EPA/600/A-97/084
Submitted for publication to The Encyclopedia of Environmental Analysis and Remediation
CHROMIUM (VI) BIOTREATMENT IN SOIL
by
Guy W. Sewell1, Hai Shen2, and P. Hap Pritchard3
1 U.S.EPA, Robert S. Kerr Environmental Research Center, P.O. Box 1198, Ada, OK 74820.
(405)436-8566, (405)436-8703 (FAX), Email:sewell@ad3100.ada.epa.gov;
2Dynamac Corporation, 3601 Oakridge Boulevard, Ada, OK 74820. (405)436-6409;
3Naval Research Laboratory, 4555 Overlook Ave. S.W., Washington, DC 20375-5321.
(202)767-3340.
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INTRODUCTION
Chromium is widely used in diverse industries and its inappropriate disposal practice has
resulted in the release of this metal into the environment (1). Chromium has become one of the
toxic metals most frequently detected in contaminated environments. The potential for adverse
human health effects has led to increased public concerns over chromium contamination.
Chromium exists in a variety of oxidation states, from 0 to +6. However, in natural environments
only hexavalent chromium Cr(VI) and trivalent chromium Cr(III) are stable species. Cr(VI) is
much more hazardous due to its carcinogenicity, mutagenicity and mobility, than the insoluble
trivalent chromium compounds. Cr(III) is considered to be relatively innocuous and even essential
to human health in minute quantities (2). Conventional chemical and electrochemical techniques
for Cr(VI) removal are all based on reduction of Cr(VI) to Cr(III) and then precipitation of it as
chromium hydroxide. The effective reduction of Cr(VI) normally requires an acidic reaction
environment (pH<3), and the complete conversion is dependent on the concentration and type of
reducing agents employed. However, applications of these techniques have limitations in terms of
cost, effectiveness and sludge production. Recently the potential for the biotreatment of Cr(VI)
wastes has received increased attention because the microbially mediated processes may offer a
cost-effective alternative to chemical treatment. There are several biological mechanisms which
may be suitable for metal treatments, including transformation, extracellular binding, complex
formation, biosorption, and intracellular accumulation (3). Considering the more immobile and
less toxic characteristics of Cr(III), the microbial reduction of Cr(VI) to Cr(III) appears to hold the
most promise for the development of an innovative biotreatment technology. This reductive
biotransformation not only leads to Cr(VI) detoxification but precipitates the metal in soils,
therefore minimizing its potential risk to human health and impacted ecosystem through decreased
toxicity and exposure.
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Cr(VI) REMEDIATION BY MICROBIAL REDUCTION
Microbial reduction of Cr(VI) to Cr(ni) is capable of occurring through two different
mechanisms. One is enzymatic reduction in which microorganisms mediate electron transport to
Cr(VI) either for anaerobic respiration or for detoxification. The other is an indirect reduction of
Cr(VI) by reduced metabolic products such as H2S. Both mechanisms have been explored for
remediation of Cr(VI) contaminated environments, with more attention on the enzymatic process.
Cr(VI) as an electron acceptor
Microbial reduction of Cr(VI) is widely observed in both aerobic and anaerobic
environments. The aerobic reduction of Cr(VI) is generally catalyzed by soluble enzymes
associated with NADH as an electron donor or a co-enzyme (Table 1). Although the physiological
function of microbially mediated electron transfer to Cr(VI) under the aerobic conditions has not
been clearly understood, aerobic reduction of Cr(VI) is considered to be a cellular defense
mechanism. This is because insoluble Cr(in), as an extracellular reduction product, is excluded
from biological cells. Despite the lack of explicit explanations for its physiological role, the use of
oxygen for microbial respiration suggests that aerobic reduction of Cr(VI) happens mainly as a side
activity of microbial metabolism. However, it is believed that anaerobic reduction of Cr( VI) occurs
when Cr(VI) acts as a terminal electron acceptor for microbial respiration (Table 1). From a
thermodynamic point of view, Cr(VI) may serve as a competitive electron acceptor for anaerobic
respiration, with the redox potential (+560 mV for CrO, 7Cr ) only slightly less favorable than
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that of Fe(III) (+760 mV for Fe3+/Fe2+) and nitrate (+740 mV for NO,YN,), and far more
favorable than that of sulfate (-230 mV for SO427H2S). Studies of the anaerobic microbial
reduction of Cr(VI) indicates that Cr(VI) can serve as a terminal electron acceptor for reoxidation of
respiratory chains during anaerobic metabolism. Further studies observed that membranes
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associated cytochrome b, c, and d and cytochrome c3 in soluble proteins are specifically involved
in the transfer of electrons to Cr(VI) by microorganisms (4).
Although Cr(VI) reduction has been extensively demonstrated under anaerobic conditions,
no cell growth has been observed to depend on Cr(VI) reduction (4). The results suggest that the
energy yielded from Cr(VI) reduction may not be conserved in a manner which supports anaerobic
cell growth. To maintain Cr(VI) reduction under anaerobic environments, the Cr(VI)-reducing
microorganisms may thus require an alternative or intermediate electron acceptor. This is
consistent with findings that anaerobic reduction of Cr(VI) commonly takes place in media
containing fermentable organic compounds or complex media like nutrient broth, soy broth, and
casamino acids. Thermodynamic calculations explain that oxidation of fermentable organic
compounds with Cr(VI) as an electron acceptor is more energetically favorable than typical
fermentation for the metabolism of glucose. As shown in Table 2, aerobic respiration is more
energetically favorable than any of the fermentative oxidations. Under aerobic conditions, thus,
Cr(VI) is reduced only via an abbreviated electron transport chain using NADH as an electron
donor (5). Cr(VI) reduction is usually enhanced with a decrease in dissolved oxygen (DO) level,
and the inhibitory effect of DO on microbial reduction of Cr(VI) has been quantitatively described
(5). Under anaerobic environments, on the other hand, complete glucose oxidation with Cr(VI) as
an electron acceptor, partial fermentation of glucose to acetate with Cr(VI) as an electron acceptor,
and fermentation with Cr(VI) as a minor electron sink are all more energetically favorable than
fermentation not involving Cr(VI) reduction.
