Project Summary

United States

Environmental Protection

Agency

National Risk Management

Research Laboratory

Ada, OK 74820

Research and Development EPA/600/SR-97/120

Bioremediation of BTEX,

Naphthalene, and Phenanthrene in

Aquifer Material Using Mixed

Oxygen/Nitrate Electron Acceptor

Conditions

October 1997

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 under these

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 acceptor conditions 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 I - 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 of these 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 after 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

Liza P. Wilson, Peter C. D'Adamo and Edward J. Bouwer
2

present) 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 - Batch Studies Under

Mixed Electron Acceptor 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 under conditions 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 prior to 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

Table 1. Summary of the Experiments Conducted in Each of the Three Research Phases

PHASE I - MICROBIAL ASSESSMENT

Sample Collection Batch Study 1 Batch Study 2

Single substrate, aquifer Single substrate, liquid

material microcosms enrichment microcosms

PHASE II - BATCH STUDIES OF MIXED OXYGEN/NITRATE ELECTRON ACCEPTOR CONDITIONS

Batch Study 1 Batch Study 2 Batch Study 3

Mixed substrates, liquid Mixed substrates, aquifer Mixed substrates, aquifer

enrichment microcosms material microcosms material microcosms,

electron acceptor

replaced over time

PHASE III - COLUMN STUDIES OF MIXED OXYGEN/NITRATE ELECTRON ACCEPTOR CONDITIONS

Column A Column B Column C

Anaerobic/microaerophilic Anaerobic/microaerophilic Aerobic single-port and

single-port injection single-port injection multi-port injection

Table 2. Aromatic Hydrocarbons Degraded Under Various Combinations of Oxygen and Nitrate in Batch Liquid Enrichment

Microcosms

Nitrate (mg/L) Oxygen (mg/L)

0 0.5 1.0 1.5 2.0 7.0 30.0

 10 T T n.t. T,N T,N B,T,E,m-X,N,P B,T,E,m-X,N,P

 50 T T T,E T,N T,N B,T,E,m-X,N,P n.t.

150 T T T,E T,N T,N B,T,E,m-X,N,P n.t.

400 T T T,E T,N T,N B,T,E,m-X,N,P n.t.

B = benzene, T = toluene, E = ethylbenzene, m-X = m-xylene, N = naphthalene and P = phenanthrene, n.t. = not tested
3

with toluene as the sole substrate suggested that in the absence of competing,

aromatic hydrocarbon substrates (i.e.,

benzene, ethylbenzene, m-xylene, naphthalene and phenanthrene), oxygen may

act as an electron acceptor during biodegradation 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 under microaerophilic conditions and was

as slow as 0.0066 mg/L-hour under oxygen saturation conditions (O2

 ~ 30 mg/L).

Batch Study 2 - Mixed Substrates - Aquifer 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 aromatic 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 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 - Aquifer 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 of the

biotic oxygen demand of biodegradation,

and 2) quantify the level of oxygen at

which aerobic degradation of mixed aromatic substrates (BTEX and 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,

m-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, 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, naphthalene, 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 significantly greater under aerobic conditions

than microaerophilic conditions. The rates

Figure 1. Substrates and dissolved oxygen remaining in Sediment Microcosm #1 with an initial oxygen concentration of 2 mg/L.

0.0

1.0

2.0

3.0

0 20 40 60 80 100 120 140

Hours

Concentration (mg/L)

Benzene Toluene Ethylbenzene

m-Xylene Naphthalene Dissolved Oxygen
4

of transformation for each of the substrates

were relatively constant under microaerophilic conditions for dissolved oxygen

concentrations ranging from 0.45 mg/Lhour 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 mixture of

microaerophilic and 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 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 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 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 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 mg O2

/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, m-xylene and

naphthalene were recalcitrant in the

absence of oxygen. Providing microaerophilic levels of oxygen (< 2 mg O2

/L) enhanced the removal of ethylbenzene,

m-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 there was a threshold oxygen concentration (1.5 mg O2

/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 rather than 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 rather than only high levels of oxygen may accomplish or yield

the following benefits: 1) low levels of

oxygen are not toxic to denitrifying bacteria allowing for facultative 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 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 denitrifying conditions will likely prove more

successful than solely aerobic remediation

for the long term remediation of aromatic

hydrocarbons.
5

Toluene

-50

-25

0

25

50

75

100

-20 0 20 40 60 80 100 120 140 160

Percent Removed

Naphthalene

-50

-25

0

25

50

75

100

-20 0 20 40 60 80 100 120 140 160

Percent Removed

Nitrate

0

20

40

60

80

100

-20 0 20 40 60 80 100 120 140 160

Days

Percent 

Consumed

Add O2

Remove NO3

Restore NO3

Remove O2

Add NO3

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).
6

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 Acceptor 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

EPA/600/SR-97/120

BULK RATE

POSTAGE & FEES PAID

EPA

PERMIT No. G-35