoring focused on aromatic hydrocarbons and production of
gaseous products (CO2 and CH4).
CONSTRUCTION AND MAINTENANCE OF MICROCOSMS AND
ENRICHMENTS
Microcosms and enrichments were prepared under strictly anaerobic conditions in an
anaerobic glove box (Coy Laboratory Products, Inc., Ann Arbor, MI). The microcosms and
enrichments were contained in glass, 250-mL, screw-cap botdes 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 syringe. The
combined volume of medium and wet solids (in microcosms) or medium and culture inoculum (in
enrichments) was 200 mL in most experiments, although 170 mL combined volume was used in
early experiments with Seal Beach and Traverse City materials. The remaining volume of the
bottles was headspace. All weighing of medium and sediment was performed in the anaerobic
glove box. Five aromatic hydrocarbons (benzene, toluene, ethylbenzene, and o- and p -xylenes)
were initially spiked at concentrations of roughly 40 to 100 |iM per compound. Sterile controls
were removed from the glove box after sealing and were autoclaved at 121°C. Replicate
7
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microcosms and controls were used in all the experiments. Incubation was carried out at 35°C in
an anaerobic glove box. Although 35°C is higher than typical aquifer temperatures, the choice of
tliis temperature was based in part on the practical consideration that experiments would probably
proceed more rapidly. In addition, the average temperature of the Pensacola aquifer was relatively
high (near 25°C).
After degradation had begun, regular re-spiking with aromatic hydrocarbons and sulfate
was performed as necessary. Aromatic hydrocarbons (in neat form) were injected directly into
active microcosms with a 10-|lL syringe when depleted (typically weekly or biweekly in Patuxent
River and Pensacola samples). For Patuxent River samples, an anaerobic solution of MgS04 was
injected via 3-mL syringe as necessary (addition was performed inside the anaerobic glove box)
and a slurry of amorphous Fe(OH)3 was occasionally added to microcosms and enrichments via
syringe.
Growth medium
The composition of the growth medium used for all microcosms and enrichments in the
ferric iron-reduction studies (Medium 1) was based on Lovley and Phillips (1986a) and is shown
in Table 1. The final pH of the medium was approximately 7.
TABLE 1. MEDIUM 1 COMPOSITION
Compound
Cone.
Cone.
(g/L)
(mM)
NaHC03
2.5
30
NH4CI
1.5
28
NaH2P04H20
0.6
4.35
NaCl
0.1
1.7
KC1
0.1
1.3
CaCl2-2H20
0.1
0.68
MgCl2-6H20
0.1
0:49
MgS04-7H20
0.1
0.41
MnCl2-4H20
0.005
0.025
Na2Mo04-2H20
0.001
0.004
The composition of the growth medium used for fermentative/methanogenic degradation
studies (Medium 2) was a modification of several previous recipes (Owen et al., 1979; Shelton
and Tiedje, 1984; Godsy, 1980; Ridgway et al., 1989; Tongetal., 1990). The composition is
8
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shown in Table 2. The final pH of the medium was approximately 7, but was later adjusted to 6
because the particular methanogenic communities operated better at a lower pH.
TABLE 2. MEDIUM 2 COMPOSITION
Cone.
Compound
Cone. (mg/L)
(mM)
KH2PO4
272
2.0
K2HPO4
348
2.0
NH4CI
535
10.0
MgS04 • 7H20
125
0.51
CaCl2 • 2H20
70
0.48
FeCl2 • 4H20
20
0.1
Trace Minerals:
H3BO3
0.6
9.7 x 10-3
ZnCl2
0.2
1.5 x 10-3
Na2Mo04 • 2H20
0.2
0.8 x 10-3
NiCl2 • 6H2O
1.5
6.3 x IO-3
MnCl2 -4H20
2.0
10.1 x IO-3
CuC12 • 2H20
0.2
1.2 x 10-3
CoC12 • 6H20
3.0
12.6 x 10"3
Na2Se03
0.04
0.2 x 10-3
Al2(S04)3 • 18H20
0.2
0.3 x 10-3
Buffer:
NaHC03
1,200
14.3
Resazurin
1.0
0.004
Reducing Agent:
FeS (amorphous*)
54.8
0.62
Vitamins:
Biotin
0.002
Folic acid
0.002
Pyridoxine hydrochloride
0.02
Riboflavin
0.005
Thiamine
0.005
Nicotinic acid
0.005
Pantothenic acid
0.005
p-Aminobenzoic a. (PABA)
0.005
Cyanocobalamin (B]2)
0.005
Thioctic (Lipoic) acid
0.005
Coenzyme M
(Mercaptoethanesulfonic a.)
0.1
*Brock and O'Dea (1977)
9
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Both media (excluding NaHC03, FeS, and vitamins, where applicable) were sterilized in
an autoclave at 121°C for 20 minutes and were then purged with an oxygen-free mixture of
N2(79%)/C02 (21%) for 45 minutes. Medium 1 was amended with NaHCC>3 immediately after
purging, and was flushed in the antechamber of an anaerobic glove box; the antechamber sequence
consisted of evacuation under vacuum, flushing with NT2, evacuation, flushing with N2, and
flushing with the glove box atmosphere [N2 (80%)/CC>2 (10%)/ H2 (10%)]. Medium 2 was
immediately sealed after autoclaving and purging with NT2 /CO2 and was taken into an anaerobic
glove box. Vitamins, FeS, and NaHCC>3 were added to the medium from sterile, anaerobic stock
solutions.
Amorphous Fe(OH)3
Iron was added to microcosms in the form of amorphous Fe(OH)3. Amorphous Fe(OH)3
was used rather than more crystalline iron phases because the rate of biological ferric iron reduction
tends to increase significandy with decreasing degree of crystalLinity (Lovley, 1987 and references
therein). This iron phase was prepared by neutralizing a 0.1 M ferric chloride solution with
sodium hydroxide. The precipitate was aged for four hours after neutralization; during this period,
the pH was adjusted periodically with sodium hydroxide to neutralize acidification resulting from
ferric iron hydrolysis. The precipitate was then rinsed with Milli-Q water in a 2-L Biichner funnel
to reduce the residual chloride concentration to a level that would correspond to less than 1 mM in a
200 mL microcosm or enrichment. The residual chloride concentration of the filtrate was
determined by measuring conductivity against NaCl standards. The amorphous Fe(OH)3 was
prepared with sterilized glassware and reagents prepared in sterilized Milli-Q water, but the iron
phase itself could not be autoclaved because the elevated heat and pressure would facilitate
crystallization, which was not desired.
Soil and sediment inocula
Patuxent River soil used for experiments PR1 and PR2 was collected at the Naval Air
Station, Patuxent River (MD) in September, 1987. The site was extensively contaminated with
aviation fuel (e.g., JP-5), The sample was collected near a hydrocarbon seep in a marshy area and
was received in water-saturated form in a plastic, screw-capped container. It was stored at 4°C
until its use in microcosms roughly two years after collection. The soil appeared to be fine-grained
and rich in organic matter, and contained some plant detritus. More recendy (in July 1990),
additional material (soil and aquifer sediment) was collected from the site and was used in
10
-------
Experiment PR3. The soil used in Experiment PR3 (Samples D7 - D10) was collected near the
hydrocarbon seep that was sampled earlier.
Samples of gasoline-contaminated aquifer material from the Seal Beach (CA) site were
collected in May, 1987 with a hollow stem auger at a depth of 2.54 m above the water table. After
collection, the contents of the auger were transferred to a 20-L plastic bucket with an air-tight snap
lid. The sediment was stored at 4°C until use.
Core samples of coarse-grained aquifer material contaminated by aviation gasoline from the
U.S. Coast Guard Air Station site in Traverse City (MI) were provided by the U.S. Environmental
Protection Agency (Ada, OK). The samples were collected aseptically with an auger under
anaerobic conditions in August, 1989. The core sample used in this study was taken from below
the water table, at 5.89-6.15 m below ground surface, from an actively methanogenic zone. It
contained very low concentrations of nitrogen and phosphorus (jig/L quantities), and 9.5 mg/L
sulfate (Stephen Hutchins, personal communication). The sample was stored at 4°C in a sealed
Mason jar until use.