Cr(VI) reduction by metabolic products
In addition to enzymatic reduction of Cr(VI), Cr(VI) can also be reduced by microbial
metabolic products, including extracellular products and intracellular reducing agents. Cr(VI) is
capable of entering bacterial cells via sulfate transport systems (6). Once Cr(VI) enters the cell it
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may react with various cellular components such as sulfhydryl groups, and be reduced to Cr(III).
It is believed that intracellular Cr(HI) can not be eliminated from cells as long as the cell membrane
remains intact. In terms of the characteristic of this redox reaction, the bacterial cell acts more like
a chemical reducing reagent. Because the microbial metabolism would gradually terminate
following continuous depletion of cellular reducing agents, the intracellular reduction of Cr(VI)
appears to have a limited capacity . Thus, this mechanism has little applicability as an effective
approach for biotreatment of Cr(VI).
However, recent progress suggests that extracellular reduction of Cr(VI) by reduced
metabolic products can be developed as a useful biotreatment process. Cr(VI) reduction readily
occurs in the presence of ferrous iron and sulfide regardless whether they are generated from
abiotic or biotic sources. The reduced Cr(ni) may precipitate in the form of chromium hydroxide
under neutral to alkaline conditions. Although extracellular reducing compounds such as cysteine,
glutathione, and other organic compounds are capable of reducing Cr(VI), most studies explored
for bioremediation of Cr(VI) are focus on the use of HLS produced from sulfate-reducing bacteria
to remediate Cr(VI). Laboratory studies indicate that sulfate-reducing bacteria alone can create
sufficient amounts of H2S to ensure Cr(VI) reduction in natural environments (7). Since
chromium sulfide is unstable, the reduced chromium is likely to deposit quickly as hydroxides.
The concept of Cr( VI) reduction by sulfate-reducing bacteria has been developed to remediate
Cr(VI)-contaminated wastewater (8). The microbial process was shown to tolerate Cr(VI)
concentrations as high as 2,500 mg/L and reduced Cr(VI) to Cr(III) as amorphous precipitates
which were attached to the bacterial surfaces. An indirect mechanism of Cr(VI) reduction, due to
the generation of FLS by the microbial consortia, was postulated. Although a successful
application of this treatment process has been demonstrated in a pilot scale, the challenges remain.
Since H2S produced from bacterial respiration is re-oxidized by Cr(VI), the system redox potential
that is required for active growth of sulfate-reducing bacteria cannot be achieved until the majority
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of Cr(VI) has been removed. To maintain the microbial activity of Cr(VI) reduction in the system,
therefore, interruption of Cr(VI) loads is repeatedly needed in practical operations. This
disadvantage will limit its service for in situ biotreatment of Cr(VI).
Cr(VI) reduction coupling to carbon oxidation
Microorganisms are able to oxidize a variety of organic compounds for aerobic reduction of
Cr(VI). The compounds serving as electron donors for Cr(VI) reduction include natural aliphatic
compounds, mainly low-molecular-weight carbohydrates, amino acids and fatty acids, as well as
alien compounds such as aromatic compounds (Table 3). Analogously, anaerobic reduction of
Cr(VI) also demonstrates versatility with regard to electron donors. In addition to fermentable
sugars, Cr(VI)-reducing anaerobes are also capable of using fermentation end products, such as
formate, acetate, pyruvate, lactate, and ethanol as electron donors (Table 3). Benzoate, a common
fermentation intermediate of aromatic compounds, has been observed to support reduction of
Cr(VI) (12). The occurrence of such a wide variety of electron donors sustaining Cr(VI) reduction
suggests that a common metabolic intermediate NADH or hydrogen, may serve as the direct
electron donor for Cr(VI) reduction, while the other compounds may merely serve as precursors to
produce these reducing agents via catabolic processes. Direct Cr(VI) reduction with hydrogen and
NADH has been observed in both cell suspensions and cell-free fractions (4,9).
In order to evaluate the feasibility of a bioprocess for treatment of Cr(VI), the mass
relationship between electron donors and Cr(VI) should be understood. During the aerobic
reduction of Cr(VI), the amount of electron donor oxidized should far exceed the amount
theoretically required for Cr(VI) reduction due to the presence of molecular oxygen as a
competitive electron sink. A good example of this is the reduction of Cr(VI) driven by phenol
oxidation (10). Theoretically, oxidation of 1 mM phenol to carbon dioxide with complete flow of
electrons to Cr(VI) should result in the reduction of 9.3 mM Cr(VI) according to following
reaction:
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3C,H,O + 28CrO *• + 122H+ = 28Cr3+ + 18HCO ' + 61E.O (1)
664 32 v '
However, a statistical analysis of the experimental data revealed that consumption of 1 mM phenol
resulted in actual reduction of 1.03 mM Cr(VI), a value much less than stoichiometrical
requirement, suggesting that molecular oxygen is a major electron acceptor during the microbial
oxidation of phenol. Therefore, to achieve aerobic biotreatment of Cr(VI), much more amounts of
carbon sources are required than that of theoretical calculations.
