United States
Environmental Protection
Agency
National Risk Management
Research Laboratory
Ada, OK 74820
Research and Development
EPA/600/SR-97/103
October 1997
v/EPA 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
sediments under mixed oxygen/
denitrifying conditions. The principal
hypothesis considered in this research
is that biodegradation of certain mono
and polycyclic aromatic hydrocarbons
occurs under mixed oxygen/denitrifying
conditions and that the rate and extent
of biodegradation is greater underthese
conditions than traditional single
electron acceptor schemes. To test this
hypothesis, laboratory experiments were
designed to compare biodegradation
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
Protection and Remediation Division,
Ada, OK, to announce key findings of
the research project that is fully
documented in a separate report of the
same title (see Project Report ordering
information at back).
Introduction
This research project included three
phases: (1) screening of site aquifer material
for microorganisms which can successfully
biodegrade model aromatic compounds
under aerobic and denitrifying conditions
(facultative anaerobes); (2) batch studies
to assess the biodegradation of the model
compounds under mixed oxygen/nitrate
electron acceptorconditions compared with
biodegradation under aerobic and
anaerobic denitrifying conditions; and (3)
aquifer material column 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 summarized in Table 1.
Methods and Results
Phase / - Microbial
Characterization 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
demonstrated that viable bacteria were
present at the site and at a variety of depths
and locations. Some ofthese bacteria could
be cultured under aerobic and anaerobic
conditions suggesting that facultative
anaerobic bacteria are present at the site.
Mineralization assay results demonstrated
that indigenous site bacteria were capable
of aerobic biodegradation of a number of
compounds including benzene, toluene,
naphthalene and phenanthrene and
anaerobic mineralization of naphthalene.
The extent of substrate mineralization under
aerobic conditions ranged from 0 to 91%.
The extent of naphthalene mineralization
afterSO days of incubation underdenitrifying
conditions was as high as 16%.
Mineralization assays conducted for 30 days
using liquid enrichments of these aquifer
bacteria (no aquifer solids were present)
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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
under anaerobic denitrifying conditions 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 nitrite).
Phase II - Under
Conditions
Batch Study 1 - Mixed Substrates -
Enrichment Microcosms - Mixed Electron
Acceptors. Batch microcosm experiments
proved to be a successful method to screen
for biodegradation of BTEX, naphthalene
and phenanthrene under varying
combinations of oxygen and nitrate. The
aromatic compounds biodegraded under
varying combinations of oxygen and nitrate
are summarized in Table 2.
Biodegradation was defined as 10% loss
of substrate relative to controls. With the
exception of toluene, oxygen appeared 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
biodegradation with increasing oxygen
concentration, the denitrifying enrichment
was sensitive to extremely high levels of
oxygen (30 mg O2/L). Although the bacteria
were able to use oxygen under these
conditions, a lag time occurred before
significant biodegradation was observed.
No lag time was observed during
biodegradation when air saturated
conditions were provided (7.6 mg O2/L)
resulting in good removal of all compounds
(except benzene in some microcosms).
Providing 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 underconditions where the oxygen
concentration is equivalent to or greater
than 1.5 mg/L. At levels of oxygen less than
1 mg/L, only toluene biodegradation was
observed. Toluene removal was not initiated
until the oxygen was nearly depleted. In this
case, the oxygen removal observed priorto
toluene biodegradation may have been due
in part to some oxygen removal or
detoxifying mechanism or to endogenous
respiration, and appeared to be required
before significant anaerobic denitrification
was observed. Results of experiments with
toluene as the sole substrate suggested
that in the absence of competing, aromatic
hydrocarbon substrates (i.e., benzene,
ethylbenzene, /??~xylene, naphthalene and
phenanthrene), oxygen may act as an
electron acceptor during biodegradation of
Table 2. Aromatic Hydrocarbons Degraded Under Various Combinations of Oxygen and Nitrate in Batch Liquid Enrichment Microcosms
Nitrate (mg/L)
10
50
150
400
T
T
T
T
0.5
T
T
T
T
1.0
n.t,
T,E
T,E
T,E
Oxygen (mg/L)
.5 2.0
T,N
T,N
T,N
T,N
T,N
T,N
T,N
T,N
7.0
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
30.0
B,T,E,m-X,N,P
n.t.
n.t.
n.t.
B = benzene, T = toluene, E = ethylbenzene, m-X = m-xylene, N = nsphthalene and P = phenanthrene, n.t. = not tested
2
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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 under microaerophilic
conditions and was as slow as 0.0066
mg/L-hour under oxygen saturation
conditions (O2 ~ 30 mg/L).
Batch Study2-MixedSubstrates-Aquifer
Material Microcosms - Mixed Electron
Acceptors. The objective of Batch Study II
was to assess the impact of sediments on
the transformation of a mixture of aromatic
hydrocarbons (BTEX, naphthalene, and
phenanthrene) under mixed electron
acceptorconditions. The results ofthe batch
microcosm experiments using sediment as
inocula under aerobic, microaerophilic and
denitrifying conditions were not
distinguishable. 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 significant abiotic oxygen demands
associated with sediments cored from
anaerobic regions of the source aquifer.
Additional oxygen demands may be
attributable to the degradation of labile
organic carbon associated with the
sediments. These results reveal that abiotic
oxygen demands must be accounted for
when batch experiments are conducted to
estimate kinetic parameters for the design
of in situ bioremediation processes.