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 contaminated by creosote and pentachloro-
phenol. The samples were obtained from an actively methanogenic, sandy zone of the aquifer,
downgradient from the contamination source, at a depth of approximately 6 m. The ground water
at this depth contained tens of mg/L of nitrogen heterocycles, simple polynuclear aromatic
hydrocarbons, and phenols (Goerlitz et al., 1985). 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.
11
-------
EXPERIMENTAL DESIGN
Many experimental trials were attempts to screen for activity of iron-reducing bacteria.
Thus, experimental design often consisted of setting up parallel series of microcosms, one with
and one without added iron. The following series of tables (Tables 3-11) describes the initial
conditions of series of microcosms and enrichments that involved iron amendments. Tables 3
through 9 are ordered chronologically with respect to the date of preparation of microcosms or
enrichments. The final table in the series (Table 11) shows amendments made to microcosms in
Experiment PR2; this table was included because the detailed history of the amendments is useful
in interpreting the data.
In the experiments with fermentative/methanogenic bacteria, two types of mixtures of
aromatic compounds were spiked into the microcosms. In the first case, the mixture consisted
only of aromatic hydrocarbons (toluene, ethylbenzene, o-xylene, and p-xylene), each at a
concentration of 40 |lM. In the second case, p-cresol (0.46 mM), a putative intermediate of
toluene transformation under anaerobic conditions (Grbic-Galic and Vogel, 1987), was added to
the hydrocarbon mixture. No other organic amendments were made and no exogenous electron
acceptors were added, except CO2.
12
-------
TABLE 3. INITIAL CONDITIONS FOR SEAL BEACH MICROCOSMS
1 1 am. Fe(OH)3
| [ca. 12-16 mM
Microcosm Autoclaved j Fe(HI)]
Sediment
(ca. 24-26 g)
Acetate (280 |iM)/
benzoate (77 (iM)
1 i x I
2 I x [
X
3 X
X
4 i i
X
X
. 5 j j
X
X
7 ! 1
X
8 i X ! X
X
9 { X | X
| v-*
! o
i
[
X
X
X
11 | ! X
X
X
12 1 X
X
13 [ IX
X
14 I |X
x
TABLE 4. INITIAL CONDITIONS FOR TRAVERSE CITY MICROCOSMS
Sediment
(ca. 24-26 g)
1 [ca. 12-16 mM
I Fe(III)]
Acetate (280 |iM)/
benzoate (77 |iM)
Microcosm
Autoclaved
13
-------
TABLE 5. INITIAL CONDITIONS FOR EXPERIMENT PR1 MICROCOSMS
(PATUXENT RIVER)
Microcosm
Autoclaved
amFe(OH)3 j ]
Tea. 12 mM S Sediment \ p-cresol
Fe(m)] \ (ca. 30 g) j (95 uM)
Fatty acid
mixture'1'
A1
X
i |
i :
A2
X
i
A6
-A8
X
1 x
X 11
x 1 X 1
A17
I x I X
A18
1 x |
r x i
X
A19
A21
X j X I X
A22
_
A24
X X j
x 1 X |
X ! X 1
X
Note: * Fatty acid mixture was 2:1:1 (molar) mixture of acetic acid, propionic acid, and
butyric acid with a total acid concentration of ca. 0.47 mM.
TABLE 6. INITIAL CONDITIONS FOR ENRICHMENTS OF EXPERIMENT PR1
Enrichment
1
1
I
:
|
Autoclaved
amFe(OH)3
[ca. 20 mM
Fe(m)1
J Fe(H[)-citrate 1
(2.5 g/L) 1
Inoculum
(15 mL)*
B1
i
1
X
X
1 x 1
B2
1
1
X 1
X
X
X
B3
i
X
X
B4
X
j |
X
B5
1
X
1 !
X
B6
—
i
x I
X
X
B8
(
i
X
1 x !
X
Note:
* Inoculum was a mixture of microcosms A19, A21, A22, and A23 taken on
Day 58 of incubation.
14
-------
TABLE 7. INITIAL CONDITIONS FOR EXPERIMENT PR2 MICROCOSMS
(PATUXENT RIVER)
Microcosm
Autoclaved
amFe(OH)3 I
[ca. 20 raM i
Fe(III)] |
Sediment
(ca. 30 g)
Nitrate (5 mM)
CI
X
1 x
C2
X
x 1
X
C3 |
X
X
C4 [
C5 1
X
1 X
—
C6
X
C8
X 1
X
C9 [
X
X
CIO I
x i
X
TABLE 8. INITIAL CONDITIONS FOR ENRICHMENTS (DAY 88) OF EXPERIMENT PR2
Enrichment
Autoclaved
amFe(OH)3 |
[ca. 2 mM
Fe(III)] I
Inoculum*
(40 g)
Hydrogen sulfide
(ca. 1.5 mM)
E4
X
X i
X
X
E6
x I
X
X
E7
X i
X
Note:
* Inoculum was Microcosm C9 taken on Day 88.
TABLE 9. INITIAL CONDITIONS FOR ENRICHMENTS (DAY 235) OF EXPERIMENT PR2
Enrichment
Autoclaved
amFe(OH)3 j j
[ca. 2 mM j FeS04 | MgS04
Fe(III)] j (ca. 2 mM) j (ca. 2 mM)
Inoculum*
(35 g)
F1
i I x
X
F2
! ! x
X
F3
X j j x
X
F4
X i ! X
X
F5
X 1 1 X
X
F6
X j 1 X
X
F7
x 1
X
"" F8 1
x
X
F9
PTO
[ X
1 x r
X
X
Note: * Inoculum was Microcosms C8 & CIO (homogenized) taken on Day 235.
15
-------
TABLE 10. INITIAL CONDITIONS OF EXPERIMENT PR3 (PATUXENT RIVER)
Microcosm | Autoclaved
am. Fe(OH)3
[ca. 10 mM Fe(III)l
Sediment (50 g)
D3 |
X
X
D4 j
X
X
D5 i
r
X
X
D6 |
X
X
D7 | X
X
X
D8 |
x
X
D9 |
X
X
DIG |
t
X
X
Note: The site of collection for soil used in Microcosms D7 - D10 was near the site of
collection for soil used in Experiments PR1 and PR2.
16
-------
TABLE 11. AMENDMENTS FOR EXPERIMENT PR2 MICROCOSMS (DAY 0 - 70)
Time
(days)
Bottle C3
Bottle C4
Bottle C5
Bottle C6
Bottle C8
Bottle C9
-Bottle G10
0
N (5mM)
amFO
(20mM)
amFO
(20mM)
amFO
(20mM)
14
N
(2.5mM)
Ac
(0.35mM)
17
T,X
24
N (5mM)
31
amFO
(10 mM)
34
amFO
(10 mM)
35
T
amFO
(lOmM)
amFO
(lOmM)
40
T
47
T
T
T
T
T
48
X (5mM)
amFO
(lOmM)
Mo
(20mM)
amFO
(lOmM)
S
(0.40mM)
amFO
(lOmM)
S
(0.80mM)
54
T
55
S
(1.2mM)
S
(1.2mM)
S
(1.2mM)
58
N (5mM)
S
(0.40mM)
S (0.40
mM),T
T
T
67
S (0.80
mM), T
S (0.80
mM), T
S (0.80
mM), T
70
N (5mM),
T
N (5mM),
T
S
(l.OmM)
S
(l.OmM)
S (1.0
mM), T
S (1.0 1
mM), T
S (1.0
mM), T
where:
Ac = acetate
amFO = amorphous Fe(OH)3
Mo = molybdate
N = nitrate
S = sulfate
T = toluene (typically 0.15 to 0.2 mM addition, but variable)
X = o -xylene (0.04 mM)
17
-------
ANALYTICAL METHODS
Aromatic hydrocarbon analysis
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 photoionization detector (10.2 eV lamp; HNU Systems, Inc., Newton, MA) and a 30-m DB-
624 megabore fused silica capillary column (3.0 |j.m film thickness; J & W Scientific, Folsom,
CA). Analyses were isothermal (65°C) and splidess. Sampling and analysis of headspace from
microcosms, enrichments, and standards was performed identically: 300 |lL of headspace was
sampled through the Mininert valve of microcosms, enrichments, or standards with a 500 |iL gas-
tight syringe that included a PTFE plunger tip and a side-port needle.
