20 Enzyme-Mediated Transformations of Heavy Metals/Metalloids Applications in Bioremediation Robert S. Dungan George E. Brown, Jr. Salinity Laboratory, USDA–ARS, Riverside, California William T. Frankenberger, Jr. University of California–Riverside, 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 5gcm Ϫ3 . 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,claycontent,ironoxidecontent,andorganicmattercontent. Copyright © 2002 Marcel Dekker, Inc. Although the ultimate goal is the complete removal of heavy metals and metalloids from water, this is not necessarily the case with contaminated soil. The most commonly used 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, although the 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 is focused on bioremedial approaches. Investigations have shown that microbiological metal transformations may be applicable in remediating heavy metals and metalloids in soil as well as in water. Novel applications in bioremediation have been designed for aquatic systems; unfortunately, relatively few applications are available for contaminated soils. Nonetheless, it is well known that the fate and transport of inorganic solutes in soils and waters 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 biologically less toxic or more easily removed from the environment or both. In natural soil and aquatic systems, heavy metal/metalloid transformations are gen- erally carried out as a direct result of microbial activities (e.g., respiration and detoxifica- tion mechanisms). However, extracellular enzymes and enzymes not directly associated with the soil and aquatic microbiota may also contribute to these transformations. In soil, a number of extracellular enzymes are produced by microorganisms; other enzyme sources include 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 enzymes can be inactivated by adsorption to organic and inorganic particles, can be denatured by physical 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/metalloid transformations. Further research attention should be applied to this area, especially with regard to bioremediation of heavy metals/metalloids. The purpose of this chapter is to review microbially mediated transformations of Se, As, Cr, and Hg and discuss, where applicable, how they are currently being applied in bioremediation approaches to detoxify soils 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, ϩ2, 0, ϩ4, and ϩ6, forming a variety of compounds. Selenate (SeO 4 2Ϫ ,Se 6ϩ ) and selenite (SeO 3 2Ϫ ,Se 4ϩ ) 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 ] 2 Se), dimethyldisele- nide (DMDSe, [CH 3 ] 2 Se 2 ), methaneselenol (CH 3 SeH), and dimethylselenenylsulfide Copyright © 2002 Marcel Dekker, Inc. (DMSeS,[CH 3 ] 2 SeS).Inorganicreducedformsincludemineralselenidesandhydrogen selenide(H 2 Se).TheenvironmentalthreatofelevatedlevelsofSeinsoilsandwatershas beenrecognizedinmanylocationsthroughoutthewesternUnitedStates(9).InCalifor- nia’sSanJoaquinValley,elevatedlevelsofSeinagriculturaldrainagewaterhavebeen linkedtothedeathanddeformityofaquaticbirds(10). Seleniumispredominantlycycledviabiologicalpathwayssimilartothatofsulfur. Likesulfur,Seundergoesvariousoxidationandreductionreactionsthatdirectlyaffect itsoxidationstateand,hence,itschemicalpropertiesandbehaviorintheenvironment. Todate,mostworkhasfocusedonreductionandmethylation/volatilizationreactionsofSe becauseoftheirpotentialapplicationinremediatingseleniferousenvironments.Currently, bioremediationstrategiesforSearemuchfurtheralongintermsofimplementationthan methodsforAs,Cr,andHg,whicharelargelystillintheexperimentalstage. A.ReductionofSelenium(VI) ThebioreductionofSetoinsolubleSe 