Under anaerobic conditions, on the other hand, Cr(VI) may be the only electron acceptor,
and thus the coupling Cr(VI) reduction to electron donor oxidation may proceed exactly according
to the stoichiometry. When H2 is used as the electron donor for Cr(VI) reduction, the molar ratio
of H2 consumption to Cr(VI) loss is 1.74±0.13 (11), a good agreement with the following
formula:
3H2 + 2CrO42~ + 10H* = 2Cr3+ + 8H2O (2)
The anaerobic biodegradation of benzoate with the transport of electrons to Cr(VI) illustrates
another stoichiometrically balanced reaction between the electron donor and acceptor (12).
Figure 1 shows the plot of the cumulative amount of benzoate degraded versus the Cr(VI) reduced.
A statistical analysis indicates a strong linear relationship between the two parameters (R2=0.98):
1.0 mM benzoate degraded = 10 mM Cr(VI) reduced. This result suggests that the benzoate
oxidation during anaerobic reduction of Cr(VI) closely follows the stoichiometrie formula:
C?H6O2 + 10CrO42- + 43H+ = 10Cr3+ + 7HCO/ + 21H2O (3)
However, predicting or even evaluating the stoichiometrie relationships under a mixed electron
donor/acceptor in the environment may prove difficult. The rate and extent of electron donor
oxidation and Cr(VI) reduction may depend on a variety of geochemical, ecological and
physiological variables associated with the particular environmental setting.
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Cr(VI) reducing microorganisms
Cr(VI) reduction has been observed with a large number of bacterial genera, most of which
are facultative and ubiquitous in the environment. All of the listed Cr(VT) reducers in Table 3 are
heterotrophic and are members of commonly occurring soil genera, such as Pseudomonas,
Bacillus, and Escherichia. Usually a higher cell density of the cultures results in a greater rate of
Cr(VI) reduction. However, cell growth is not necessarily required for Cr(VI) reduction to occur.
Mass balance analysis indicates that Cr(VI) is ultimately and quantitatively reduced to Cr(IH)
although Cr(V) has been detected as a transient intermediate. Facultative microorganism such as
Agrobacterium, Bacillus, Escherichia, and Pseudomonas can reduce Cr(VI) in the presence of
oxygen and after the available oxygen is consumed. Others like Enterobacter can reduce Cr(VI)
only under anaerobic conditions, and immediately lose the ability to reduce Cr(VI) in the presence
of molecular oxygen despite good growth under aerobic conditions. The sulfate reducer
Desulfovibrio, an obligatory anaerobe, is also capable of using Cr( VI) as a terminal electron
acceptor (11). In addition, Cr(VI) is reduced by microbial consortia in soils, under both aerobic
and anaerobic conditions. The mixed cultures reportedly reduce Cr(VI) to a greater extent than
individual pure cultures. The dominance of facultative microorganisms in Cr(VI) reduction has a
distinct advantage for biotreatment of soils in which the location of the oxic-anoxic interface may
vary as a result of climate variations, human actions, and other biotic activities.
BIOPROCESS CONTROL PARAMETERS
In addition to the selection or cultivation of the appropriate bacterial strains, the bioprocess
parameters that control microbial activities for Cr(VI) reduction must be evaluated and optimized
for growth of the organisms, enzyme generation, and occurrence of Cr(VI) reduction. This
process optimization will facilitate the systematic design and operation of the treatment unit.
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Cr(VI) concentration
Cr(VI) may inhibit microbial metabolism, as demonstrated by decreases in cell growth,
carbon utilization and Cr(VI) reduction rate (4). At high concentrations of Cr(VI), bacterial
cultures show no significant cell growth, and a net decrease in active cells during the Cr(VI)
reduction process. The simultaneous loss of the capabilities for glucose utilization and Cr( VI)
reduction have been observed in a bacterial culture with high concentrations of Cr(VI). Studies
using various pure cultures demonstrated that the time required for equivalent Cr(VI) reduction
increased with the increase in the initial Cr(VI) concentration. However, some microorganisms
have the ability to effectively reduce Cr(VI), even at a concentration as high as 500 mg/L. The
adverse effects of high Cr(VI) concentrations on Cr(VI) reduction suggest that a suitable process
design may be important to minimize the impact of high Cr(VI) concentrations.
Electron donors
The rate and extent of Cr(VI) reduction is dependent on the type and concentration of
electron donors. In pure culture Enterobacter HOI, the use of complex electron donors such as
casamino acids, tryptone, or yeast extract resulted in faster Cr(VI) reduction than single compound
electron donors such as low-molecular weight acids, sugars or tricarboxylic acid cycle
intermediates (13). Microorganisms may also utilize endogenous electron reserves for Cr(VI)
reduction when external carbon sources become depleted. A benzoate initiated system showed that
Cr(VI) reduction proceeded continuously in the absence of measurable external electron donor
(benzoate), but at a significantly lower rate (12). In addition, native organic matter such as grass
or cow manure have been tested for the ability to support biological Cr(VI) reduction in soils. In
soils with a low organic matter content, Cr(VI) reduction was only increased slightly when the soil
suspension was amended with 10% manure (14), suggesting that the absence of adapted microbial
populations in the soil may limit the rate of Cr(VI) reduction. However, it has been reported that
amendments with 2.2% cattle manure in an enriched system immediately increased the percentage
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of Cr(VI) removal from 51% to 98% (15). A further comparison demonstrated that yeast extract,
grass and other readily degradable organic matter supported even more rapid rates of microbial
Cr(VI) reduction (16).
