Enzymes in the Environment: Activity, Ecology and Applications - Chapter 20 doc

During the tricarboxylicacid TCA cycle, reduced nicotinamide-adenine dinucleotide NADH and succinate areused as electron donors to reduce SeO4 ⫺and SeO3 ⫺.. Selenate reduction to SeO3 ⫺i

20 Enzyme-Mediated Transformations

5 g cm⫺3 Although this encompasses a large percentage of the metals, only several heavymetals/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 theNational Priority (Superfund) sites ranked on the National Priorities List (NPL) containheavy metals that are designated as a threat or problem to the environment (4) Sinceheavy metals/metalloids cannot be degraded (i.e., biologically or chemically) they areamong 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, andother industrial processes), animal manure, and sewage sludge (5) Surprisingly, someinorganic fertilizers contain significant quantities of heavy metal impurities Sewagesludge, which is often used as a soil conditioner, contains useful quantities of organicmatter, N, and P; however, it often contains heavy metals The metals are chelated by theorganic 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) easilyexchangeable 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

Although the ultimate goal is the complete removal of heavy metals and metalloidsfrom water, this is not necessarily the case with contaminated soil The most commonlyused remedial techniques to deal with heavy metal/metalloid-contaminated soil are land-filling and solidification (7) Solidification involves a process in which the contami-nated soil is stabilized, fixed, solidified, or encapsulated into a solid material by the addi-tion of a resin or some other chemical compound that acts as a cement However, althoughthe contaminants are immobilized in the matrix, they are not destroyed, and, as a result,there is major concern over the stability of the contaminants in the solidified matrix Addi-tional remedial technologies include soil washing, soil flushing, acid extraction, and vitri-fication.

In an effort to find economically viable remedial technologies, much attention isfocused on bioremedial approaches Investigations have shown that microbiological metaltransformations may be applicable in remediating heavy metals and metalloids in soil aswell as in water Novel applications in bioremediation have been designed for aquaticsystems; unfortunately, relatively few applications are available for contaminated soils.Nonetheless, it is well known that the fate and transport of inorganic solutes in soils andwaters can be controlled by biochemical processes such as oxidation, reduction, methyla-tion, and demethylation (8) As a consequence of these reactions mediated by microorgan-isms, heavy metals and metalloids can exist in chemical states (i.e., soluble phase, insolu-ble nonaqueous phase such as mineral precipitants, or gaseous phase) that are biologicallyless toxic or more easily removed from the environment or both

In natural soil and aquatic systems, heavy metal/metalloid transformations are erally carried out as a direct result of microbial activities (e.g., respiration and detoxifica-tion mechanisms) However, extracellular enzymes and enzymes not directly associatedwith the soil and aquatic microbiota may also contribute to these transformations In soil,

gen-a number of extrgen-acellulgen-ar enzymes gen-are produced by microorggen-anisms; other enzyme sourcesinclude plant seeds, fungal and bacterial endospores, protozoan cysts, and plant roots, all

of which contribute to free enzymes found in soil and in some cases water Free enzymescan be inactivated by adsorption to organic and inorganic particles, can be denatured byphysical and chemical factors, or can serve as growth substrates for other microorganisms.Although many background enzymes can be found in natural soil and aquatic systems,very little research has been conducted on their involvement in heavy metal/metalloidtransformations Further research attention should be applied to this area, especially withregard to bioremediation of heavy metals/metalloids The purpose of this chapter is toreview microbially mediated transformations of Se, As, Cr, and Hg and discuss, whereapplicable, how they are currently being applied in bioremediation approaches to detoxifysoils and waters

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 (SeO4 ⫺, Se6 ⫹) and selenite (SeO3 ⫺, Se4 ⫹) are the most commonions found in soil solution and natural waters Organic Se-containing compounds includeSe-substituted amino acids, such as selenomethionine, selenocysteine, and selenocystine,and volatile methyl species such as dimethylselenide (DMSe, [CH3]2Se), dimethyldisele-nide (DMDSe, [CH3]2Se2), methaneselenol (CH3SeH), and dimethylselenenylsulfide

(DMSeS, [CH3]2SeS) Inorganic reduced forms include mineral selenides and hydrogenselenide (H2Se) The environmental threat of elevated levels of Se in soils and waters hasbeen recognized in many locations throughout the western United States (9) In Califor-nia’s San Joaquin Valley, elevated levels of Se in agricultural drainage water have beenlinked to the death and deformity of aquatic birds (10).

