United States
Environmental Protection
Agency
National Risk Management
Research Laboratory
Ada, OK 74820
Research and Development
EPA/600/SR-97/120
October 1997
Project Summary
Bioremediation of BTEX,
Naphthalene, and Phenanthrene in
Aquifer Material Using Mixed
Oxygen/Nitrate Electron Acceptor
Conditions
Liza P. Wilson, Peter C. D'Adamo and Edward J. Bouwer
The goal of the research described
herein was to examine the feasibility of
biodegradation of mono and polycyclic
aromatic hydrocarbons typically present
in a manufactured gas processing (MGP)
site groundwater and subsurface sedi-
ments under mixed oxygen/denitrifying
conditions. The principal hypothesis
considered in this research is that bio-
degradation of certain mono and
polycyclic aromatic hydrocarbons oc-
curs under mixed oxygen/denitrifying
conditions and that the rate and extent
of biodegradation is greater under these
conditions than traditional single elec-
tron acceptor schemes. To test this
hypothesis, laboratory experiments
were designed to compare biodegrada-
tion under mixed electron acceptor
conditions with biodegradation under
single electron acceptor schemes.
This Project Summary was developed
by EPA's National Risk Management
Research Laboratory's Subsurface Pro-
tection and Remediation Division, Ada,
OK, to announce key findings of the
research project that is fully docu-
mented in a separate report of the same
title (see Project Report ordering infor-
mation at back).
Introduction
This research project included three
phases: (1) screening of site aquifer ma-
terial for microorganisms which can
successfully biodegrade model aromatic
compounds under aerobic and denitrify-
ing conditions (facultative anaerobes); (2)
batch studies to assess the biodegrada-
tion of the model compounds under mixed
oxygen/nitrate electron acceptor condi-
tions compared with biodegradation un-
der aerobic and anaerobic denitrifying
conditions; and (3) aquifer material col-
umn studies to confirm the findings of the
batch studies and to better simulate mixed
oxygen/denitrifying remediation in the
subsurface. Specific experiments included
in each phase of the research are sum-
marized in Table 1.
Methods and Results
Phase I - Microbial Characteriza-
tion and Assessment
For in situ biodegradation with mixtures
of oxygen and nitrate to be successful,
bacteria that are capable of using both
oxygen and nitrate as electron acceptors
must be present at the remediation site.
A survey of the MGP site sediments dem-
onstrated that viable bacteria were
present at the site and at a variety of
depths and locations. Some of these bac-
teria could be cultured under aerobic and
anaerobic conditions suggesting that fac-
ultative anaerobic bacteria are present at
the site. Mineralization assay results dem-
onstrated that indigenous site bacteria
were capable of aerobic biodegradation
of a number of compounds including ben-
zene, toluene, naphthalene and
phenanthrene and anaerobic mineraliza-
tion of naphthalene. The extent of
substrate mineralization under aerobic
conditions ranged from 0% to 91%. The
extent of naphthalene mineralization af-
ter 80 days of incubation under
denitrifying conditions was as high as
16%. Mineralization assays conducted for
30 days using liquid enrichments of these
aquifer bacteria (no aquifer solids were
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Table 1. Summary of the Experiments Conducted in Each of the Three Research Phases
PHASE I - MICROBIAL ASSESSMENT
Sample Collection
Batch Study 1
Single substrate, aquifer
material microcosms
Batch Study 2
Single substrate, liquid
enrichment microcosms
PHASE II - BATCH STUDIES OF MIXED OXYGEN/NITRATE ELECTRON ACCEPTOR CONDITIONS
Batch Study 1
Mixed substrates, liquid
enrichment microcosms
Batch Study 2
Mixed substrates, aquifer
material microcosms
Batch Study 3
Mixed substrates, aquifer
material microcosms,
electron acceptor
replaced over time
PHASE III - COLUMN STUDIES OF MIXED OXYGEN/NITRATE ELECTRON ACCEPTOR CONDITIONS
Column A
Anaerobic/microaerophilic
single-port injection
Column B
Anaerobic/microaerophilic
single-port injection
Column C
Aerobic single-port and
multi-port injection
present) under anaerobic denitrifying con-
ditions did not yield mineralization of the
target compounds. The biodegradation
rates were so slow that 30 days was not
long enough to observe mineralization of
the target substrates. Notwithstanding the
lack of mineralization of the model
compounds in the liquid enrichment
microcosms, sufficient evidence of
denitrifying activity was observed in the
culture fluids (conversion of nitrate to ni-
trite).
Phase II - Batch Studies Under
Mixed Electron Acceptor Condi-
tions
Batch Study 1 - Mixed Substrates - En-
richment Microcosms - Mixed Electron
Acceptors. Batch microcosm experiments
proved to be a successful method to
screen for biodegradation of BTEX, naph-
thalene and phenanthrene under varying
combinations of oxygen and nitrate. The
aromatic compounds biodegraded under
varying combinations of oxygen and ni-
trate are summarized in Table 2.
