Article

pubs.acs.org/est

Anaerobic Conversion of Chlorobenzene and Benzene to CH4 and

CO2 in Bioaugmented Microcosms
Xiaoming Liang,† Cheryl E. Devine,† Jennifer Nelson,‡ Barbara Sherwood Lollar,§ Stephen Zinder,‡
and Elizabeth A. Edwards†,*
†
Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON M5S 3E5, Canada
‡
Department of Microbiology, Cornell University, Ithaca, New York 14853, United States
§
Department of Earth Sciences, University of Toronto, Toronto, ON M5S 3B1, Canada
*
S Supporting Information

ABSTRACT: Chlorobenzene is a widespread groundwater contaminant found at many industrial

sites. Reductive dechlorination of chlorobenzene requires input of electron donor and results in
problematic accumulation of benzene, which is more toxic than chlorobenzene. We hypothesized
that coupling a culture capable of reductive dechlorination of chlorobenzene to benzene with a
second benzene-degrading methanogenic culture would completely detoxify chlorobenzene. To
this end, active chlorobenzene-dechlorinating microcosms that were producing benzene were
inoculated with a previously described enriched methanogenic benzene-degrading consortium.
The combination resulted in the transformation of chlorobenzene via benzene to the nontoxic
degradation products, CO2 and CH4. Sustainable degradation of chlorobenzene and benzene was
observed in the microcosms and was further confirmed by shifts in the carbon isotopic ratios of
chlorobenzene and benzene during degradation. Moreover, we could show that benzene derived electrons fueled chlorobenzene
dechlorination removing the need to provide exogenous electron donor. The results have promising implications for sustainable
bioremediation of sites contaminated with chlorinated benzenes and benzene.

■ INTRODUCTION
Chlorinated benzenes are ubiquitous groundwater contami-
nants because of their intensive use in the production of
pesticides, herbicides, and chemical intermediates.1,2 Incom-
plete dechlorination of higher chlorinated benzenes often
results in the accumulation of chlorobenzene (herein referred
to as monochlorobenzene; MCB) in situ.2,3 Microbial
degradation of lindane, one of the most widely used pesticides,
can also lead to the formation of MCB.4 Toxicity studies have
demonstrated that MCB can cause severe damage to the liver
and kidney.5 As a result, MCB is listed as a priority pollutant by
the United States Environmental Protection Agency and the
maximum contaminant level (MCL) in drinking water is
currently regulated at 100 μg/L.5
Both aerobic and anaerobic biodegradation processes
contribute to the natural attenuation of MCB in the
environment. Under aerobic conditions, the initial attack of
MCB aromatic ring is catalyzed by dioxygenases with the
formation of 3-chlorocatechol as an intermediate.6,7 While pure
strains capable of anaerobically degrading MCB have not yet
been described, anaerobic reductive dechlorination of MCB to
benzene was observed in sediment microcosms,8−10 and an
organism responsible for MCB dechlorination in sediment
microcosms from a site historically contaminated with
chlorobenzenes was recently identified as a Dehalobacter sp.11
Unfortunately, reductive dechlorination of MCB results in the
problematic accumulation of benzene, which is a known human
carcinogen12 with a MCL (5 μg/L13), significantly lower than
MCB (100 μg/L5). Reductive dechlorination of MCB to
benzene can therefore have an adverse effect, hence the
motivation for this study to investigate the coupled effects of
reductive dechlorination of MCB and biodegradation of
benzene.
The anaerobic mineralization of benzene has been
extensively studied,14 and can occur under nitrate-reduc-
ing,15−17 perchlorate-reducing,15 sulfate-reducing,18,19 iron-
reducing,20−23 and methanogenic conditions.16,24−27 Phenol
and benzoate have been detected as intermediates during
anaerobic benzene degradation in iron-reducing,28 sulfate-
reducing,27 and methanogenic enrichment culture conditions.29
However, Kunapuli et al.23 suggested that the formation of
phenol could be an artifact resulting from the oxidation of
benzene after exposure to air during sampling and analysis
procedures. Methane and carbon dioxide were identified as the
final degradation products in these benzene-degrading meth-
anogenic enrichment cultures.16,29
Previous studies have described the complete biodegradation
of chlorinated aromatic compounds under anaerobic conditions
by combining two different cultures or microcosms togeth-
er.30−32 For instance, both 3-chlorobenzoate (3CB) and
benzoate were totally degraded in an anaerobic consortium
Received: October 22, 2012
Revised: January 17, 2013
Accepted: January 29, 2013
Published: January 29, 2013

© 2013 American Chemical Society 2378 dx.doi.org/10.1021/es3043092 | Environ. Sci. Technol. 2013, 47, 2378−2385
Environmental Science & Technology
containing both 3CB and benzoate degraders.30 The rate of
benzoate degradation with the addition of both 3CB and
benzoate as substrates was 50% higher than that with benzoate
added alone.30 Measurement of hydrogen flux indicated that
3CB-degrading bacterium Desulfomonile tiedjei consumed
hydrogen produced from a benzoate-degrader (strain BZ-2),30
and the rate of benzoate degradation rate was enhanced by the
lower H2 concentration.30 Yang et al.31 recently demonstrated
the anaerobic biodegradation of pentachlorophenol (PCP)
under anaerobic conditions via phenol to CO2 and CH4 by
coupling a PCP-degrading enrichment culture with a phenol-
degrading methanogenic enrichment culture. The present study
investigates the potential for the complete anaerobic biode-
gradation of MCB via benzene to CO2 and CH4 by combining
a MCB-degrading culture8 with a benzene-degrading methano-
genic culture.16
The principles and applications of compound-specific isotope
analysis (CSIA) have been reviewed extensively elsewhere.33
Briefly, the preferential cleavage of bonds containing exclusively
a light isotope (e.g., 12C) relative to those containing a heavy
isotope (e.g., 13C) results in the enrichment of the heavy
isotope in the remaining substrate during chemical reaction.