Selenium is predominantly cycled via biological pathways similar to that of sulfur.Like sulfur, Se undergoes various oxidation and reduction reactions that directly affectits oxidation state and, hence, its chemical properties and behavior in the environment

To date, most work has focused on reduction and methylation/volatilization reactions of Sebecause of their potential application in remediating seleniferous environments Currently,bioremediation strategies for Se are much further along in terms of implementation thanmethods for As, Cr, and Hg, which are largely still in the experimental stage

A Reduction of Selenium(VI)

The bioreduction of Se to insoluble Se0has been extensively investigated as a techniquefor removing Se from contaminated water Selenium undergoes dissimilatory microbialreduction, whereby SeO4 ⫺is reduced to Se0as the terminal electron acceptor in respiratory

metabolism Macy (11) isolated Thauera selenatis, a SeO4 ⫺, NO3 ⫺, and NO2 ⫺respiringbacterium, from seleniferous sediments The reduction of SeO4 ⫺to SeO3 ⫺and NO3 ⫺to

NO2 ⫺by T selenatis occurs through the use of separate terminal reductases, a SeO4 ⫺and

NO3 ⫺reductase, respectively The complete reduction of SeO4 ⫺to Se0only occurs whenthe organism is grown in the presence of both SeO4 ⫺and NO3 ⫺ During the tricarboxylicacid (TCA) cycle, reduced nicotinamide-adenine dinucleotide (NADH) and succinate areused as electron donors to reduce SeO4 ⫺and SeO3 ⫺ The electrons are then transferredvia an electron transport system that is part of a dehydrogenase, which is loosely bound

to the cytoplasmic membrane Selenate reduction to SeO3 ⫺involves a periplasmic SeO4 ⫺

reductase, whereas SeO3 ⫺produced during the respiration of SeO4 ⫺and NO3 ⫺is believed

to be reduced via a periplasmic NO2 ⫺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 SeO4 ⫺and NO3 ⫺as terminal electron acceptors during obic growth and can reduce SeO4 ⫺to Se0 under growth conditions and in washed-cellsuspensions under microaerophilic conditions Although strain SLD1a-1 respires SeO4 ⫺

anaer-anaerobically, the complete reduction of SeO3 ⫺ to Se0 does not occur unless NO3 ⫺ ispresent, suggesting that NO3 ⫺is necessary for the reduction of SeO3 ⫺to Se0(13) Orem-

land and associates (14) isolated a strictly anaerobic motile vibrio (Sulfurospirillum

barnesii strain SES-3) that grows in the presence of either SeO4 ⫺or NO3 ⫺while usinglactate as an electron donor It was determined that the reduction of SeO4 ⫺and NO3 ⫺

ions is achieved by separate inducible enzyme systems Although growth was not observed

on SeO3 ⫺, washed-cell suspensions of SES-3 could reduce SeO3 ⫺to Se0

a periplasmic NO2 ⫺reductase (16) or reduced aerobically as a detoxification mechanism,

Figure 1 Hypothetical model of selenate reduction to elemental selenium by Thauera selenatis involving a periplasmic selenate reductase, cytochrome C551, and nitrite reductase (From Ref 11.)

independently of dissimilatory reduction (15) However, in a 1998 study, the reduction

of SeO3 ⫺ by Bacillus selenitireducens was linked to its respiration (17) Selenite was

reduced to Se0by aerobically grown Salmonella heidelberg (18) and by resting cells of

Streptococcus faecalis and Streptococcus faecium (19) Two common soil bacterial strains, Pseudomonas fluorescens and Bacillus subtilis, apparently reduced SeO3 ⫺to Se0 via adetoxification mechanism independent of NO2 ⫺and SeO3 ⫺(20,21) Yanke and coworkers

(22) found that Clostridium pasteurianum utilized the constitutive enzyme hydrogenase

(I) as a SeO3 ⫺reductase In addition, the enzyme was found to reduce not only SeO3 ⫺

but also tellurite (TeO3 ⫺) Selenite reduction ceased when the enzyme was exposed to