Standards for headspace analyses were prepared by spiking a methanolic stock solution of
the aromatic analytes into a Mininert-sealed bottle that contained 200 mL of water. The transfer of - -
the methanolic stock solution was made with a gas-tight 500-|iL syringe. The amount of stock
solution (and correspondingly, the mass of each analyte) added to the standard bottle was
determined gravimetrically by weighing the syringe immediately before and after spiking. The
aqueous concentration of aromatic compounds in standards was estimated by using Henry's Law
constants obtained from the literature (Mackay and Shiu, 1981), and Equation 2 below, which was
derived from the definition of Henry's Law (Equation 1) and a mass balance expression.
Eq. 1 He — Cg/Cw
Eq. 2 Cw = nrr/CVw + Hc*Vg)
where:
He = Henry's Law constant (dimensionless)
Cg = Gaseous solute concentration (moles/L)
Cw = Aqueous solute concentration (moles/L)
Vg = Volume of headspace in bottle (L)
Vw = Volume of water in bottle (L)
mj = Total amount of solute added to standard (moles).
18
-------
The aqueous concentration of aromatic compounds in microcosms and enrichments was
. determined by comparison of peak areas to standards. Note that the mass of analyte sorbed to
solids was not represented in this measurement.
Based on detailed studies of Henry's constants of other volatile organic compounds that
entailed spiking with methanolic stock solutions (Gossett, 1985), it was assumed in the present
study that the presence of methanol at the applicable methanol concentrations would not
significantly affect the gas/liquid partitioning of the analytes in the standards.
There was a noteworthy shortcoming of the headspace analyses in microcosms, which
related to the presence of sediment. In microcosms, the calculated amount of aromatic substrate
degraded was underestimated when the substrate was completely consumed (a regular occurrence
in some Patuxent River microcosms). This underestimate resulted because the toluene that was
sorbed to sediment immediately after spiking would progressively desorb and be degraded but
would not be accounted for in the headspace analysis that immediately followed spiking.
Controlled sorption experiments were not performed to quantify this effect However, it is
roughly estimated that the reported amount of toluene consumed in Patuxent River microcosms
(Experiment PR2) could be lower by as much as 15 to 25 percent based upon (1) the known
amount of pure toluene spiked into Patuxent River microcosms, (2) the corresponding aqueous
concentrations as determined by headspace analysis, and (3) the assumption that the mass of
toluene that sorbed just after spiking eventually desorbed and was degraded by the time of the next
analysis, when toluene was undetectable. In contrast, the relatively low concentrations of solids in
enrichments minimize this concern for enrichment cultures. Thus, toluene consumption reported in
Patuxent River enrichments should be considered to be more accurate than in microcosms in terms
of potential sorption effects.
Fe(II) Analysis
HCl-extractable Fe(TI) was measured as described by Lovley and Phillips (1986b). 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 HC1. 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; 3-(2-pyridyl)-5,6-
diphenyl-l^^-triazine-p^'-disulfonic acid, monosodium salt] in 50 mM HEPES buffer [4-(2-
hydroxyethyl)-l-piperazine-ethanesulfonic acid] (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
19
-------
562 nm was measured with an HP Model 8451A diode array spectrophotometer (Hewlett-Packard
Company, Palo Alto, CA). The advantages of first dissolving culture medium in HC1 are that: (1)
Fe(II) in poorly crystalline precipitates (such as amorphous ferrous sulfide) could be determined in
addition to soluble Fe(ll), which is important because precipitates were undoubtedly the
predominant Fe(II) phases in the systems studied, and (2) under the moderately oxidizing
conditions of analysis, Fe(H) is thermodynamically stable at acidic pH but not at neutral pH.
Standards were prepared by dissolving a known amount of ammonium iron (II) sulfate
hexahydrate (stored in the dark in an anaerobic glove box) in 0.5 M HC1, and were analyzed as
described above.
Sulfate and nitrate analysis
Sulfate and nitrate in filtered culture medium were determined by ion chromatography
(Dionex Series 4000i with a Nelson Analytical Chromatography Software system) equipped with
an HPIC-AS4A column (Dionex, Sunnyvale, CA), an anion micro membrane suppressor, and a
conductivity detector. Analyses were isocratic, with a sodium bicarbonate (0.75 mM) / sodium
carbonate (2.2 mM) eluant. Ions were identified and quantified by comparing retention times and
peak areas to those of external standards.
Gas chromatography/mass spectrometry (GC/MS) analysis
GC/MSD (mass selective detector) analyses for potential metabolites were performed on
samples from some microcosms. An aliquot of 2.75 mL of culture liquid was acidified and
extracted with 1 mL of diethyl ether. Derivatization was not attempted. Analyses were performed
on an HP Model 5890 GC with a 60-m DB-5 column (J & W Scientific, Folsom, CA) coupled
with a 5970 Series MSD (Hewlett-Packard Company, Palo Alto, CA). The temperature program
was as follows: 40°C (hold 5 min) to 120°C (at 2°C/min) and then to 200°C (at 4°C/min). The
mass scanning range was from 50 to 250 amu.
14C Analysis
14C-labeled toluene was used in the experiments with methanogenic Pensacola microcosms
and suspended cultures. [14C-ring]toluene and [14C-methyl]toluene were purchased from Sigma
Co. (St. Louis, MO). 14C-Activity in liquid and gaseous samples from the cultures was
determined on a Tricarb Model 4530 scintillation spectrometer (Packard Instrument Co., Downers
20
-------
Grove, IL). Counting efficiency corrections were made using the external standard channels ratio
method (Bell and Hayes, 1958). Three separate 1-mL liquid samples were counted for-each
analysis (Grbic-Galic and Vogel, 1987). All of them were mixed with 10 mL of liquid scintillation
cocktail (Universol TM Biodegradable-Nontoxic-Nonflammable, 1CN Biomedicals, Inc., Irvine,
CA), but one was pretreated with 1 mL of IN HC1, the other with 1 mL of IN NaOH, whereas the
third received no pretreatment. The pretreated samples were nitrogen-stripped before adding the
scintillation cocktail. These measurements were used to determine the 14C-activity in the volatile,
nonvolatile, and CO2 fractions, as described by Grbic-Galic and Vogel (1987).
21
-------
SECTION 5
RESULTS AND DISCUSSION
This section will focus primarily on studies performed with Patuxent River material,
including microcosm experiments (Experiments PR1 and PR2) as well as enrichment cultures.
Microcosms containing Patuxent River material and enrichment cultures of these microcosms
showed evidence of toluene degradation, sulfate reduction, and ferric iron reduction. In addition,
the presence of amorphous Fe(OH)3 had a marked effect on toluene degradation rates.
Although some degradation of aromatic substrates (e.g., toluene and ethylbenzene) was
observed in Seal Beach and Traverse City microcosms, the activity could not be sustained and the
presence of iron appeared to have little or no effect on degradation rates. Data for these materials
will be summarized briefly in this section.
The studies of Pensacola microcosms showed that toluene and o-xylene were completely
degraded under the conditions of methanogenic fermentation. The effects of p-cresol addition and
other factors on the length of the acclimation period to toluene were investigated. Stable
methanogenic consortia that were enriched from the microcosms degraded toluene, o-xylene, and
/71-xylene.
SULFATE REDUCTION AND FERRIC IRON REDUCTION IN PATUXENT
RIVER SOILS
Experiment PR1 - Iron's relationship to toluene degradation rates
Results of the first experiment with Patuxent River material (Experiment PR 1) 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 controls (A6 and
A8), (2) three microcosms without added amorphous Fe(OH)3, and (3) four microcosms with
added amorphous Fe(OH)3. Although rates of toluene degradation for microcosms with and
22
-------
0.20
Controls (A6,8)
¦ - - w/o Fe(OH)3 (A17-19)
*• with Fe(OH)3 (A21 -24)
0.15
£
£
V
c
-------
without added iron were very similar for the first 3 to 4 weeks of incubation, the microcosms with
added iron clearly had faster toluene degradation rates starting at Day 30. These differences in
rates continued throughout the next 30 days of incubation, after which toluene degradation
effectively ceased (based on later evidence, it is likely that this cessation of activity was due to
depletion of sulfate, the electron acceptor).