Isotope fractionation typically can be described by the Rayleigh
model
R = R 0f (ε /1000) (1)
where R is the isotope ratio of the remaining substrate at any
time, R0 is its isotopic ratio at time zero, f is the fraction of the
remaining substrate at a given time (i.e., C/C0), and ε is the
enrichment factor. The enrichment factor (ε) for MCB
dechlorination to benzene (ε = −5.0 ± 0.2 ‰) was recently
determined in microcosms prepared from sediments from the
DuPont Chambers Works site.34 The ε value for methanogenic
degradation of benzene in the cultures studied herein (ε = −0.8
± 0.2 ‰) was also previously reported. 35 These laboratory
enrichment factors are consistent with field data. Kaschl et al.7
observed that an enrichment of 4.5 ‰ in MCB at the fringe of
a plume, and Stelzer et al.36 documented 13C enrichment of the
overall weighted isotope signature for total chlorinated
benzenes in field samples.
The first objective of the present work was to examine the
feasibility of this combination strategy for the sustainable
transformation of MCB via benzene to CO2 and CH4. The
second objective was to test if benzene-derived electrons from
the fermentation could fuel MCB dechlorination without
providing exogenous electron donor. Both kinetic and
compound specific stable isotope analyses were carried out
during the course of MCB/benzene degradation by the
combined consortium. In addition, the electron balance
between MCB, benzene, and methane was examined in order
to better understand the complex interactions between MCB-
dechlorinating and benzene-degrading microorganisms.
■ EXPERIMENTAL SECTION
Chemicals. The following chemicals were obtained from
Sigma-Aldrich (St. Louis, MO): MCB (99.5%), benzene
(99.8%), bicarbonate (99.5%), sodium lactate (99.0%), and
yeast extract.
MCB-Dechlorinating Sediment Cultures. Sediment
samples from the DuPont Chambers Works site (Salem
County, New Jersey) historically impacted with chlorinated
benzenes and anilines were used to prepare microcosms as
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described in Fung et al.8 and are referred to herein as the CW
(Chambers Works) sediment cultures. Briefly, these were
prepared in 120 mL serum bottles sealed with Teflon-coated
butyl rubber stoppers and contained 50 mL of anaerobic
deionized water and 20 g of wet sediment from the DuPont
Chambers Works site. The microcosms were flushed with 70%
N2/30%CO2 and amended with 1 g/L of sodium bicarbonate as
a buffer, 0.2 g/L of yeast extract as an electron donor, and neat
MCB (49.3 μmol/bottle) as an electron acceptor. To promote
acclimation to MCB, each fresh sediment culture bottle was
inoculated with 2 mL slurry from a previously prepared active
CW sediment culture,8 to which six or more MCB doses (49.3
μmol/bottle) had been amended to increase biodegradation
rate. Each amendment of MCB corresponds to an approximate
aqueous concentration of 700 μM.
Benzene-Degrading Methanogenic Enrichment Cul-
tures. These cultures were derived from microcosms prepared
with sediment and groundwater collected from an oil refinery in
Oklahoma37 and are referred to as the OR (Oil Refinery)
cultures. Several bottles of OR culture have been maintained
anaerobically on benzene as the sole source of organic carbon
and energy over 14 years. The cultures are capable of degrading
benzene at concentrations up to 1 mM, and degradation rates
as high as 33 μmol/L/day are typical in some.16 The cultures
used in these experiments were degrading benzene at typical
rates of around 5−13 μmol/L/day.16
Combination of CW Sediment Cultures with OR
Enrichment Cultures. Two culture combination experiments
were carried out. In the first culture combination experiment,
60 mL of OR culture was centrifuged anaerobically and
resuspended in 10 mL residual medium prior to inoculation
into a 120 mL serum bottle containing approximately 50 mL
CW sediment culture. Two replicate bottles (#1 and #2, Figure
S1 of the Supporting Information) were prepared this way, and
each was amended with neat MCB (∼30 μmol/bottle) as
electron acceptor and yeast extract (0.1 g/L) as electron donor.
When first MCB and then benzene were depleted, both neat
MCB (∼30 μmol/bottle) and benzene (∼15 μmol/bottle)
were added again. After these substrates were consumed, the
two replicate bottles (#1 and #2, Figure S1 of the Supporting
Information) were each split into two new subculture bottles
(#1 into A1 and B1, #2 into A2 and B2, Figure S1 of the
Supporting Information) each with an approximately equal
volume of blended culture (∼30 mL). To compare methane
production, the subculture bottles A1 and A2 were amended
with both MCB and benzene, whereas the subculture bottles B1
and B2 were amended with benzene only. After approximately
60 days, aliquots of 10 mL from the 30 mL subculture bottles
A1 and A2 (amended with both MCB and benzene) were
transferred anaerobically to new 60 mL serum bottles
containing 10 mL fresh medium (A1→A3, A2→A4, Figure
S1 of the Supporting Information). Bottles A1, A2, A3, and A4
were each amended with both MCB (∼15 μmol/bottle) and
benzene (∼7.5 μmol/bottle). These amendments correspond
to about 400 μM MCB and 200 μM benzene (aqueous
concentrations). Methane production was measured over
repeated amendments of MCB and benzene in all bottles.
