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Applied and Environmental Microbiology, April 2001, p. 1517-1521, Vol. 67, No. 4
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.4.1517-1521.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Relationship of Hydrogen Bioavailability to
Chromate Reduction in Aquifer Sediments
Tamara L.
Marsh and
Michael J.
McInerney*
Department of Botany and Microbiology,
University of Oklahoma, Norman, Oklahoma 73019
Received 12 June 2000/Accepted 4 January 2001
 |
ABSTRACT |
Biological Cr(VI) reduction was studied in anaerobic sediments from
an aquifer in Norman, Okla. Microcosms containing sediment and mineral
medium were amended with various electron donors to determine those
most important for biological Cr(VI) reduction. Cr(VI) (about 340 µM)
was reduced with endogenous substrates (no donor), or acetate was
added. The addition of formate, hydrogen, and glucose stimulated
Cr(VI) reduction compared with reduction in unamended controls. From
these sediments, an anaerobic Cr(VI)-utilizing enrichment was obtained
that was dependent upon hydrogen for both growth and Cr(VI) reduction.
No methane was produced by the enrichment, which reduced about 750 µM
Cr(VI) in less than six days. The dissolved hydrogen concentration was
used as an indicator of the terminal electron accepting process
occurring in the sediments. Microcosms with sediments, groundwater, and
chromate metabolized hydrogen to a concentration below the detection
limits of the mercury vapor gas chromatograph. In microcosms without
chromate, the hydrogen concentration was about 8 nM, a concentration
comparable to that under methanogenic conditions. When these microcosms
were amended with 500 µM Cr(VI), the dissolved hydrogen concentration
quickly fell below the detection limits. These results showed that the hydrogen concentration under chromate-reducing conditions became very
low, as low as that reported under nitrate- and manganese-reducing conditions, a result consistent with the free energy changes for these
reactions. The utilization of formate, lactate, hydrogen, and glucose
as electron donors for Cr(VI) reduction indicates that increasing the
availability of hydrogen results in a greater capacity for Cr(VI)
reduction. This conclusion is supported by the existence of an
enrichment dependent upon hydrogen for growth and Cr(VI) reduction.
| |
INTRODUCTION |
More than 170,000 tonnes of
chromium wastes are released annually, mainly due to industrial
practices, including electroplating, leather tanning, pigment
manufacture, corrosion inhibition, and fungicide production
(11). These industries generate large quantities of waste,
which must be treated before discharge. The widespread use of chromium
as well as the improper disposal of by-products and wastes has led to
areas of serious environmental contamination, with chromium presently
being listed as one of the contaminants in 635 Superfund sites (U. S. Environmental Protection Agency, Office of Health and Environmental
Assessment [http://www.epa.gov/ngispgm3/iris]).
Chromium can exist in six valence states, from 0 to +6, but is
generally encountered as the trivalent [Cr(III)] or hexavalent [Cr(VI)] form (24). Trivalent chromium, an essential
trace element in the human diet, has relatively low toxicity
and is nearly insoluble at neutral pH. Thus, it is
nearly immobile in the environment (3). Conversely,
hexavalent chromium is acutely toxic, mutagenic, teratogenic, and
carcinogenic. In addition, Cr(VI) is soluble and, thus, highly mobile
in the environment (5, 8). Focusing on its toxicity and
exposure potential, the U.S. Environmental Protection Agency recently
designated chromium, as well as its compounds, as one of seventeen
chemicals posing the greatest threat to human health (U. S. Environmental Protection Agency, Office of Health and Environmental Assessment).
The valence state and relative solubility of chromium are dependent on
a variety of environmental conditions (redox potential, pH, and
temperature) and the presence of other organic and inorganic molecules
(14, 23). Oxidation-reduction (redox) reactions can
greatly influence the fate and mobility of these organic and inorganic
compounds in both pristine and contaminated aquifers. Many of the
significant redox reactions taking place in aquifers, such as nitrate
reduction, Fe(III) reduction, sulfate reduction, and methane
production, are microbially catalyzed. Lovley and Goodwin
(18) have proposed that H2
concentrations in groundwater may indicate which terminal electron
accepting process (TEAP) is dominant at a given site, with each TEAP
having a characteristic range for its dissolved hydrogen concentration.
