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Enzyme-Mediated Transformations
of Heavy Metals/Metalloids
Applications in Bioremediation
Robert S. Dungan
George E. Brown, Jr. Salinity Laboratory, USDA–ARS, Riverside, California
I. INTRODUCTION
A major emphasis has been placed on the bioremediation of organic compounds (1) and
their fate and transport throughout the environment (2,3). However, another important
class of chemicals polluting our environment are inorganic, particularly heavy metals and
metalloids. Heavy metals are elements of the periodic table with a density of more than
5 g cm!3. Although this encompasses a large percentage of the metals, only several heavy
metals/metalloids are regarded as of environmental concern, including selenium (Se), arse-
nic (As), chromium (Cr), and mercury (Hg). In the United States, more than 50% of the
National Priority (Superfund) sites ranked on the National Priorities List (NPL) contain
heavy metals that are designated as a threat or problem to the environment (4). Since
heavy metals/metalloids cannot be degraded (i.e., biologically or chemically) they are
among the most intractable pollutants to remediate.
The contamination of soils and waters with heavy metals/metalloids usually occurs
by direct application from sources, including mine waste, atmospheric deposition (a result
of metal emissions to the atmosphere from metal smelting, fossil fuel combustion, and
other industrial processes), animal manure, and sewage sludge (5). Surprisingly, some
inorganic fertilizers contain significant quantities of heavy metal impurities. Sewage
sludge, which is often used as a soil conditioner, contains useful quantities of organic
matter, N, and P; however, it often contains heavy metals. The metals are chelated by the
organic matter and are released upon its decomposition. Heavy metal/metalloid cations
in soil may be present as several different forms: (1) ions in soil solution; (2) easily
exchangeable ions; (3) organically bound; (4) coprecipitated with metal oxides, car-
bonates, phosphates, or secondary minerals; or (5) ions in primary minerals (6). As a re-
sult, the heavy metal form is highly influenced by soil properties such as pH, oxidation–
reduction (redox) state, clay content, iron oxide content, and organic matter content.
II. SELENIUM
Selenium belongs to group VIA of the periodic table and has been classified as a metalloid.
In the environment Se exits in four oxidation states, "2, 0, "4, and "6, forming a variety
of compounds. Selenate (SeO 42!, Se 6") and selenite (SeO 32!, Se 4") are the most common
ions found in soil solution and natural waters. Organic Se-containing compounds include
Se-substituted amino acids, such as selenomethionine, selenocysteine, and selenocystine,
and volatile methyl species such as dimethylselenide (DMSe, [CH 3] 2Se), dimethyldisele-
nide (DMDSe, [CH 3] 2Se 2), methaneselenol (CH 3SeH), and dimethylselenenylsulfide
A. Reduction of Selenium(VI)
The bioreduction of Se to insoluble Se 0 has been extensively investigated as a technique
for removing Se from contaminated water. Selenium undergoes dissimilatory microbial
reduction, whereby SeO 42! is reduced to Se 0 as the terminal electron acceptor in respiratory
metabolism. Macy (11) isolated Thauera selenatis, a SeO 42!, NO 3!, and NO 2! respiring
bacterium, from seleniferous sediments. The reduction of SeO 42! to SeO 32! and NO 3! to
NO 2! by T. selenatis occurs through the use of separate terminal reductases, a SeO 42! and
NO 3! reductase, respectively. The complete reduction of SeO 42! to Se 0 only occurs when
the organism is grown in the presence of both SeO 42! and NO 3!. During the tricarboxylic
acid (TCA) cycle, reduced nicotinamide-adenine dinucleotide (NADH) and succinate are
used as electron donors to reduce SeO 42! and SeO 32!. The electrons are then transferred
via an electron transport system that is part of a dehydrogenase, which is loosely bound
to the cytoplasmic membrane. Selenate reduction to SeO 32! involves a periplasmic SeO 42!
reductase, whereas SeO 32! produced during the respiration of SeO 42! and NO 3! is believed
to be reduced via a periplasmic NO 2! reductase (Fig. 1).
