Journal of Industrial Microbiology, 119951114,85-91995 Society for Indu;;tnal M,uo,'Jlolo y :J1694l46!~

Bioremediation of organic and metal contaminants with dissimilatory

metal reduction

Derek R. Lovley ,

W'aler Re~ource~ Divi.~ion. (f.S' Geological Survey. 4_i'O Nalio/llli Cenler. Re~tlJn. Vif.,~in~a 22092. US)

,(Received 22 March 1994: accepted 12 May 1994)

SUMMARY

Dissimilatory "1etal reduction has the potential to be a helpful mechanism lor both intrinsic and engineered bioremediation of contaminated environments

Dissimilatory Fe(1111 reduction is an important intrinsic process for removing organic contaminants from aquifers contaminated with petroleum or landfill leachate

Stimulation of micrpbial Fe(I!!) reduction can enhance the degradation of organic contaminants in ground water. Dissimilatory reduction of uranium, selenium,

chromium, technetiJm. and possibly other metals, can convert soluble metal species to insoluble forms that can readIly be removed from contaminated waters or

waste streams. Red~ction of mercury can volatilize mercury from waters and soils Despite its potential there has ~s yet been limited applied research into the

use of dissimilatory metal reduction as a bioremediation tool.

INTRODUCTION TABLE I

jReaclions. ex~mplifying how dissimil.alory metal reduction can beInvolved In blOrelnedlallon 0 contaminantsThe possibility of using bioremediation to restore environ-

ments contami~ated with organics is well known [21]. Biore-

mediation may either.be 'intrinsic bioremediation' which nat-urally takes place in contaminated environments or engineered[29]. For non-c!hlorinated organics, the common engineeringpractice is to enlhance aerobic degradation of the contaminants

by ensuring that the supply of O2 and inorganic nutrients doesnot limit the rate of contaminant degradation [100]. Nitratehas been considered as an alternative electron acceptor for

engineered biorlc';mediation of ground water but has not foundwidespread use! This is because most engineered bioremedi-ation of non-clilorinated organics deals with petroleum con-tamination and benzene. the most important petroleum-relatedground water ~ontaminant, does not appear to be readilydegraded with nitrate as the electron acceptor

[6,ll,35,48,49,$5}. Recent studies have demonstrated thatFe(llI) may be an important electron acceptor in the intrinsicbioremediation ! of ground waters contaminated with non-chlorinated org-.nics [7,67.74]. Furthermore, techniques for sti-mulating the acti vity of Fe(IlI)-reducing microorganisms tostimulate conta/llinant degradation have been developed [72].

In contrast to organics, metal contaminants are not 'biodeg-radable'. However,. a number of important contaminant metalsand metalloids are either less soluble or more volatile in thereduced state than they are in the oxidized state (Table I).Thus, dissimilatory metal reduction may be a useful techniqueto precipitate or volatilize metal contaminants from pollutedwaters or waste streams. The purpose of this review is to sum-

ProductsReaction Reactants

number

O.1:idation of organic conlaminant~ coupled to Fe(llli ri'dJlction

I. Toluene+36 (Fe(IIII+21 H2O7HCO) +36 Fe([[)-.-43 H

Reduction of soluble metaltl) an insoluble form

2 U(VI)+H,," U(IV)+2 H

3. Cr(VI)+3/2 H, Cr(III)+3 H4. Se(VI)+[3 H,I" I Se(O)+6 H+

5. Pb(II)+[H,i I Pb(O)+2 H+

6. Tc(VIl)+[3/2 H,J j Tc(IV)+3 H~

Reduction of ~oluble metal t( a volatile form

7. Hg(II)+[H,1 Hg(O)+2 H

"H, designates that molecular H, has been demonstrated to be an elec-tron donor for reduction of the metal. However, microorganisms canalso use organic electron donors for reduction of these metals.b[H,1 designates two electrons are donated from various organic elec-tron donors; molecular H, has not been shown to be an electron donorfor reduction of these metals in pure culture.

marize recent studies which have investigated the potential forusing the metabolism of dissimilatory metal-reducing microor-ganisms as a tool for bioremediation of both organic and inor-ganic wastes.

