Dissimilatory Fe(III) and Mn(IV) reducing prokaryotes

Dissimilatory Fe(III) and Mn(IV) reducing prokaryotes

Dissimilatory Fe(III)- and Mn(IV)-Reducing Prokaryotes DEREK LOVLEY Introduction Dissimilatory Fe(III) reduction is the process in which microorganisms transfer electrons to external ferric iron [Fe(III)], reducing it to ferrous iron [Fe(II)] without assimilating the iron A wide phylogenetic diversity of microorganisms, including archaea as well as bacteria, are capable of dissimilatory Fe(III) reduction Most microorganisms that reduce Fe(III) also can transfer electrons to Mn(IV), reducing it to Mn(II) As detailed in the next section, dissimilatory Fe(III) and Mn(IV) reduction is one of the most geochemically significant events that naturally takes place in soils, aquatic sediments, and subsurface environments Dissimilatory Fe(III) and Mn(IV) reduction has a major influence not only on the distribution of iron and manganese, but also on the fate of a variety of other trace metals and nutrients, and it plays an important role in degradation of organic matter Furthermore, dissimilatory Fe(III)-reducing microorganisms show promise as useful agents for the bioremediation of sedimentary environments contaminated with organic and/or metal pollutants Despite their obvious environmental significance, Fe(III) and Mn(IV)-reducing microorganisms are among the least studied of any of the microorganisms that carry out important redox reactions in the environment The Fe(III)- and Mn(IV)-reducing microorganisms are also of intrinsically interesting because they have unique metabolic characteristics Foremost is the ability of these microorganisms to transfer electrons to external, highly insoluble electron acceptors such as Fe(III) and Mn(IV) oxides, as well as extracellular organic compounds such as humic substances Furthermore, microbiological and geological evidence suggests that dissimilatory Fe(III) reduction was one of the earliest forms of microbial respiration Thus, insights into Fe(III) reduction mechanisms may aid in understanding the evolution of respiration in microorganisms Significance of Fe(III)- reducing Microorganisms and Mn(IV)- Some claims for the significance of Fe(III)-reducing microorganisms may be exaggerated, such as the assertion that "if it were not for the bacterium GS-15 [a Fe(III)-reducing microorganism] we would not have radio and television today" (Verschuur, 1993) However, it is also clear that Fe(III)-reducing microorganisms are of vitally important to the proper functioning of a variety of natural ecosystems and have practical applications Detailed reviews of the literature covering many of these aspects of Fe(III) and Mn(IV) reduction are available (Lovley, 1987a; Lovley, 1991a; Lovley, 1993a; Nealson and Saffarini, 1994; Lovley, 1995a; Lovley et al 1997c) Therefore only highlights of the significance of Fe(III)-reducing microorganisms, abstracted from these reviews, will be briefly summarized here Oxidation of Organic Matter in Anaerobic Environments Microbial oxidation of organic matter coupled to the reduction of Fe(III) and Mn(IV) is an important mechanism for organic matter oxidation in a variety of aquatic sediments, submerged soils, and in aquifers Depending on the aquatic sediments or submerged soils considered, Fe(III) and/or Mn(IV) reduction have been estimated to oxidize anywhere from 10% to essentially all of the organic matter oxidation in the sediments (Lovley, 1991a; Canfield et al., 1993; Lovley, 1995b; Lovley et al., 1997c) An important factor that enhances the significance of Fe(III) and Mn(IV) reduction in aquatic sediments is bioturbation which leads to the reoxidation of Fe(II) and Mn(II) so that each molecule of iron and manganese can be used as an electron acceptor multiple times prior to permanent burial In deep pristine aquifers, there are often extensive zones exist in which Fe(III) reduction is the predominant mechanism for organic matter oxidation (Chapelle and Lovley, 1992; Lovley and Chapelle, 1995c) The ability of Fe(III)-reducing microorganisms to outcompete sulfate-reducing and methanogenic microorganisms for electron donors during organic matter degradation is an important factor limiting the production of sulfides and methane in some submerged soils, aquatic sediments, and the subsurface (Lovley, 1991a; Lovley, 1995b) A model for the oxidation of organic matter in sedimentary environments in which Fe(III) reduction is the predominant terminal electron-accepting process has been suggested (Lovley et al., 1997c) This model is based upon the known physiological characteristics of Fe(III)- and Mn(IV)-reducing microorganisms available in pure culture as well as on studies on the metabolism of organic matter metabolism by natural communities of microorganisms living in various sedimentary environments in which Fe(III) reduction is the terminal electron-accepting process (TEAP) In this model (Fig 1), complex organic matter is hydrolyzed to simpler components by the action of hydrolytic enzymes from a variety of microorganisms Fermentative microorganisms are the principal consumers of fermentable compounds such as sugars and amino acids and these compounds are converted primarily to fermentation acids and, possibly to hydrogen Acetate is by far the most important fermentation acid