O2and CuSO4, potent inhibitors of hydrogenase (I) activity

B Methylation of Selenium

The methylation of Se is a biological process and is thought to be a protective mechanismused by microorganisms to detoxify their surrounding environment The methylation andsubsequent volatilization of Se may constitute important steps in the transport of Se fromcontaminated terrestrial and aquatic environments Bacteria and fungi are the predominantSe-methylating organisms isolated from soils, sediments, and waters (23) The predomi-nant Se gas produced by most microorganisms is DMSe (24), although other volatile Secompounds, such as DMDSe, DMSeS, and methaneselenol, may also be produced in lesseramounts Although the biological significance of Se methylation is not clearly understood,once volatile Se compounds are released to the atmosphere and diluted, Se has lost itshazardous potential

The first report of microbially derived gaseous Se was discovered by Challenger

and North (25) during their studies of pure cultures of Penicillium brevicaule (previously named Scopulariopsis brevicaulis) They found that the fungus was able to convert both

SeO4 ⫺and SeO3 ⫺to DMSe while growing on bread crumbs Several reports that followed

over the years identified many other fungi capable of methylating Se, including Penicillium sp., Fusarium sp., Schizopyllum commune, Aspergillus niger, Alternaria alternata, and

Acremonium falciforme (26) Abu-Erreish et al (27) noticed the production of volatile Se

in seleniferous soils appeared to be related to fungal growth The addition of a fungal

inoculum, Candida humicola, to soil caused the rate of Se volatilization to double (28).

However, the addition of chloramphenicol to soil reduced the amount of Se volatilizedfrom a soil by 50%, suggesting that bacteria also play an important role in Se methylation

To date, only a few bacterial genera capable of methylating Se have been identified

Chau et al (29) isolated three bacteria (Aeromonas sp., Flavobacterium sp., and

Pseudo-monas sp.) from lake sediment that were capable of methylating SeO3 ⫺to DMSe and

DMDSe A strain of Corynebacterium sp., isolated from soil, formed DMSe from SeO4 ⫺,SeO3 ⫺, Se0, selenomethionine, selenocystine, and methaneseleninate (methaneseleninic

acid) (30) Aeromonas veronii, isolated from seleniferous agricultural drainage water, was

active in volatilizing DMSe and lesser amounts of methaneselenol, DMSeS, and DMDSe

(31) McCarty et al (32) identified two phototrophic bacterial species, Rhodospirillum

rubrum S1 and Rhodocyclus tenuis, that produced DMSe and DMDSe in the presence of

SeO4 ⫺ Enterobacter cloacae SLD1a-1, the SeO4 ⫺and SeO3 ⫺reducing bacterium, duces DMSe from SeO4 ⫺, SeO3 ⫺, Se0, dimethylselenone [(CH3)2SeO2], selenomethio-nine, 6-selenopurine, and 6-selenoinosine (33) The methylation of Se by algae has alsobeen confirmed by Fan et al (34), who isolated a euryhaline green microalga species of

pro-Chlorella from a saline evaporation pond that was able to transform SeO3 ⫺aerobicallyinto DMSe, DMDSe, and DMSeS

In general, the formation of alkylselenides from Se oxyanions involves a reductionand methylation step; however, the pathway by which these reactions occur is still highlydebated Challenger (35) postulated that the formation of DMSe occurs through successivemethylation and reduction steps, in which dimethylselenone was suspected to be the lastintermediate prior to the formation of DMSe (Fig 2) Reamer and Zoller (36) identifiedDMDSe and dimethylselenone in addition to DMSe as products from soil and sewagesludge amended with either SeO3 ⫺or Se0 It was then suggested that Challenger’s pathway

Figure 2 Proposed mechanism for the methylation of selenium by fungi (From Ref 35.)