Biodegradation was defined as 10%
loss of substrate relative to controls. With
the exception of toluene, oxygen ap-
peared to be the key to removal of the
aromatic hydrocarbons. Increased levels
of oxygen yielded an improvement in the
extent of compound removal in the batch
microcosms. Despite the improved bio-
degradation with increasing oxygen
concentration, the denitrifying enrichment
was sensitive to extremely high levels of
oxygen (30 mg O2/L). Although the bacte-
ria were able to use oxygen under these
conditions, a lag time occurred before
significant biodegradation was observed.
No lag time was observed during bio-
degradation when air saturated conditions
were provided (7.6 mg O2/L) resulting in
good removal of all compounds (except
benzene in some microcosms). Provid-
ing oxygen in excess of the stoichiometric
requirements for aerobic biodegradation
did not necessarily yield a greater extent
of biodegradation of aromatic compounds.
It appears that the rate of biodegradation
of all compounds may be enhanced by
providing a lower level of oxygen (i.e., 7
to 8 mg O2/L) which may be less toxic to
facultative anaerobic bacteria.
Under microaerophilic conditions, the
enrichment was able to use oxygen to
degrade naphthalene without any lag time
suggesting that the enzymes for aerobic
biodegradation (oxygenases) are easily
induced under conditions where the oxy-
gen concentration is equivalent to or
greater than 1.5 mg/L. At levels of oxy-
gen less than 1 mg/L, only toluene
biodegradation was observed. Toluene
removal was not initiated until the oxy-
gen was nearly depleted. In this case,
the oxygen removal observed prior to tolu-
ene biodegradation may have been due
in part to some oxygen removal or detoxi-
fying mechanism or to endogenous
respiration, and appeared to be required
before significant anaerobic denitrification
was observed. Results of experiments
Table 2. Aromatic Hydrocarbons Degraded Under Various Combinations of Oxygen and Nitrate in Batch Liquid Enrichment
Microcosms
Nitrate fmg/L^
:LQ
Oxygen (mg/L)
1,5 ZQ
LQ
30.0
10
50
150
400
T
T
T
T
T
T
T
T
n.t.
T,E
T,E
T,E
T,N
T,N
T,N
T,N
T,N
T,N
T,N
T,N
B,T,E,m-X,N,P
B,T,E,m-X,N,P
B,T,E,m-X,N,P
B,T,E,m-X,N,P
B,T,E,m-X,N,P
n.t.
n.t.
n.t.
B = benzene, T = toluene, E = ethylbenzene, m-X = m-xylene, N = naphthalene and P = phenanthrene, n.t. = not tested
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with toluene as the sole substrate sug-
gested that in the absence of competing,
aromatic hydrocarbon substrates (i.e.,
benzene, ethylbenzene, m-xylene, naph-
thalene and phenanthrene), oxygen may
act as an electron acceptor during biodeg-
radation of toluene. The rate of oxygen use
was slow at high initial levels of oxygen
and faster at microaerophilic levels. Zero
order rates for oxygen consumption
ranged from 0.016- 0.032 mg/L-hour un-
der microaerophilic conditions and was
as slow as 0.0066 mg/L-hour under oxy-
gen saturation conditions (O2 ~ 30 mg/L).
Batch Study 2 - Mixed Substrates - Aqui-
fer Material Microcosms - Mixed Electron
Acceptors. The objective of Batch Study
2 was to assess the impact of sediments
on the transformation of a mixture of aro-
matic hydrocarbons (BTEX, naphthalene,
and phenanthrene) under mixed electron
acceptor conditions. The results of the batch
microcosm experiments using sediment as
inocula under aerobic, microaerophilic and
denitrifying conditions were not distinguish-
able. Only toluene was degraded and
mineralization of toluene occurred under
denitrifying conditions. Residual oxygen
was consumed in the microcosms within
24 hours of experimental setup. Oxygen
consumption was presumably due to sig-
nificant abiotic oxygen demands associated
with sediments cored from anaerobic re-
gions of the source aquifer. Additional
oxygen demands may be attributable to
the degradation of labile organic carbon
associated with the sediments. These re-
sults reveal that abiotic oxygen demands
must be accounted for when batch experi-
ments are conducted to estimate kinetic
parameters for the design of in situ
bioremediation processes.