Batch Study 3- Mixed Substrates -A quifer
Material Microcosms - Electron Acceptor
Replenished Over Time. The primary goals
of Batch Study 3 were to: 1) satisfy the
abiotic demand for oxygen in the
microcosms to allow for study ofthe biotic
oxygen demand of biodegradation, and 2)
quantify the level of oxygen atwhich aerobic
degradation of mixed aromatic substrates
(BTEXand naphthalene)was inhibited and
denitrification was initiated in batch
sediment microcosms. The results of this
set of experiments revealed that both
oxygen and nitrate were utilized as terminal
electron acceptors under microaerophilic
conditions (O2 concentration < 2 mg/L).
Concurrent use of oxygen and nitrate as
terminal electron acceptors occurred when
aqueous oxygen concentrations were below
2.0 mg/L. Toluene was degraded under
denitrifying conditions while benzene,
ethylbenzene, /77-xylene and naphthalene
were degraded using oxygen as the electron
acceptor. Denitrifying activity and toluene
transformation 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 additional 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, /77-xyleneandtoluenewere
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, naphthalene,
and /77-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 significantly
greater under aerobic conditions than
microaerophilic conditions. The rates of
transformation for each of the substrates
were relatively constant under
microaerophilic conditions for dissolved
oxygen concentrations ranging from
3.0
D)
C
o
^5
5
•+->
c
0)
o
c
o
o
n- Toluene
Naphthalene
Ethylbenzene
Dissolved Oxygen
1.0 --
0.0
20
40
60 80
Hours
100
120
140
Figure 1 - Substrates and dissolved oxygen remaining in Sediment Microcosm #1 with an initial oxygen concentration of 2 mg/L.
3
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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 levels also
controlled denitrification as well as the rate
and extent of substrate removal. Providing
a mixtureof microaerophilicand denitrifying
conditions did not necessarily improve
biodegradation 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 ill - Simulation of in Situ
Treatment in Soil Columns
Under 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 contaminated
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 O7 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 occurred
under aerobic conditions (~ 8.6 mg O2/L) in
the presence of nitrate. Excellent toluene
removal was also achieved in all columns
with all levels of oxygen (i.e., 0, 1, 2 and
8.6 mgO2/L) except in the absence of nitrate
underscoring the importance of nitrate to
toluene remediation in these sediments. By
increasing the concentration of oxygen, the
number of compounds and the extent of
their biodegradation was enhanced.
Benzene, ethylbenzene, /77-xylene and
naphthalene were recalcitrant in the
absence of oxygen. Providing
microaerophilic levels of oxygen
(<_2 mg O2/L) enhanced the removal of
ethylbenzene, /77-xylene and naphthalene
but fully aerobic 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
naphthalene that was converted to carbon
dioxide and intermediates was unaffected
by oxygen concentration. Therefore, oxygen
concentration probably controls the initial
step(s) in naphthalene breakdown and may
not be involved in the mineralization of the
resulting intermediates or the decay of
microbial cells. Naphthalene 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 liquid enrichment
microcosm studies which concluded that
therewasathreshold oxygen concentration
(1.5mgO2/L) below which naphthalene
removal did not occur. 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, toluene 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
illustrated in Figure 2. Nitrate consumption
and nitrite production increased in the
aerobic column in response to an increase
in the influent toluene concentration.
Furthermore, sampling along the length of
the aerobic column revealed that the extent
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 denitrifying 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 concentrations
may have been lower than in the bulk pore
space. These data support the belief that
nitrate may enhance mineralization by
acting as an alternative electron acceptor
or simply by stimulating additional cell
formation. Regardless of its role, aerobic
bioremediation is enhanced by the addition
of nitrate in aquifer sediments 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 removal
than anaerobic denitrifying conditions
except in the case of toluene where oxygen
did not provide any benefit in terms of the
extent of toluene removal. There were no
benefits to providing microaerophilic levels
of oxygen (< 2 mg/L) in combination with
nitrate when compared with higher levels of
oxygen (7 and 30 mg O2/L). Moderate, yet
aerobic levels of oxygen in combination
with nitrate ratherthan high concentrations
(30 mg O2/L) resulted in comparable
substrate removal and faster kinetics.
Providing low levels of oxygen in
combination with nitrate during in situ
bioremediation ratherthan only high levels
of oxygen may accomplish or yield the
following benefits: 1) low levels of oxygen
are nottoxicto denitrifying bacteria allowing
forfacultative use of both oxygen and nitrate
as electron acceptors; 2) low levels of
oxygen are less expensive to maintain in
the subsurface; and 3) it is easierto 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
denitrifying conditions will likely prove more
successful than solely aerobic remediation
for the long term remediation of aromatic
hydrocarbons.
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•o
0)
o
0?
0)
o
tl
Q.
±QQ
Toluene
tee
Restore NO3
Remove O2
-20 0 20 40 60 80 lo 10 14/3 160
i i . i
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,
Maryland 21218
Stephen R. Hutchins is the EPA Project Officer (see below).
The complete report, entitled "Bioremediation ofBTEX, Naphthalene, andPhenanthrene
in Aquifer Material Using Mixed Oxygen/Nitrate Electron Acceptor Conditions," (Order
No. PB95-X; Cost: $X.OO, 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:
U.S. Environmental Protection Agency
National Risk Management Research Laboratory
Subsurface Protection and Remediation Division
P.O. Box 1198
Ada, OK 74820
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