The dissolved hydrogen concentrations reported for specific
terminal electron accepting processes are as follows: methanogenesis, 7 to 10 nM; sulfate reduction, 1 to 1.5 nM; Fe(III) reduction, 0.2 nM;
Mn(IV) or nitrate reduction, <0.05 nM (detection limit)
(18).
Many bacterial strains have been shown to mediate reduction of Cr(VI)
to Cr(III) both aerobically (7, 13, 15, 16) and
anaerobically (17, 19, 20 25, 28, 30, 32); however, few
studies have examined the in situ potential of microbial reduction in
aquifers (12). The stimulation of existing microbial
populations with bioavailable electron donors may result in microbial
chromium reduction, potentially preventing the migration, and reducing the toxicity, of Cr(VI) in aquifers. However, it is not clear whether
highly reducing conditions (e.g., sulfate reducing, iron reducing, or
methanogenic) are needed, or whether microbial Cr(VI) reduction will
occur under more oxidized conditions (aerobic or nitrate reducing).
Electron donors used as stimulants for microbially mediated Cr(VI)
reduction in aquifers, as well as the prevailing H2 concentration during Cr(VI) reduction, were
assessed in this study.
| |
MATERIALS AND METHODS |
Sample collection and microcosm construction.
Subsurface
sediments from an aquifer underlying the municipal landfill in Norman,
Okla., were collected by digging to the top of the water table (5 to 6 ft below the surface) with a post-hole digger and collecting aquifer
material in sterile glass jars. The jars were filled to capacity,
sealed, and transported to the laboratory, where they were stored in an
anaerobic chamber at room temperature until used.
Microcosms were prepared in an anaerobic glove box (Coy Laboratory
Products, Inc., Ann Arbor, Mich.) by placing 5 g (wet weight) of
sediment into sterile serum bottles (160-ml capacity) and adding 50 ml
of sterile, anaerobic mineral medium. The composition of the mineral
medium has been previously described by Tanner (27) and
was prepared according to the procedure described by Balch and Wolfe
(4). No reductant was added to the microcosms. All serum
bottles were sealed with butyl rubber stoppers, and the gas headspace
was exchanged with 80% N2-20%
CO2 (
125 kPa); the final pH of the microcosms
was 7.2. Each microcosm typically had an initial Cr(VI) concentration
of approximately 500 µM, added from a sterile stock of
K2CrO4, which was
replenished after complete reduction. Triplicate microcosms were used
for each treatment, and heat-killed controls were included for each
experiment. The heat-killed controls were autoclaved twice at 121°C
for 20 min and then amended with 20 mg of
HgCl2 per liter. All incubations were carried out
at room temperature in the dark. Samples were collected regularly from
each microcosm.
Effect of electron donor.
To determine the effect of
electron donor additions, microcosms were incubated without a donor
until the rate of Cr(VI) reduction slowed in order to exhaust any
endogenous electron donor present in the sediment. After 161 days, the
microcosms were amended with donor and reamended with Cr(VI) before an
additional 360-day incubation period began. During this second
incubation period, microcosms were reamended with Cr(VI) on one
occasion, 217 days after donor addition. Statistical differences
between the mean Cr(VI) reductions for each donor, compared with the no
donor control, were determined with the Student t test
(P = 0.05).
Enrichment for Cr(VI)-reducing, H2-utilizing
consortium.