Enterobacter cloacae strain SLD1a-1, a facultative anaerobe isolated by Losi and
Frankenberger (12), operates under mechanisms very similar to that of T. selenatis. E.
cloacae strain SLD1a-1 uses SeO 42! and NO 3! as terminal electron acceptors during anaer-
obic growth and can reduce SeO 42! to Se 0 under growth conditions and in washed-cell
suspensions under microaerophilic conditions. Although strain SLD1a-1 respires SeO 42!
anaerobically, the complete reduction of SeO 32! to Se 0 does not occur unless NO 3! is
present, suggesting that NO 3! is necessary for the reduction of SeO 32! to Se 0 (13). Orem-
land and associates (14) isolated a strictly anaerobic motile vibrio (Sulfurospirillum
barnesii strain SES-3) that grows in the presence of either SeO 42! or NO 3! while using
lactate as an electron donor. It was determined that the reduction of SeO 42! and NO 3!
ions is achieved by separate inducible enzyme systems. Although growth was not observed
on SeO 32!, washed-cell suspensions of SES-3 could reduce SeO 32! to Se 0. A Pseudomonas
stutzeri isolate could only reduce SeO 42! and SeO 32! to Se 0 under aerobic conditions, a
limitation that was speculated to be a detoxification mechanism (15).
The biological reduction of SeO 32! to Se 0 also occurs, but only the reduction of
SeO 42! supports anaerobic growth. Although a number of SeO 32! reducing bacteria have
been isolated and described metabolically, it is still unclear which reductive processes are
involved. In the literature it has been reported that SeO 32! can be reduced anaerobically by
a periplasmic NO 2! reductase (16) or reduced aerobically as a detoxification mechanism,
3. Cell-Free Systems
Adams et al. (54,58) treated mine water containing 0.62 mg L !1 SeO 42! by using an immo-
bilized cell-free preparation of Pseudomonas stutzeri. Tests were conducted by using a
single-pass bioreactor with a retention time of 18 hours. The cell-free extracts were pre-
pared by disrupting the cells then immobilizing the lysate in calcium alginate beads. The
immobilized enzyme preparation performed for approximately 4 months, achieving efflu-
ent levels below 10 µg L !1. Another cell-free system was used to treat mining process
solution containing cyanide and Se. The system contained cell-free extracts of P. pseudoal-
caligenes, P. stutzeri, CN-oxidizing, and Se-reducing microbes combined and immobi-
lized in calcium alginate beads. Tests were conducted in single-pass 1-in-diameter columns
with a retention time of 9 to 18 hours. The system was capable of simultaneously removing
cyanide and Se (initial concentrations of 102 and 31.1 mg L !1, respectively) to concentra-
tions of 1.0 and 1.6 mg L !1, respectively.
III. ARSENIC
Arsenic (As) is a metalloid of group VA of the periodic table and exists in four oxidation
states, "5, "3, 0, and !3. It occurs naturally in the environment as well through anthrop-
ogenic discharge in a variety of chemical states. Arsenic forms alloys with various metals
and covalently bonds with carbon, hydrogen, oxygen, and sulfur (59). Arsenate (AsO 43!),
a biochemical analog of phosphate, is transported by highly specific energy-dependent
membrane pumps into the cell during assimilation of phosphate, whereas arsenite (AsO 2!)
has a high affinity for thiol groups of proteins, resulting in the inactivation of many en-
zymes. Its similarity to phosphorus and its ability to form covalent bonds with sulfur are
two reasons for As toxicity. The poisonous character of As make it an effective herbicide
and insecticide. The ubiquity of As in the environment, its biological toxicity, and its
redistribution are factors evoking public concern.