The replicability of the effect of amorphous Fe(OH)3 on toluene degradation rate is
apparent in Figure 2, in which individual microcosms from Experiment PR1 are represented in
terms of cumulative toluene degradation over time. The presence/absence of added iron appears to
be the primary variable that distinguishes the toluene degradation rates of these seven microcosms,
despite the fact that other variables were applicable (i.e., the presence of additional substrates, such
as /7-cresol, in certain microcosms; see Table 5). Because the range of variables measured was
limited in this preliminary experiment, a more detailed assessment is not possible (e.g.,
determination of the electron acceptor). Dissolved/colloidal Fe(II) (i.e., that which could pass
though a 0.2 Jim filter) was measured, but equilibrium modeling suggested that solid forms of
Fe(II) would predominate over dissolved forms in aqueous systems with the composition of the
growth medium. Also, sulfate was not measured regularly in this experiment. In the follow-up
experiment using the same soil inoculum (Experiment PR2), particulate Fe(II) and sulfate (the
suspected electron acceptor) were also measured.
Experiment PR2 - Interrelationships among toluene, sulfate, and iron
In Experiment PR2, as in Experiment PR1, the presence of amorphous Fe(OH)3 had an
effect on toluene degradation, but the effect was qualitatively different in the two experiments.
Toluene and sulfate concentrations versus time are shown in Figure 3a and b for the first 47 days
of incubation of five microcosms and two controls. As shown in Figure 3a, 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 CIO) initiated toluene degradation within a few days in each of those
microcosms. Two microcosms that did not receive amorphous Fe(OH)3 over the period shown
(Microcosms C5 and C6) did not degrade toluene until roughly 40 days later than the iron-amended
microcosms (as discussed later in this section).
Sulfate reduction was observed in all active microcosms (Figure 3b) but, over the period
shown, was most rapid in the microcosms without added iron (Microcosms C5 and C6). Sulfate
24
-------
Without Fe(OH)3
----- A17
— A18
Aj9
With added Fe(OH)3
0 1 0 20 30. 40 50
Time (days)
Figure 2. Cumulative toluene disappearance vs. time in Experiment PR1 microcosms
25
-------
0 20
0.15
S
E
C 0. 1 0
-------
reduction in the microcosms with added iron had virtually ceased between Days 24 and 33, before
rapid toluene degradation began. Once toluene degradation began in the Patuxent River
microcosms, the rate of sulfate reduction increased and there was a strong correlation between
sulfate disappearance and toluene disappearance. Figure 4 depicts this correlation for Microcosm
C8 over the first three months of incubation. Similarly strong regressions were observed for the
other active microcosms. The ratio of sulfate consumed/toluene consumed in Microcosm C8 was
4.2 (Day 81); similar ratios (4.1 to 4.2) were observed for the other iron-amended bottles, C9 and
CIO. These values, which were corrected for the amount of sulfate reduced before toluene
degradation began, approximate the theoretical ratios ranging from 4.0 (toluene oxidation to
bicarbonate with bacterial cell growth; Equation 4) to 4.5 (toluene oxidation to bicarbonate with no
cell growth; Equation 3); the estimation of cell growth in Equation 4 was derived using methods
described by McCarty (1971, 1975).
Eq. 3 C7H8 + 4.5 SO42- + 3 H20 = 2.25 H2S + 2.25 HS~ + 7 HCO3- + 0.25 H+
AG0' = -49 kcal/reaction
Eq. 4 C7Hg + 4.03 S042' + 0.19 NH4+ + 0.75 CO2 + 3.18 H2O
= 2.02 H2S + 2.02 HS* + 0.19 C5H7O2N (cells) + 6.8 HCO3- + 0.94 H+
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. As
further evidence of the link between toluene degradation and sulfate reduction, it was apparent that
toluene degradation ceased when sulfate was depleted, and conversely, sulfate reduction ceased
when toluene was depleted (such data are shown for an enrichment in the following section).
The reduction of Fe(lll) [i.e., the appearance of Fe(Il)] followed a similar trend as the
disappearance of toluene and sulfate (shown for Microcosm C8 in Figure 5). Note that the Fe(ll)
concentration in Figure 5 represents the amount of iron reduced since the initiation of toluene
degradation, not the absolute concentration. The available data cannot be used to demonstrate
conclusively whether iron reduction was directly coupled with toluene oxidation (where ferric iron
would have served as a terminal electron acceptor for toluene) or coupled with sulfate reduction
(where ferric iron would have served as the electron acceptor for the oxidation of biogenic
hydrogen sulfide). However, the observed stoichiometry gives some insight into this issue. The
ratio of Fe(lll) reduced/toluene consumed can be used to investigate the possibility that ferric iron-
reducing bacteria are oxidizing toluene. As shown in Table 12, the stoichiometry of iron reduction
27
-------
y = 0.05 + 4.35x RA2 = 0.996
o
o.o
0.8
1.0
1.2
0.2
0.4
0.6
Cumulative toluene removal (mM)
Figure 4. Sulfate vs. toluene consumed in Microcosm C8
(Experiment PR2; Day 0 to 85).
28
-------
Fe(Il) (diss, and panic.)
Sulfate
Toluene
Figure 5. Cumulative Fe(II) appearance and sulfate arid toluene
disappearance in Microcosm C8 (Experiment PR2).
29
-------
is not consistent with plausible biological processes [e.g., toluene oxidation to bicarbonate with
amorphous Fe(OH)3 being reduced to siderite; Equation 51 because the ratio is too low. Notably,
the ratio of 36 shown for the iron reduction reaction is universal for any iron phases in the absence
of cell growth, and would be the same for any Fe(Iir) phase being reduced to any Fe(II) phase in
conjunction with toluene oxidation to carbon dioxide. Thus, two lines of stoichiometric evidence
argue against biotic ferric iron reduction: (1) the ratio of Fe(III) reduced/toluene consumed and (2)
the ratio of sulfate consumed/toluene consumed (which suggests the predominant role of sulfate
reducers). Nonetheless, it is possible that ferric iron reducers were syntrophically associated with
sulfate reducers and were performing only small steps in toluene oxidation rather than complete
oxidation to carbon dioxide.
TABLE 12. THEORETICAL AND OBSERVED STOICHIOMETRY OF
IRON REDUCTION AND TOLUENE OXIDATION
Fe(EII) reduced/
Reaction
toluene consumed
Observed (Microcosm C8 - Day 90)
12
Fe(OH)3 to FeC03 (without cells; Eq. 5)
36
Fe(OH)3 to FeC03 (with cells)
21
Eq. 5 C7Hg + 36 Fe(OH)3 (s)+ 29 HC03" = 36 FeC03 (s) + 58 H20 + 29 OH"
The ratio of Fe(ID)reduced/sulfate consumed provides an indication of the likelihood that
ferric iron reduction resulted from an oxidation-reduction reaction between ferric iron and biogenic
hydrogen sulfide. Table 13 presents the observed ratio (Microcosm C8) along with the theoretical
ratios for various possible reactions between amorphous Fe(OH)3 and hydrogen sulfide. The
reactions were chosen based on studies of the abiotic, anoxic or oxygen-limited reactions of
goethite or amorphous Fe(OH)3 with hydrogen sulfide (Pyzik and Sommer 1981, Rickard 1974,
Bemer 1964) in which the observed products included ferrous sulfide, elemental sulfur, and, in
Pyzik and Sommer (1981), thiosulfate. It is assumed in Table 13 and the related discussion that
''sulfate consumed" is equivalent to "sulfide present" in the microcosms, that is, complete reduction
of sulfate to sulfide is assumed; this assumption is well-founded based on the literature. Certain
pairs of reactions in Table 13 (Equations 6 and 7; Equations 8 and 9) have the same sulfide
oxidation product (i.e., elemental sulfur or thiosulfate) but different proportions of available sulfide
relative to iron. For example, in Equations 6 and 8, there is sufficient sulfide present to precipitate
30
-------
TABLE 13. THEORETICAL AND OBSERVED STOICHIOMETRY OF
IRON REDUCTION VIA HYDROGEN SULFIDE
Reaction
Fe(IIT) reduced/
sulfate consumed
Observed (Microcosm C8 - Day 90)
2.6
Sulfide to S° (Eq. 6)
0.67
Sulfide (limited) to S° (Eq. 7)
2
Sulfide to thiosulfate (Eq. 8)
0.8
Sulfide (limited) to thiosulfate (Eq. 9)
4
Sulfide (limited) to thiosulfate (Eq. 10)
2.6
Eq. 6 4 Fe(OH)3 (s) + 3 H2S + 3 HS" = 2S° + 4 FeS (s) + 9 H2O + 3 OH"
Eq. 7 4 Fe(OH)3 (s) + H2S + HS" = 2 S° + 4 Fe2+ + 3 H20 + 9 OH"
Eq- 8 8 Fe(OH)3 (s) + 5 H2S + 5 HS" = S2032- + 8 FeS (s) +18 H20 + 3 OH"
Eq. 9 8 Fe(OH)3 (s) + H2S + HS" = S2032" + 8 Fe2+ + 6 H20 + 15 OH'
Eq. 10 Fe(OH)3 (s) +0.19 H2S+0.19 HS" +0.87 HCOr
= 0.125 S2032" + 0.13 FeS (s) + 0.87 FeC03 (s) + 1.79 H20 + 0.81 OH"
Eq. 11 C7H8 + 11.8 Fe(OH)3 (s) + 4.5 S042" + 3.3 HC03"
= 1.48 S2032" + 1.54 FeS (s) + 10.3 FeC03 (s) + 18.5 H20 + 9.34 OH~
all reduced iron as ferrous sulfide, whereas in Equations 7 and 9 there is sufficient sulfide present
only to reduce all available ferric iron, but not to precipitate any of the reduced iron. Intermediate
amounts of sulfide would result in intermediate ratios. Equation 10 represents a reaction that
agrees with the observed stoichiometry. In this reaction, there is insufficient sulfide present to
precipitate all reduced iron as FeS, as was apparendy the case in Experiment PR2 (compare y axes
in Figure 5). The remainder of the reduced iron is precipitated as FeC03 in Equation 10, as would
be expected based on equilibrium considerations. A combination of Equation 10 and Equation 3
results in an overall reaction (Equation 11) that is consistent with the observed stoichiometry in
Microcosm C8 in terms of iron, sulfate, and toluene; this suggests, but does not prove, that toluene
is being oxidized to carbon dioxide by sulfate-reducing bacteria and that the resulting sulfide is
responsible for the observed iron reduction. The abiotic reaction proposed for hydrogen sulfide
31
-------
and amorphous Fe(OH)3 is only one of a number of possible reactions that could explain the
stoichiometry.