Intrinsic bioremediation of organic contaminant.~ by Fe( //1)-

reducing microorganismsThe contamination of shallow, oxic ground water with pet-

roleum products or landfill leachate typically results in theCorrespondence to: D.R. Lovley, Water Resources Division. US Geo-

logical Survey, 430 National Center, Reston, V A 22092, USA.

Bioremediation of organic and metal contaminants

development of anoxic conditions Prior to the developmentof anoxic conditions, Fe(lll) is generally the most abundampotential electron acceptor for organic matter oxidation inmost soils and sediments [601. Studies with pure culture'i andhighly purified enrichments have demonstrated that a widevariety of monoaromatic compounds can be oxidized to carbondioxide with Fe(lll) serving a,; the sole electron acct:ptor[58,62,671 and geochemical evidence has suggested that thistype of metabolism (reaction I, Table IJ is an important mech-anism for the intrinsic bioremediation of organic contami-nants [7.34,67.74,101].

For example, in the anoxic zone of a petroleum-contami-nated aquifer there was a loss of aromatic hydrocarbons whichwas accompanied by an accumulation of isotopically-light car-bon dioxide [67). This suggested that the isotopically-light aro-matic hydrocarbons were being oxidized to carbon dioxide.The accumulation of Fe(ll) in the ground water and depletionof Fe(III) from the aquifer sediments suggested that the aro-

matic hydrocarbon oxidation was coupled to Fe(lll) reduction.

Geochemical modelling suggested that, in the portion of theaquifer closest to the hydrocarbon contamination, ca. half ofthe contaminant decomposition might be linked to Fe(IIIJreduction [71. Since Fe(III) oxides have been depleted fromthe sediment near the source of contamination, the overall con-tribution of Fe(III) reduction to aromatic hydrocarbondecomposition within the whole of the anoxic plume may begreater than 50c/c.

In an aquifer contaminated with landfill leachate, mostclasses of organic contaminants monitored (monoaromatichydrocarbons. substituted benzenes. napthalenes.

chlorophenols) persisted as the ground water moved throughzones in which methanogenesis and sulfate reduction were the

terminal electron accepting process [74J. However, the con-taminants were removed from the ground water as it entered azone further downgradient where microbially-reducible Fe(IIIJoxides were still available and Fe(lll) reduction was the pre-dominant terminal electron accepting process. This suggested

that the loss of contaminants was the result of contaminantoxidation coupled to Fe(III) reduction [74]. However, directevidence that these contaminants were being oxidized to car-bon dioxide by Fe(III)-reducing microorganisms was not pro-vided.

Fe(lII) reduction is an important process for the oxidationof naturally occurring organic matter in aquatic sediments[4,26,63]. Thus, there is the potential for Fe(III) reduction to

be important in intrinsic bioremediation of organic contami-nants in some sediments. Fe(III)-reducing enrichment culturescapable of degrading a variety of monoaromatic contaminantcompounds were readily established with freshwater bottomsediments from the Potomac River [58]. However, the role ofFe(lII) reduction in oxidizing contaminant organics in aquaticsediments has yet to be investigated in detail.

Dissimilatory Fe(lII) reduction may also affect the fate of

some organic contaminants through an indirect, non-enzymaticmechanism. The reduction of Fe(lII) oxides by dissimilatoryFe(III)-reducing microorganisms may result in the formationof ultrafine-grained magnetite [60,70]. Fe(II) associated withthis magnetite has been shown to nonenzymatirally reduce 4-

chloronitrobenzene to 4-chloroanilinc

posed that this type of reaction may

nitroreductive capacity of anoxic soil;