Both oxidation and methylation are microbial transformations involved in the redis-
tribution and global cycling of As. Oxidation involves the conversion of toxic AsO 2! to
less toxic AsO 43!. Arsenite is much more toxic to aquatic microbiota of agricultural drain-
age water and evaporation pond sediments than any other As species (60). Bacterial meth-
ylation of inorganic As is coupled to the formation of methane in methanogenic bacteria
and may serve as a detoxification mechanism. The mechanism involves the reduction of
AsO 43! to AsO 2!, followed by methylation to dimethylarsine. Fungi are also able to trans-
form inorganic and organic As compounds into volatile methylarsines. The pathway pro-
ceeds aerobically by AsO 43! reduction to AsO 2! followed by several methylation steps
producing trimethylarsine. Currently, a number of microbially mediated oxidation and
methylation reactions are being studied in the interest of developing bioremediation tech-
niques for detoxifying As-contaminated soil and water.
B. Oxidation of Arsenic(III)
Currently, a number of microbially mediated oxidation reactions are being studied in the
interest of developing bioremediation techniques for detoxifying As-contaminated soil and
water. Bacillus, Thiobacillus, and Pseudomonas spp. that have been isolated can oxidize
AsO 2! to the less toxic AsO 43!. In addition, a strain of Alcaligenes faecalis obtained from
raw sewage was capable of oxidizing AsO 2! (70). Osborne and Ehrlich (71) isolated a
similar AsO 2!-oxidizing soil strain of A. faecalis whose oxidation process was induced
by AsO 2! and AsO 43!. The use of respiratory inhibitors prevented further oxidation of
AsO 2!, indicating that oxygen served as the terminal electron acceptor. Studies suggested
that the oxidation of AsO 2! involved an oxidoreductase with a bound flavin that passed
electrons from AsO 2! to O 2 by way of cytochrome c and cytochrome oxidase (71). Indirect
evidence suggested that the organism may be able to derive maintenance energy from
the oxidation of AsO 2! (72). Ilyaletdinov and Abdrashitova (73) isolated Pseudomonas
arsenitoxidans from a gold and arsenic ore deposit that was capable of growing autotrophi-
cally with AsO 2! as the soil energy source.
Anderson et al. (74) found that A. faecalis strain NCIB 8687 contained an inducible
AsO 2 -oxidizing enzyme that was located on the outer surface of the plasma membrane,
!
a finding that suggested that AsO 2! oxidation occurred in its periplasmic space. The 85-
kD enzyme was a molybdenum-containing hydroxylase with a pterin cofactor and inor-
ganic sulfide, one atom of molybdenum, and five or six atoms of iron. The enzyme cata-
lyzed the oxidation of AsO 2! when both azurin and cytochrome c were used as electron
acceptors. Oxidation of AsO 2! by heterotrophic bacteria plays an important role in detoxi-
fying the environment, catalyzing as much as 78% to 96% of the AsO 2! to AsO 43! (75).
C. Methylation of Arsenic
1. Bacterial Methylation
Bacterial methylation of inorganic As has been studied extensively using methanogenic
bacteria. Methanogenic bacteria are a morphologically diverse group consisting of coccal,
bacillary, and spiral forms but are unified by the production of methane as their principal
metabolic end product. They are present in large numbers in anaerobic ecosystems, such
as sewage sludge, freshwater sediments, and composts where organic matter is decompos-
Figure 5 Fungal methylation pathway for the formation of trimethylarsine. (From Ref. 35.)
3. Algal Methylation
Arsenic is also metabolized into various methylated forms by freshwater algae; however,
there is some question about whether biomethylation of As in freshwater is a widespread,
common process. Arsenite is methylated by at least four freshwater species of green algae:
Ankistrodesmus sp., Chlorella sp., Selenastrum sp., and Scenedesmus sp. (88). All four
species methylated AsO 2! when present in media at 5000 mg L !1, approximately the
same level of AsO 2! used to control aquatic plants in lakes (89). The levels of recovered
methylated As species were quite high on a per gram dry weight basis. Each of these
organisms transformed AsO 2! to methylarsonic acid and dimethylarsinic acid, and all,
except Scenedesmus sp., produced detectable levels of trimethylarsine oxide. Unlike for
fungi, volatile methylarsines were not produced (89); instead, limnetic (freshwater) algae
like marine algae synthesize lipid-soluble As compounds. Freshwater algae grown in me-
dia amended with 1.0 to 3.0 µg L !1 AsO 43! synthesized lipid-soluble As compounds to
levels approximately equal to those of marine algae (90,91).