A Cr(VI)-reducing, H2-utilizing
enrichment was obtained from aquifer sediment and serially transferred
in a mineral salts medium with an 80% H2-20%
CO2 (125 kPa) headspace and an initial Cr(VI)
concentration of approximately 750 µM. The mineral medium contained
the following components (per liter of deionized water): Tanner's
metal solution (27), 5 ml; Tanner's vitamin solution (27), 10 ml; Pfennig's mineral solution
(22), 10 ml; yeast extract, 0.1 g; and
NaHCO3, 10 g. The enrichment was maintained by transferring a 1 to 5% inoculum to fresh media bimonthly.
Determination of H2 concentration under
Cr(VI)-reducing conditions.
Microcosms were constructed as
described above, with 5 g (wet weight) of sediment, 50 ml of
groundwater and 500 µM Cr(VI). Hydrogen was added with a Hamilton
syringe to give an initial soluble H2
concentration of approximately 300 nM (0.8%). Microcosms were
stored in a stationary position at room temperature in the dark.
Analytical methods.
The Cr(VI) concentration was determined
colorimetrically by reaction with diphenylcarbazide in acid solution,
having a detection limit of approximately 5 µM Cr(VI) (9,
31). The coefficient of variation was 4.7%. Hydrogen was
quantitated with a mercury vapor reduction gas analyzer
(26). Fatty acids were analyzed with a high-performance
liquid chromatograph (HPLC) equipped with a Bio-Rad Aminex HPX-87H
column (300 by 7.8 mm) and an isocratic mobile phase of 0.016 N
H2SO4 at a flow rate of 0.9 ml/min (2). Benzoate was analyzed with an HPLC equipped
with an Alltech Econosphere C18 column (250 by
4.6 mm; reversed phase) and a UV detector set at 254 nm (Alltech Inc.,
Deerfield, Ill.). The HPLC was operated at a flow rate of 1.0 ml/min
with a mobile phase of 80% (vol/vol) sodium acetate (50 mM, pH 4.5)
and 20% (vol/vol) acetonitrile. At the conclusion of each experiment,
methane production was measured by gas chromatography (6).
| |
RESULTS |
Effect of electron donor.
The addition of formate, hydrogen,
and glucose to aquifer sediments stimulated Cr(VI) reduction compared
to that in unamended controls (Table 1).
Cr(VI) reduction in microcosms amended with lactate, benzoate, and
acetate was not significantly different from that observed in
microcosms with no exogenous donor. Little loss of Cr(VI) occurred in
heat-killed controls, indicating that reduction of Cr(VI) in viable
microcosms was microbiologically mediated.
Measurement of the donors revealed that formate (2.1 mM) was completely
utilized and that the 1.6 mM lactate added was converted
to about 1.2 mM acetate. Hydrogen, initially 80% of the gas headspace,
was below
detection limits as measured by mercury vapor gas chromatography
at the
conclusion of the experiment. Glucose (approximately 58
mM) was
converted to about 80 mM acetate, 4 mM propionate, 9 mM
isobutyrate,
and 3 mM butyrate. Taking into account methane production
(
1.8 mmol)
and an assumed production of CO
2 (one
CO
2 molecule
produced for every two carbon
compounds produced) resulted in
a glucose carbon recovery of about
91%. Approximately 62 µM of
the 639 µM benzoate added was used and
no depletion of acetate
(2.1 mM) occurred during the 360-day incubation
period. At the
conclusion of the experiment, methane was measured.
Microcosms
supplied with H
2 produced 0.84 mmol of
methane, and glucose-amended
microcosms produced 1.8 mmol of methane.
Lactate- and benzoate-amended
microcosms produced 0.04 mmol of methane;
0.05 mmol of methane
was formed when formate was the donor. Little
methane (0.001 mmol)
was detected in acetate-amended and
non-acetate-amended microcosms.
Cr(VI) reduction accounted for about
80% of the electrons available
from benzoate and about 16 to 17% of
the electrons available from
lactate and formate. Glucose- and
hydrogen-amended microcosms
were primarily fermentative and
methanogenic, respectively, with
these processes accounting for the
majority of the electrons
available.