could be modified to include the production of DMDSe through an alternate pathwaywhereby methaneseleninic acid is reduced to methaneselenol or methaneselenenic acid orboth, to produce DMDSe Doran (24) proposed that SeO3 ⫺is reduced via Se0to a selenidefrom before it is methylated to form methaneselenol and finally DMSe Although meth-aneselenol and methaneselenide were not tested for as intermediates, evidence in support

of Doran’s pathway comes from Bird and Challenger (37), who detected small amounts

of methaneselenol emitted from actively methylating fungal cultures Doran’s pathway isalso markedly similar to findings of studies conducted with mammals, which demonstratedthat methaneselenol is an intermediate in the methylation of Se to DMSe (38,39) Cookeand Bruland (40) proposed a pathway for the formation of DMSe from SeO4 ⫺and SeO3 ⫺

in natural waters Apparently both Se oxyanions are reduced and assimilated into theintermediate selenomethionine [CH3Se(CH2)2CHNH2COOH], which is then methylated

to produce methylselenomethionine [(CH3)2Se⫹(CH2)2CHNH2COOH] Finally, selenomethionine is hydrolyzed to DMSe and homoserine

methyl-The biosynthesis of methionine from homocysteine is an important transformation

in the methylation of Se During the activated methyl cycle homocysteine is methylated viathe coenzyme methylcobalamin (CH3B12; derivative of vitamin B12), yielding methionine.Methylcobalamin has been isolated from bacteria (41) and is believed to donate methylgroups to Se, resulting in the formation of volatile alkylselenides Thompson-Eagle

et al (42) found that the addition of methylcobalamin promoted the methylation ofSeO4 ⫺ McBride and Wolfe (43) found that cell-free extracts of a Methanobacterium sp.

methylated SeO4 ⫺when methylcobalamin was present Cell-free extracts of E cloacae

SLD1a-1 catalyzed the formation of DMSe from SeO3 ⫺or Se0 when methylcobalamin

was the methyl donor (33) In addition, S-adenosylmethionine has been identified as a

cofactor in the microbial methylation of inorganic Se (30) Doran (24) found that

cell-free extracts of the soil Corynebacterium sp were able to methylate SeO3 ⫺or Se0when

S-adenosylmethionine was present Drotar and associates (44) identified an

S-adenosyl-methionine-dependent selenide methyltransferase in cell-free extracts of Tetrahymena

thermophila, which reportedly produced methaneselenol from Na2Se Although there issome understanding of the pathway by which Se oxyanions are transformed to DMSe,neither of the pathways elucidates the mechanism of the reaction Clearly more work isneeded to understand the biochemical characteristics of Se methylation

C Bioremediation of Seleniferous Water and Sediment

Since the 1990s attention has been given to the development of an effective remediationtechnology for the permanent removal of Se oxyanions from seleniferous soil and water

A majority of the focus has been applied to contaminated agricultural drainage water,which has been responsible for a number of well-documented ecotoxicological problems.Since Se undergoes microbial transformations, their application may be potentially useful

as bioremediation strategies Several different bioremedial approaches have been or arebeing developed; they include a variety of bioreactors utilizing bacteria with the ability

to reduce the toxic, soluble Se oxyanions to insoluble Se0 These systems are designed

to remove Se from contaminated wastewater (industrial or agricultural) before release intothe environment Because of the high SeO4 ⫺to SeO3 ⫺ratio of most agricultural drainagewaters of the western United States, removal of mainly SeO4 ⫺ must be considered inthese systems Another means to remove Se from contaminated soil and water involvesstimulation of the indigenous microorganisms that volatilize Se This process has provedeffective as an in situ treatment for seleniferous soils and sediments in the San JoaquinValley, California (45,46)

1 Bioreduction of Selenium Oxyanions to Elemental Selenium

The use of Thauera selenatis, a SeO4 ⫺respiring bacterium, in a biological reactor system

to remediate both SeO4 ⫺and SeO3 ⫺ions from contaminated water has been described

by Macy and associates (47), Lawson and Macy (48), and Cantafio and coworkers (49).The latest pilot scale system, which consisted of a series of four medium-packed tanks,was used to treat seleniferous agricultural drainage water (49) Using acetate as the electrondonor, Se oxyanion and NO3 ⫺concentrations were reduced by 98% An earlier systemincluded the use of two bioreactors in series; the first was an aerobic sludge blanket reactorand the second a fluidized bed reactor (47) Once again acetate was used as the electrondonor and the growth of the organism was found to be dependent on the presence of

NH4Cl The SeO4 ⫺, SeO3 ⫺, and NO3 ⫺levels were all reduced by 98% in the influent Asimilar system, later used to remediate SeO3 ⫺from oil refinery wastewater, reduced the