IV. CHROMIUM
Chromium (Cr) has many industrial uses, and, as a result, large volumes of Cr waste in
various chemical forms are discharged into the environment. Chromium can exist in oxida-
tion states ranging from !2 to "6. However, only "3 and "6 are normally found within
the range of pH and redox potentials common in environmental systems. Hexavalent Cr
(Cr 6" ) forms chromate (CrO 42! ) and dichromate (Cr 2O 72! ), which are toxic and muta-
genic, soluble over a wide pH range, and mobile in the environment. Hexavalent Cr can
easily cross the membranes of eukaryotic and prokaryotic cells, and enters the cells by
SO 42! transport pathways (92). Once in the cytosol, Cr 6" may be reduced to Cr 3", which
in turn reacts with deoxyribonucleic acid (DNA) (93). The trivalent form (Cr 3") is virtually
nonmobile, largely because it precipitates as oxides and hydroxides at pH # 5 (94,95),
and considerably less toxic, since biological membranes do not allow its passing (96).
Thus, the reduction of Cr 6" to Cr 3" represents a viable remediation and detoxification
strategy.
Traditional techniques for remediating chromate (CrO 42!) from contaminated water
involved reducing Cr 6" to Cr 3" by chemical or electrochemical means at pH # 5, followed
by precipitation and finally filtration or sedimentation (97). The discovery of microorgan-
isms that preferentially reduce Cr 6" has led to applications in the bioremediation field that
are potentially more cost-effective than traditional techniques. Russian researchers were
the first to propose using Cr 6"-reducing bacterial isolates in the removal of CrO 42! from
various industrial effluents (98,99). Since then a wide variety microorganisms that reduce
Cr 6" have been isolated from CrO 42!-contaminated waters and sediments. Through the
A. Reduction of Chromium(VI)
Although the microbial reduction of Cr 6" to Cr 3" is not known to be a plasmid-determined
process, it may act as an additional mechanism of resistance to CrO 42!. Bopp and cowork-
ers (100) isolated a CrO 42!-resistant strain of Pseudomonas fluorescens and determined
that the resistance was plasmid-conferred but later reported (101) that CrO 42! reduction
was associated with a constitutive, membrane-associated enzyme. It was subsequently
shown that CrO 42! reduction and plasmid-mediated CrO 42! resistance were independent
processes, since CrO 42!-sensitive and -resistant P. fluorescens were equally able to reduce
CrO 42! (93,101).
The direct biological reduction of CrO 42! is an enzyme-mediated reaction that takes
place under both aerobic (102–104) and anaerobic (99,105,106) conditions. Some bacteria
are capable of reducing CrO 42! under both aerobic and anaerobic conditions (107,108).
Although it was reported that some facultative anaerobic bacteria are capable of using
Cr 6" as the sole terminal electron acceptor during respiratory metabolism (99,106), Lovley
(109) concluded that Cr 6" reduction had not been shown to yield energy to support growth.
To date, enzymes involved in the microbial reduction of CrO 42! have not been identified,
but a few have been characterized in species of Bacillus (104,108), Enterobacter (110),
Streptomyces (111), and Pseudomonas (101,103,112).
Chromate reductase activities have been associated with the cell membrane
(101,110) and with the soluble protein fraction (103,104,108,111). Crude cell-free extracts
of Pseudomonas fluorescens LB300 readily reduced Cr 6" with glucose as the electron
donor (101). The addition of NADH to the S 32 supernatant fraction (32,000 $ g for 20 min)
prepared from the crude cell-free extract exhibited increased CrO 42!-reducing activity.
