Appl Microbiol Biotechnol (1997) 48: 687692

Springer-Verlag 1997

MINI-REVIEW

H. L. Ehrlich

Microbes and metals

Received: 21 May 1997 / Received revision: 2 September 1997 / Accepted: 3 September 1997

Abstract Many base metals and a few precious metals as well as some metalloids can be enzymatically or nonenzymatically concentrated and dispersed by microbes in their environment. Some of these activities are commercially exploited or have a potential for it. This article summarizes these activities and the commercial or potentially commercial use of some of them.

Introduction
Microbes encounter metals and metalloids of various kinds in the environment and it is, therefore, not surprising that they should interact with them, sometimes to their benet, at other times to their detriment. Of particular practical interest are the base metals including vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, molybdenum, silver, cadmium and lead; the precious metals gold and silver; and the metalloids arsenic, selenium, and antimony. In nature, these metals and metalloids exist mostly as cations, oxyanions, or both in aqueous solution, and mostly as salts or oxides in crystalline (mineral) form or as amorphous precipitates in insoluble form. A few, like iron, copper, and gold, may also exist in the metallic state in nature, but the rst two of these only very rarely. All microbes, whether prokarotic or eukaryotic, employ metal species for structural functions and/or catalytic functions. The alkali metals Ca and Mg serve structural as well as catalytic functions. The metals V, Cr, Mn Fe, Co, Ni, Cu, Zn, Mo, and W, and the metalloid Se may participate in catalytic functions. For such uses, low environmental concentrations are sucient.
H. L. Ehrlich Department of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180-3590 USA Tel.: +1 518 276 8428 Fax: 518 276 2344 e-mail: ehrlih@rpi.edu

Some prokaryotes can employ metal species that can exist in more than one oxidation state, among them Cr, Mn, Fe, Co, Cu, As, and Se, as electron donors or acceptors in their energy metabolism. For this function, the metals or metalloids must occur in suciently high concentration locally to meet the organisms' demand. Microbial interactions with small quantities of metals or metalloids do not exert a major impact on metal or metalloid distribution in the environment, whereas interactions with larger quantities, as are required in energy metabolism for instance, have a noticeable impact. Some of the latter interactions are commercially exploited or have a potential for it. More detailed treatment of various aspects of the subject of this brief review can be found in a number of articles and books. Some of these include two special issues of the Journal of Industrial Microbiology (nos. 24, vol. 14, 1995) devoted to specic aspects of microbial interactions with metals; reviews by Lovley 1987, 1991, 1993; and books by Beveridge and Doyle 1989; Ehrlich 1996; Ehrlich and Brierley 1990; and Gaylarde and Videla 1995.

Types of interaction
The way microbes interact with metals depends in part on whether the organisms are prokaryotic or eukaryotic. Both types of microbes have the ability to bind metal ions present in the external environment at the cell surface or to transport them into the cell for various intracellular functions. On the other hand, only the prokaryotes (eubacteria and archaea) include organisms that are able to oxidize Mn(II), Fe(II), Co(II), Cu(I), 0 2A AsOA 2 , Se or SeO3 , or reduce Mn(IV), Fe(III), Co(III), 2A 2A A AsO4 , SeO4 , or SeO2 3 on a large scale and conserve energy in these reactions (see Ehrlich 1996). Some microbes may reduce metal ions such as Hg2+ or Ag+ to Hg0 and Ag0 respectively, but do not conserve energy from these reactions (Summers and Sugarman 1974). Some prokaryotes and eukaryotes may form metabolic

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products, such as acids or ligands, that dissolve base metals contained in minerals, such as Fe, Cu, Zn, Ni, Co and others. Others may form anions, such as sulde or carbonate, that precipitate dissolved metal ions (see Ehrlich 1996). Some prokaryotes may methylate some metal and metalloid compounds, producing corresponding volatile metal derivatives (see below, and Summers and Silver 1978; Beveridge and Doyle 1989; Ehrlich 1996).