1~41. [t has been pro

account for the genera

and sediments 1441

Potential/fIr ell,l,'ineered bi(lremediat;(ln (If (lrganic contami-

nant.~ with Fe( III I-reducers

In some instances it may be possible to manipulate theactivity of Fe(III)-reducing microorganisms in subsurfaceenvironments in order to accelerate the rate of organic con-taminant degradation. For example. the activity of Fe(I!I)-reducing microorganisms can be stimulated by increasing theavailability of Fe(lII) for microbial Fe(!!!) reduction [72]. TheFe(lII) in aquifer sediments is primarily in the form of insol-uble Fe(III) oxides. !n culture, Fe(III)-reducing microorgan-isms reduce soluble, chelated forms of Fe(III) much faster thaninsoluble Fe(lII) oxides [60J. Fe(II!) reduction was stimu!atedwhen the Fe(lII) ligand. nitrilotriacetic acid (NT A), was addedto sediments from a petroleum-contaminated aquifer in whichFe(lII) reduction was the terminal electron accepting process.Stimulation of Fe(lII) reduction enhanced the degradation ofboth toluene and benzene. Studies with '4C-labelled tolueneand benzene indicated that the compounds were being com-

pletely oxidized to carbon dioxide, presumably with Fe(lII) asthe electron acceptor. After an adaptation period, the rates ofbenzene and toluene degradation in the anoxic NT A-amendedsediments were comparable to rates of degradation that havepreviously been observed in oxic aquifer material. This wassurprising, because although microorganisms have been shownto degrade aromatic hydrocarbons under anoxic conditions[19,33,41,55,114], the rates of anoxic degradation were muchslower than under oxic conditions. Aromatic hydrocarbons,especially highly toxic benzene, tend to persist in anoxicground water [1,17,38,57,80,100,109).

These results demonstrate that O~ is not necessary for rapiddegradation of benzene in ground y,.ater. This may be usefulinformation because the introduction of O~ into ground waterto stimulate bioremediation of petroleum contaminants can betechnically difficult and expensive [57,80,82,100,109]. Theaddition of Fe(III) chelates to contaminated aquifers which stillcontain Fe(III) oxides should be relatively simple. Further-more, it was found that the addition of soluble chelated Fe(III)could stimulate aromatic hydrocarbon degradation in contami-nated aquifer material from which Fe(lII) oxides had beendepleted [72]. Thus, the stimulation of microbial Fe(III)reduction by enhancing the availability of Fe(III) shows poten-tial as a novel approach to engineered bioremediation of pet-roleum-contaminated ground waters.

The finding that dissimilatory Fe(lII) reduction can beimportant in the intrinsic bioremediation of organic contami-nants and that this process could be manipulated to enhancecontaminant degradation emphasizes the need to learn moreabout this relatively unexplored process. The metabolism oftoluene by Geobacter metallireducens [62,67] is the onlyexample of a dissimilatory Fe(III)-reducing microorganism inpure culture that can oxidize an aromatic hydrocarbon. Littleis known about this metabolism. Furthermore, for benzene, the

most important aromatic hydrocarbon contaminant, the organ-

Bioremediation of organic and metal contaminants

isms responsible and their mode of metabolism are com.

pletely unknown.

Microbial reduction of toxic meta/y and metalloid,yWith the po~,ible exception of Mn(IV) which is generally

present at level$ about IO-fold less than Fe(lll) in soils, theconcentrations of metals other than Fe(III) are too low forthem to serve as significant oxidants for organic matter in sedi-

mentary environments. However, microbial reduction of theseless abundant m~tals can greatly affect their fate and mobility.

um-contaminated soils (l)21 The discovery that [he c, cyto-chrome is the U(Vlj reducta,e in Desu(t()l'ibriu 1!lIf~aris sug-

gests the possibility of developing cell-free fixed enzymesystems and/or engineered organisms with enhanced U(Vlj-

reducing capacity for treating uranium-contaminated waters

[71 J.Microbial U(Vlj reduction offers several advantages over

other technologies for uranium removal such as ion exchangeand biosorption [64 J Advantages include: I) the ability to pre-

cipitate uranium from U(VI)-carbonate complexes; 2) therecovery of uranium in a highly concentrated and pure form;3) high uranium removal per amount of biomass; 4) the poten-tialto simultaneously treat organic contaminants and uraniumby using the organic as an electron donor for U(VI) reduction:and 5) the potential for in .\"itu remediation of groundwater.However, for small scale applications such as the removal ofuranium from drinking water supplies for homes, technicallyless complex methods such as ion exchange resins [104J arelikely to be more practical [641.