0 hasbeenextensivelyinvestigatedasatechnique forremovingSefromcontaminatedwater.Seleniumundergoesdissimilatorymicrobial reduction,wherebySeO 4 2Ϫ isreducedtoSe 0 astheterminalelectronacceptorinrespiratory metabolism.Macy(11)isolatedThaueraselenatis,aSeO 4 2Ϫ ,NO 3 Ϫ ,andNO 2 Ϫ respiring bacterium,fromseleniferoussediments.ThereductionofSeO 4 2Ϫ toSeO 3 2Ϫ andNO 3 Ϫ to NO 2 Ϫ byT.selenatisoccursthroughtheuseofseparateterminalreductases,aSeO 4 2Ϫ and NO 3 Ϫ reductase,respectively.ThecompletereductionofSeO 4 2Ϫ toSe 0 onlyoccurswhen theorganismisgrowninthepresenceofbothSeO 4 2Ϫ andNO 3 Ϫ .Duringthetricarboxylic acid(TCA)cycle,reducednicotinamide-adeninedinucleotide(NADH)andsuccinateare usedaselectrondonorstoreduceSeO 4 2Ϫ andSeO 3 2Ϫ .Theelectronsarethentransferred viaanelectrontransportsystemthatispartofadehydrogenase,whichislooselybound tothecytoplasmicmembrane.SelenatereductiontoSeO 3 2Ϫ involvesaperiplasmicSeO 4 2Ϫ reductase,whereasSeO 3 2Ϫ producedduringtherespirationofSeO 4 2Ϫ andNO 3 Ϫ isbelieved tobereducedviaaperiplasmicNO 2 Ϫ 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 4 2Ϫ and NO 3 Ϫ as terminal electron acceptors during anaer- obic growth and can reduce SeO 4 2Ϫ to Se 0 under growth conditions and in washed-cell suspensions under microaerophilic conditions. Although strain SLD1a-1 respires SeO 4 2Ϫ anaerobically, the complete reduction of SeO 3 2Ϫ to Se 0 does not occur unless NO 3 Ϫ is present, suggesting that NO 3 Ϫ is necessary for the reduction of SeO 3 2Ϫ 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 4 2Ϫ or NO 3 Ϫ while using lactate as an electron donor. It was determined that the reduction of SeO 4 2Ϫ and NO 3 Ϫ ions is achieved by separate inducible enzyme systems. Although growth was not observed on SeO 3 2Ϫ , washed-cell suspensions of SES-3 could reduce SeO 3 2Ϫ to Se 0 .APseudomonas stutzeri isolate could only reduce SeO 4 2Ϫ and SeO 3 2Ϫ to Se 0 under aerobic conditions, a limitation that was speculated to be a detoxification mechanism (15). The biological reduction of SeO 3 2Ϫ to Se 0 also occurs, but only the reduction of SeO 4 2Ϫ supports anaerobic growth. Although a number of SeO 3 2Ϫ 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 3 2Ϫ can be reduced anaerobically by a periplasmic NO 2 Ϫ reductase (16) or reduced aerobically as a detoxification mechanism, Copyright © 2002 Marcel Dekker, Inc. Figure 1 Hypothetical model of selenate reduction to elemental selenium by Thauera selenatis involving a periplasmic selenate reductase, cytochrome C 551 , and nitrite reductase. (From Ref. 11.) independently of dissimilatory reduction (15). However, in a 1998 study, the reduction of SeO 3 2Ϫ by Bacillus selenitireducens was linked to its respiration (17). Selenite was reduced to Se 0 by 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 SeO 3 2Ϫ to Se 0 via a detoxification mechanism independent of NO 2 Ϫ and SeO 3 2Ϫ (20,21). Yanke and coworkers (22) found that Clostridium pasteurianum utilized the constitutive enzyme hydrogenase (I) as a SeO 3 2Ϫ reductase. In addition, the enzyme was found to reduce not only SeO 3 2Ϫ Copyright © 2002 Marcel Dekker, Inc. butalsotellurite(TeO 3 2Ϫ ).Selenitereductionceasedwhentheenzymewasexposedto O 2 andCuSO 4 ,potentinhibitorsofhydrogenase(I)activity. B.MethylationofSelenium ThemethylationofSeisabiologicalprocessandisthoughttobeaprotectivemechanism usedbymicroorganismstodetoxifytheirsurroundingenvironment.Themethylationand subsequentvolatilizationofSemayconstituteimportantstepsinthetransportofSefrom contaminatedterrestrialandaquaticenvironments.Bacteriaandfungiarethepredominant