In the second culture combination experiment, 50 mL of OR
culture was directly inoculated (without centrifuging to
concentrate as in the first experiment) into 50 mL of CW
sediment culture in two replicate 160 mL serum bottles (#3
and #4, Figure S1 of the Supporting Information). Neat MCB
(50 μmol/bottle) and benzene (∼50 μmol/bottle) were added
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simultaneously to the blended cultures. When MCB and donor for MCB dechlorination in the CW sediment culture, the
benzene were both depleted, the experimental bottles were effect of yeast extract on benzene degradation was examined.
spiked with only MCB (∼50 μmol/bottle) without benzene. Benzene degradation was significantly delayed by the addition
Benzene (∼25 μmol/bottle) was not added until 30 days after of 0.1 g/L yeast extract (part B of Figure S2 of the Supporting
the amendment of MCB. Information) during which methane accumulated presumably
A larger volume (scaled-up) of blended culture (Bottle #5, from yeast extract fermentation (part B of Figure S2 of the
Figure S1 of the Supporting Information) was prepared by Supporting Information). Benzene degradation only began after
adding 100 mL OR culture to 150 mL CW culture into a 500 an 80-day lag presumably once methanogenesis from added
mL bottle. This bottle contained approximately 10% sediment. yeast extract was complete (part B of Figure S2 of the
Subsequently, a second blended culture (Bottle #6, Figure S1 of Supporting Information). In sterile control incubations, there
the Supporting Information) was prepared by combining 720 was no significant change in benzene concentration over 110
mL CW sediment (preacclimated to MCB) with 100 mL OR days (part B of Figure S2 of the Supporting Information).
culture and 180 mL medium. These scaled-up cultures were Combined Degradation of MCB and Benzene. In the
initially amended with both MCB and benzene for the first two first combined culture experiments to which 0.1 g/L yeast
feedings, and were subsequently amended with MCB only extract was added once initially, the dechlorination of 23
(without benzene) to determine if dechlorination could be μmoles MCB stiochiometrically to benzene took about 40 days
sustained without exogenous electron donor. (part A of Figure 1). Benzene was not degraded until after Day
Control cultures to test activity of cultures on individual
substrates were also prepared using CW sediment culture and
OR enrichment culture in parallel with coculture experiments.
One of the benzene-amended OR culture controls was also
amended with yeast extract (0.1 g/L). Abiotic controls with
MCB and benzene in sterile defined mineral medium38 were
also prepared. All bottles were incubated statically in the dark at
room temperature in an anaerobic chamber (Coy Products,
Grass Lake, MI) supplied with a gas mix of 10% H2, 10% CO2,
and 80% N2.
Analytical Methods. Concentrations of MCB, benzene,
and methane were quantified by gas chromatography (GC)
with headspace analysis using methods given in Liang et al.34
Three point external calibration curves were prepared daily.
Relative standard deviations for samples and standards using
this method were typically ±5%. Compound-specific carbon
isotope analysis for MCB and benzene was conducted by direct
injection of headspace samples into a Hewlett-Packard 6890
GC interfaced with a combustion oven in line with a Finnigan
Mat Delta plus XL isotope ratio mass spectrometer. The
combustion oven temperature was controlled at 980 °C. The
GC was equipped with a VOCOL capillary column (60 m ×
0.25 mm ×1.5 μm), and the GC temperature was set at 40 °C,
immediately ramped to 210 °C at 25 °C/min, and held for 8
min. Carbon isotope ratios are reported as δ13C with respect to
the international standard V-PDB (in units of ‰).39 The total
analytical uncertainty for δ13C of MCB and benzene was less
than 0.5 ‰.34

■ RESULTS AND DISCUSSION

MCB Dechlorination in the CW Sediment Cultures.
The transformation of MCB to benzene in replicate positive
control cultures was nearly completed by 60 days although a
short lag time (∼14 days) was observed at the beginning of the
incubation (part A of Figure S2 of the Supporting Information).
The observed rate of MCB degradation was similar to that
previously reported.8,11 There was no significant change in
MCB concentration in the sterile control over 40 days (part A
of Figure S2 of the Supporting Information).
Benzene Mineralization in the OR Enrichment Culture
with and without Yeast Extract. Benzene was stoichio-
metrically degraded to methane in the control OR culture
within approximately 60 days with a maximum degradation rate
of about 16 μmol/L/day (part B of FIgure S2 of the Supporting
Information), which is comparable to the previously reported
rate 33 ± 28 μmol/L/day.16 Since yeast extract was the electron
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Figure 1. Microbial degradation of MCB and benzene in the blended
cultures (CW + OR). Each curve shows the mean value with error bars
representing the range for duplicate bottles. Panel A (Bottles #1 and
#2, Figure S1 of the Supporting Information). Arrows indicate
amendments of 0.5 mM MCB and 0.1g/L yeast extract (Day 0) and
0.5 mM MCB and 0.25 mM benzene (Day 125). The dashed lines
divide the degradation process into phase 1 (simultaneous degradation
of MCB and benzene) and phase 2 (benzene degradation only); Panel
B (Bottles #3 and #4, Figure S1 of the Supporting Information).