Interpretation of the role of iron is complicated by results for Microcosm C6, which
received no iron additions. Toluene degradation began in Microcosm C6 around Day 70 and has
continued at a rate comparable to bottles that received additional iron (e.g., Microcosm C8; Figure
6). It should be noted that, although iron was not added to Microcosm C6, significant indigenous
reduced iron was present in this microcosm: a relatively constant concentration of ca. 20 mM Fe(ll)
was measured in Microcosm C6 over several months. Since toluene degradation commenced in
Microcosm C6, the correlation between toluene and sulfate disappearance has been strong (r2 >
0.98), although the ratio of sulfate consumed/toluene consumed (ca. 5.1) is somewhat higher than
that observed for the iron-amended microcosms (i.e., C8, C9, and CIO). The higher ratio may
reflect the consumption of an additional electron donor that was present in the sediment, as
indicated by the fact that sulfate was reduced in Microcosm C6 for roughly 70 days before toluene
degradation began (Figure 3b). The microflora of Microcosm C6 have not been further studied,
although comparisons of this assemblage with assemblages of enrichments that responded to iron
addition could enhance our understanding of the role of iron in toluene-degrading, sulfate-reducing
microorganisms.
Patuxent River enrichments - Reproduction of trends seen in microcosms
Attempts to make enrichment cultures from microcosms in Experiment PR1 were
unsuccessful, however, active enrichments were generated from microcosms of Experiment PR2.
Like the Patuxent River microcosms, enrichments generated from Microcosm C9 (see Table 8)
demonstrated a strong relationship between the presence of amorphous Fe(OH)3 and toluene
degradation rate (Figure 7). Several interesting features are apparent in Figure 7: (1) Enrichment
E6, which initially received 1.5 mM sulfide in addition to 2mM amorphous Fe(OH)3, appeared to
degrade toluene considerably more slowly than Enrichment E7, which initially received the same
amount of iron but no sulfide, and (2) the addition of amorphous Fe(OH)3 to both enrichments on
Day 32 markedly increased toluene degradation rates, especially in Enrichment E7, in which
toluene degradation had slowed to the rate of Enrichment E6 after Day 20. Considerably lower
concentrations of Fe(HI) were spiked into the Patuxent River enrichments than in the microcosms,
and there is some evidence in Figure 7 that additional iron amendments were required to sustain
toluene degradation rates (see Enrichment E7 between Days 20 and 30).
32
-------
3.0
Microcosm C8 (with iron)
Microcosm C6 (w/o iron)
2.5-
2.0-
u
c
o
O
J2
3
s
6
0.5-
0.0
60
20
80
100
0
40
120
1 40
1 60
Time (days)
Figure 6. Cumulative toluene disappearance vs. time in
Microcosms C6 and C8 (Experiment PR2).
33
-------
Initial conditions:
E6- 1.5 mM sulfide,
2 mM am. Fe(0H)3
E7 - 2 mM am. Fe(OH)3
4 mM Fe(OH)3
4 mM Fe(OH)3
4 mM Fe{0H)3
Figure 7. Cumulative toluene disappearance vs. time in two enrichments
from Experiment PR2.
34
-------
The strong relationship between Fe(II) appearance, toluene disappearance, and sulfate
disappearance that was demonstrated for microcosms (Figure 5) was also evident in enrichments
(data for Enrichment E7 are shown in Figure 8). Notably, the sulfate consumed/toluene consumed
ratios in Enrichments E6 and E7 (ranging from 3.5 to 3.7; Day 84) were approximately 20 percent
lower than the ratios observed for the microcosms. The lower ratio in enrichments may have been
due to the lower concentrations of solids in the enrichments, which resulted in headspace analyses
that were more representative of true toluene consumption because they were largely unaffected by
sorption (recall discussion in Analytical Methods section). However, another set of enrichments
(see Table 9) had typical ratios in the range of 3.9 to 4.2 (by Day 53), which is consistent with the
ratios observed in Patuxent River microcosms. It is possible that the variability observed in these
ratios was a result of analytical variability, or was related to variability among microbial
assemblages in different systems (e.g., sulfate reducers may have played a different metabolic role
in toluene degradation in different microcosms and enrichments).
As further evidence of the close linkage between toluene degradation and sulfate reduction,
a controlled test of the effect of sulfate depletion on toluene degradation and the effect of toluene
depletion on sulfate reduction was performed with Enrichment F9. The results are shown in
Figure 9. When sulfate was depleted (Days 53 to 56), toluene was not appreciably degraded.
After sulfate was spiked into the enrichment (Day 56), concurrent toluene degradation and sulfate
reduction occurred until Day 60, when toluene was depleted. In the absence of toluene (Days 60 to
63), sulfate reduction was not apparent. The ratio of sulfate consumed/toluene consumed during
this test was approximately 4.4 (between Days 53 and 60).
Experiment PR3 - Additional Patuxent River microcosms
As shown in Table 10, eight microcosms were generated with recently collected soil and
aquifer sediment from various portions of the Patuxent River site. Some of the Patuxent River
microcosms have shown toluene degrading activity (particularly D6 and D9), but the activity
ceased when sulfate was depleted. In order to see if ferric iron reducers were present that could
carry out toluene degradation, sulfate was not replenished but amorphous Fe(OH)3 was added.
Based on the cessation of toluene-degrading activity over weeks of monitoring, either ferric iron
reducers were not present or their growth was extremely slow.
35
-------
1)
o
S3
OJ
%
OJ
_>
—
e
3
u
1 0.0
8.0 -
6.0
0.0
Fe(ll) (diss, and particulate)
Sulfate
Toluene
2.0
-6.0
o
u
OJ
CL.
&
.55
•3
,cg
«u
>
=3
£
3
u
1 .5
i.o-
0.5
2
s
D-
5/5
-------
0.30
Toluene
Sulfate
0.25-
0.20-
0.15-
0.10-
-0.5
0.05-
0.00
-f 0.0
63
53
57
59
61
55
Time (days)
Figure 9. 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.