Se-methylatingorganismsisolatedfromsoils,sediments,andwaters(23).Thepredomi- nantSegasproducedbymostmicroorganismsisDMSe(24),althoughothervolatileSe compounds,suchasDMDSe,DMSeS,andmethaneselenol,mayalsobeproducedinlesser amounts.AlthoughthebiologicalsignificanceofSemethylationisnotclearlyunderstood, oncevolatileSecompoundsarereleasedtotheatmosphereanddiluted,Sehaslostits hazardouspotential. ThefirstreportofmicrobiallyderivedgaseousSewasdiscoveredbyChallenger andNorth(25)duringtheirstudiesofpureculturesofPenicilliumbrevicaule(previously namedScopulariopsisbrevicaulis).Theyfoundthatthefunguswasabletoconvertboth SeO 4 2Ϫ andSeO 3 2Ϫ toDMSewhilegrowingonbreadcrumbs.Severalreportsthatfollowed overtheyearsidentifiedmanyotherfungicapableofmethylatingSe,includingPenicillium sp.,Fusariumsp.,Schizopyllumcommune,Aspergillusniger,Alternariaalternata,and Acremoniumfalciforme(26).Abu-Erreishetal.(27)noticedtheproductionofvolatileSe inseleniferoussoilsappearedtoberelatedtofungalgrowth.Theadditionofafungal inoculum,Candidahumicola,tosoilcausedtherateofSevolatilizationtodouble(28). However,theadditionofchloramphenicoltosoilreducedtheamountofSevolatilized fromasoilby50%,suggestingthatbacteriaalsoplayanimportantroleinSemethylation. Todate,onlyafewbacterialgeneracapableofmethylatingSehavebeenidentified. Chauetal.(29)isolatedthreebacteria(Aeromonassp.,Flavobacteriumsp.,andPseudo- monassp.)fromlakesedimentthatwerecapableofmethylatingSeO 3 2Ϫ toDMSeand DMDSe.AstrainofCorynebacteriumsp.,isolatedfromsoil,formedDMSefromSeO 4 2Ϫ , SeO 3 2Ϫ ,Se 0 ,selenomethionine,selenocystine,andmethaneseleninate(methaneseleninic acid)(30).Aeromonasveronii,isolatedfromseleniferousagriculturaldrainagewater,was activeinvolatilizingDMSeandlesseramountsofmethaneselenol,DMSeS,andDMDSe (31).McCartyetal.(32)identifiedtwophototrophicbacterialspecies,Rhodospirillum rubrumS1andRhodocyclustenuis,thatproducedDMSeandDMDSeinthepresenceof SeO 4 2Ϫ .EnterobactercloacaeSLD1a-1,theSeO 4 2Ϫ andSeO 3 2Ϫ reducingbacterium,pro- ducesDMSefromSeO 4 2Ϫ ,SeO 3 2Ϫ ,Se 0 ,dimethylselenone[(CH 3 ) 2 SeO 2 ],selenomethio- nine,6-selenopurine,and6-selenoinosine(33).ThemethylationofSebyalgaehasalso beenconfirmedbyFanetal.(34),whoisolatedaeuryhalinegreenmicroalgaspeciesof ChlorellafromasalineevaporationpondthatwasabletotransformSeO 3 2Ϫ aerobically intoDMSe,DMDSe,andDMSeS. Ingeneral,theformationofalkylselenidesfromSeoxyanionsinvolvesareduction andmethylationstep;however,thepathwaybywhichthesereactionsoccurisstillhighly debated.Challenger(35)postulatedthattheformationofDMSeoccursthroughsuccessive methylationandreductionsteps,inwhichdimethylselenonewassuspectedtobethelast intermediatepriortotheformationofDMSe(Fig.2).ReamerandZoller(36)identified DMDSe and dimethylselenone in addition to DMSe as products from soil and sewage sludge amended with either SeO 3 2Ϫ or Se 0 . It was then suggested that Challenger’s pathway Copyright © 2002 Marcel Dekker, Inc. 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 pathway whereby methaneseleninic acid is reduced to methaneselenol or methaneselenenic acid or both, to produce DMDSe. Doran (24) proposed that SeO 3 2Ϫ is reduced via Se 0 to a selenide from 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 is also markedly similar to findings of studies conducted with mammals, which demonstrated that methaneselenol is an intermediate in the methylation of Se to DMSe (38,39). Cooke and Bruland (40) proposed a pathway for the formation of DMSe from SeO 4 2Ϫ and SeO 3 2Ϫ in natural waters. Apparently both Se oxyanions are reduced and assimilated into the intermediate selenomethionine [CH 3 Se(CH 2 ) 2 CHNH 2 COOH], which is then methylated to produce methylselenomethionine [(CH 3 ) 2 Se ϩ (CH 2 ) 2 CHNH 2 