Cr(VI)-reducing, H2-utilizing consortium.
From
these sediments a Cr(VI)-reducing, H2-utilizing
enrichment was obtained, which was maintained sediment-free for over two years. This enrichment was able to reduce approximately 750 µM
Cr(VI) in 6 days under growing conditions when hydrogen was supplied as
the donor (Fig. 1). Some growth, but no
chromium reduction, occurred in the absence of hydrogen.
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FIG. 1.
Growth and chromate reduction by
H2-utilizing, Cr(VI)-reducing enrichment. (Error bars
represent the standard deviation of triplicate samples; if there are no
error bars, then standard deviation is less than the width of the data
point.)
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|
Growth and Cr(VI) reduction by the enrichment were completely inhibited
in the presence of 10 mM formaldehyde. The enrichment
was able to grow
and reduce Cr(VI) when either 10 mM bromoethanesulfonic
acid (BESA), an
inhibitor of methanogenic bacteria, or 10 mM molybdate,
an inhibitor of
sulfate-reducing bacteria, was present. However,
molybdate appeared to
partially inhibit growth of the enrichment,
since the optical densities
were not equivalent to those of cultures
with no inhibitor present. In
addition, cultures grown with molybdate
required an additional day to
completely reduce the Cr(VI). No
methane was produced by the enrichment
at any time. Based on the
lack of methane production and tolerance to
BESA it was concluded
that methanogenic bacteria were not important
members of this
consortium. The fact that molybdate had little effect
on growth
or Cr(VI) reduction argues against the possibility that
Cr(VI)
reduction was the result of the cycling of low levels of sulfate
to S
2
, which could then react with Cr(VI).
Neither growth nor Cr(VI)
reduction was affected by the addition of 40 mM NaCl, which was
included as an ionic strength control (data not
shown).
H2 concentration under Cr(VI)-reducing conditions.
The dissolved hydrogen concentration in the presence of Cr(VI) was
below detection limits (Fig. 2). This has
also been observed when either nitrate or manganese serves as the
terminal electron acceptor (18). After 34 days, bottles
were reamended with hydrogen and the concentrations again fell to below
detection limits. No loss of Cr(VI) could be measured due to the small
amount of added hydrogen, and no methane was detected when Cr(VI) was
present. Microcosms to which no chromate was added had a dissolved
hydrogen concentration of 5.6 nM, which falls in the range reported for methanogenesis as the terminal electron accepting process (5 to 10 nM)
(18). Upon reamendment with hydrogen, the concentration of
dissolved hydrogen fell to 9.7 nM and was stable for over 50 days. The
addition of 500 µM Cr(VI) to microcosms that previously had not
received Cr(VI) caused the hydrogen concentration to drop from 9.7 nM
to below detection levels, as was found for the other Cr(VI)-amended
microcosms (Fig. 2). Stoichiometric balances between hydrogen
consumption and Cr(VI) reduction were not possible since the amount of
hydrogen added would result in very little Cr(VI) reduction. However,
previous experiments showed a decrease in hydrogen concentration
concomitant with Cr(VI) reduction.
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FIG. 2.
Hydrogen concentrations in aquifer microcosms in the
presence and absence of Cr(VI). (The lower graph has an expanded
axis of the upper graph.) Symbols:  , heat-killed control with
Cr(VI);  , heat-killed control without Cr(VI);  , live incubation
with Cr(VI);  , live incubation without Cr(VI).
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DISCUSSION |
Cr(VI) is a strong oxidant that can be reduced abiotically in the
presence of electron donors commonly found in soils, such as aqueous
Fe(II), ferrous iron minerals, reduced sulfur, and soil organic matter
(10, 24). In addition to abiotic reduction, many
microorganisms have been shown to mediate reduction of Cr(VI) to the
trivalent form (17, 33). However, under the conditions used in this study, namely a mixed culture with sediment, it would be
difficult to determine whether Cr(VI) reduction occurred by direct
enzymatic reduction of the chromate anion or by the continual production of small amounts of Fe(II) or S2,
which can then abiotically react with Cr(VI). However, the
establishment of a sediment-free enrichment in medium with very low
levels of sulfate and Fe(II) is consistent with the conclusion that
direct microbial reduction of Cr(VI) occurred. Also, the addition of molybdate to the enrichment resulted in slight inhibition of Cr(VI) reduction, suggesting that sulfur cycling was not an important mechanism for Cr(VI) reduction in the enrichment.