Batch Study 3 - Mixed Substrates - Aqui-
fer Material Microcosms - Electron
Acceptor Replenished Over Time. The pri-
mary goals of Batch Study 3 were to: 1)
satisfy the abiotic demand for oxygen in
the microcosms to allow for study of the
biotic oxygen demand of biodegradation,
and 2) quantify the level of oxygen at
which aerobic degradation of mixed aro-
matic substrates (BTEX and naphthalene)
was inhibited and denitrification was initi-
ated in batch sediment microcosms. The
results of this set of experiments revealed
that both oxygen and nitrate were utilized
as terminal electron acceptors under mi-
croaerophilic conditions (O2 concentration
< 2 mg/L). Concurrent use of oxygen
and nitrate as terminal electron accep-
tors occurred when aqueous oxygen
concentrations were below 2.0 mg/L.
Toluene was degraded under denitrifying
conditions while benzene, ethylbenzene,
m-xylene and naphthalene were degraded
using oxygen as the electron acceptor.
Denitrifying activity and toluene transfor-
mation were observed in the presence of
slightly higher bulk solution dissolved
oxygen concentrations than observed in
the liquid enrichment microcosms (Batch
Study 1). The sediments likely exerted
additional oxygen demand such that ad-
ditional oxygen was required to achieve
the same results as were observed in
Batch Study 1. The presence of sediments
may have resulted in microsite dissolved
oxygen concentrations below that of the
bulk solution.
Naphthalene, m-xylene and toluene
were preferentially degraded to a greater
extent and at a faster rate than benzene
and ethylbenzene. Significant benzene
and ethylbenzene biotransformation did
not typically occur until toluene, naphtha-
lene, and m-xylene were removed from
the microcosms (Figure 1).
A zero-order rate model (independent
of substrate concentration) provided the
best fit to the experimental data. The rate
of substrate transformation was signifi-
cantly greater under aerobic conditions
than microaerophilic conditions. The rates
3.0
2.0
Toluene
Naphthalene
Ethylbenzene
Dissolved Oxygen
D)
C
o
c
0)
o
c
o
o
0 20 40 60 80 100 120 140
Hours
Figure 1. Substrates and dissolved oxygen remaining in Sediment Microcosm #1 with an initial oxygen concentration of 2 mg/L.
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of transformation for each of the substrates
were relatively constant under microaero-
philic conditions for dissolved oxygen
concentrations ranging from 0.45 mg/L-
hour to 1.1 mg/L-hour. Oxygen
concentration controlled biodegradation
of this suite of aromatic hydrocarbons in
batch sediment microcosms. Oxygen lev-
els also controlled denitrification as well
as the rate and extent of substrate re-
moval. Providing a mixture of
microaerophilic and denitrifying conditions
did not necessarily improve biodegrada-
tion when compared with oxygen alone as
long as oxygen was maintained between
0.45 and 1.1 mg/L. Denitrification appears
to play a role in substrate removal only
when the supply of oxygen is limited and
finite.
Phase III - Simulation of In Situ
Treatment in Soil Columns
Under Mixed Electron Acceptor
Conditions
The overall objective of this research
was to evaluate the biodegradability of a
mixture of aromatic compounds using
mixed oxygen/nitrate electron acceptors
under conditions which simulate a con-
taminated groundwater aquifer. This was
accomplished using saturated sediment
columns. Biodegradation of BTEX and
naphthalene was evaluated under the
following electron acceptor conditions:
Microaerophilic - 2 mg/L O2 and 150 mg/L NO3
Microaerophilic - 1 mg/L O2 and 150 mg/L NO3
Aerobic - 8.6 mg/L O2 and 55 mg/L NO3
As with the batch microcosm studies,
the most successful biodegradation of the
mixture of aromatic hydrocarbons oc-
curred under aerobic conditions (~ 8.6
mg O2/L) in the presence of nitrate. Ex-
cellent toluene removal was also
achieved in all columns with all levels of
oxygen (i.e., 0, 1, 2 and 8.6 mg O2/L) ex-
cept in the absence of nitrate underscoring
the importance of nitrate to toluene
remediation in these sediments. By in-
creasing the concentration of oxygen, the
number of compounds and the extent of
their biodegradation was enhanced. Ben-
zene, ethylbenzene, m-xylene and
naphthalene were recalcitrant in the
absence of oxygen. Providing microaero-
philic levels of oxygen (<_2 mg O2/L) en-
hanced the removal of ethylbenzene,
m-xylene and naphthalene but fully aero-
bic conditions (> 7 mg O2/L) allowed for
some removal of all compounds with
naphthalene and toluene completely
transformed (> 95%).