Arrows indicate amendments of 0.5 mM MCB and 0.5 mM benzene
(Day 0), 0.5 mM MCB (Day 142), and 0.25 mM benzene (Day 172).
70 (part A of Figure 1) and was completely degraded by Day
120. It is likely that remaining yeast extract delayed the onset of
benzene degradation as occurred in the incubations with
benzene and yeast extract (part B of Figure S2 of the
Supporting Information). The second feeding to these
cocultures (Day 125) consisted of both MCB (∼30 μmol/
bottle) and benzene (∼15 μmol/bottle) without yeast extract.
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Figure 2. Sustained degradation in the blended cultures (CW + OR). Bottles A1 and A2 were amended with both MCB (0.5 mM) and benzene
(0.25 mM). Bottles B1 and B2 were amended with only benzene (0.25 mM). Solid arrows indicate number of times substrates were reamended.
Measurements were not performed for feedings #4 (Panels: B1 and B2) and #11 (Panel B2). The cultures were purged with N2/CO2 (80:20 vol) on
Day 60 and Day 120.
MCB was dechlorinated to benzene at a similar rate to that of
the first feeding (∼6 μmol/L/day) even in the absence of yeast
extract. Benzene degradation began right away this time; the
observed maximum benzene concentration (26 μmol/bottle)
on day 176, was noticeably less than the sum of added MCB
and benzene (19 + 13 = 32 μmol/bottle). This indicated that
reductive dechlorination of MCB to benzene and anaerobic
mineralization of benzene to CH4 and CO2 occurred
simultaneously.
To assess methane production in more detail and interpret
electron balances in the blended culture, we divided the
degradation cycle into two phases, the first where both MCB
and benzene were present and being degraded (phase 1), and
the second where MCB was depleted and only benzene was
being degraded (part A of Figure 1). The maximum benzene
degradation rate observed in phase 2 was faster than in our
control cultures (25 μmol/L/day vs 16 μmol/L/day)
suggesting beneficial effects of the CW sediment on the
benzene culture.
In the second combined culture experiments, MCB and
benzene were amended simultaneously initially, and both were
depleted by Day 140 (part B of Figure 1). On Day 142, only
MCB was reamended to the bottles. This second dose of MCB
was not dechlorinated. After waiting 30 days to see if
dechlorination would begin, we then added benzene to the
duplicate cultures (Day 172), and very shortly thereafter, MCB
dechlorination resumed (part B of Figure 1). This suggested
that benzene-derived electrons (likely as hydrogen) were
needed for MCB dechlorination.
Sustainable Degradation of MCB and Benzene.
Degradation of both MCB and benzene was sustained for
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over 12 months in two subcultures (bottles A1 and A2, Figure
S1 of the Supporting Information) amended with both MCB
(∼15 μmol/bottle) and benzene (∼7.5 μmol/bottle) (parts A1
and A2 of the Figure 2). Methane accumulated as degradation
of MCB and benzene proceeded. The blended cultures were
purged with 80% N2/20% CO2 immediately before doses on
Day 60 and Day 135. To compare methane production, two
parallel 30 mL combined cultures were only amended with
benzene (parts B1 and B2 of Figure 2). The maximum rate of
benzene degradation observed in these combined cultures fed
only benzene (31 μmol/L/day) was twice as fast as that
observed in the OR culture used as the inoculum (part B of
Figure S2 of the Supporting Information). Some components
(e.g., nutrients and/or minerals) in the CW sediment culture
appeared to stimulate benzene-degrading microorganisms.
Similarly, previous studies also demonstrated that the addition
of autoclaved soil slurry enhanced the performance of
chlorinated phenol-degrading enrichment cultures.31,40
Compound Specific Isotope Analysis (CSIA). No
significant change was observed in the carbon isotope
signatures for MCB and benzene in abiotic control bottles
(Figure S3 of the Supporting Information). In the combined
cultures amended with both MCB and benzene, the values of
δ13C for MCB became more enriched as biodegradation
proceeded from an initial value of −25.1 ‰ and increasing to
−3.5 ‰ when approximately 6% of MCB remained in the
culture (Table S1 of the Supporting Information, part A of
Figure 3). During this time, δ13C values for benzene decreased
from the initial −27.1 ‰ to −29.1 ‰ when 45% of the MCB
was degraded (f = 0.55) reflecting the accumulation of 12C in
the product benzene as a result of MCB dechlorination. As
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Figure 3. Carbon isotope signature of (A) MCB and Benzene added
together in the blended cultures (CW + OR) (Bottles A1 and A2) and
(B) Benzene alone in blended cultures (CW + OR) (Bottles B1 and
B2) and OR culture. Error bars on f (= C/C0) represent ±7% total
uncertainty calculated by propagation of error in concentration (text),
and error bars on δ13C represent ±0.5‰ (reproducibility and
accuracy) and are smaller than the plotted symbols.
both MCB and benzene were further degraded, the carbon
isotope values of benzene became enriched in 13C to −19.4 ‰
(f = 0.05) (Table S1 of the Supporting Information, part A of
Figure 3). The solid curve in part A of Figure 3 represents the
expected δ13C enrichment trend for MCB using the Rayleigh
model41 with the previously reported enrichment factor for
MCB (ε = −5.0 ± 0.2 ‰).34 The experimental δ13C values for
MCB were similar to the predicted Rayleigh enrichment trend
but diverged at low fraction remaining (f < 0.3). A possible
explanation is inaccurate measurement of the fraction
remaining because of sorption of MCB to the sediments.