REDUCTIV PRECIPITATION OF URANIUM

REDUCTIVE PRECIPITATION OF CHROMIUM

Irrigation pr&ctices [22,113], the mining and processing ofuranium [75], and natural phenomena [1041 can result in unde-sirably high conpentrations of dissolved uranium in waters andwaste streams. This dissolved uranium is generally in the formof U(VI) carbonate complexes [64]. Although U(VI) is highlysoluble in most natural waters, U(IV) is highly insoluble [56].

Therefore, microbial reduction of U(VI) to U(IV) (reaction 2,Table I) could potentially be used to precipitate uranium from

contaminated waters and waste streams.Several microorganisms are known to enzymatically reduce

U(VI). These include Geobacter metallireducens andShe~'anella putrefaciens which can conserve energy to supportgrowth by coupling the oxidation of acetate (G.metallireducens) or H2 (S. putrejacien5) to the reduction ofU(VI) [68]. Several Desulfovibrio species also reduce U(VI)but do not conserve energy to support growth from this metab-olism [65,69,71]. To date, evaluation of the practical use of

microbial U(VI) reduction has focused on the Desulfovibriospecies because they are easy to mass culture and can be storedaerobically in a freeze-dried state without losing their capacityfor U(VI) reduction [64].

Microbial U(VI) reduction effectively precipitated uraniumfrom solution under defined conditions in which the U(VI) waspresent in the form of a U(VI)-carbonate complex. Both G.

metallireducens and D. desulfuricans rapidly converted high

concentrations (ca. I mM) of U(VI) to U(IV) and precipitatedall of the uranium from solution within several hours[40,64,65]. With both organisms, the U(IV) precipitate was allextracellular and was in the form of the mineral uraninite(UO2). The uraninite readily settled to the bottom of the incu-

bation vessels or could be removed with filtration. Alterna-tively, the U(VI)-reducing microorganisms could be main-

tained separately from the bulk of the contaminated water byplacing the U(VI)-reducers within a dialysis sac [64]. TheU(VI) diffused into the sac, the U(VI)-reducers converted theU(VI) to U(IV), and the U(IV) precipitated in the bottom ofthe sac. Of a wide variety of potentially inhibiting anions andcations that were evaluated, only high concentrations(>20 ILM) of copper inhibited U(VI) reduction.

D. desulfuricans readily removed uranium from a varietyof contaminated waters [64J. These included acidic (pH 4.0)and pH-neutral uranium mine drainage waters and uranium-

contaminated groundwaters from the Department of Energy'sHanford site located near Richland, Washington. Uranium wasalso precipitated from the soil washings of a variety of urani-

Manufacturing processes, domestic wastewater, and thedumping of sewage sludge are major sources of chromiumcontamination [28,81,91 J. Cr(VI) and Cr(lIl) are the principalforms of chromium found in natural waters [9I,96J. Cr(VI) ishighly soluble and toxic whereas Cr(lIl) is much less toxicand tends to form insoluble hydroxides [8I,9I,96J. Cr(III) isalso quickly immobilized in sediments by adsorption whereasCr(VI) is not [81]. Therefore, reduction of Cr(VI) to Cr(lIl)(reaction 3, Table I) is a potentially useful process tor theremediation of chromium-contaminated waters and waste

streams.A wide variety of microorganisms can enzymatically

reduce Cr(VI) to Cr(III) [61]. In those instances in which themechanisms for Cr(VI) have been investigated, it is clear thatthe Cr(VI) reduction is an enzymatically catalyzed reaction[20,54,111,112]. Cr(VI) reduction may be a fortuitous reactionthat is catalyzed by enzymes that have other physiologicalfunctions [27,50]. Energy conservation linked to Cr(VI)reduction has never been conclusively demonstrated [61].