Competing electron acceptors
Observation of Cr(VI) reduction by facultative bacteria has shown that the presence of
molecular oxygen can suppress Cr(VI) reduction, but does not completely terminate the transport
of electrons to Cr(VI). A reduced level of dissolved oxygen may result in an increase in the rate of
Cr(VI) reduction and the highest rate for Cr(VI) reduction is always obtained under anaerobic
conditions with facultative microorganisms. An uncompetitive inhibition behavior for dissolved
oxygen toward Cr(VI) reduction has been shown using the Lineweaver-Burk method, and the
anaerobic reduction of Cr(VI) showed a maximum specific reduction rate approximately twice that
observed under aerobic conditions (5). On the other hand, the aerobic bacteria usually do not have
the capability for anaerobic reduction of Cr(VI) and the strict anaerobes will completely lose their
activity in the presence of molecular oxygen.
Sulfate and nitrate have not been reported to inhibit aerobic reduction of Cr(VI) (4). Cr(VI)
reduction was not inhibited with sulfate up to 96 mg/L and nitrate at 12 mg/L in the culture of
Pseudomonas, while Cr(VI) reduction in Bacillus was not affected even with concentrations of
sulfate and nitrate up to 1000 mg/L. However, under anaerobic conditions, a sulfate-reducing
culture of Desulfovibrio was shown to reduce Cr(VI) in the presence of 5 g/L sulfate, but
concentrations of 24 mg/L sulfate or 300 mg/L nitrate inhibited anaerobic reduction of Cr(VI) in
Enterobacter. Cr(VI) reduction in anaerobic cultures of Escherichia coli was resistant to sulfate and
nitrate up to levels of 8 g/L. In the studies with soil microcosms containing mixed native
populations, Cr(VI) reduction was not affected by the presence of nitrate up to a concentration of
300 mg/L (12). Analysis of the microcosm system indicated that Cr(VI) and nitrate were
concurrently used as the electron acceptors for benzoate oxidation without preference.
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Thermodynamic calculations also illustrate the oxidation of benzoate with transport of electrons to
Cr(VI) or nitrate yields very close standard free energies (AG° =-499 and -510 kcal/mole benzoate,
with Cr(VI) and nitrate as electron acceptors, respectively), supporting the concept of parallel
transfer of electrons to Cr(VI) and nitrate in mixed or complex microbial system.
Other toxic metals and organic toxicants
High concentrations of heavy metals such as lead, mercury, copper, cadmium, and others
are known to inhibit microbial metabolism, Cr(VI)-reducing microorganisms have been shown to
be susceptible to certain heavy metal ions (4,9). Cr(VI) reduction in Enterobacter was completely
inhibited by 30 mg/L of Zn2+, and 30 mg/L of Cu2+ decreased the reduction rate to 70% of the
activity observed in the culture without added metals. An addition of 50 mg/L Zn2+ or 190 mg/L
Cu in the culture of Escherichia coli also reduced the reduction rate to approximate 80% of the
activity observed in the absence of the metals, while Cd2+ or Pb2+ levels up to 20 mg/L showed no
inhibition of Cr(VI) reduction. Cultures of Demlfovibrio reduced Cr(VI) at the same rate
regardless of the presence of 11 metals (0.1 mM each) including nickelous chloride, cuprous
chloride, zinc chloride, magnesium sulfate, vanadyl sulfate, sodium vanadate, sodium molybdate,
and sodium selenate (11). Strong inhibition of Cr(VI) reduction in Pseudomonas by Hg2+ and
Ag2+ was shown to be noncompetitive, with an inhibitory constant (K.) equal to 20 |iM for both
metal ions. The same strain also reduced Cr(VI) without interference from the reduced product,
Cr(III), at a concentration of 10 mg/L. In addition to metal ions, aromatic compounds were also
observed as co-contaminants in Cr(VI) polluted streams and sites. Phenol and p-cresol at 5 mM
and 2-chlorophenol at 2 mM severely inhibited Cr(VI) reduction and cell growth. Anaerobic
cultures seem to be more susceptible to toxicity effects than the aerobic cultures. Toxicity studies
using phenol, p-cresol and 2-chlorophenol indicated that the concentrations that caused 50%
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decreases in rates of Cr(VI) reduction under aerobic conditions are nearly 1.5 times more toxic
under anaerobic conditions.
Soil characteristics
Soils are composed of organic matter, inorganic matrix, soil atmosphere, water, plant roots
and living microbial populations. The nature and the percentage of these contents may influence
the performance of Cr(VI) biotreatment. In addition to the factors regulating abiotic Cr(VI)
transport and fate processes in soils (such as sorption, anion exchange, and chemical
transformation), parameters governing the rate and extent of Cr(VT) biotransformation in soils may
include soil moisture, organic matter content, nutrient availability, redox potential, pH and salinity.