δ13C values were also measured in the parallel combined culture
bottles that were spiked with benzene only. The enrichments in
δ13C values for benzene in these incubations (part B of Figure
3) followed the Rayleigh curve predicted from previously
published ε value for the OR culture (ε = −0.8 ± 0.2 ‰).35
Benzene in the cultures amended with MCB (part A of Figure
3) became significantly more enriched than benzene incubated
alone (part B of Figure 3) clearly proving degradation of
benzene derived from MCB. To evaluate MCB degradation at
field sites, the enrichment factor (ε = −5.0 ± 0.2 ‰)34 should
be used according to the U.S. EPA Guide,33 which suggested
that ε values should be reported from well-defined laboratory
studies.
Compositional and isotope analyses confirmed that the
degradation pathway for the removal of MCB in the combined
cultures was reductive dechlorination of MCB to benzene,
followed by anaerobic transformation of benzene to CO2 and
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methane. Previous studies36,42,43 discussed an alternative
pathway for MCB degradation under anaerobic conditions:
direct mineralization of MCB without accumulation of benzene.
This pathway was suggested because 13CO2 was formed over
the course of degradation but 13CH4 was not.36,42,43 However,
because of the presence of benzene as an impurity in the
labeled [13C6]−MCB, no definitive conclusions could be made.
Electron Balance in the Combined Cultures. There are
30 electron equivalents per mole of benzene, 8 electron
equivalents per mole of methane, and 2 electron equivalents per
mole of MCB dechlorinated to benzene. In the absence of
MCB, and ignoring cell growth, 3.75 (=30/8) moles of
methane would be produced from one mole of benzene (Figure
4). If all benzene came from MCB dechlorination, and benzene
Figure 4. Electron balance for MCB dechlorination and methano-
genesis from benzene. The right panel illustrates the stoichiometry for
methane production from benzene alone. The left panel depicts the
situation when one mole of MCB is dechlorinated to one mole of
benzene, and 2 electron equivalents from benzene are required to fuel
the dechlorination of one mole of MCB. If both benzene and MCB are
amended to the culture, the electrons equivalents from benzene that
can be used for dechlorination may vary from 2 to 6 electron
equivalents (possibly even higher) depending on the rates of MCB
dechlorination and intermediates of benzene oxidation.
fueled MCB dechlorination, then only 3.5 (28/8) moles of
methane would be produced from one mole of MCB, because 2
electrons from benzene would be used for dechlorination. If
MCB transformation to benzene is faster than methanogenesis
from benzene (for example because hydrogen is more
effectively utilized by dechlorinating bacteria), then the
methane production will be further depressed as more electrons
are diverted to dechlorination (phase 1, Figure 4).
We tabulated methane production in all our culture bottles
(Table S2 of the Supporting Information) for each dechlori-
nation/methanogenesis cycle, separated into phase 1 (both
MCB and benzene present) and phase 2 (only benzene
remaining) as illustrated in part A of Figure 1. We also
tabulated the changes in MCB and benzene concentrations for
the same time intervals (Table S2 of the Supporting
Information). Then, we predicted from stoichiometry the
theoretical methane produced (ΔCH4) and compared this to
the actual change in methane we measured (Table S2 of the
Supporting Information). To compare each time interval on the
same basis, we calculated the ratio of the change in methane
concentration (ΔCH4) to the sum of the change of MCB plus
change in benzene concentration (ΔMCB + ΔBenzene), where
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ΔBenzene can sometimes be positive if dechlorination is faster
than methanogenesis (i.e., benzene is produced) (Table S2 of
the Supporting Information). This ratio is predicted to range
from 3.0 (for example if 3 mols H2 from benzene is used for
dechlorination) to 3.75 (when there is no MCB) (Figure 4).
Specifically, we calculated ΔCH4 /(ΔMCB + ΔBenzene) in
phase 1 and phase 2 over multiple feedings in four experimental
bottles (A1, A2, A3, and A4), in which the combined cultures
were spiked with both MCB and benzene simultaneously, as
well as in duplicate bottles (B1 and B2) that were spiked with
benzene only (ΔMCB = 0). The data for bottles A1, A2, B1,
and B2 are also plotted in Figure 2.
The measured molar ratios of methane produced to net
change of benzene and MCB in phase 1 ranged from 2.6 to 4.3
(3.27 ± 0.53, n = 8), while the ratios of methane produced to
benzene degraded in phase 2 vary from 3.7 to 5.0 (4.37 ± 0.56,
n = 8) (Table S2 of the Supporting Information, Figure 5).
Figure 5. Methane molar yields during MCB dechlorination and
benzene degradation. Numbers beside dashed lines (3.75, 3.75, and
3.42) represent the theoretical methane production calculated from
measured substrate depletion, and error bars on the ratios represent
95% confidence intervals. The calculations were repeated for many
feedings (n), and P values shown represent the probability that there is
no significant difference between the two groups tested (text).