Although all studies on microbial Cr(VI) reduction appearto have been conducted with the potential of microbial Cr(VI)reduction as a remediation tool in mind, the applied aspectsof this process have been most extensively studied in Entero-bacter cloacae strain HO I. Initial studies with E. cloacae dem-onstrated that high concentrations of Cr(VI) (as high as10 mM) could be rapidly reduced, but the Cr(lIl) was difficultto remove from the culture [531. For example, only ca. 30%of the Cr(III) that was produced from Cr(VI) reduction couldbe removed with centrifugation. Most of the soluble chromiumcould be removed from solution with Cr(VI) reduction if thecells of E. cloacae were placed on one side of an anion-exchange membrane which permitted CrO4- but not Cr3+ topass through [52]. With this method, E. cloacae could reducean initial concentration of I mM Cr(VI) down to ca. 0.04 mMdissolved chromium within 55 h.

However, the successful removal of dissolved chromium

Bioremediation of organic and metal contaminants

release of other trace metals. A number ()f CriVI/-reducing

microorganisms reduce Cr(VI) aer{)bically [20,46,50J. -rhus. ifa technique was devi'ied to 'itimulate CriVI/ reduction in 'iuchaerobic CriV[)-reducing microorganisms thi'i might be a usefulstrategy for in 'iitu immobilization.

Enzymatic treatment of Cr(VI) might also be advantageousin mixed-waste situations where organic contaminants and

CriVI) could be treated simultaneously by coupling the oxi-dation of the organic contaminants to the reduction of CriVI).

Furthermore, when there are other metal contaminants such asuranium that are not easily chemically reduced (see above). itmight be practical to use metal-reducing microorganisms toreduce CriVI) and the other metals simultaneously

REDUCTIVE PRECIPITATION OF SELENIUM

durin~ growth in heterotrophic growth medium could not bematched in contaminated waters. Cr(VI) was not reduced whena t:ell suspen'iion of t'. cloa(,(le was added directly to a chro-

mate-(!ontaining industrial effluent 1.85]. The Cr(Vlj could bereduced if the effluent was diluted at least two-fold and sup-

plemented with a nutrient solution containing peptone, meatextract. urea. and phosphate [851. It was speculated that heavymetals were responsible for the inhibition of Cr(Vl) reductionin the undiluted effluent. Further testing of heavy metal tox-

icity indicated that the following metal ions inhibited Cr(VI)reduction: Hg2+ > Ag + > Cu2. > Zn2+> Ni2. > CO2+ >

Mn2+with Hg2i- inhibiting Cr(VI) reduction at concentrationsas low! as 1 jJ.M [43]. Cr(VI) was not reduced in an industrial

effluent that contained Cu2+(390 jJ.M), Mn2+(480 jJ.M) and

Zn2+(().3 jJ.M) [43]. Even when the heavy metals wereremovtd, only about 40% of the Cr(VI) could be reduced. Theresidual inhibition of Cr(VI) reduction was attributed to highsulfate concentrations (60 mM) in the waste as similar concen-trations of sulfate added to nutrient medium also inhibited

Cr(VI) reduction by about 50%. These studies indicate thatfurther investigation is required before E. cloacae can be

applied to the remediation of real-world Cr(Vl) contamination.

De.~ulfovibrio vulgari,'i might be more useful than E.cloacae for chromate bioremediation because sulfate and avariety of metals had no effect on Cr(VI) reduction by D. vul-f?aris [166]. An additional benefit was that D. vulgaris couldreduceICr(VI) in a simple mineral medium with H2 serving asthe el~ctron donor. It did not need the rich heterotrophic

medium required by E. cl()acae. Freeze-dried cells of D. \'ul-garis that had been stored at room temperature could be usedfor Cr~VI) reduction. As with U(VI) reduction, the c, t:yto-chrome functions as a Cr(VI) reductase in D. vulgari'i [66].