COOH]. Finally, methyl- selenomethionine is hydrolyzed to DMSe and homoserine. The biosynthesis of methionine from homocysteine is an important transformation in the methylation of Se. During the activated methyl cycle homocysteine is methylated via the coenzyme methylcobalamin (CH 3 B 12 ; derivative of vitamin B 12 ), yielding methionine. Methylcobalamin has been isolated from bacteria (41) and is believed to donate methyl groups to Se, resulting in the formation of volatile alkylselenides. Thompson-Eagle et al. (42) found that the addition of methylcobalamin promoted the methylation of SeO 4 2Ϫ . McBride and Wolfe (43) found that cell-free extracts of a Methanobacterium sp. methylated SeO 4 2Ϫ when methylcobalamin was present. Cell-free extracts of E. cloacae SLD1a-1 catalyzed the formation of DMSe from SeO 3 2Ϫ or Se 0 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 SeO 3 2Ϫ or Se 0 when 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 Na 2 Se. Although there is some 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 is needed to understand the biochemical characteristics of Se methylation. Copyright © 2002 Marcel Dekker, Inc. C. Bioremediation of Seleniferous Water and Sediment Since the 1990s attention has been given to the development of an effective remediation technology 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 are being developed; they include a variety of bioreactors utilizing bacteria with the ability to reduce the toxic, soluble Se oxyanions to insoluble Se 0 . These systems are designed to remove Se from contaminated wastewater (industrial or agricultural) before release into the environment. Because of the high SeO 4 2Ϫ to SeO 3 2Ϫ ratio of most agricultural drainage waters of the western United States, removal of mainly SeO 4 2Ϫ must be considered in these systems. Another means to remove Se from contaminated soil and water involves stimulation of the indigenous microorganisms that volatilize Se. This process has proved effective as an in situ treatment for seleniferous soils and sediments in the San Joaquin Valley, California (45,46). 1. Bioreduction of Selenium Oxyanions to Elemental Selenium The use of Thauera selenatis, a SeO 4 2Ϫ respiring bacterium, in a biological reactor system to remediate both SeO 4 2Ϫ and SeO 3 2Ϫ 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 electron donor, Se oxyanion and NO 3 Ϫ concentrations were reduced by 98%. An earlier system included the use of two bioreactors in series; the first was an aerobic sludge blanket reactor and the second a fluidized bed reactor (47). Once again acetate was used as the electron donor and the growth of the organism was found to be dependent on the presence of NH 4 Cl. The SeO 4 2Ϫ , SeO 3 2Ϫ , and NO 3 Ϫ levels were all reduced by 98% in the influent. A similar system, later used to remediate SeO 3 2Ϫ from oil refinery wastewater, reduced the Se oxyanion concentration by 95%. Although Macy (11) has shown that this organism can reduce both SeO 4 2Ϫ and NO 3 Ϫ simultaneously, NO 3 Ϫ must be present in the system for SeO 4 2Ϫ to be completely reduced to Se 0 , since the NO 2 Ϫ reductase only catalyzes the reduction of SeO 3 2Ϫ when denitrification is occurring. The algal–bacterial selenium removal system (ABSRS) is another process used to remove soluble Se and NO 3 Ϫ from drainage water (50). The influent is first directed toward high-rate ponds where microalgae are grown; removal of some NO 3 Ϫ 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 discharged into an anoxic unit where bacteria use the algae as a C and energy source and subsequently reduce the SeO 4 2Ϫ and SeO 3 2Ϫ to Se 0 , and NO 3 Ϫ to N 2 gas. Although near-complete