Although the ratios do not perfectly match the predicted values,
they are reasonably close (Figure 5). Most notably, the ratios in
phase 1 are statistically significantly lower than those in phase 2
(paired t test, P < 0.0014, Table S3 of the Supporting
Information, Figure 5). When the combined cultures were
amended with benzene only, the experimentally measured
ratios ranged from 3.0 to 4.8 (3.97 ± 0.40, n = 15), and were
not significantly different (t test, P > 0.05) from the ratios
measured during phase 2 in bottles A1, A2, A3, and A4 (Table
S3 of the Supporting Information, Figure 5). However, the
ratios measured for phase 1 (both MCB and Benzene) were
also significantly different from ratios measured in benzene-fed
only cultures (t test, P < 0.0024) (Table S3 of the Supporting
Information, Figure 5). These data provide yet another line of
evidence that electrons derived from benzene fermentation fuel
MCB dechlorination. Further confirmation comes from data in
larger volume (scaled-up) blended cultures (Bottles #5 and #6,
Figure S1 of the Supporting Information). The first scaled-up
culture (Bottle #5, Figure S1 of the Supporting Information)
was initially amended with both MCB and benzene (feedings
#1 and #2, Figure 6). The sustained degradation of both MCB
and benzene continued when the scaled-up culture was
subsequently amended with MCB only (feedings #3−5, Figure
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Figure 6. Microbial degradation of MCB and benzene in the scaled-up
blended culture. Arrows indicate amendments of MCB and benzene
(first two feedings). For the last three feedings (#3, #4, and #5), only
MCB (148 μmol/bottle, which corresponds to an approximate
aqueous concentration of 500 μM) was added at each feeding. The
initial concentrations of MCB and benzene at each feeding were
estimated based on the amount of MCB or benzene added.
6). The second scaled-up culture (Bottle #6, Figure S1 of the
Supporting Information) was amended in the same manner,
and sustained degradation of MCB via benzene continued
without adding benzene during at least four successive feedings
of MCB without benzene (data not shown). Because no
external electron donor was added to these cultures, it appears
that either a small amount of residual benzene or the decay of
biomass provided the electrons required to initiate MCB
dechlorination after each feeding, and that newly produced
benzene then fueled further MCB dechlorination.
Implications for Contaminated Sites. Chlorinated
benzenes are widespread groundwater contaminants found at
many industrial sites. Complete dechlorination of chlorinated
benzenes results in problematic accumulation of benzene,
which is more toxic than parent compounds. In this work, we
developed microcosms containing microbes capable of both
MCB dechlorination and benzene degradation. Sustainable
degradation of MCB and benzene was achieved and the final
products are nontoxic compounds CO2 and CH4. The cultures
have the ability to use benzene fermentation products as
electron donors for further dechlorination of MCB to benzene,
creating a feedback loop that means no (or very little) external
electron donor needs to be added for the process to occur. This
essentially solves the two major challenges associated with
conventional bioremediation approaches for MCB-contami-
nated sites: (1) accumulation of more toxic benzene, and (2)
substantial cost of chemical reagents that are required as
electron donors for MCB dechlorination. Because fast
degradation of benzene was achieved in the combined novel
cultures, our findings can also have promising implications for
sustainable anaerobic bioremediation of benzene-contaminated
sites.
■
*
ASSOCIATED CONTENT
S Supporting Information
Additional data that can be found in the Supporting
Information include: a flowchart of culture combination
experiments (Figure S1); degradation profiles of MCB in CW
sediment with yeast extract as electron donor (Figure S2);
degradation profiles of benzene in the OR culture in the
presence and absence of yeast extract (Figure S2); carbon
isotope signatures of MCB and benzene in abiotic controls
dx.doi.org/10.1021/es3043092 | Environ. Sci. Technol. 2013, 47, 2378−2385
Environmental Science & Technology
(Figure S3); carbon isotope data (Table S1); data for electron
balance (Table S2); and a summary of methane production
data and statistics (Table S3).This material is available free of
charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*Phone: (416) 946-3506, fax: (416) 978-8605, e-mail:
elizabeth.edwards@utoronto.ca.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We thank Sarah McRae (University of Toronto) for
maintaining scaled-up cultures. This research was supported
by the Government of Canada through Genome Canada and
the Ontario Genomics Institute, the Natural Science and
Engineering Research Council of Canada (NSERC) Strategic
Projects Program and the Canada Research Chairs Program to
B.S.L., and a grant from the DuPont Corporate Remediation
Group to S.H.Z.
■
REFERENCES
(1) Field, J.; Sierra-Alvarez, R. Microbial degradation of chlorinated
benzenes. Biodegradation 2008, 19 (4), 463−480.
(2) Adrian, L.; Görisch, H. Microbial transformation of chlorinated
benzenes under anaerobic conditions. Res. Microbiol. 2002, 153 (3),
131−137.
(3) Adrian, L.; Szewzyk, U.; Wecke, J.; Görisch, H. Bacterial
dehalorespiration with chlorinated benzenes. Nature 2000, 408
(6812), 580−583.
(4) Phillips, T.; Seech, A.; Lee, H.; Trevors, J. Biodegradation of
hexachlorocyclohexane (HCH) by microorganisms. Biodegradation
2005, 16 (4), 363−392.
(5) http://water.epa.gov/drink/contaminants/basicinformation/
chlorobenzene.cfm (Accessed October 21, 2012).