Levels of interaction
Metabolic/enzymatic Uptake of trace metals and their subsequent incorporation into metalloenzymes or utilization in enzyme activation occurs in all microbes (Wackett et al. 1989). Some examples of metalloenzymes are nitrogenase (Mo/Fe or sometimes V/Fe, or Fe only) (Orme-Johnson 1992; Robson et al. 1986), cytochromes (Fe) and cytochrome oxidase aa3 (Fe, Cu) (Wackett et al. 1989), superoxide dismutases (Fe, Mn, Cu or Zn) (Fridovich 1978), bacteriochlorophyll (Mg) (Scheer 1991), iron-sulfur proteins (Wackett et al. 1989), CO dehydrogenase with Ni in anaerobic bacteria and Mo in aerobic bacteria (Ferry 1995), NADP-dependent formate dehydrogenase (W/Se/Fe) (Yamamoto et al. 1983), and formate dehydrogenase H (Mo/Se/Fe) (Boyington et al. 1997). For uptake, these metals must be in ionic form. Uptake may require genetically determined and controlled transport mechanisms (Silver and Walderhaug 1992). In some cases, uptake may be fast, nonspecic, and constitutive, as for instance with the CorA Mg2+ exchange system and the Mgta and Mgtb Mg2+ uptake systems in Salmonella typhimurium (Snavely et al. 1989). Since ferric iron in the environment, at around neutral pH, exists mainly in a water-insoluble form, its uptake under aerobic conditions requires the microbial formation of ligands, called siderophores, to render the ferric iron soluble (Neilands 1974). A number of microbes are able to use some metals or metalloids as electron donors or acceptors in energy metabolism. They include eubacteria and archaea (see Ehrlich 1996). Depending on the element, the metal species may be in simple ionic form or in the form of oxyanions. As energy sources, oxidizable metals or metalloids may satisfy the entire energy demand of an organism (chemolithotrophs). For example, the eubacteria Thiobacillus ferrooxidans and Leptospirillum ferrooxidans and the archaea Acidianus brierleyi and Sulfolobus acidocaldarius are able to obtain all their energy for growth from the oxidation (FeII) to Fe(III) (for summary see Ehrlich 1996); Stibiobacter senarmontii from the oxidation of Sb2O3 to Sb2O5 (Lyalikova et al. 1976); and Pseudomonas arsenitoxidans from the oxidaA tion of AsO2A to AsO3 4 (Ilyaletdinov and Abrashitova 1981). Some oxidized metal species may serve as terminal electron acceptors in anaerobic respiration by heterotrophs and, depending on the organism, this may

enable them to mineralize the organic carbon that serves as reductant. Some anaerobic, hydrogen-oxidizing autotrophic bacteria also use oxidized metal species as terminal electron acceptors in their respiration. Examples of anaerobic respiration in which an oxidized metal or metalloid species serves as terminal electron acceptor include Fe(III) reduction to Fe2+, Fe3O4, or FeCO3 (Lovley and Phillips 1988; Coleman et al. 1993) and MnO2 reduction to Mn2+ or MnCO3 with acetate by the eubacterium Geobacter metallireducens (Lovely and 0 A 2A Phillips 1988), and SeO2 4 and SeO3 reduction to Se by the eubacterium Thauera selenatis in the presence of nitrate (Rech and Macy 1992; DeMoll-Decker and Macy 1993). The archeon Sulfolobus sp. has been shown A to reduce MoO2 4 to a lower oxidation state (Brierley and Brierley 1982). Aerobic reduction of MnO2 to A Mn2+ by a few marine eubacteria and of CrO2 to 4 Cr(III) by the eubacterium Pseudomonas uorescens LB300 as part of respiration has also been observed (Ehrlich 1996; Wang and Shen 1995). The use of metals or metalloids as electron donors or acceptors in energy metabolism of eukaryotes is unknown. Enzymatic microbial detoxication of harmful metals or metalloids is a third type of interaction. In this process, a toxic metal species may be converted to a less toxic or non-toxic entity by enzymatic oxidation or re3A duction. The bacterial oxidation of AsOA 2 to AsO4 by a A strain of Alcaligenes faecalis, and reduction of CrO2 4 to Cr(OH)3 by P. uorescens LB300 or Enterobacter clocae are examples of such redox reactions (Ehrlich 1996; Wang and Shen 1995). The detoxication in the foregoing two examples is part of the respiratory process of the organisms. In some other instances, detoxication may be by enzymatic reduction that is not part of the respiratory process, as in mercury detoxication (Robinson and Tuovinen 1984). In general, mercury-resistant bacteria produce the enzyme mercuric reductase, which catalyzes the conversion of Hg2+to volatile Hg0. Mercuric reductase formation is induced by Hg2+ in all organisms tested except in Thiobacillus ferrooxidans, in which the enzyme is constitutive (Robinson and Tuovinen 1984). Still other detoxication processes involve enzymatic or non-enzymatic methylation of metals and metalloids such as Sn, Hg, Pb, As, and Se (Chau et al. 1976; Frankenberger and Karlson 1992; Guard et al. 1981; Hallas et al. 1982; Summers and Silver 1978; Trevors 1992; Wong et al. 1975). When microbes cannot detoxify harmful metals, they often have other genetically determined defenses against them (Ji and Silver 1995; Silver 1992). These defenses include modication or elimination of membrane transport systems into the cell for the harmful metal or metalloid species, or eux systems (molecular pumps) for their removal from the cell interior if taken up. Anaerobic, enzymatically catalyzed biocorrosion of metals is another example of metal/microbe interaction. In the original concept, as formulated by von Wolzogen Ku hr and van der Vlugt (1934), sulfate-reducing bacteria promote biocorrosion of cast-iron metal surfaces