produced (Lovley and Phillips, 1989a) Acetate also may be produced as the result of incomplete oxidation of some sugars by some Fe(III)reducing microorganisms (Coates et al., 1999a) Other Fe(III)-reducing microorganisms oxidize the acetate and other intermediary products Some Fe(III)reducing microorganisms also can oxidize aromatic compounds and long-chain fatty acids Thus, through the activity of diverse microorganisms, complex organic matter can be oxidized to carbon dioxide with Fe(III) serving as the sole electron acceptor A similar model probably is probably appropriate for organic matter oxidation in sediments in which Mn(IV) reduction is the TEAP This model emphasizes that acetate is likely to be the major electron donor for Fe(III) or Mn(IV) reduction in environments in which naturally occurring, complex organic matter is the major substrate for microbial metabolism However, when otherwise organic-poor environments, such as sandy aquifers, are contaminated with a specific class of organic compounds, such as aromatics, then these contaminants may be the most important direct electron donors for Fe(III) or Mn(IV) reduction Fig Proposed pathways for organic matter degradation in mesophilic environments in which Fe(III) reduction is the predominant terminal electronaccepting process Influence on Metal and Nutrient Geochemistry and Water Quality The reduction of Fe(III) to Fe(II) is one of the most important geochemical changes as anaerobic conditions develop in submerged soils and aquatic sediments (Ponnamperuma, 1972) The Fe(II) produced as the result of Fe(III) reduction is the primary reduced species responsible for the negative redox potential in many anaerobic freshwater environments The reduction of Fe(III) oxides and of the structural Fe(III) in clays typically results in a change in soil color from the redyellow of Fe(III) forms to the green-gray of Fe(II) minerals (Lovley, 1995c) The oxides of Fe(III) and Mn(IV) oxides bind trace metals, phosphate, and sulfate, and Fe(III) and Mn(IV) reduction is associated with the release of these compounds into solution (Lovley, 1995a) Also, typically the pH, ionic strength of the pore water, and the concentration of a variety of cations are increased (Ponnamperuma, 1972; 1984) All of these changes influence water quality in aquifers and can affect the growth of plants in soils The solubility of Fe(II) and Mn(II) is greater than that of Fe(III) and Mn(IV) and thus Fe(III) and Mn(IV) reduction result in an increase in dissolved iron and manganese in pore waters Undesirably high concentrations of iron and manganese may be toxic to plants (Lovley, 1995b) and are particularly significant in groundwaters sources of drinking water, being one of the most prevalent groundwater quality problems (Anderson and Lovley, 1997) Most of the Fe(II) and Mn(II) produced from microbial Fe(III) and Mn(IV) reduction is found in solid phases, often in the form of Fe(II) and Mn(II) minerals of geochemical significance (Lovley, 1995c) The most intensively studied mineral that is formed during microbial Fe(III) reduction is the magnetic mineral magnetite (Fe3O4) (Lovley et al., 1987c; Lovley, 1990a; Lovley, 1991a) The magnetite produced during microbial Fe(III) reduction can be an important geological signature of this activity For example, large quantities of magnetite at depths up to 6.7 km below the Earth's surface provided some of the first evidence for a deep, hot biosphere (Gold, 1992) The massive magnetite accumulations that comprise the Precambrian Banded Iron Formations provide evidence for the possible activity of Fe(III)-reducing microorganisms on early Earth Formation of magnetite as the result of microbial Fe(III) reduction may contribute to the magnetic remanence of soils and sediments The magnetic anomalies that aid in the localization of subsurface hydrocarbon deposits may result from the activity of hydrocarbon-degrading Fe(III) reducers Formation of other Fe(II) and Mn(II) minerals such as siderite (FeCO 3) and rhodochrosite (MnCO3) also may provide geological signatures of microbial Fe(III) and Mn(IV) reduction As detailed below, many Fe(III)- and Mn(IV)-reducing microorganisms can use other metals and metalloids as electron acceptors Microbial reduction of the soluble oxidized form of uranium, U(VI), to insoluble U(IV) may be an important mechanism for the formation of uranium deposits and the reductive sequestration of uranium in marine sediments, the process which prevents dissolved uranium from building up in marine waters (Lovley et al., 1991a; Lovley and Philips 1992) Reduction of other metals such as vanadium, molybdenum, copper, gold, and silver, as well as metalloids such as selenium and arsenic, can affect the solubility and fate of these compounds in a variety of sedimentary environments and may contribute to ore formations (Lovley, 1993a; Oremland, 1994a; Newman et al., 1998; Kashefi and Lovley, 1999) Bioremediation of Organic and Metal Contaminants Iron [Fe(III)]-reducing microorganisms have been shown to play a major role in removing organic contaminants from polluted aquifers For example, Fe(III)-reducing microorganisms naturally remove aromatic hydrocarbons from petroleumcontaminated aquifers (Lovley et al., 1989b; Lovley, 1995c; Lovley, 1997a; Anderson et al., 1998) and this process