(6) Werlen, C.; Kohler, H.-P. E.; van der Meer, J. R. The broad
substrate chlorobenzene dioxygenase and cis-chlorobenzene dihydro-
diol dehydrogenase of Pseudomonas sp. strain P51 are linked
evolutionarily to the enzymes for benzene and toluene degradation.
J. Biol. Chem. 1996, 271 (8), 4009−4016.
(7) Kaschl, A.; Vogt, C.; Uhlig, S.; Nijenhuis, I.; Weiss, H.; Kastner,
M.; Richnow, H. H. Isotopic fractionation indicates anaerobic
monochlorobenzene biodegradation. Environ. Toxicol. Chem. 2005,
24 (6), 1315−1324.
(8) Fung, J. M.; Weisenstein, B. P.; Mack, E. E.; Vidumsky, J. E.; Ei,
T. A.; Zinder, S. H. Reductive dehalogenation of dichlorobenzenes and
monochlorobenzene to benzene in microcosms. Environ. Sci. Technol.
2009, 43 (7), 2302−2307.
(9) Masunaga, S.; Susarla, S.; Yonezawa, Y. Dechlorination of
chlorobenzenes in anaerobic estuarine sediment. Water Sci. Technol.
1996, 33, 173−180.
(10) Nowak, J.; Kirsch, N. H.; Hegemann, W.; Stan, H. J. Total
reductive dechlorination of chlorobenzenes to benzene by a
methanogenic mixed culture enriched from Saale river sediment.
Appl. Microbiol. Biotechnol. 1996, 45 (5), 700−709.
(11) Nelson, J. L.; Fung, J. M.; Cadillo-Quiroz, H.; Cheng, X.;
Zinder, S. H. A role for Dehalobacter spp. in the reductive
dehalogenation of dichlorobenzenes and monochlorobenzene. Environ.
Sci. Technol. 2011, 45 (16), 6806−6813.
(12) Dean, B. J. Recent findings on the genetic toxicology of
benzene, toluene, xylenes, and phenols. Mutat. Res. 1985, 154 (3),
153−181.
(13) http://water.epa.gov/drink/contaminants/basicinformation/
benzene.cfm (Accessed October 21, 2012).
2384
Article
(14) Lovley, D. R. Anaerobic benzene degradation. Biodegradation
2000, 11, 107−116.
(15) Coates, J. D.; Chakraborty, R.; Lack, J. G.; O’Connor, S. M.;
Cole, K. A.; Bender, K. S.; Achenbach, L. A. Anaerobic benzene
oxidation coupled to nitrate reduction in pure culture by two strains of
dechloromonas. Nature 2001, 411 (6841), 1039−1043.
(16) Ulrich, A. C.; Edwards, E. A. Physiological and molecular
characterization of anaerobic benzene-degrading mixed cultures.
Environ. Microbiol. 2003, 5 (2), 92−102.
(17) Burland, S. M.; Edwards, E. A. Anaerobic benzene
biodegradation linked to nitrate reduction. Appl. Environ. Microbiol.
1999, 65, 529−533.
(18) Lovley, D. R.; Coates, J. D.; Woodward, J. C.; Phillips, E. J. P.
Benzene oxidation coupled to sulfate reduction. Appl. Environ.
Microbiol. 1995, 61, 953−958.
(19) Phelps, C. D.; Kerkhof, L. J.; Young, L. Y. Molecular
characterization of a sulfate-reducing consortium which mineralizes
benzene. FEMS Microbiol. Ecol. 1998, 27 (3), 269−279.
(20) Kunapuli, U.; Lueders, T.; Meckenstock, R. U. The use of stable
isotope probing to identify key iron-reducing microorganisms involved
in anaerobic benzene degradation. ISME J 2007, 1 (7), 643−653.
(21) Lovley, D. R.; Woodward, J. C.; Chapelle, F. H. Rapid anaerobic
benzene oxidation with a variety of chelated Fe(III) forms. Appl.
Environ. Microbiol. 1996, 62 (1), 288−291.
(22) Jahn, M. K.; Haderlein, S. B.; Meckenstock, R. U. Anaerobic
degradation of benzene, toluene, ethylbenzene, and o-xylene in
sediment-free iron-reducing enrichment cultures. Appl. Environ.
Microbiol. 2005, 71, 3355−3358.
(23) Kunapuli, U.; Griebler, C.; Beller, H. R.; Meckenstock, R. U.
Identification of intermediates formed during anaerobic benzene
degradation by an iron-reducing enrichment culture. Environ. Micro-
biol. 2008, 10 (7), 1703−1712.
(24) Grbić-Galić, D.; Vogel, T. M. Transformation of toluene and
benzene by mixed methanogenic cultures. Appl. Environ. Microbiol.
1987, 53 (2), 254−260.
(25) Kazumi, J.; Caldwell, M. E.; Suflita, J. M.; Lovley, D. R.; Young,
L. Y. Anaerobic degradation of benzene in diverse anoxic environ-
ments. Environ. Sci. Technol. 1997, 31, 813−818.
(26) Weiner, J. M.; Lovley, D. R. Rapid benzene degradation in
methanogenic sediments from a petroleum-contaminated aquifer.
Appl. Environ. Microbiol. 1998, 64, 1937−1939.