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anaerobically through cathodic depolarization. In this model, an iron surface exposed to aqueous moisture undergoes the spontaneous reaction: Fe0 2H2 O 3 Fe2 2OH H2 with the reaction Fe0 3 Fe2 2e at anodic regions, and the reaction 2H2 O 2e 3 2OH H2 3 at cathodic regions. The H2 generated in a cathodic region was thought to accumulate at the iron surface where it was generated, its build-up causing passivation (polarization) of the surface; i.e., its build-up stopped further corrosion. Sulfate-reducing bacteria, when using this hydrogen in their reduction of sulfate, as illustrated by the reaction
4H2 SO2 4 H 3 HS 4H2 O

1 2

were thought to depolarize the surface, thereby promoting continuation of the corrosion process. The sulde they generate could react with the Fe2+ produced at anodic areas, which would also help to promote corrosion if iron sulde did not precipitate on the iron surface as a uniform lm that would passivate the iron surface as long as the lm was undisturbed. Although some past experimentation seemed to lend support to this model, the general view now is that anaerobic biocorrosion is the result of several dierent microbiological reactions. Metal surfaces are often colonized by biolms and their activity has to be taken into account. These biolms consist of a consortium of dierent types of bacteria, often including aerobic, facultative and anaerobic bacteria, with specic locations in the biolm. Metabolic products released by one consortium member in the biolm and not consumed by any of the other members may be corrosive to the metal or act as chemical cathodic depolarizers besides the H2-consuming activity of the sulfate-reducing bacteria at the bottom of the biolm (Videla 1995). Metabolic/non-enzymatic Prokaryotic and eukaryotic microbes are capable accumulating metals by binding them as cations to the cell surface in a passive process (Beveridge and Doyle 1989; Gadd 1993). Even dead cells can bind metal ions. Depending on conditions, such binding may be selective or non-selective. In some cases, if the cell surface becomes saturated by a metal species, the cell may subsequently act as a nucleus in the formation of a mineral containing the metal (Macaskie et al. 1987, 1992; Schultze-Lam et al. 1996). Some bacteria and fungi can promote selective and non-selective leaching of one or more metal constituents from an ore or other rock with metabolic products such as acids and/or ligands produced by them (see Ehrlich