However, no CrO 42! reductase activity occurred in the S 150 fraction (150,000 $ g for 40
min), suggesting that some or all of the enzymes necessary for the transfer of electrons
from NADH to CrO 42! are membrane-bound. Wang et al. (110) found the addition of
NADH did not increase the CrO 42! reductase activity of Enterobacter cloacae HO1, proba-
bly because the membrane vesicles were not accessible to NADH. It was observed that
membrane vesicles reduced by NADH and later exposed to Cr 6" oxidized the c- (c 548 ,
c 549 , and c 550 ) and b- (b 555 , b 556 , and b 558 ) type cytochromes (113). The cytochrome c 548
was found to be involved specifically in the electron transfer to CrO 42!. In Desulfovibrio
vulgaris, the c 3 cytochrome reportedly functioned as a Cr 6" reductase when H 2 was used
as the electron donor (114). Chromate reducing activity occurred in the membrane-bound
and soluble protein fraction; the fastest Cr 6" reduction occurred in the soluble protein
fraction. The Cr 6" reductase activity was lost when the soluble protein fraction was passed
over a cation-exchange column that specifically removed the c 3 cytochrome. The ability
to reduce Cr 6" was restored when cytochrome c 3 was added back to the soluble protein
fraction.
Chromate-reducing activity in Pseudomonas putida PRS2000 was associated with
the soluble protein fraction, but the addition of either NADH or reduced nicotinamide-
adenine dinucleotide phosphate (NADPH) to cell-free extracts was required for Cr 6"
reduction. The Cr 6"-reducing enzyme in cell-free extracts of Pseudomonas ambigua
G-1 required NADH but not NADPH as the hydrogen donor (102). Similar results were
1. Bioreduction of Chromium
The major focus of Cr 6" bioreduction is aimed at developing bioreactors, which con-
tain Cr 6"-reducing bacteria immobilized on inert matrices within the reactor. After the
bioreduction phase, Cr 3" precipitates are removed by a settling or filtration phase. With
this type of system, CrO 42!-contaminated water is pumped through the reactor along
with various C sources and nutrient supplements. Advantages include its potential cost-
effectiveness and absence of chemical reductants; the major disadvantage is that the low-
est achievable Cr concentration obtained in batch experiments so for is around 1 mg L !1
(115,116). Unfortunately, an effluent concentration of 1 mg L !1 Cr would be 20 times
higher than the Environmental Protection Agency (EPA) drinking water standard. Also
reaction rates may also be slow, since CrO 42! must diffuse into direct contact with the
cells, which maximizes its toxicity effects as well. Since optimal bioreduction has been
correlated with the exponential growth phase of the bacteria (115), conditions within the
bioreactor must be adjusted to maintain high growth rates. As a result, bioreactors of this
design are probably better suited to treatment of wastewater prior to discharge rather than
in situ applications because flow rates are expected to be relatively slow, and pumping
of groundwater can significantly raise costs.
Losı̀ et al. (117) in 1994 investigated a land application method for the treatment
of Cr 6"-contaminated groundwater. The method consisted of using contaminated water
high in Cr 6" to irrigate an alfalfa field (supplemented with an organic amendment), where
reduction, precipitation, and immobilization would take place. In a greenhouse experiment,
Cr 6"-contaminated water was passed through soil columns that were either amended with
cattle manure or grown with alfalfa. The influent concentration to the columns was 1 mg
L !1 Cr 6", and effluent levels were consistently below 0.02 mg L !1. It was found that the
dominant removal mechanism was reduction, followed by precipitation. Lower O 2 levels
favored the reduction reaction, and subsequent experiments determined that indigenous
microorganisms, in conjunction with a readily available C source (i.e., cattle manure),
were largely responsible for the Cr 6" reduction reactions.
V. MERCURY
Mercury (Hg) is noted for being one of the few metals that exist as a liquid at ambient
temperatures and for being a potent human neurotoxin. Natural (e.g., volcanic eruptions)
and anthropogenic (e.g., fossil fuel combustion) activities release large amounts of metallic
Hg (Hg 0) into the biosphere; however, it readily undergoes biotic and abiotic conver-
sion to organic forms such as methylmercury (MeHg, CH 3Hg "). Methylmercury is water-
soluble and fat-soluble; therefore it poses a threat to aquatic organisms such as fish and
especially to fish consumers. Mercury pollution first received a great deal of publicity from
the infamous Minamata Bay incident in Japan, where direct discharge of Hg-contaminated
industrial waste led to extremely high levels of MeHg in fish, which, when eaten by
humans, caused physical impairments and death. Aquatic organisms found in surface wa-
ters in the United States also have elevated levels of Hg, particularly in the Florida Ever-
glades, which are believed to be the result of bioaccumulation within the food chain.