removal of SeO 4 2Ϫ and NO 3 Ϫ occurred at times in field experiments, it was speculated that since the project was run for an insufficient amount of time, steady-state reducing conditions could not be established. Since substantial reduction of SeO 4 2Ϫ to SeO 3 2Ϫ was occurring, use of FeCl 3 was applied to precipitate out inorganic SeO 3 2Ϫ , thereby reducing the soluble Se levels. Oremland (52) has also described a process similar to the ABSRS. This process involves using a two-stage reaction, which uses algae in the first aerobic stage to deplete the NO 3 Ϫ concentrations below 62 mg L Ϫ1 . The water is then transferred to an anoxic Copyright © 2002 Marcel Dekker, Inc. reactor containing SeO 4 2Ϫ reducing bacteria where SeO 4 2Ϫ is reduced to insoluble Se 0 .In 7 days, the influent SeO 4 2Ϫ concentration of 56 mg Se L Ϫ1 was reduced by more than 99%. EPOC AG (Binnie California) conducted studies on the removal of Se from agricul- tural drainage water using a pilot-scale two-stage biological process (53). The system consisted 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 µgL Ϫ1 of soluble selenium. When the effluent was further processed through a soil column the soluble Se concentration was less than 10 µgL Ϫ1 . Owens (53) describes a pilot-scale biological system that utilized an upflow anaero- bic sludge blanket reactor. The C source used in the system was methanol, which was added at a dosage of 250 mg L Ϫ1 . Most of the C added to the system was used during denitrification; thus, enough methanol must be added to support both denitrification and Se reduction. Denitrification is important to the process since Se reduction does not occur until the NO 3 Ϫ is removed. It was reported that the reactor was able to remove 94% of the soluble Se, with a final effluent concentration of 29 µgL Ϫ1 obtained. 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 Ϫ1 . The bioreac- tor system consisted of a rotating biological contactor (RBC) and was able to remove 97% of the Se within 4 hours. A bench-scale RBC system was also tested on mining process waters, and using Pseudomonas stutzeri, with molasses (1 g L Ϫ1 ) 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 would stimulate Se volatilization (55). Water columns in the evaporation ponds were treated with a single casein application of 0.2 g L Ϫ1 pond water. The evaporation pond water Se concentration was reported as high as 2.9 mg L Ϫ1 . Unamended pond water evolved volatile Se at low rates of 0.1 µgSeL Ϫ1 d Ϫ1 , whereas casein amended pond water produced emission rates of 2.2 µgSeL Ϫ1 d Ϫ1 . After 142 days, the casein amended pond water lost 38% 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; the addition of cattle manure resulted in the removal of 58% after 22 months (56). The initial mean plot soil Se concentration in the top 15 cm was 11.4 mg kg Ϫ1 . The background emission rate of volatile Se averaged 3.0 µgSem Ϫ2 h Ϫ1 , whereas the cattle manure treated plot promoted an average emission rate of 54 µgSem Ϫ2 h Ϫ1 . As reported in other Se volatilization studies, the parameters that enhanced Se volatilization were moisture, high temperatures, 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 was dissipated 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 Ϫ1 . Since no pattern of Se depletion was correlated with rainfall events or temperature, it was speculated that leaching dominated during the winter months, because most rainfall occurred during the winter, Copyright © 2002 Marcel Dekker, Inc. whereas volatilization was dominant during the summer months. The addition of C amend- ments had no significant effect greater than that of the moisture-only treatment, a finding that 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). 