1996). The acids may be organic or inorganic. Groudev and Groudeva (1986) were able to leach aluminum from clays with oxalic and citric acids formed by Aspergillus niger. Alibhai et al. (1991) were able to leach nickel selectively from low-grade Greek laterites with citric acid produced by various species of Aspergillus and Penicillium. The process discriminated against iron, probably because of a higher anity of citric acid for nickel than for iron. Microbes may excrete inorganic metabolic products such as sulde, carbonate, or phosphate ions in their respiratory metabolism and with them precipitate toxic metal ions as a form of non-enzymatic detoxication (Macaskie et al. 1987; see also Ehrlich 1996). To be effective, the precipitation must decrease the concentration of the dissolved metal species below their inhibitory level. Under some circumstances, microbes may cause nonenzymatic corrosion of metals like aluminum or iron or some metal alloys through formation and release of corrosive metabolic products (Edyvean 1995). These products are chiey organic and inorganic acids. Natural occurrences of metal/microbe interactions In nature, noticeable microbial interaction with metals frequently manifests itself through metal immobilization or mobilization (Ferris et al. 1989; Ghiorse and Ehrlich 1992; Ehrlich 1996). Metal immobilization may be through cellular sequestration and accumulation, or through extra-cellular precipitation. Metal mobilization results from dissolution of insoluble metal-containing phases. Bioleaching of metals from ores is a practical example (see Ehrlich and Brierley 1990). These processes are central to controlling biological availability of metals in soils, sediments, and water. Extra-cellular metal accumulations that result from microbial respiratory metabolism or through metal binding to microbial cell surfaces include some sedimentary iron and manganese oxide deposits, some iron and manganese carbonate deposits, a few metal sulde deposits (most, however, have a hydrothermal origin), and some gold deposits (Beveridge and Koval 1981; Beveridge et al. 1982; Doyle 1991; Ferris 1991; Ghiorse and Ehrlich 1992; Schultze-Lam et al. 1996). An example of a site where mercury-resistant bacteria likely contribute to gradual detoxication of a naturally metal-polluted environment through reduction of mercuric ion and volatilization of Hg0 is the Fiora River in southern Tuscany, Italy (Baldi et al. 1989). With exception of some rare natural occurrences, iron and copper in their metallic state are found in the environment only because human beings have placed them there. Even though articially introduced, such metals and others are subject to biocorrosion by some members of the microbial ora indigenous at the site of emplacement. The ease and extent to which these metals corrode depends, in part, on whether they are pure or alloyed (Pope et al. 1984).

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Practical applications
At present, the most important biotechnological applications or potential applications of metal/microbe interactions are in bioleaching or biobeneciation of ores, and in bioremediation of metal-polluted sites and mineralization of polluting organic matter. Bioleaching of Cu from suldic ores has been practised empirically for many centuries. The microbiological basis for the process was not clearly recognized until the 1950s. The copper solubilization is brought about by the action of various extremely acidophilic bacteria that are naturally associated with the ore and which act by either attacking oxidizable components of the ore mineral directly, or by generating an oxidizing agent, usually ferric ion in acid solution, which attacks the ore and dissolves the copper (see Ehrlich 1996). Two bacteria that have been identied as playing important roles in the process are Thiobacillus ferrooxidans and Leptospirillum ferrooxidans, the former capable of Fe and S oxidation, the latter only of Fe oxidation (Sand et al. 1992). Using the mineral covellite (CuS) as an example, these two types of bacterial action can be summarized as follows, Direct bacterial action X
CuS+2O2 3 Cu2 SO2 4 bacteria

Indirect bacterial actionX 2Fe2 2H 0X5O2 3 2Fe3 H2 O 2Fe3 CuS 3 Cu2 S 2Fe2
S H2 O 1X5O2 3 2H SO2 4 bacteria abiological bacteria

6 7 8

Sand et al. (1995) propose that no distinction exists between direct and indirect bacterial action because iron is involved in both. However, the iron in indirect action is free ferric iron in the bulk solution that acts as oxidant, as shown in Eq 7, whereas the iron in direct action is bound in the cell envelope and acts as an electron shuttle by undergoing reversible oxidation/reduction (see Ingledew 1982). The above reactions occur best in a pH range between 1.5 and 2.5. The copper solubilized in bioleaching used to be recovered by re-reducing it to the metallic state with scrap iron. However, the resultant copper needed purication by smelting. Nowadays, the solubilized copper is recovered in high purity by electrolysis or, if accompanied by other metals in solution, by dierential solvent extraction followed by electrolysis. Until recently, bioleaching of copper has been practised economically only in situ, or in heaps or dumps of low-grade ores or tailings containing less than around 0.3%0.5% (by weight) Cu. Recently, however, pilot-scale experiments by Rio Algom Ltd. at their Cerro Colorado mine in Chile have demonstrated the economic feasibility of bioleaching high-grade ores in heaps (approx. 1.2% Cu). This pro-