To optimize microbial transformations of Cr(VI) in situ, these parameters must be adjusted to
enhance development of large populations of Cr(VI)-reducing microorganisms and to bring these
organisms into intimate contact with Cr(VI),
Soil moisture strongly influences microbial activity in the soil. Generally speaking, soil
moisture at 70% to 80% of field capacity allows for rapid movement of air into the soil, and thus
maintains optimal aerobic metabolism. When soils become excessively dry, microbial activity can
be inhibited or terminated. In soils saturated with water, the available oxygen can be quickly
consumed, which limits aerobic activities but may enhance the anaerobic transformations of
Cr(VI). Microorganisms in soil require carbon sources for Cr(VI) reduction and may need other
nutrients for cell growth. Soil organic matter is generally composed of 25% to 35% readily
decomposed organic materials or compounds which have a short life in soils, while the other 65%
to 75% is composed of humic materials which are generally resistant to microbial degradation. The
widespread observation that Cr(VI) reduction is enhanced by organic amendments suggest that the
biodegradable carbon sources are a limiting factor for microbial reduction of Cr(VI) in soils (16).
Addition of easily degradable organic matter may result in the competition for this resource
between Cr(VI)-reducing and other microorganisms, and may also cause shortages of other
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nutrients due to the increased mierobial biomass. Animal manures may supply both microbial
carbon sources and certain other nutrients. The finding that the amendment of cow manure
improved microbial reduction of Cr(VI) suggests that it may serve as an economical nutrient
resource for the biotreatment of Cr(VI) in soils.
The redox potential of soils generally varies from -0.3V to +0.8V dependent on the rates of
soil aeration and microbial respiration. Because Cr(VI) reduction occurs over a redox potential
range from -0.24V to +0.25V (4), the redox potential of soils does not appear to be a critical
limiting factor for occurrences of Cr(VI) reduction. However, a low redox potential environment
in soils following depletion of oxygen will definitely promote anaerobic reduction of Cr(VI). A
pH range of 6 to 8 has been shown to support microbial reduction of Cr(VI) (4). Soil pH may be
lowered by addition of ferrous or aluminum sulfate, whereas it can be raised by addition of
agriculture limes. Salinity also affects microbial activities. High salinity soils with an electrical
conductivity (EC) value greater than 8 dSm will restrict activities of many microorganisms, while
soils with a value of 2 dSm"1 or less may not be a problem to most microbial metabolism. The
effect of salinity on microbial reduction of Cr(VI) has not been established but may also follow this
general rule.
APPLICATION OF Cr(VI) BIOTREATMENT
The remediation of Cr(VI) contaminated soils is a complex and challenging task. In situ
microbial cleanup of contaminants has been successfully utilized for more than 30 years to restore
polluted sites including soils and groundwater. Currently, most applications of biotreatment have
focused on oxidation transformations of organic wastes. The application of reductive
biotransformation for bioremediation is less well developed and, to date, has been used most
successfully for the biotreatment of chlorinated organic compounds and for nitrate removal. The
extensive occurrence of Cr(VI) reducing microorganisms in both contaminated and uncontaminated
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soils and sediments is evidence for the potential of in situ biotreatment processes. The occurrence
of microbial reduction of Cr(VI) in a neutral pH range may offer the best promise for in situ
bioremediation of Cr(VI). In situ treatment would concentrate chromium on the soils as Cr(ni),
which has greatly reduced environmental mobility and biological availability. Laboratory and field
research is currently underway to develop effective reductive biotreatment techniques for Cr(VI)
contaminated soil and water.
The biotreatment of Cr(VI) in soils may be enhanced through two approaches. One is
stimulating native microorganisms for Cr(VI) reduction by adjusting environmental conditions, and
the other is altering the microbial population by inoculating seed organisms. Both approaches have
been employed to accelerate bioremediation of petroleum contaminated sites. Laboratory and field
evidence also demonstrates the capability of these approaches for enhancing microbial treatment of
Cr(Vl).
Enhancement of indigenous microorganisms
The site environment can be adjusted to activate or enhance microbial reduction of Cr(VI)
by supplying carbon sources, essential nutrients, and possibly other electron acceptors for
stimulating bacterial growth. In soils with abundant organic materials, native microorganisms may
use those easily degradable organic compounds as electron donors to reduce Cr(VI). As shown in
Figure 2, significant reduction of Cr(VI) was observed in the absence of external organic
compounds (Sewell and Shen, unpublished data). However, the addition of an appropriate
electron donor further enhanced Cr(VI) reduction in the microcosms (Figure 2). Based on the
observed curves of Cr(VI) reduction, sucrose was the most effective electron donor for bacterial
transformation of Cr(VI), followed by lactate and acetate, and then the native organic compounds
in soil. On the other hand, in oligotrophic soils, the addition of external electron donors may play
an essential role in stimulating Cr(VI) reduction. Soils with both native microorganisms and
inoculant showed insignificant Cr(VI) reduction in the absence of added electron donors (14).
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When amended with carbon sources, however, the soil microorganisms rapidly reduced Cr(VI) to
Cr(IH). The rate of Cr(VI) reduction was also dependent on the nature of carbon sources. The
addition of the easily decomposable yeast extract yielded a higher rate of Cr(VI) reduction, while a
much lower rate of Cr(VI) reduction was obtained when grass and cow manure were used as
carbon sources. Field test results also confirmed the requirement of carbon sources for effective
biotreatment of Cr(VI) (15). The amount of Cr(VI) reduced was observed to be proportional to the
cow manure loading and Cr(VI) removal percentages as high as 98% have been reported. The cow
manure serving as the electron donor appeared to be a limiting factor for microbial reduction of
Cr(VI) because its loading decided the extent of Cr(VI) reduction in the biotreatment system.