3. Cell-Free Systems Adams et al. (54,58) treated mine water containing 0.62 mg L Ϫ1 SeO 4 2Ϫ 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 µgL Ϫ1 . 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 Ϫ1 , respectively) to concentra- tions of 1.0 and 1.6 mg L Ϫ1 , respectively. III. ARSENIC Arsenic (As) is a metalloid of group VA of the periodic table and exists in four oxidation states, ϩ5, ϩ3, 0, and Ϫ3. 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 4 3Ϫ ), 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 Ϫ ) 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 Ϫ to less toxic AsO 4 3Ϫ . 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 4 3Ϫ to AsO 2 Ϫ , 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 4 3Ϫ reduction to AsO 2 Ϫ 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. Copyright © 2002 Marcel Dekker, Inc. A. Reduction of Arsenic(V) It is known that a certain number of bacteria reduce As 5ϩ to As 3ϩ as a detoxification mechanism; however, the significance in the biogeochemical cycling of As is not clear. In addition to reductive detoxification, which may occur under both aerobic and anaerobic conditions, dissimilatory reduction of AsO 4 3Ϫ may contribute to the reduction of As 5ϩ to As 3ϩ in anaerobic sediments (61,62). Dowdle et al. (61) found that As 5ϩ was reduced to As 3ϩ in anoxic salt marsh sediment slurries when the electron donor was lactate, H 2 ,or glucose. The addition of the respiratory inhibitor/uncoupler dinitrophenol, rotenone, or 2-heptyl-4-hydroxyquinoline N-oxide blocked the reduction of As 5ϩ , suggesting that the reduction of As 5ϩ in sediments proceeds through a dissimilatory process. To date, several SAsO 4 3Ϫ respiring organisms have been isolated and characterized: Sulfurospirillum arsenophilus strain MIT-13 (63), S. barnesii strain SES-3 (14,64), Desul- fotomaculum auripigmentum strain OREX-4 (65), and Chrysiogenes arsenatis strain BAL- 1T (66). The only common electron acceptor of these organisms is fumurate, and studies have shown that strain MIT-3, strain SES-3, and strain BAL-1T respire NO 3 Ϫ and AsO 4 3Ϫ but not SO 4 2Ϫ , whereas strain OREX-4 can grow on SO 4 2Ϫ but not NO 3 Ϫ . The mechanisms by which electrons are passed to AsO 4 3Ϫ during dissimilatory reduction and reductive detoxification differ. The reductive detoxification of AsO 4 3Ϫ occurs when reduced dithiols transfer elec- trons for the ArsC enzymes (67), whereas the respiratory AsO 4 3Ϫ reductase in strain SES- 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 AsO 4 3Ϫ , 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.) Copyright © 2002 Marcel Dekker, Inc. [...]... 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 2ϩ in the periplasm The Hg 2ϩ is then transported through the inner membrane to the cytoplasm by the membrane-bound protein merT In the cytoplasm, Hg 2ϩ is reduced to Hg 0 by a soluble, NADH-dependent, flavin adenine dinucleotide (FAD)-containing mercuric... immobilization of Cr in chromate-contaminated soils and groundwater Such an application involves the production of H 2S in groundwater or deep within the soil profile by in situ stimulation of SO 42Ϫ-reducing bacteria through additions of SO 42Ϫ and other nutrients This type of process was effective in the removal of Cr from Cr 6ϩ-containing tannery wastes entering Otago Harbor in New Zealand ( 120) V MERCURY... reduction In Streptomyces sp 3M, reduction of CrO 42Ϫ was observed in the soluble protein fraction and was NADH- and NADPH-dependent; however, higher reduction rates were obtained with NADH (111) B