Regardless of the mechanism of Cr(VI) reduction, our studies show the
potential for stimulating Cr(VI) reduction by naturally occurring
microorganisms. Addition of electron donors that increase the
bioavailable hydrogen for microbial use, such as formate, hydrogen, and
glucose, resulted in a greater extent of Cr(VI) reduction. Benzoate,
which is thermodynamically more difficult to degrade with hydrogen
production, and acetate, which may be degraded without any hydrogen
production, did not stimulate Cr(VI) reduction to the extent of the
other electron donors. Thus, the addition of suitable electron donors
may be an effective method for treating chromium contamination in
aquifers owing to stimulation of organisms indigenous to the
aquifer. The existence of a Cr(VI)-reducing enrichment that is
dependent upon H2 for both growth and Cr(VI) reduction supports the conclusion that increasing the availability of
hydrogen promotes greater reduction of Cr(VI).
This study documents the dissolved hydrogen concentration during
Cr(VI)-reducing conditions. Our results indicate that very low hydrogen
concentrations occur under Cr(VI)-reducing conditions, as has been
reported for nitrate- and manganese-reducing conditions (18). Furthermore, the dissolved hydrogen concentration in
methanogenic microcosms fell below the detection limits within 2 days
of Cr(VI) addition. The fact that very low hydrogen concentrations were observed in Cr(VI)-amended microcosms, similar to that reported for
nitrate- and manganese-reducing conditions, is logical given the Gibbs
free energy changes for these reduction reactions when coupled to
hydrogen oxidation (Table 2) (1,
29). The Gibbs free energy change for Cr(VI) reduction is less
favorable than for nitrate or manganese reduction but much more
favorable than for sulfate reduction or methanogenesis. This is
also consistent with our previous experiments, in which Cr(VI) was
added in combination with NO3,
Fe(III), and SO42
(21). Cr(VI) reduction was found to occur simultaneously
with nitrate reduction but prior to iron or sulfate reduction. When Cr(VI) was present, no Fe(II) or S2 production
was observed. However, in the absence of Cr(VI), nitrate utilization
was followed by Fe(II) production and, later, sulfate reduction.
Understanding the processes that stimulate naturally occurring
microorganisms to reduce Cr(VI) is essential to improving present remediation strategies for contaminated sites by optimizing
Cr(VI)-reducing conditions. For example, addition of electron donors
that make more hydrogen available for microbial use, thereby resulting
in a greater extent of Cr(VI) reduction, may offer a straightforward approach to the treatment of Cr(VI)-contaminated aquifers. Second, the
very low H2 concentrations observed during Cr(VI)
reduction (Fig. 2) and the observation that Cr(VI) reduction occurs
before iron or sulfate reduction (21) suggest that highly
reducing conditions (e.g., iron-reducing, sulfate-reducing, or
methanogenic conditions) will not likely be required for Cr(VI) reduction.
| |
ACKNOWLEDGMENTS |
Support for this research was provided by the U.S. Department of
Energy (grants DE-FG03-96ER202/14 and DE-FG02-97ER62478).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Botany and Microbiology, University of Oklahoma, 770 Van Vleet Oval, Norman, OK 73019. Phone: (405) 325-6050. Fax: (405) 325-7619. E-mail:
McInerney{at}ou.edu.
| |
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Applied and Environmental Microbiology, April 2001, p. 1517-1521, Vol. 67, No. 4
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.4.1517-1521.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