Se oxyanion concentration by 95% Although Macy (11) has shown that this organismcan reduce both SeO4 ⫺and NO3 ⫺simultaneously, NO3 ⫺must be present in the systemfor SeO4 ⫺to be completely reduced to Se0, since the NO2 ⫺reductase only catalyzes thereduction of SeO3 ⫺when denitrification is occurring

The algal–bacterial selenium removal system (ABSRS) is another process used toremove soluble Se and NO3 ⫺from drainage water (50) The influent is first directed towardhigh-rate ponds where microalgae are grown; removal of some NO3 ⫺results About 10%

of the N is removed in the high-rate ponds, a proportion that supports that algae are made

up of 9.2% N by dry weight (51) After this step, the biomass suspension is dischargedinto an anoxic unit where bacteria use the algae as a C and energy source and subsequentlyreduce the SeO4 ⫺and SeO3 ⫺to Se0, and NO3 ⫺to N2gas Although near-complete removal

of SeO4 ⫺and NO3 ⫺occurred at times in field experiments, it was speculated that sincethe project was run for an insufficient amount of time, steady-state reducing conditionscould not be established Since substantial reduction of SeO4 ⫺to SeO3 ⫺was occurring,use of FeCl3was applied to precipitate out inorganic SeO3 ⫺, thereby reducing the soluble

Se levels

Oremland (52) has also described a process similar to the ABSRS This processinvolves using a two-stage reaction, which uses algae in the first aerobic stage to depletethe NO3 ⫺ concentrations below 62 mg L⫺1 The water is then transferred to an anoxic

reactor containing SeO4 ⫺reducing bacteria where SeO4 ⫺is reduced to insoluble Se In

7 days, the influent SeO4 ⫺concentration of 56 mg Se L⫺1 was reduced by more than99%

EPOC AG (Binnie California) conducted studies on the removal of Se from tural drainage water using a pilot-scale two-stage biological process (53) The systemconsisted of an upflow anaerobic sludge blanket reactor followed by a fluidized-bed reac-tor A crossflow microfilter was used after the biological reactors for the removal of partic-ulate Se The effluent concentration from the system averaged less than 30 µg L⫺1 ofsoluble selenium When the effluent was further processed through a soil column thesoluble Se concentration was less than 10µg L⫺1

agricul-Owens (53) describes a pilot-scale biological system that utilized an upflow bic sludge blanket reactor The C source used in the system was methanol, which wasadded at a dosage of 250 mg L⫺1 Most of the C added to the system was used duringdenitrification; thus, enough methanol must be added to support both denitrification and

anaero-Se reduction Denitrification is important to the process since anaero-Se reduction does not occuruntil the NO3 ⫺is removed It was reported that the reactor was able to remove 94% ofthe soluble Se, with a final effluent concentration of 29µg L⫺1obtained

Adams et al (54) conducted a pilot study in which Escherichia coli was used to

treat a weak acid effluent from a base metal smelter containing 30 mg Se L⫺1 The tor system consisted of a rotating biological contactor (RBC) and was able to remove97% of the Se within 4 hours A bench-scale RBC system was also tested on mining

bioreac-process waters, and using Pseudomonas stutzeri, with molasses (1 g L⫺1) as the C source,97% of the Se was removed in 6-hour retention time

2 Selenium Volatilization in the Field

Field studies were performed on the Sumner Peck Ranch (Fresno County, California)evaporation pond water in an effort to determine whether the addition of casein wouldstimulate Se volatilization (55) Water columns in the evaporation ponds were treatedwith a single casein application of 0.2 g L⫺1pond water The evaporation pond water Seconcentration was reported as high as 2.9 mg L⫺1 Unamended pond water evolved volatile

Se at low rates of 0.1 µg Se L⫺1 d⫺1, whereas casein amended pond water producedemission rates of 2.2µg Se L⫺1d⫺1 After 142 days, the casein amended pond water lost38% of the initial Se inventory