When native microorganisms are incapable of coupling oxidation of the supplied carbon
sources to Cr(VI) reduction, the addition of an alternative electron acceptor may provide assistance
(12). Microcosm tests observed that native microorganisms in oligotrophic soils were unable to
reduce Cr(VI) when benzoate was provided as the carbon source. However, it has been noted that
nitrate or oxygen may act as an initial stimulator for linkage of benzoate oxidation and Cr(VI)
reduction. After depletion of nitrate or dissolved oxygen, the microorganisms still retained the
capacity for benzoate degradation linked to Cr(VI) reduction. Since denitrifying organisms in the
microcosms alone did not have the capability to reduce Cr(VI), Cr(VI) reduction in this system is
probably attributed to microbial consortia, possibly including both denitrifers and Cr(VI) reducers.
According to these findings, the oxic-anoxic conditions in soils may easily facilitate microbial
reduction of Cr(VI) allowing a wide spectrum of carbon sources to serve as electron donors.
Other nutrients are reported to have less effect on Cr(VI) reduction in soils. This is not
surprising since Cr(VI) reduction occurs without necessarily being coupled to the growth of
microbial cells, and the microbial cells retain the normal ability to reduce Cr(VI) even without
additions of nitrogen and phosphorous (4). Because Cr(VI) toxicity may lead to cell inactivation
and loss of Cr(VI) reduction capacity, the stimulation of microbial growth to generate fresh cells in
soils may be required under operational conditions. In addition to carbon sources, alternative
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electron acceptors may also be critical to applications requiring cell growth, since significant
growth of cells has not been observed during Cr(VI) reduction. Oxygen and nitrate appear to be
the most appropriate electron acceptors for promoting microbial growth in Cr(VI) biotreatment
applications. Oxygen could be supplied by sparging air in a batch mode or as a continuous feed.
The batch mode addition is more economical and may also give better Cr(VI) reduction
performance. This is because the feeding patterns result in cyclic changes in soil redox that may
optimize aerobic cell growth and anaerobic Cr(VI) reduction. Nitrate is much more soluble than
oxygen, hence it may be more economical to sustain microbial growth under denitrifying rather
than aerobic conditions. This is especially true for biotreatment of Cr(VI) in deep soils because of
the difficulties in delivering a significant mass of soluble oxygen to the contaminated zone, and the
potential for degassing, and reactions with inorganic reducing compounds.
Inoculation of acclimated microorganisms
Another approach for enhancing Cr(VI) biotreatment is the addition of adapted microbes
into Cr(VI) contaminated soils. There is little information describing the use of inoculation to
promote microbial reduction of Cr(VI). One successful example reported the introduction of an
adapted enrichment into microcosms that contained Cr(VI) and benzoate as the sole electron donor
and acceptor (12). In the inoculated microcosms, Cr(VI) was rapidly reduced with concurrent
degradation of benzoate following a lag period of 4 days for both Cr(VI) and benzoate. This
biological activity was extended to more than 50 days following repeated addition of Cr(VI) and
benzoate, demonstrating that the inoculant was capable of survival under the new environmental
conditions. Although the inoculated microcosms continued to consume Cr(VI) and benzoate, the
analogous microcosms which contained no inoculation showed no decreases in the amount of
Cr(VI) and benzoate throughout the same period. In another case, a pure culture of Pseudomonas,
a common strain known to be capable of Cr( VI) reduction, was inoculated into soil microcosms
amended with Cr(VI) (16). The inoculant, however, failed to provide any better reduction of
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Cr(VI) than was achieved by simply adding organic matter. Apparently, the addition of organic
matter allowed native Cr(VI)-reducing microorganisms to accelerate the activity of Cr(VI)
reduction, probably outcompeting the introduced organisms. Hence, microbial inoculation may be
necessary only if the native Cr(VI)-reducing organisms are missing or are inactivated by high
levels of Cr(VI). Many factors have been recognized to restrict the applicability of inoculation in
bioremediation (17). In the case of Cr(VI) biotreatment, these limitations may include the delivery
of the inoculum to the contaminated zone, adverse microbial interactions such as competition and
predation from native organisms, and antibacterial substances in soils.
Advantages and disadvantages of in situ biotreatment
When biotreatment of Cr(VI) is carried out in situ, costs may be substantially reduced by
eliminating the large energy inputs for excavation and shipment of contaminated soils.
Conceptually, in situ treatment also reduces the risks associated with soil movement to workers
and local residents. Where soil organic compounds are rich, Cr(VI) reduction may occur by soil
microorganisms as natural attenuation. If electron donors become a limiting factor for microbial
Cr(VI) reduction in soils, natural organic matter from economical sources can be added to improve
microbial activity. Under certain conditions, microbial consortia can even utilize soil co-
contaminants such as aromatic compounds as electron donors for Cr(VI) reduction, accomplishing
simultaneous cleanup of metal and organic contaminants. Since Cr(VI) reduction by indigenous
microbes produces no hazardous metabolites and occurs at a neutral range of pH, this biotreatment
process should result in minimal impacts on the soil ecosystem.
Despite the potential advantages, in situ biotreatment of Cr(VI) requires detailed site
examinations for geochemical, hydraulic, and microbial characterization. High levels of Cr(VI)
may repress or inhibit microbial activities including Cr(VI) reduction. The rate and extent of
microbial Cr(VI) reduction are greatly affected by temperature, soil moisture, co-contaminant and
other conditions. In addition, extended growth of microbes can also plug the soil and thus reduce
nutrient circulation. However, it should be emphasized that the biotreatment process may not
16
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necessarily replace other treatment technologies, but may be combined with them or be used as a
supplemental polishing process. The microbial process, together with widely reported abiotic
reduction processes, may offer an efficient and cost-effective approach for in situ remediation of
Cr(VI) contaminated soils.