In another study investigating the potential practical appli-

cation of microbial Cr(VI) reduction, a mixed microbial t:om-

munity that was established as a biofilm on rotating plasticdisks readily reduced Cr(VI) in a synthetic waste water [28].Cr(VI) concentrations of as high as 200 mg L ~ I were eftec-

tively treated to levels of 2-3 mg L -I in the final effluent The

microorganisms responsible for the Cr(VI) reduction werenot determined.

It seems doubtful that enzymatic Cr(VI) reduction wouldbe easier and cheaper than non-enzymatic methods for treatingwaste waters. For example, Fe(rI) rapidly reduces Cr(VI) at

circumneutral pH, even under aerobic conditions., and the

Cr(lli)-Fe(lli) hydroxides that form maintain dissolved chro-mium concentrations below the 10-6 M drinking water limit

[32]. However, enzymatic Cr(VI) reduction may have someadvantages over a non-enzymatic process for the in sitll immo-bilization of chromium contamination. For example. a pro-posed in .'iitu technique for preventing Cr(VI) migration is toform geochemical barriers of inorganic reductants such aspyrite or other Fe(II)-containing minerals [91]. However. thelong-term in .'iitu reduction of Cr(VI) with inorganic reductantssuch as Fe(ll) and sulfide would require anoxic conditions tobe effective. Generating and maintaining anoxic conditions

may be technically difficult, especially in unsaturated soils.Furthermore, the generation of anaerobic conditions in groundwater may result in other contamination problems such as the

Selenium contamination is associated with metal retinin"'"[83), fly ash waste [2], and agricultural drainage waters in theWestern United States, most notably the highly publicizedKesterson National Wildlife Refuge [94]. The predominantredox states of selenium in natural environments are Se(VI)

(selenate, SeO42-), Se(IV) (selenite, SeO32-), Se(O) (elementalselenium), and Se(-II) (selenide) [30]. The first three of thesecan potentially serve as electron acceptors for microbial

metabolism. The reduction of highly soluble selenate to insol-uble elemental selenium (reaction 4, Table I) is a potentialmechanism to remove selenate from contaminated waters

The capacity to reduce selenate to elemental selenium iswide-spread in various genera of bacteria [18,25,51. 79J. butmicroorganisms with the capacity to conserve energy to sup-port growth from selenate reduction have only recently beendescribed. Two of these organisms, strains SES-l [88] andSES-3 [87, I06] reduce selenate to elemental selenium.

Another, Thauera selenati.~ only reduces selenate to selenite

[78). However, many organisms reduce selenite to elementalselenium [30,87] and T. selenatis can be combined with a sel-enite reducer to produce elemental selenium [77].

Microbial reduction of selenate and selenite to insolubleelemental selenium has been observed in a variety of soils and

aquatic sediments [31,79,88,105]. This metabolism intrinsi-cally bioremediates the agricultural drainage water that is col-lected in evaporation ponds and may remove selenate fromground waters [87,89,90]. Estimates of in situ selenatereduction in the sediments of an evaporation pond suggestedthat microbial selenate reduction could remove all of the selen-ate in the overlying water within several months [89].

Furthermore, it is possible to engineer conditions toenhance microbial selenate reduction. For example, whenexposed soil at Kesterson Reservoir was flooded with sel-enium-free water in order to generate anoxic conditions, 66-110% of the dissolved selenium that had been present in theupper 1.22 m of soil was immobilized [59], A limitation to thistechnique is that bottom-feeding organisms may bring some ofthe immobilized elemental selenium back into the food chain[73]. Furthermore, the soils must be maintained in an anoxicstate because once the soils are oxidized the sequestered sel-enium may become highly mobile [3].