In dewatered evaporation pond sediments at the Sumner Peck Ranch, 32% of the

Se in the top 15 cm was removed with the application of water plus tillage alone; theaddition of cattle manure resulted in the removal of 58% after 22 months (56) The initialmean plot soil Se concentration in the top 15 cm was 11.4 mg kg⫺1 The backgroundemission rate of volatile Se averaged 3.0µg Se m⫺2h⫺1, whereas the cattle manure treatedplot promoted an average emission rate of 54 µg Se m⫺2 h⫺1 As reported in other Sevolatilization studies, the parameters that enhanced Se volatilization were moisture, hightemperatures, aeration, and an available C source The highest gaseous Se flux was re-corded in the summer months and the lowest flux occurred in the winter

Over a 100-month period at Kesterson Reservoir, 68%–88% of the total Se wasdissipated from the top 15 cm of seleniferous soil (46) The soil Se concentration varied

in each of the plots from approximately 40 to 60 mg Se kg⫺1 Since no pattern of Sedepletion was correlated with rainfall events or temperature, it was speculated that leachingdominated during the winter months, because most rainfall occurred during the winter,

whereas volatilization was dominant during the summer months The addition of C ments had no significant effect greater than that of the moisture-only treatment, a findingthat suggests that tillage and irrigation prevailed over the effects of the amendments How-ever, cattail roots providing C were disked into all plots at the onset of this investiga-tion (57).

amend-3 Cell-Free Systems

Adams et al (54,58) treated mine water containing 0.62 mg L⫺1SeO4 ⫺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 pared by disrupting the cells then immobilizing the lysate in calcium alginate beads Theimmobilized enzyme preparation performed for approximately 4 months, achieving efflu-ent levels below 10µg L⫺1 Another cell-free system was used to treat mining process

pre-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 columnswith a retention time of 9 to 18 hours The system was capable of simultaneously removingcyanide 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 oxidationstates,⫹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 metalsand covalently bonds with carbon, hydrogen, oxygen, and sulfur (59) Arsenate (AsO4 ⫺),

a biochemical analog of phosphate, is transported by highly specific energy-dependentmembrane pumps into the cell during assimilation of phosphate, whereas arsenite (AsO2 ⫺)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 aretwo reasons for As toxicity The poisonous character of As make it an effective herbicideand insecticide The ubiquity of As in the environment, its biological toxicity, and itsredistribution are factors evoking public concern

Both oxidation and methylation are microbial transformations involved in the tribution and global cycling of As Oxidation involves the conversion of toxic AsO2 ⫺toless toxic AsO4 ⫺ 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 bacteriaand may serve as a detoxification mechanism The mechanism involves the reduction ofAsO4 ⫺to AsO2 ⫺, 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 AsO4 ⫺reduction to AsO2 ⫺followed by several methylation stepsproducing trimethylarsine Currently, a number of microbially mediated oxidation andmethylation reactions are being studied in the interest of developing bioremediation tech-niques for detoxifying As-contaminated soil and water

As3⫹in anaerobic sediments (61,62) Dowdle et al (61) found that As5⫹was reduced to

As3⫹in anoxic salt marsh sediment slurries when the electron donor was lactate, H2, orglucose The addition of the respiratory inhibitor/uncoupler dinitrophenol, rotenone, or

2-heptyl-4-hydroxyquinoline N-oxide blocked the reduction of As5 ⫹, suggesting that thereduction of As5 ⫹in sediments proceeds through a dissimilatory process

To date, several SAsO4 ⫺respiring organisms have been isolated and characterized:

Sulfurospirillum arsenophilus strain MIT-13 (63), S barnesii strain SES-3 (14,64), fotomaculum auripigmentum strain OREX-4 (65), and Chrysiogenes arsenatis strain BAL-

Desul-1T (66) The only common electron acceptor of these organisms is fumurate, and studieshave shown that strain MIT-3, strain SES-3, and strain BAL-1T respire NO3 ⫺and AsO4 ⫺

but not SO4 ⫺, whereas strain OREX-4 can grow on SO4 ⫺but not NO3 ⫺ The mechanisms

by which electrons are passed to AsO4 ⫺ during dissimilatory reduction and reductivedetoxification differ

The reductive detoxification of AsO4 ⫺occurs when reduced dithiols transfer trons for the ArsC enzymes (67), whereas the respiratory AsO4 ⫺reductase in strain SES-

elec-3 appears to utilize prosthetic groups such as Fe:S clusters (68) Additionally, a b-type

cytochrome is present in the membrane when it is grown on AsO4 ⫺, and it may be involved

in the transfer of electrons Fig 3 is the proposed biochemical pathway by which strain

Figure 3 Biochemical model of arsenate respiration in Sulfurospirillum barnesii strain SES-3.