ACKNOWLEDGMENTS
We thank Robert Powell (Powell and Associate, Inc.) for helpful comments and
suggestions on the manuscript.
17
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BIBLIOGRAPHY
(1) C.D,Palmer, and R.W, Puls, Natural Attenuation of Hexavalent Chromium in Ground
Water and Soils, EPA/540/S-94/505, US EPA, Washington D.C., (1994).
(2) E. Nieboer and A. A. Jusys, "Biological Chemistry of Chromium" in J.O. Nriagu and E.
Nieboer, eds., Chromium in the Natural and Human Environments, John Wiley & Sons,
Inc., New York, 1988, pp. 21-78.
(3) G.N. Gadd and C. White, Trends in Biotechnol., 11, 353-359 (Nov. 1993).
(4) Y. Wang and H. Shen, /. Ind. MicrobioL, 14, 158-163 (Jan. 95).
(5) H. Shen and Y. Wang, Appl. Environ. MicrobioL, 59, 3771-3777 (Nov. 1993).
(6) C. Cervantes and S. Silver, Plasmid, 27, 65-71 (Jan. 1992).
(7) R.H. Smillie, K. Hunter and M. Loutit, Wat. Res., 15, 1351-1354 (Dec. 1981).
(8) L. Fude, B. Harris, M.M. Urrutia and T.J. Beveridge, Appl. Environ. MicrobioL, 60,
1525-1531 (May 1993).
(9) D.R. Lovley, Annu. Rev. MicrobioL, 47, 263-290 (Feb. 1993).
(10) H. Shen and Y. Wang, Appl. Environ. MicrobioL, 61, 2754-2758 (Jul. 1995).
(11) D.R. Lovley and E.J.P. Phillips, Appl. Environ. MicrobioL, 60, 726-728 (Feb. 1994).
(12) H. Shen, P.H. Pritchard and G.W. Sewell, Environ. Sci. TechnoL, 30, 1667-1674 (May
1996).
(13) H. Ohtake, E. Fujii and K. Toda, J. Gen. Appl. MicrobioL, 36, 203-208 (Mar. 1990).
(14) RJ. Bartlett and J.M. Kimble, J. Environ. QuaL, 5, 383-386 (May 1976).
(15) M.E. Losi, A. Amrhein and W.T. Frankenberger,Jr, /. Environ. QuaL, 23, 1141-1150
(Nov. 1994).
(16) F.R. Cifuentes, W.C. Lindemann and L.L Barton, Soil Sci., 161, 233-241 (Apr. 1994).
(17) P.H. Pritchard, Current Opinion in Biotechnol., 3, 232-243 (Mar. 1992).
18
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Figure 1. Cumulative Cr(VI) reduced versus cumulative benzoate degraded in an active enrichment
of soil microorganisms with Cr(VI) and benzoate as the sole electron acceptor and donor. The
results were reported by Shen et al (12),
Figure 2. Microbial reduction of Cr(VI) in microcosms constructed with soils from Norman
Landfill, Norman, Oklahoma,(unpublished data)
19
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Table 1. Properties of reductase involved in Cr(VI) reduction
Property Aerobic reduction Anaerobic
reduction
Location
Soluble + +
Membrane - +
Co-factor
NADH +/- +
Cytochrome - +
Phosphorylation occurrence + 4-7-
Inhibition
Oxygen - +
Nitrate - +/-
Sulfate - +/-
Electron donors
NADH + +
Hydrogen ? +
Sugars + +
Fatty acids + +
Endogenous reserves + +
+ Positive result
- Negative result
-------
Table 2. Energy potentially available from various pathways for glucose metabolism (5)
No
1
2
3
4
5
6
7
8
9
Reactant
C6HI206 H
C6HI206 H
C6H1206 H
C6HI206 H
C6H1206 -
C6HI206
C6H1206
C6H1206 -
C6H1206 -
h6O2
h 8Cr042 + 34H +
hH20
h 2.7H2O
h2H2O
I- 2.7Cr042 + 9.3H +
I- 0.67CrO42- + 0.3H+
Product
6C02 + 6H20
8Cr3+ + 6HCO3- + 20H2O
CH3COO + CH3CH2COO + HCO3 + H2 + 3H+
0.67CH3COO + 0.67CH3CH2CH2COO- + 2HCO3 + 2.7H2 -1
2CH3CH2OH + 2HCO3 + 2H +
OOCCH2CH2COO2 + CH3COO +H2 + 3H +
2CH3CHOHCOO + 2H +
2.7Cr3+ + 2CH3COO + 2HCO3 + 6.7H2O
0.67Cr3+ + CH3COO +CH3CH2COO + HCO3 + 1.67H2O
AG°'
(KJ/electron transferred)
-121
-83
-71
- 3.3H+ -60
-57
-66
-50
-109
-96
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Table 3. Genera of microorganisms capable of reducing Cr(VI)*
Genera
Substrate/redox condition/source
Achromobacter
Aeromonas
Agrobacterium
Bacillus
Desulfovibrio
Enterobacter
Escherichia
Micrococcm
Pseudomonas
Escherichia and Pseudomonas
Undefined soil organisms
acetate, glucose/anaerobic/undefined
galactose, fructose, mannose, melibiose, sucrose, lactose, cellobiose, arabinose,
mannitoldulcitol, sorbitol, glycerol/anaerobic/sewage
glucose, fructose, maltose, lactose, mammitol, glycerol/aerobic, anaerobic/soil
acetate, glucose/aerobic, anaerobic/soil
H2/anaerobic /undefined
acetate, glycerol, glucose, casamino acid, malate, oxalate, pyruvate/anaerobic/sewage
acetate, glucose/aerobic, anaerobic/sewage
acetate, glucose/aerobic, anaerobic/undefined
peptone, glucose, ribose, fructose, glycerol, fumarate, lactate, acetate, succinate, butyrate,
ethylene,/aerobic, anaerobic/sewage, sediment, soil
phenol, chlorophenol, cresol, dimethylphenols, benzene, toluene/aerobic/soil, sewage
lactate, acetate, sucrose, ethanol, benzoate, grass, cow manure/anaerobic/soil
* Based on references (4, 11- 16)
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Figure 1. Cumulative Cr(VI) reduced versus cumulative benzoate degraded in an active
enrichment of soil microorganisms with Cr(VI) and benzoate as the sole electron acceptor and
donor. The results were reported by Shen et al (12).