37
-------
DISCUSSION OF THE ROLE OF IRON IN TOLUENE DEGRADATION
(PATUXENT RIVER EXPERIMENTS)
The data presented in this report indicate the importance of iron in either initiating or
accelerating toluene degradation, but do not explicitly indicate how iron is responsible for these
effects. Five hypotheses have been formulated that could explain the nature of iron's effect; these
hypotheses are described below and are discussed in relation to the available data.
Hypothesis 1- Reduction of sulfide toxicity
Fe(III) could be reducing sulfide toxicity in two ways: by serving as an electron acceptor
in the oxidation of hydrogen sulfide (e.g., to elemental sulfur) and by precipitating sulfide as
ferrous sulfide once the iron has been reduced to ferrous form (both processes are illustrated by
Equation 6). Sulfide toxicity is often referred to in the literature but is not extensively documented.
Postgate (1984) invoked sulfide toxicity to explain the linear rather than exponential growth that
has been observed in batch cultures of sulfate-reducing bacteria; it was postulated that sulfide
inhibits growth, and thus reduces the specific growth rate (typically represented as (!) over time as
sulfide is produced.
Hypothesis 2 - Iron as a limiting nutrient
Iron might be a limiting nutrient that is required for toluene degradation. The concentration
of soluble (biologically available) ferrous iron was decreased considerably by precipitation as
ferrous sulfide. Note that the millimolar concentrations of Fe(II) presented in the Results and
Discussion section are largely particulate, not dissolved.
Postgate (1984) noted that "Desutfovibrio shows an exceptionally high requirement for
inorganic iron". As a quantitative indication of this requirement,.Postgate (1956) reported that D.
desulfuricans required on the order of 10 |iM iron for optimal growth with lactate. It is likely that
the iron requirement of sulfate-reducing bacteria is related to the synthesis of iron-rich proteins
(e.g., cytochromes c and ferredoxin). For example, Tsuji and Yagi (1980), using D. vulgaris,
found that the amount of cytochrome c3 in iron deficient (11 nM iron) medium was as high as in
iron-rich (50 nM iron) medium, whereas other iron-rich proteins were present at much lower
concentrations in the iron-deficient bacteria. Thus, cytochrome C3 was presumably critical for
bacterial metabolism, as it was produced preferentially in iron-deficient cultures. Postgate (1965),
citing unpublished data, noted that Desulfovibrio sp. changed from linear to exponential growth
38
-------
when chelators were added (e.g., citrate or EDTA). This finding is at odds with the assumption
discussed previously (Hypothesis 1) that sulfide toxicity is responsible for such growth patterns.
In general, it is difficult to experimentally distinguish between the effects of sulfide toxicity and
iron limitation in sulfate-reducing cultures, as both are related to the interaction between iron and
sulfide.
Hypothesis 3 - Oxidation of sulfide to preferable electron acceptors
Ferric iron may oxidize sulfide to thiosulfate (Pyzik and Sommer, 1981) or sulfite; these
sulfur oxyanions are intermediates (or potential intermediates, in the case of thiosulfate; e.g.,
Akagi, 1981) in sulfate reduction and both may be used as electron acceptors for the oxidation of
aromatic compounds (Widdel, 1988; Cypionika et al., 1985). The use of these sulfur species
would be energetically preferable to sulfate because the energy-requiring activation of sulfate to
form APS (adenosine-5'-phosphosulfate) would not be necessary (e.g., Widdel, 1988; Badziong
and Thauer, 1978).
Hypothesis 4 - Presence of ferric iron-reducing bacteria
Iron-reducing bacteria may be performing the initial (but incomplete) oxidation of toluene
or in some other way may be engaged in a syntrophic relationship with sulfate-reducers. Recently,
ferric iron-reducing cultures have been isolated that can mineralize toluene and can also degrade
potential toluene intermediates, such as p-cresol (Lovley and Lonergan, 1990). Thus, the
existence of such organisms is plausible in the Patuxent River samples.
Hypothesis S - Presence of fermentative bacteria
• Fermentative bacteria that are highly dependent on iron could be performing the initial
oxidation of toluene. This iron dependence differs from Hypothesis 2 in the sense that the bacteria
being considered are not the predominant species involved in overall toluene degradation (i.e.,
sulfate reducers).
Since the experiments performed for this project were not designed to test the hypotheses
stated above, the available data are of limited use in evaluating their validity. At this time, none of
the hypotheses can be definitively ruled out. However, some speculation based on the data is
warranted. Perhaps the most striking data regarding the effect of iron on toluene degradation was
39
-------
the initiation of toluene degradation after the second addition of amorphous Fe(OH)3 in Experiment
PR2 (Figure 3). From the sulfate data, it is clear that sulfate reducers were active in these
microcosms well before toluene degradation began. Thus, the addition of iron must have either
allowed these sulfate reducers to switch substrates (to toluene) or promoted the very rapid
development of iron-dependent sulfate reducers or fermenters, or iron-reducing bacteria. The
observations are consistent with Hypothesis 2; it is plausible that iron was a limiting nutrient that
was required for producing respiratory or other iron-containing enzymes specific to toluene
degradation in sulfate reducers. However, it is difficult to explain why the iron addition at Day 0
was not sufficient to supply the needed iron.
The stimulation of iron-reducing bacteria (Hypothesis 4) is also a plausible explanation for
the rapid initiation of toluene degradation in Experiment PR2; however, other data argue against the
presence of iron-reducers. For example, the addition of amorphous Fe(OH)3 to Microcosm C5
(Experiment PR2) on Day 48, when sulfate had been depleted, did not result in the initiation of
toluene degradation over a ten-day period, after which sulfate was again added (data not shown). -
In addition, Experiment PR3 results suggested that, if iron reducers were present, they must have
had very slow growth rates, which is not consistent with the observation of rapid initiation of
toluene degradation in Experiment PR2. This discussion applies equally well to iron-reducing
bacteria that could perform only a minor, initial toluene degradation and those that could completely
oxidize toluene (the latter have been ruled out by stoichiometry).
The observation of the rapid initiation of toluene degradation by iron addition (Experiment
PR2) also does not strongly support Hypotheses 1 or 3 (Le., iron reduced sulfide toxicity or
generated preferable electron acceptors) because these mechanisms would tend to accelerate toluene
degradation rates (via enhanced growth rates) once degradation had begun, but would not
necessarily be expected to initiate toluene degradation. With regard to sulfide toxicity, it seems
unlikely that the ca. 0.4 mM of sulfide generated before toluene degradation began in Microcosms
C8, C9, and CIO would have resulted in significant toxicity, especially in the presence of the 20
mM Fe(HI) that was added initially.
40
-------
Preliminary experiment to examine the role of iron
¦ A preliminary test with Patuxent River enrichments was performed in an attempt to narrow
down the list of plausible hypotheses for the role of iron in toluene degradation. The test was
predicated on the fact that certain of the hypotheses stated above (namely Hypotheses 3 and 4)
apply only to ferric iron, whereas the remaining hypotheses could apply to ferric or ferrous iron.
Thus, if ferrous iron were added to the enrichments and had the same apparent effect as ferric iron,
Hypotheses 3 and 4 (i.e., oxidation to preferable electron acceptors and the presence of ferric iron-
reducing bacteria) could be ruled out. The experimental design (summarized in Table 9) consisted
of initially adding either ferrous iron (as FeSO-*) or ferric iron fas amorphous Fe(OH)3] and
observing the relative toluene degradation rates. Live controls consisted of enrichments that were
not amended with iron (unfortunately, the parent microcosms had a considerable amount of iron,
particularly ferrous iron, that carried into the enrichments and may have compromised their role as
controls).
The results for the first 40 days of incubation are shown in Figure 10, which shows
average cumulative toluene degradation vs. time for the three different initial conditions. For the
first three weeks of incubation, toluene degradation rates were extremely consistent among all
enrichments (error bars represent one standard deviation). After Day 20, the Fe(II)-amended
enrichments continued to degrade toluene as rapidly as it was supplied and variability remained
very low; in contrast, the Fe(III)-amended enrichments began to degrade toluene less rapidly and
with far greater variability. The trend for FeQTTj-amended enrichments was not unlike the trend for
enrichments shown in Figure 7. The results are difficult to explain if one assumes that the same
microbial assemblages were present in all enrichments, especially when the controls are
considered. If all enrichments were assumed to contain the same assemblages, it would appear that
ferric iron was actually inhibitor}'. It is possible that the different initial conditions provided
selective pressure for different microbial assemblages, and the Fe(II)-amended and control
enrichments may have had similar assemblages that were unlike the Fe(lH)-amended assemblages.