(From Ref 68.)

SES-3 reduces AsO4 ⫺to AsO2 ⫺when grown on lactate It was postulated that strain

SES-3 contains an AsO2 ⫺-efflux system similar to that of other bacteria, which would enablestrain SES-3 to cope with the AsO2 ⫺produced in the cytoplasm Therefore, the flow ofelectrons could be generated from a cytoplasmically oriented lactate dehydrogenase to theAsO4 ⫺reductase and could occur through the use of a proton-pumping intermediate (e.g.,menaquionone) or through diffusion of H2(formed by a cytoplasmic hydrogenase) throughthe membrane to the outside Hydrogen in the periplasm would be oxidized by a hydro-genase, allowing electrons to flow back to the AsO4 ⫺reductase through membrane-boundelectron carriers White (69) proposed a similar model for the generation of the protonmotive force during dissimilatory reduction of SO4 ⫺ Although the reduction of As5 ⫹is

of environmental interest because As3 ⫹is more mobile and toxic than As5 ⫹, additionalwork is clearly needed to understand the environmental significance of dissimilatoryAsO4 ⫺reduction

B Oxidation of Arsenic(III)

Currently, a number of microbially mediated oxidation reactions are being studied in theinterest of developing bioremediation techniques for detoxifying As-contaminated soil and

water Bacillus, Thiobacillus, and Pseudomonas spp that have been isolated can oxidize

AsO2 ⫺to the less toxic AsO4 ⫺ In addition, a strain of Alcaligenes faecalis obtained from

raw sewage was capable of oxidizing AsO2 ⫺(70) Osborne and Ehrlich (71) isolated asimilar AsO2 ⫺-oxidizing soil strain of A faecalis whose oxidation process was induced

by AsO2 ⫺and AsO4 ⫺ The use of respiratory inhibitors prevented further oxidation ofAsO2 ⫺, indicating that oxygen served as the terminal electron acceptor Studies suggestedthat the oxidation of AsO2 ⫺involved an oxidoreductase with a bound flavin that passedelectrons from AsO2 ⫺to O2by way of cytochrome c and cytochrome oxidase (71) Indirect

evidence suggested that the organism may be able to derive maintenance energy fromthe oxidation of AsO2 ⫺ (72) Ilyaletdinov and Abdrashitova (73) isolated Pseudomonas

arsenitoxidans from a gold and arsenic ore deposit that was capable of growing

autotrophi-cally with AsO2 ⫺as the soil energy source

Anderson et al (74) found that A faecalis strain NCIB 8687 contained an inducible

AsO2 ⫺-oxidizing enzyme that was located on the outer surface of the plasma membrane,

a finding that suggested that AsO2 ⫺oxidation occurred in its periplasmic space The

85-kD enzyme was a molybdenum-containing hydroxylase with a pterin cofactor and ganic sulfide, one atom of molybdenum, and five or six atoms of iron The enzyme cata-lyzed the oxidation of AsO2 ⫺when both azurin and cytochrome c were used as electron

inor-acceptors Oxidation of AsO2 ⫺by heterotrophic bacteria plays an important role in fying the environment, catalyzing as much as 78% to 96% of the AsO2 ⫺to AsO4 ⫺(75)

detoxi-C Methylation of Arsenic

1 Bacterial Methylation

Bacterial methylation of inorganic As has been studied extensively using methanogenicbacteria Methanogenic bacteria are a morphologically diverse group consisting of coccal,bacillary, and spiral forms but are unified by the production of methane as their principalmetabolic end product They are present in large numbers in anaerobic ecosystems, such

as sewage sludge, freshwater sediments, and composts where organic matter is

decompos-ing (76) Under anaerobic conditions, the biomethylation of As only proceeds to arsine, which is stable in the absence of O2but is rapidly oxidized under aerobic conditions.

dimethyl-It has been shown that at least one Methanobacterium sp is capable of methylating

inor-ganic As to produce volatile dimethylarsine Arsenate, AsO2 ⫺, and methylarsonic acid(methanearsonic acid) can serve as substrates in dimethylarsine formation Inorganic arse-nic methylation is coupled to the CH4biosynthetic pathway and may be a widely occurringmechanism for As detoxification