Figure 2. Microbial reduction of Cr(VI) in microcosms constructed with soils from Norman
Landfill, Norman, Oklahoma.
23
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I o.io
$-1
W)
0>
03
O
N
c
(D
0.08
0.06
0.04
£ 0.02
u
0.0
J Regression result (R2=0.98, n=l 15)
Benzoate degraded = 10.0 Cr(VI) reduced
95% Confidence intervals
Experimental data
.0
0.2
0.4
0.6
0.8
1.0
Cumulative Cr(VI) reduced, mM
-------
bb
S
•N
fi
o
03
o
fi
O
O
u
sucrose (2 mM)
lactate (1 mM)
acetate (1 mM)
none
sterile control
0
0
Incubation time, days
-------
TECHNICAL REPORT DATA
1. REPORT NO.
IPA/600/A-97/084
2.
4. TITLE AND SUBTITLE
CHROMIUM (VI) BIOTREATMENT IN SOIL
7. AUTHOR (S)
Guy W. Sewell*
Hai Shen2
P. Hap Pritchard1
9. PERFORMING ORGANIZATION NAME AND ADDRESS
HJ.S. EPA, NRMRL, SPED; P.O. Box 1198; Ada, OK 74820
'Dynamac Corporation; 3601 Oakridge Boulevard; Ada, OK 74820
"Naval Research Lab.; 4555 Overlook Ave, S.W.;
Washington DC 20375-5321
12. SPONSORING AGENCY NAME AND ADC
U.S. EPA
NATIONAL RISK MANAGEMENT RESEARCH
SUBSURFACE PROTECTION AND REMEDIAL
P.O. BOX 1198; ADA, OK 74820
15. SUPPLEMENTARY NOTES
Submitted to: The Encyclopedia of
RESS
LABORATORY
ION DIVISION
3
5. REPORT DATE
6, PERFORMING ORGANIZATION CODE
8. PERFORMING ORGANIZATION REPORT NO.
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
13. TYPE OF REPORT AND PERIOD COVERED
Book Chapter
14, SPONSORING AGENCY CODE
EPA/600/15
Environmental Analysis and Remediation
16. ABSTRACT
Chromium is widely used in diverse industries and its inappropriate disposal practice has resulted in the
release of this Metal into the environment. Chronium has become one of the toxic metals most frequently
detected in contaminated environments. The potential for adverse human health effects has led to increased
public concerns over chromium contamination. Chromium exists in a variety of oxidation states, from 0 to +6.
However, in natural environments only hexavalent chromium Cr(VI) and trivalent chromium CR(III) are stable
species. Cr(VI) is much more hazardous due to its carcinogenicity, mutagenicity and mobility, than the
insoluble trivalent chromium compounds. Cr(III) is considered to be relatively innocuous and even essential to
human health in minute quantities (2). Conventional chemical and electrochemical techniques for Cr(VI) removal
are all based on reduction of Cr(VI) to Cr(III) and then precipitation of it as chromium hydroxide. The
effective reduction of Cr(Vl) normally requires an acidic reaction environment (pH<3) , and the complete
conversion is dependent on the concentration and type of reducing agents employed. However, applications of
these techniques have limitations in terms of cost, effectiveness and sludge production. Recently the
potential for the biotreatment of Cr(VI) wastes has received increased attention because the microbially
mediated processes may offer a cost-effective alternative to chemical treatment. There are several biological
mechanisms which may be suitable for metal treatments, including transformation, extracellular binding, complex
formation, biosorption, and intracellular accumulation (3) . Considering the more immobile and less toxic
characteristics of Cr(III), the microbial reduction of Cr(VI) to Cr(III) appears to hold the most promise for
the development of an innovative biotreatment technology. This reductive biotransformation not only leads to
Cr(VI) detoxification but precipitates the metal in soils, therefore minimizing its potential risk to human
health and impacted ecosystem through decreased toxicity and exposure.
17.
A. DESCRIPTORS
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
KEY WORDS AND DOCUMENT ANALYSIS
B. IDENTIFIERS/OPEN ENDED TERMS
19. SECURITY CLASS (THIS REPORT)
UNCLASSIFIED
20. SECURITY CLASS (THIS PAGE)
UNCLASSIFIED
C. COSATI FIELD, GROUP
21. NO. OF PAGES
29
22. PRICE
EPA FORM 2220-1
(REV.4-77)
PREVIOUS EDITION IS OBSOLETE
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