Selenate may also be treated in ex situ treatment processes

Bioremediation of organic and metal contaminantsDR Lovlev

In which selenate-contaminated waters are passed through soilc(J!umns or bio-reactors which contain selenate-reducing

microorganisms [5,37,,51.76.891, A cost evaluation of the useof selenate- and selenite-reducing microorganisms to remove

",clenium from waste waters concluded that the microbial pro-cess could potentially be cheaper than chemical reduction if anutrient cheape~ than the peptone that was used to culture the

organisms could be found [5.1, The requirement for a cheapcarbon and enefgy source for selenate reducers might be met

by first growing algae in the nutrient-rich water [37.89). Thistechnique also serves to remove the nitrate which can inhibitselenate reduction by some organisms. However. nitrate is notalways a concem as some selenate reducers continue to reduceselenate despiteithe presence of nitrate in contaminated waters[761. Oremland, [87J raises the point of whether the value ofthe irrigated crqps justifies the cost of water treatment and thewater consumed by irrigation,

REDUCTIVE PRECIPITATION OF LEAD

The reduction of soluble Pb(lI) to insoluble Pb(O) (reaction5, Table I) is a potential mechanism for removing lead fromsolution. When P,eudomonas maltophilia was grown aerob-ically in an organic-rich medium in the presence of I mM leadacetate, dissolvtd lead decreased over time and there was an

accumulation of a gray-black, lead-containing colloid [99].The colloids had a diameter of 175 nm and had a stronglynegative zeta potential. The colloidal lead aggregated andsettled out of solution. Several techniques other than settlingby gravity tor removing the lead colloid from water are beinginvestigated [99]. However, whether the lead precipitation wasactually the res~lt of reduction of Pb(lI) to Pb(O) was not dem-onstrated.

REDUCTIVE PRECIPITATION OF TECHNETIUM

Technetium is a long-lived (half-life, 2.15 x 105 years)

radioactive contaminant in the environment that is a by pro-duct of fission reactions during atomic explosions and innuclear power stations [110]. The oxidized form of technetiumis the highly soluble pertechnetate ion (TcO4-; Tc(VII» [110].Tc(IV), the reduced form of technetium, is highly insoluble[IIO}. Thus, reduction of technetium (reaction 6, Table 1) cangreatly reduce its mobility in the environment [102].

The role of microorganisms in enzymatically reducing tech-netium in the environment has not been adequately studied.There are several studies with pure cultures and enrichmentsthat suggest that microorganisms may enzymatically reducetechnetium [45,93]. There do not appear to have been anystudies on the potential for using microorganisms to removetechnetium from contaminated waters or waste streams. How-ever, it seems possible that microbial technetium reductioncould be used for bioremediation in a manner similar to that

described for uranium [681.

reduce ionic Hg(ll) to Hg(()) (reaction 7, Table I) during aero-bic growth as a detoxitic~tion mechanism [23,97,1071.

Whereas Hg(ll) is highly sol~ble. Hg(O) is volatile and thuscan be readily vented or extracted from contaminated environ-ments or waste streams 142,X4.1081.

Removal of mercury through reductive volatilization ofmercury may be an intrinsic remediation process in bothaquatic and terrestrial environments [16,39,841. Althoughsome of this Hg(ll) reduction is clearly microbiological, insome environments non-biological mechanisms may also beimportant [12,14,16,95J. The microbial community typicallyresponds to mercury contamination with an enhanced capacityfor Hg(ll) reduction [8,10,12-16.86,981.

It may be possible to stimulate rates of microbial Hg(II)reduction as an engineering approach to in ,~itu bioremediationof contaminated soils and water [16,39,84 J, This could poten-tially be accomplished by the addition of Hg(II) reducers, sti-mulating the growth of indigenous populations, amplifying thenumber of mer operons in indigenous microorganisms, or byincreasing the percentage of indigenous mer operons that areexpressed, However. a limitation on the use of microbialHg(ll) reduction for in ~itu bioremediation is that most of theHg(lI) is typically unavailable for microbial reduction[16,95,103J, Another concern for in situ treatment is that,unless special recovery systems are developed, the mercuryremoved from the contaminant site will be transported in theatmosphere and deposited elsewhere.