(27) Caldwell, M. E.; Suflita, J. M. Detection of phenol and benzoate
as intermediates of anaerobic benzene biodegradation under different
terminal electron-accepting conditions. Environ. Sci. Technol. 2000, 34
(7), 1216−1220.
(28) Botton, S.; Parsons, J. Degradation of BTX by dissimilatory
iron-reducing cultures. Biodegradation 2007, 18 (3), 371−381.
(29) Ulrich, A. C.; Beller, H. R.; Edwards, E. A. Metabolites detected
during biodegradation of 13C6-benzene in nitrate-reducing and
methanogenic enrichment cultures. Environ. Sci. Technol. 2005, 39,
6681−6691.
(30) Dolfing, J.; Tiedje, J. M. Kinetics of two complementary
hydrogen sink reactions in a defined 3-chlorobenzoate degrading
methanogenic co-culture. FEMS Microbiol. Ecol. 1991, 86 (1), 25−32.
(31) Yang, S.; Shibata, A.; Yoshida, N.; Katayama, A. Anaerobic
mineralization of pentachlorophenol (PCP) by combining PCP-
dechlorinating and phenol-degrading cultures. Biotechnol. Bioeng. 2009,
102 (1), 81−90.
(32) Li, Z.; Yang, S.; Inoue, Y.; Yoshida, N.; Katayama, A. Complete
anaerobic mineralization of pentachlorophenol (PCP) under con-
tinuous flow conditions by sequential combination of PCP-
dechlorinating and phenol-degrading consortia. Biotechnol. Bioeng.
2010, 107 (5), 775−785.
(33) Hunkeler, D.; Meckenstock, R. U.; Sherwood Lollar, B.;
Schmidt, T. C.; Wilson, J. T., A Guide for Assessing Biodegradation
and Source Identification of Organic Ground Water Contaminants
Using Compound Specific Isotope Analysis (CSIA). In EPA 600/R-
08/148, United States Environmental Protection Agency: Ada, OK,
2008.
dx.doi.org/10.1021/es3043092 | Environ. Sci. Technol. 2013, 47, 2378−2385
Environmental Science & Technology Article

(34) Liang, X.; Howlett, M. R.; Nelson, J. L.; Grant, G.; Dworatzek,
S.; Lacrampe-Couloume, G.; Zinder, S. H.; Edwards, E. A.; Sherwood
Lollar, B. Pathway-dependent isotope fractionation during aerobic and
anaerobic degradation of monochlorobenzene and 1,2,4-trichloroben-
zene. Environ. Sci. Technol. 2011, 45 (19), 8321−8327.
(35) Mancini, S. A.; Devine, C. E.; Elsner, M.; Nandi, M. E.; Ulrich,
A. C.; Edwards, E. A.; Sherwood Lollar, B. Isotopic evidence suggests
different initial reaction mechanisms for anaerobic benzene bio-
degradation. Environ. Sci. Technol. 2008, 42 (22), 8290−8296.
(36) Stelzer, N.; Imfeld, G.; Thullner, M.; Lehmann, J.; Poser, A.;
Richnow, H.-H.; Nijenhuis, I. Integrative approach to delineate natural
attenuation of chlorinated benzenes in anoxic aquifers. Environ. Pollut.
2009, 157 (6), 1800−1806.
(37) Nales, M.; Butler, B. J.; Edwards, E. A. Anaerobic benzene
biodegradation: A microcosm survey. Bioremed. J. 1998, 2 (2), 125−
144.
(38) Edwards, E. A.; Wills, L. E.; Reinhard, M.; Grbić-Galić, D.
Anaerobic degradation of toluene and xylene by aquifer micro-
organisms under sulfate-reducing conditions. Appl. Environ. Microbiol.
1992, 58 (3), 794−800.
(39) Coplen, T. B.; Brand, W. A.; Gehre, M.; Gröning, M.; Meijer, H.
A. J.; Toman, B.; Verkouteren, R. M. After two decades a second
anchor for the VPDB δ13C scale. Rapid Commun. Mass Spectrom. 2006,
20 (21), 3165−3166.
(40) Ehlers, G. A.; Rose, P. D. An integrated anaerobic/aerobic
bioprocess for the remediation of chlorinated phenol-contaminated
soil and groundwater. Water Environ. Res. 2006, 78 (7), 701−709.
(41) Mariotti, A.; Germon, J. C.; Hubert, P.; Kaiser, P.; Letolle, R.;
Tardieux, A.; Tardieux, P. Experimental determination of nitrogen
kinetic isotope fractionation: Some principles; illustration for the
denitrification and nitrification processes. Plant Soil 1981, 62, 413−
430.
(42) Nijenhuis, I.; Stelzer, N.; Köstner, M.; Richnow, H.-H. Sensitive
detection of anaerobic monochlorobenzene degradation using stable
isotope tracers. Environ. Sci. Technol. 2007, 41 (11), 3836−3842.
(43) Martínez-Lavanchy, P.; Dohrmann, A.; Imfeld, G.; Trescher, K.;
Tebbe, C.; Richnow, H.-H.; Nijenhuis, I. Detection of monochlor-
obenzene metabolizing bacteria under anoxic conditions by DNA-
stable isotope probing. Biodegradation 2011, 22 (5), 973−982.

2385 dx.doi.org/10.1021/es3043092 | Environ. Sci. Technol. 2013, 47, 2378−2385