Cell-free extracts of Methanobacterium sp strain MOH, when incubated under

an-aerobic conditions with AsO4 ⫺, methylcobalamin, H2, and adenosine triphosphate (ATP),produced volatile dimethylarsine (43) Fig 4 shows the pathway by which dimethylarsine

is produced by Methanobacterium sp., which involves the reduction of AsO4 ⫺to AsO2 ⫺

with subsequent methylation by a low-molecular-weight cofactor coenzyme M (CoM).CoM has been found in all methane bacteria examined and chemically is 2,2′-dithiodi-ethane sulfonic acid (76) Methylarsonic acid added to cell-free extracts is not reduced

to methylarsine but requires an additional methylation step before reduction However,dimethylarsinic acid (cacodylic acid) is reduced to dimethylarsine even in the absence of

a methyl donor (43) Under anaerobic conditions, whole cells of methanogenic bacteriaalso produce dimethylarsine as a biomethylation end product of As, but not heat-treated

cells, indicating that this is a biotic reaction Cell-free extracts of Desulfovibrio vulgaris

strain 8303 also produced a volatile As derivative, presumably an arsine, when incubatedwith AsO4 ⫺ (43) The reaction occurred in the absence of exogenous methyl donors;however, the addition of methylcobalamin greatly stimulated the reaction

Interestingly, another study indicated that resting cell suspensions of Pseudomonas and Alcaligenes spp incubated with either AsO4 ⫺and AsO2 ⫺under anaerobic conditions

produced arsine, but no other As intermediates were formed (77) An Aeronomonas sp and a Flavobacterium sp isolated from lake water were capable of methylating As to dimethylarsinic acid, and Flavobacterium sp additionally methylated dimethylarsinic acid

derived from inorganic and organic As species The volatilized As dissipates from thecells, effectively reducing the As concentration to which the fungus is exposed The impor-tance of fungal metabolism of As dates back to the early 1800s, when a number of poison-ing incidents in Germany and England were caused by trimethylarsine gas (35) Sincethen, several species of fungi that are able to volatilize As have been identified The fungus

Penicillium brevicaule produces trimethylarsine when grown on bread crumbs containing

either methylarsonic acid or dimethylarsinic acid A biochemical pathway for sine production was proposed by Challenger in 1945 (Fig 5)

trimethylar-In 1973 studies, three different fungal species, Candida humicola, Gliocladium

ro-seum, and Penicillium sp., were reported as capable of converting methylarsonic acid and

dimethylarsinic acid to trimethylarsine (79) In addition, C humicola used AsO4 ⫺andAsO2 ⫺as substrates to produce trimethylarsine Cell-free extracts of C humicola trans-

formed AsO4 ⫺into AsO2 ⫺, methylarsonic acid to dimethylarsinic acid and trimethylarsineoxide, and dimethylarsinic acid to methylarsonic acid and trimethylarsine oxide (80) Al-though trimethylarsine formation from inorganic As and methylarsonic acid is inhibited

by the presence of phosphate, its synthesis from dimethylarsinic acid is increased in thepresence of phosphate (81) More recently, Huysmans and Frankenberger (82) isolated a

Penicillium sp from agricultural evaporation pond water capable of producing

trimethylar-sine from methylarsonic acid and dimethylarsinic acid

Methylation of As is thought to occur via the transfer of the carbonium ion from

S-adenosylmethionine (SAM) to As Incubation of cells with an antagonist of methionine

inhibited the production of arsines, thus supporting the role of methionine as a methyldonor (83) The addition of either methanearsonic acid or dimethylarsinic acid to cell-free extracts yields trimethylarsine oxide (80) Further reduction of trimethylarsine oxide

to trimethylarsine requires the presence of intact cells (84) Various arsenic thiols ine, glutathione, and lipoic acid) are thought to be involved in the reduction step of trimeth-ylarsine oxide to trimethylarsine (85,86) The final reduction step is inhibited by severalelectron transport inhibitors and uncouplers of oxidative phosphorylation (84,87) Preincu-

(cyste-Figure 5 Fungal methylation pathway for the formation of trimethylarsine (From Ref 35.)