Biomagnification can result when Hg concentrations in lakes and streams may be undetect-
able. Concentrations in fish tissues can be far greater than levels in surrounding water,
particularly when MeHg is present.
In the environment, microorganisms are involved in the transformation of inorganic
and organic mercury compounds, mostly as a detoxification mechanism. The microbial
formation of volatile Hg 0 or dimethylmercury [(CH 3 ) 2 Hg], neither of which is soluble,
A. Reduction of Mercury(II)
Numerous microorganisms avoid Hg toxicity by reducing ionic Hg (Hg 2") to volatile Hg 0,
a potentially useful application to remove Hg from Hg-contaminated water. The reduction
of Hg 2" to Hg 0 can be mediated by a number of microorganisms, including enteric bacteria,
Pseudomonas sp., Staphylococcus aureus, Thiobacillus ferrooxidans, Streptomyces sp.,
and Cryptococcus sp. (121). The ability of bacteria to reduce Hg 2" is linked to Hg resis-
tance (mer) operons (122). The hypothesized plasmid-mediated detoxification mechanism
is shown in Fig. 6. The plasmid codes for a protein (merP) that initially binds to Hg 2" in
the periplasm. The Hg 2" is then transported through the inner membrane to the cytoplasm
by the membrane-bound protein merT. In the cytoplasm, Hg 2" is reduced to Hg 0 by a
soluble, NADH-dependent, flavin adenine dinucleotide (FAD)-containing mercuric reduc-
tase. Mercuric reductase is active in the presence of excess thiols (R-SH) such as mercapto-
tethanol, dithiothreitol, glutathione, or cysteine. Intracellular Hg 0 is then eliminated from
the cell by enhanced diffusion.
B. Methylation of Mercury
It was initially believed that methanogens were important methylators of Hg (123); how-
ever, this may not be the case after all. The methylation of Hg was not detected in whole
cells of methanogens or in methanogenic sewage sludge (124), and the addition of a spe-
cific methanogen inhibitor to lake sediments did not alter the production of MeHg (125),
suggesting that methanogens are not active in Hg methylation.
C. Bioremediation of Mercury
1. Bioaccumulation
A number of naturally occurring Hg biotransformations may have applications in the bio-
remediation of Hg-contaminated soil and water. Mercury-resistant bacteria have been iso-
lated by Glombitza at the Akademie der Wissenschaften (137,138). These Hg-resistant
bacteria can accumulate up to 2%–4% by weight of nonvolatile Hg from aerated solutions
containing 50–100 mg L !1 Hg 2" (139). Biomass can be suspended in a feed solution
within a bioreactor and provided a C source (e.g., methanol or acetate) plus additional
nutrients. The Hg-laden biomass is separated from solution and thermally decomposed at
400°–500°C to recover the Hg distillate.
2. Biosorption
Another technique being investigated to remediate Hg is biosorption. One such technol-
ogy, which employs immobilized algae, is marketed by Bio-Recovery Systems under the
name Alga-SORB (139). The product is prepared by heating the algae (Chlorella vulgaris)
to 300°–500°C and immobilizing them on a silicate-based matrix. Alga-SORB shows a
strong adsorption for Hg 2" independent of pH over a range of 2–6. Mercury adsorption
occurs as a result of interactions with ‘‘soft’’ ligands (forms covalent complexes with
‘‘soft’’ functional groups that contain N or S) present in the cell wall. In an E.P.A.-
Figure 7 Proposed bioreactor design for the treatment of Hg-contaminated water. (From Ref.
142.)
VI. CONCLUSION
REFERENCES