The extent of naphthalene removal was
a function of oxygen concentration and
increased with an increase in oxygen
concentration. The proportion of naph-
thalene that was converted to carbon
dioxide and intermediates was unaffected
by oxygen concentration. Therefore, oxy-
gen concentration probably controls the
initial step(s) in naphthalene breakdown
and may not be involved in the mineral-
ization of the resulting intermediates or
the decay of microbial cells. Naphtha-
lene removal was observed in the column
which received 2 mg O2/L but not in the
column which received 1 mg O2/L. These
results support the findings of batch liq-
uid enrichment microcosm studies which
concluded that there was a threshold oxy-
gen concentration (1.5 mg O?/L) below
which naphthalene removal did not oc-
cur. However, for batch electron acceptor
replenishment studies (Phase II, Batch
Study 3), naphthalene was transformed
at aqueous oxygen concentrations less
than 0.5 mg/L.
Toluene and naphthalene removal
ceased once nitrate was removed from
the microaerophilic columns. When nitrate
was restored to the column influent, tolu-
ene and naphthalene removal continued
(with 2 mg O2/L) providing further evidence
that nitrate is required for biodegradation of
toluene and naphthalene in these aquifer
sediments. The role of O2 and NO3 in the
removal of toluene and naphthalene in a
microaerophilic sediment column is illus-
trated in Figure 2. Nitrate consumption
and nitrite production increased in the
aerobic column in response to an increase
in the influent toluene concentration. Fur-
thermore, sampling along the length of
the aerobic column revealed that the ex-
tent of toluene transformation could be
correlated to the consumption of nitrate
and the production of nitrite along the
length of the column in the presence of
aqueous pore oxygen concentrations
greater than 2 mg/L. Substantial denitrify-
ing activity was observed in the aerobic
column in the presence of pore dissolved
oxygen concentrations as high as 5 mg/
L. Either aerobic levels of oxygen did not
inhibit denitrifying activity, or denitrifying
activity occurred in microsites or within a
biofilm where dissolved oxygen concen-
trations may have been lower than in the
bulk pore space. These data support the
belief that nitrate may enhance mineral-
ization by acting as an alternative electron
acceptor or simply by stimulating addi-
tional cell formation. Regardless of its
role, aerobic bioremediation is enhanced
by the addition of nitrate in aquifer sedi-
ments harboring denitrifying bacteria.
Conclusions
The results of these experiments have
important implications for in situ
bioremediation. Providing some level of
oxygen resulted in better substrate re-
moval than anaerobic denitrifying
conditions except in the case of toluene
where oxygen did not provide any ben-
efit in terms of the extent of toluene
removal. There were no benefits to pro-
viding microaerophilic levels of oxygen
(< 2 mg/L) in combination with nitrate
when compared with higher levels of oxy-
gen (7 and 30 mg O2/L). Moderate, yet
aerobic levels of oxygen in combination
with nitrate rather than high concentra-
tions (30 mg O2/L) resulted in comparable
substrate removal and faster kinetics.
Providing low levels of oxygen in com-
bination with nitrate during in situ
bioremediation rather than only high lev-
els of oxygen may accomplish or yield
the following benefits: 1) low levels of
oxygen are not toxic to denitrifying bacte-
ria allowing for facultative use of both
oxygen and nitrate as electron acceptors;
2) low levels of oxygen are less expen-
sive to maintain in the subsurface; and 3)
it is easier to maintain a low residual
oxygen concentration in the subsurface
than a high concentration due to the many
oxygen demands/sinks. An in situ
bioremediation scheme which combines
moderate aerobic (7 mg/L O2) and deni-
trifying conditions will likely prove more
successful than solely aerobic remediation
for the long term remediation of aromatic
hydrocarbons.
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d>
o
d>
o:
+rf
o>
o
0>
Q.
Toluene
0 140 1(50
o>
o
i
^Naphthalene
Add NO
l^3\
-torn
... Restore NO3
Nitrate J^ Remove 02
-20 0 20 40,60 80 1(JO 120 14p 160
• Days , , ,
Figure 2. Percent toluene and naphthalene removed and nitrate consumed in Column A over time under anaerobic, mixed
oxygen/nitrate and aerobic conditions (2 mg/L oxygen and 150 mg/L nitrate).
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Liza P. Wilson, Peter C. D'Adamo and Edward J. Bouwer are with Department of
Geography and Environmental Engineering, The Johns Hopkins University,
Baltimore, MD 21218
Stephen R. Hutchins is the EPA Project Officer (see below).
The complete report, entitled "Bioremediation of BTEX, Naphthalene, and
Phenanthrene in Aquifer Material Using Mixed Oxygen/Nitrate Electron Ac-
ceptor Conditions," (Order No. PB98-106446; Cost: $35.00, subject to change)
will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Subsurface Protection and Remediation Division
National Risk Management Research Laboratory
U.S. Environmental Protection Agency
Ada, OK 74820
United States
Environmental Protection Agency
Center for Environmental Research Information
Cincinnati, OH 45268
Official Business
Penalty for Private Use $300
BULK RATE
POSTAGE & FEES PAID
EPA
PERMIT No. G-35
EPA/600/SR-97/120
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