Bioremediation of Chromium-Contaminated Water The bioremediation of CrO 42Ϫ-contaminated water is currently proceeding along two fronts: indirect microbial reduction, by stimulating sulfur-reducing bacteria... for the Cr 6ϩ reduction reactions Copyright © 200 2 Marcel Dekker, Inc Another application of bioreduction was demonstrated by Komori et al (118) In this study, anaerobic Cr-reducing bacteria were contained within dialysis tubing and subsequently submerged in contaminated water Chromate that diffused through the tubes was reduced and precipitated within the dialysis tubing A laboratory study employing... producing trimethylarsine 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 methyl donor (83) The addition of either methanearsonic acid or dimethylarsinic... (84,87) Preincu- Figure 5 Fungal methylation pathway for the formation of trimethylarsine (From Ref 35.) Copyright © 200 2 Marcel Dekker, Inc bation of cells with trimethylarsine oxide increases the rate of conversion to trimethylarsine, suggesting an inducible system (84) In addition, the rate of transformation of AsO 43Ϫ to trimethylarsine is increased by preconditioning the cells with dimethylarsinic... methylarsonic acid to dimethylarsinic acid and trimethylarsine oxide, and dimethylarsinic acid to methylarsonic acid and trimethylarsine oxide (80) Although trimethylarsine formation from inorganic As and methylarsonic acid is inhibited by the presence of phosphate, its synthesis from dimethylarsinic acid is increased in the presence of phosphate (81) More recently, Huysmans and Frankenberger (82) isolated... to be involved specifically in the electron transfer to CrO 42Ϫ In Desulfovibrio vulgaris, the c 3 cytochrome reportedly functioned as a Cr 6ϩ 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 6ϩ reduction occurred in the soluble protein fraction The Cr 6ϩ reductase activity was lost when the soluble... vulgaris strain 8303 also produced a volatile As derivative, presumably an arsine, when incubated with AsO 43Ϫ (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 AsO 43Ϫ and AsO 2Ϫ under... methylcobalamin was found to catalyze the transfer of a methyl group to homocysteine, resulting in the formation of methionine (134) In Neurospora crassa, the formation of MeHg was stimulated by the addition of homocysteine but was inhibited by the addition of methionine (135) It was proposed that the fungus first complexes Hg 2ϩ with homocysteine or cysteine, which is then methylated and finally cleaved by a transmethylase . 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. Inc. ensurestheremovalofHgfromtheenvironmentthroughatmosphericdissipation.Current studiesarenowfocusingonbiologicalreductionandmethylationreactionsasaremedial approachtoimmobilizeHg. A.ReductionofMercury(II) NumerousmicroorganismsavoidHgtoxicitybyreducingionicHg(Hg 2ϩ )tovolatileHg 0 , apotentiallyusefulapplicationtoremoveHgfromHg-contaminatedwater.Thereduction ofHg 2ϩ toHg 0 canbemediatedbyanumberofmicroorganisms,includingentericbacteria, Pseudomonassp.,Staphylococcusaureus,Thiobacillusferrooxidans,Streptomycessp., andCryptococcussp.(121).TheabilityofbacteriatoreduceHg 2ϩ islinkedtoHgresis- tance(mer)operons(122).Thehypothesizedplasmid-mediateddetoxificationmechanism isshowninFig.6.Theplasmidcodesforaprotein(merP)thatinitiallybindstoHg 2ϩ in the periplasm. The Hg 2ϩ is then transported through the inner membrane to the cytoplasm by the membrane-bound protein merT. In the cytoplasm,. H 2 ,or glucose. The addition of the respiratory inhibitor/uncoupler dinitrophenol, rotenone, or 2-heptyl-4-hydroxyquinoline N-oxide blocked the reduction of As 5ϩ , suggesting that the reduction of As 5ϩ in