Notably, the Fe(II)-amended enrichments and controls began to slow down after Day 45 and were
clearly stimulated by the addition of more Fe(II) (data not shown). Thus, these enrichments had a
response to ferrous iron, possibly negating Hypotheses 3 and 4; however, these results cannot
necessarily be applied to the earlier experiments with Patuxent River microcosms because different
microbial assemblages may have been present.
41
-------
No iron added (n=2)
Fc(III) added (n=4)
Fe(H) added (n=4)
0.9
0.6
0.3
0.0
0
1 0
20
30
40
Time (days)
Figure 10. Test of the effect of iron oxidation state on toluene degradation
using Patuxent River enrichments. Error bars represent one standard deviation.
42
-------
NITRATE REDUCTION IN PATUXENT RIVER MICROCOSMS
One of the Patuxent River microcosms (C3; Experiment PR2) was amended with nitrate on
Day 0 (Table 7). As shown in Figure 11a, almost immediate disappearance of toluene and nitrate
was observed in this microcosm. Nitrate was likely the electron acceptor for toluene because
Fe(E) and sulfate data (discussed later) indicate that iron and sulfate were not electron acceptors in
this microcosm. The cumulative ratio of nitrate consumed/toluene consumed was about 35 at Day
70; the theoretical ratio when toluene degradation is coupled with the reduction of nitrate to
nitrogen gas is 7.2. Thus, nitrate must have been participating in reactions (either biotic or abiotic)
in addition to toluene oxidation. A very interesting trend was observed with sulfate in this
microcosm (see Figure 1 lb): sulfate concentration increased from control levels, plateaued from
Day 47 to 58, and then continued to rise, whereas control levels of sulfate were constant. The
primary source of the sulfate was probably the oxidation of sulfide (which was present in the
controls as well as in the active, sulfate-reducing microcosms, and thus must have been present at
Day 0). The shape of the sulfate curve in Figure lib suggests that a second source of reduced
sulfur may have been oxidized starting after Day 58.
Nitrate may have been the electron acceptor in a reaction with sulfide; this reaction could
explain the additional sink for nitrate mentioned above. The oxidation of reduced sulfur species
may have been carried out by chemolithotrophic bacteria (e.g., Thiobacillus denitrificans). The
observed stoichiometry is consistent with the reduction of nitrate to nitrite by hydrogen sulfide:
The theoretical ratio of [sulfate produced/nitrate consumed] in the above equation is 0.25, whereas
the observed ratio was 0.256. Note that the amount of nitrate used in the observed ratio was
corrected for the biological oxidation of toluene; that is, the amount of nitrate that would
theoretically be required to completely oxidize the toluene was subtracted from the total amount of
nitrate consumed. This correction of the amount of nitrate consumed (and the associated
assumptions) are not of great significance in relation to the overall amount of nitrate consumed.
Notably, the thermodynamically favorable reduction of nitrate to nitrogen gas in conjunction with
sulfide oxidation
8 N03- + H2S + HS- = 8 N02- + 2 S042" + 3 H+
AG0 (pH 7) = - 232 kcal.
I6NO3- + 5H2S + 5HS" + H+ = 8N2 + IOSO42- + 8H20
AG0' (pH 7) = - 1777 kcal
43
-------
Toluene
Nitrate
20 40 60
Time (days)
Toluene
Sulfate
2
£
a
<2
3
C/3
20 40 60
Time (days)
Figure 11. Toluene and nitrate (a) or toluene
and sulfate (b) vs. time in Microcosm C3.
Arrows indicate amendments of toluene and/or nitrate.
44
-------
has a [sulfate produced/nitrate consumed] ratio of 0.63, which is not consistent with the observed
ratio.
The total Fe(II) concentration in this microcosm was below control levels, suggesting some
oxidation of Fe(I3). However, the concentration of Fe(II) remained relatively constant
(8.49±0.91), suggesting that Fe(II) oxidation was not an ongoing process (or was balanced with
Fe(III) reduction).
ATTEMPTS TO ENRICH IRON-REDUCERS IN SEAL BEACH AND TRAVERSE
CITY MICROCOSMS
Seal Beach Microcosms
After a lag period of one to three weeks, toluene degradation was observed in five of the
eight Seal Beach microcosms. Of these five microcosms, two contained added amorphous
Fe(OH)3 and three did not. The toluene degradation Tates were relatively slow (Figure 12) and .
activity could not be maintained over time. The electron acceptor in these samples is unknown, but
the available data suggest that sulfate, not ferric iron, was the electron acceptor. Sulfate was not
measured in this study, however, active microcosms turned noticeably darker (blacker) by Day 25,
suggesting the possibility of ferrous sulfide formation. Dissolved Fe(II) (not dissolved plus
particulate, as the culture medium was filtered through a 0.2 ^m filter during the Seal Beach and
Traverse City experiments) did not increase significantly in Seal Beach microcosms, and never
exceeded one mole percent of the iron initially added. Although a microcosm with added iron
showed a slightly greater extent of toluene degradation than the average of three microcosms
without iron (Figure 12), the significance of this trend relative to experimental error is debatable.
Traverse City Microcosms
Degradation of ethylbenzene was clearly apparent in five of the eight active microcosms,
and toluene degradation was also observed in two of these microcosms. Of the five microcosms
with apparent degradation, two contained added amorphous Fe(OH)3 and three did not. However,
as observed for the Seal Beach microcosms, there was no significant appearance of dissolved
Fe(II) or clear effect on substrate degradation that could be attributed to the presence of iron. In
Figure 13, toluene and ethylbenzene degradation are shown for one microcosm without added iron
and one with added iron.
45
-------
0.08
Controls (Boules 2,3,9,10)
w/o iron (Bottles 5,6,7)
w/ iron (Bottle 13)
0.06
s
0.04
0.02
0.00
0
50
1 00
1 50
200
Time (days)
Figure 12. Toluene concentration vs. time in Seal Beach microcosms.
Bars represent one standard deviation.
46
-------
a
— Control (Boulcs 16,17,24)
—*— w/iron (Bottle 26)
w/o iron (Bottle 20)
0.08
o
c
3
0.06
0.04
0.02-
0.00
20
1 40
50
80
1 1 0
Time (days)
0.08
—O.
0.06
o
c
a
c
£
•5
a
0.04
0.02
^ 1*1 ¦
50
0.00
20
80
140
1 1 0
Time (days)
Figure 13. Toluene (a) and ethylbenzene (b) vs. time
in two Traverse City microcosms. Arrows indicate
amendments of toluene or ethylbenzene.
47
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METHANOGENIC DEGRADATION OF TOLUENE AND o-XYLENE BY
MICROORGANISMS FROM THE PENSACOLA AQUIFER
Pensacola microcosms were initially fed mixtures of benzene, toluene, ethylbenzene,
-------
100
80 -
¥ 6°
"*T
G
O
S 40
20
~ * a % \\V'
/ / / / /
/ / / / /
. \ N \ \
~ ~ ~ ~
B
co2
ffl
Volatile
~
Nonvolatile
initial
final
Figure 14. 14C mass balance in suspended cultures fed 14C-labeled toluene. The
first column represents the initial distribution of the label; the volatile portion is toluene.
The second column shows the distribution of 14C after all the toluene was degraded.
The large CO2 fraction confirms complete degradation of toluene. The nonvolatile
fraction contains degradation intermediates and cell-bound 14C; this fraction was
found to decrease with time. Both ring-labeled and methyl-labeled toluene yielded
the same Tesults.
49
-------
mass balance in suspended cultures that degraded L14C]toluene. After several refeedings, the
cultures stabilized and increased their degradation rate (complete degradation of toluene and
o-xylene in one to two weeks of incubation; see Figure 15). The substrates were continuously
converted to stoichiometric amounts of CO2 and CH4 (Edwards and Grbic-Galic, 1990). Note that
the data presented in Figure 15 (and in the remaining figures in this report, except Figure 21)
represent the averages of two or three replicates. In these figures, the maximum difference
between replicates was 25%, and most replicates agreed within 10%.