cess bypasses the need for preparing ore concentrate in an expensive undertaking involving ore milling and separation by otation before extracting the copper pyrometallurgically. Uranium can be extracted from uraninite ore through bioleaching by indirect action with T. ferrooxidans. In this case, ferric iron in acidic solution, which the organisms generate when oxidizing pyrite (FeS2), oxidizes insoluble UO2 to soluble UO2+ 2 . The uranium is recovered from solution by solvent extraction or by adsorption to an ion-exchange resin (McCready and Gould 1990). The extraction of metals such as Co, Mo, Ni, Pb, and Zn from suldic ores by bioleaching is technically feasible. Gencor (South Africa) is currently developing the BioNIC process to recover nickel from low-grade sulde ores (Dew and Miller 1997). Signet Technology Inc. (Denver, Colorado) and Signet Engineering Pty Ltd. (Perth, Western Australia) are currently developing a process to recover cobalt from pyrite ore from the Kasese Cobalt Project in Uganda (Briggs and Millard 1997). Sandstro m et al. (1997) are working on recovery of zinc from a Swedish zinc-containing ore. The principle of bioleaching can be applied as biobeneciation, a process in which an ore is enriched in its valuable metal component(s) by selectively removing undesirable components. Such a process is currently applied on an industrial scale to suldic gold ores (Olson 1994; Bell and Quan 1997). When the nely disseminated gold in such ores is encapsulated in sulde minerals that include, especially, pyrite and arsenopyrite (FeAsS), it is not readily accessible to the usual chemical extractants of gold, namely cyanide or thiourea. Partial removal of the pyrite and arsenopyrite through biooxidation with T. ferrooxidans frees the gold enough to allow ecient subsequent extraction by application of solutions of cyanide or thiourea. Various microbially reducible metals, especially ferric iron in complexed form to keep it soluble at circumneutral pH, can be used as terminal electron acceptors in in situ anaerobic bioremediation of sites polluted with toxic organics (Lovley 1995). In this instance, the oxidized metal species serve as electron acceptors in a mineralization process. It is also technically feasible to remediate in situ sites polluted with uranyl ion (UO2+ 2 ), in which the oxidation state of U is +6, by promoting its anaerobic reduction by appropriate bacteria to insoluble UO2, in which the oxidation state of U is +4 (Lovley and Phillips 1992; Phillips et al. 1995). Further, it is possible to bioremediate in situ sites polluted with chromate or dichromate [Cr(VI)] by stimulating reduction of the Cr(VI) to Cr(III) by bacteria (Wang and Shen 1995). If the Cr concentration at the polluted site is below about 10 mM, enzymatic reduction with a suitable electron donor is possible. If the Cr concentration exceeds this limit, it is possible to stimulate appropriate bacteria at the perimeter of the polluted site to generate a chemical reductant of chromate, such as H2S or Fe(II), with subsequent hydraulic transfer of the reductant to

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the polluted site. In whatever manner it is formed, Cr(III) tends to form insoluble Cr(OH)3, CrO(OH), or other insoluble oxides. Thus, the reduction of Cr(VI) to Cr(III) is a form of immobilization of chromium. A variety of bacteria are known to be able to reduce Cr(VI) to Cr(III) enzymatically, some only anaerobically and others both aerobically and anaerobically (DeLeo and Ehrlich 1994; Wang and Shen 1995). No bacteria are known to date that oxidize Cr(III) to Cr(VI) enzymatically. Recently it was shown that it is technically feasible to clean-up sites polluted with selenate and selenite by stimulating bacteria that reduce selenate and selenite to Se0 in a reactor, a form of immobilization (Cantao et al. 1996). The selenium can be recovered for industrial use. Fungi can convert oxidized selenium to volatile methylated selenides for escape into the atmosphere, a removal by volatilization (Frankenberger and Karlson 1992). Metals may also be removed from polluted waters by biosorption to living or non-living biomass (Beveridge and Doyle 1989; Ehrlich and Brierley 1990; Pethkar and Paknikar 1997). The source of the biomass may be bacteria, fungi, or algae. In some cases, the biomass is slightly modied to enhance its sorption capacity (e.g., Brierley et al. 1986). Bosecker (1993) found that, among various lamentous fungi, Penicillium funiculosum was able to extract more than 50% Ni, and 75% Zn from test solutions containing 100 mg lA1 of a metal at pH 6.6 and 6.5 respectively. The sorptive capacity of the biomass can usually be regenerated after saturation by eluting the sorbed metals. If the recovered metals have value, they can be marketed and at least some of the cost of treatment of the polluted waters recovered. Removal of metals by sorption from polluted ground water usual requires a pump-and-treat process.

Conclusion
Since microbial encounters with metals are unavoidable in the environment, it is not surprising that microbes have developed means to put some metals to use for their benet, and to develop defenses against them when they are harmful. Some microbes can signicantly aect the distribution of metals in the environment. As discussed above, some of the microbial interactions with metals are industrially exploited or at least have a technological potential for such exploitation.

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