The potential for ex situ remediation of mercury-contami-nated waters and waste streams has also been investigated, Forexample, mercury that was chemically precipitated from liquidmining and chemical wastes and then redissolved in nutrientmedium could be volatilized with the mercury-resistant micro-organism Pseudomonas K62 [108]. The chemical pretreatmentwas necessary because salts in the industrial waste could have

inhibited Hg(lI) reduction. The chemical pretreatment that wasused in precipitating the mercury was expensive and a cheaperalternative method would be required for microbial Hg(lI)reduction to be used on an industrial scale,

Hansen and coworkers [42] found that a mixed culturereactor incubated at 37 DC and fed mercury-amended raw sew-age at an influent detention time of I day, continuouslyremoved 88o/c of the 70 mg L ~ I Hg(II) in the influent. If the

effluent was allowed to incubate an additional day at roomtemperature, then 98% removal of mercury was achieved. Thefate of the mercury was not determined but it was assumed tobe removed in the effluent air stream, It was considered thatthe Hg(O) in the effluent air might be recovered through con-

densation,In a similar manner, a pure culture of Pseudomonas putida

FB I removed mercury when grown under continuous culture

conditions on an organic-rich medium [9]. Effluent mercuryconcentrations could be maintained below the upper legal limitfor mercury in liquid wastes.

Immobilized Hg(II)-reducing microorganisms in small lab-oratory columns effectively removed mercury from solution,depositing elemental mercury in particles about 1-5 ILm indiameter outside the cells [24,36], The effluent from the col-umn typically had mercury concentrations of less than

REDUCTIVE VOLATILIZATION OF MERCURY

Mercury is the most common metal contaminant that is ahuman health concern [84]. A number of microorganisms,

Bioremediation of organic and metal contaminants

DR Lov!e\

9050 /Lg L '. Mercury retention was improved when pure strains

were ~sed instead of a natural consortia. In a stirred tank con-

figuration the elemental mercury was released into the almos-

phere. Mercury reductase activity could be enhanced throughgenetic engineering to generate strains which constitutively

pr(>du~ed Hg(II)-reductase [24]. Furthermore, organisms withenhanced capacity for cleaving mercury from organomercurialpollut~nts and then reducing the released Hg(ll) to Hg(O) canbe engineered [47].

Several studies have suggested that microbial Hg(II)reductjon might be a better method of mercury removal thanchemi~al methods which can be expensive and result in the

I. ..gener~tlon of a mercury-containing sludge [9,421. However,side Ijy side comparisons between biological and chemicalmethods have not been presented and microbial Hg(II)reduction has only been tested at the bench scale. Thus,although the microbial mechanisms and genetics for microbialHg(II) reduction are better understood than for any other typeof mi9robial metal reduction, it appears that even for this pro-

cess, ~ubstantial additional research will be required before itwill b~ apparent whether microbial metal reduction can beused ~s a bioremediation tool.

CONCLUSION

Thtf studies summarized here demonstrate that microbialmetal reduction has the potential to be a useful technique forthe biorestoration of environments contaminated with organicsand/or'metals. As recently reviewed [61], microorga,nisms mayenzymatically reduce other metals such as vanadium, molyb-denum, copper, gold, and silver. These redox changes canaffect 'the solubility of these metals as well and could poten-tially be applied in bioremediation strategies. [n comparisonwith the vast amount of research that has been done on theaerobic degradation of organic contaminants, there has been

relatively little investigation into the use of microbial metalreduction for bioremediation. More basic research into thispotentially useful type of metabolism is required, as areattem~ts to try to scale-up techniques that have shown promiseon a laboratory scale.

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