The stable mixed cultures (both primary and secondary enrichments) were maintained on
•toluene and o-xylene as sole carbon sources for over a year. They degraded toluene and o-xylene
relatively rapidly. The cultures have been acclimated to an additional compound, m-xylene, which
was degraded only slowly, after the other two compounds were completely depleted. The original
microcosms and the first transfers from the microcosms, which still contained a minor amount of
aquifer solids, were relatively active and degraded toluene and o-xylene (fed at a concentration of
50 p.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). Subsequent transfers demonstrated no activity. It is suspected that the solids were
necessary for attachment of the microorganisms, or that they contained a trace nutrient which might
have been essential for the transformations.
Since the original headspace in the microcosms contained 10% molecular hydrogen, which
could have influenced the activity of fermentative or obligate proton-reducing acetogens
participating in the hydrocarbon transformation, the effect of different concentrations of hydrogen
on toluene degradation and methane production was tested. In this experiment, 40-mL vials sealed
with Mininert valves and containing 20 mL of Medium 2 were inoculated with 10 mL of stable
mixed culture. The headspace (10 mL) was exchanged with gases containing different
concentrations of hydrogen and each vial was spiked with toluene. The results are summarized in
Table 14. As is evident from the table, 70% of H2 in the headspace did not strongly inhibit toluene
degradation, although degradation in the absence of H2 in the headspace was the fastest. Some of
the effects might have been due to differences in the initial CO2 concentration in the headspace,
which might have influenced the pH. However, the data strongly suggest that the toluene
50
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0.08'
0.06-
i
rt
h
e
v
u
a
0.04 -
0.02-
Toluene sterile control
o-xylene sterile control
o-xylene
Toluene
o.oo
1 0
Time (days)
Figure 15. Degradation of toluene and o-xylene in suspended mixed culture. The
degradation of both compounds proceeded simultaneously in mixed cultures amended
with both compounds.
51
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TABLE 14. INFLUENCE OF H2 CONCENTRATION IN THE HEADSPACE ON TOLUENE
DEGRADATION AND METHANE PRODUCTION IN SUSPENDED METHANOGENIC
CULTURES DEGRADING TOLUENE AS THE SOLE SUBSTRATE
Time
(Days)
10% h2,
10% co2,
80% N2
NO H2
21.3%'
co2,
78.7%
N2
70% H2,
30% C02
10% h2,
10% C02,
80% N2
NO
Toluene
(Positive
Control)
10% h2.
10% C02,
80% N2,
Sterile
Control
1
TOL:4.00a
CH4: 0.058
TOL. 4.05
CH4: 0.014
TOL: 4.26
CH4: 0.045
CH4: 0.061
CH4: 0.005
5
TOL: 1.79
CH4: 0.299
TOL: 0.38
CH4: 0.082
TOL: 0.84
CH4: 1.18
CH4: 0.529
CH4: 0.005
7
TOL: 0.85
CH4: 0.349
TOL: <0.01
CH4: 0.106
TOL: 0.35
CH4: 2.64
CH4: 0.784
CH4: 0.006
11
TOL: 0.01
CH4: 0.362
TOL: -b
CH4:0.104
TOL. 0.54
CH4: 4.06
CH4: 0.814
CH4: b
a Toluene concentration is expressed in mg/L, and methane concentration as percentage in
the headspace. Values are averages of three replicates.
b Not measured.
transformers were not affected by high concentrations of hydrogen, and also that the methanogens
were performing well even in the complete absence (initially) of hydrogen in the atmosphere.
The degradation of toluene and o-xylene in suspended mixed cultures was associated with
cell growth. Cell counts were determined by staining with acridine orange and observation under
the cpifluorescence microscope. 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 toluene degradation were both dependent on the initial cell count, as shown in Figures 16
and 17.
We have studied the influence of some environmental factors (temperature, pH, presence of
exogenous electron acceptors, presence of increased concentrations of sulfide) on the performance
52
-------
c
o
to
c
V
CJ
c
o
y
-------
of these cultures, and the results are summarized in Figures 18, 19, and 20. The cultures degraded
toluene faster at 35°C than at 20°C. pH 6 was more favorable than pH 7, and pH 8 appeared to be
unfavorable. This is consistent with the conditions in the contaminated aquifer, where a
groundwater pH of 6 or below was predominant (E.M. Godsy, personal communication). The
addition of exogenous electron acceptors (nitrate or sulfate) slowed down toluene degradation,
indicating that the active community consisted of fermentative and methanogenic bacteria that were
acclimated to the conditions of methanogenic fermentation. Sulfide also slowed the transformation
down, in accordance with other reports that sulfide may be inhibitory to methanogens (Oremland,
1988).
In an attempt to isolate pure cultures from the mixed cultures, roll tubes were prepared
using the same mineral medium with toluene and o-xylene, with the addition of 2% agar. The
headspace in the roll tubes consisted of N2 and CO2 or H2/CO2. Colonies were observed after
three to four weeks of incubation. Some were transferred to liquid medium with toluene and
o-xylene. No activity toward aromatic substrates has been observed; however colonies transferred
into liquid medium with H2 in the headspace produced methane.
. Some of the Pensacola aquifer material, previously used in experiments of degradation of
creosote constituents (courtesy of E.M. Godsy, U.S. Geological Survey) was used to set up new
microcosms for studying adaptation to toluene degradation under various environmental
conditions. The following conditions were tested: 1) toluene (0.08 mM) alone, 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 results are presented in
Figure 21. Each data point in Figure 21 is the average of four replicate microcosms. The sediment
used in this experiment was homogenized to the extent possible by mixing in a large beaker inside
a glove box before distribution into bottles. As a result of homogenization, replicates in this
experiment were reproducible, with a maximum coefficient of variation of 20%. The lag time
before the onset of toluene degradation was about 50 days for both static and agitated microcosms.
This lag time was increased to about 100 days if p-cresol was present, indicating thatp-cresol was
utilized as a preferential substrate. Toluene degradation started 50 days after all thep-cresol had
been depleted; it is possible that the onset of toluene degradation required an enrichment of a
different microbial population, containing the active enzymes. The p-cresol results conflict with
the initial results obtained with the microcosms (see above). The difference is probably due to the
fact that the aquifer material used for these adaptation studies had already undergone an enrichment
step during creosote-degradation studies (E.M. Godsy, personal communication), so that specific
54
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.0
15
Time (days)
Figure 18. Effect of temperature on toluene degradation in suspended culture.
e
o
c3
i-i
a
CL>
a
c
c
u
c
a>
D
>
"53
5 10
Time (days)
Figure 19. Effect of pH on toluene degradation in suspended culture.
55
-------
.2
3
Lh
w
a
-------
0.8
0.6 "
0.4-
0.2
0.0
175
Time (days)
Figure 21. The length of the acclimation lag in toluene-degrading methanogenic
microcosms under different environmental conditions.
Symbols:
---Q---toluene concentration in killed biological control microcosms;
O toluene in microcosms incubated statically;
• toluene in microcosms incubated shaken;
B toluene and ¦— p-cresol in microcosms amended with
^ both toluene and p-CTesol;
^ toluene and acetate in microcosms amended with
both toluene and acetate.
57
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microbial populations were enriched, whereas others were reduced or eliminated. The addition of
acetate prolonged the acclimation lag: acetate-amended microcosms did not adapt to toluene
degradation for more than 100 days after the depletion of acetate. This finding supports the
hypothesis 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 community on the aquifer material.
Attempts to detect some intermediates of toluene and xylene transformation using HPLC
and GC/MS were unsuccessful because of the low concentrations involved. The simultaneous
adaptation method ( Stanier, 1947) is being used to determine which potential intermediates can be
degraded by the enrichment culture.
In summary, active fermentative/methanogenic communities capable of degrading
alkylbenzenes (i.e., toluene, xylenes) were successfully enriched from a methanogenic zone of a
creosote-contaminated aquifer. The aromatic hydrocarbons were completely mineralized in a
medium containing inorganic nutrients and vitamins, in the absence of accessory organic substrates
and any electron acceptors other than CC>2- The conditions favorable for this degradation (the
absence of oxygen, nitrate, and sulfate; the presence of solids; acidic pH) reflect the conditions in
the zone of the aquifer from which the inoculum was obtained. The results demonstrate the
significance of natural biotransformation processes in anoxic aquifers contaminated by aromatic
hydrocarbons.
58
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