can be artificially enhanced with compounds that make Fe(III) more available for microbial reduction (Lovley et al., 1994a; Lovley, 1997a) The Fe(II)-minerals formed as the result of microbial Fe(III) reduction can be important reductants for the reduction of nitroaromatic contaminants (Heijman et al., 1993; Hofstetter et al., 1999) Minerals containing Fe(II) also may serve to reductively dechlorinate some chlorinated contaminants (Fredrickson and Gorby, 1996) The ability of Fe(III)-reducing microorganisms to substitute other metals and metalloids in their respiration may be exploited for remediation of metal contamination (Lovley, 1995a; Lovley, 1995b; Fredrickson and Gorby, 1996; Lovley and Coates, 1997b) Reduction of soluble U(VI) to insoluble U(IV) can effectively precipitate uranium from contaminated groundwaters and surface waters Microbial uranium reduction can be coupled with a simple soil-washing procedure to concentrate uranium from contaminated soils Iron [Fe(III)]-reducing microorganisms can precipitate technetium from contaminated waters by reducing soluble Tc(VII) to insoluble Tc(IV) Soluble radioactive Co(III) complexed to EDTA can be reduced to Co(II) which is less likely to be associated with the EDTA found in contaminated groundwaters and more likely to adsorb to aquifer solids Some Fe(III) reducers convert soluble, toxic Cr(VI) to less soluble less toxic Cr(III) Reduction of soluble selenate to elemental selenium can effectively precipitate selenium in sediments or remove selenate from contaminated waters in bioreactors A Possible Early Form of Microbial Respiration Iron [Fe(III)] reduction may have been one of the earliest forms of microbial respiration (Vargas et al., 1998) Biological evidence for this hypothesis is the finding from 16S rRNA phylogenies that all of microorganisms that are the most closely related to the last common ancestor of extant microorganisms are Fe(III)-reducing microorganisms All of the deeply branching bacteria and archaea that have been examined can oxidize hydrogen with the reduction of Fe(III) Several that have been examined in more detail can conserve energy to support growth from this metabolism Of most interest in this regard is Thermotoga maritima, which was previously considered to be a fermentative organism because it could not conserve energy to support growth from the reduction of other commonly considered electron acceptors However, T maritima it does grow via Fe(III) respiration This result and the apparent conservation of the ability to reduce Fe(III) in all these deeply branching organisms suggests that the last common ancestor was a hydrogenoxidizing, Fe(III)-reducing microorganism The concept that Fe(III) reduction is an early form of respiration agrees with geological scenarios that suggest the presence of large quantities of Fe(III) on prebiotic Earth (Cairns-Smith et al., 1992; de Duve, 1995) and elevated hydrogen levels (Walker, 1980)—conditions that would be conducive to the evolution of a hydrogen-oxidizing, Fe(III)-reducing microorganism The large accumulations of magnetite in the Precambrian iron formations (discussed above) indicate that the accumulation of Fe(III) on prebiotic Earth was biologically reduced early in the evolution of life on Earth This and other geochemical considerations suggest that Fe(III) reduction was the first globally significant mechanism for organic matter oxidation (Walker, 1987; Lovley, 1991a) Fe(III)- and Mn(IV)- reducing Microorganisms Available in Pure Culture Dissimilatory Fe(III)- and Mn(IV)-reducing microorganisms can be separated into two major groups, those that support growth by conserving energy from electron transfer to Fe(III) and Mn(IV) and those that not Early investigations on Fe(III) and Mn(IV) reduction in pure culture were conducted exclusively with organisms that are not considered to be conservers of energy from Fe(III) or Mn(IV) reduction (Lovley, 1987a) However, within the last decade, a diversity of microorganisms has been described in which Fe(III) and Mn(IV) reduction are linked to respiratory systems capable of ATP generation It is these Fe(III)- and Mn(IV)-respiring microorganisms (abbreviated here as FMR) that are likely to be responsible for most of the Fe(III) and Mn(IV) reduction in many sedimentary environments (Lovley, 1991a) A brief description of the known metabolic and phylogenetic diversity of dissimilatory Fe(III)- and Mn(IV)-reducing microorganisms follows Fermentative Fe(III)- and Mn(IV)- reducing Microorganisms Many microorganisms which grow via fermentative metabolism can use Fe(III) or Mn(IV) as a minor electron acceptor during fermentation (Table 1) Growth is possible in the absence of Fe(III) or Mn(IV) In this form of Fe(III) and Mn(IV) reduction, most of the electron equivalents in the fermentable substrates are recovered in organic fermentation products and hydrogen Typically, less than 5% of the reducing equivalents are transferred to Fe(III) or Mn(IV) (Lovley, 1987a; Lovley and Phillips, 1988b) However, significant amounts of Fe(II) and Mn(II) can accumulate in cultures of these fermentative organisms when Fe(III) or Mn(IV) is provided as a potential electron sink Although thermodynamic calculations have demonstrated that fermentation with Fe(III) reduction [electron transfer to Fe(III)] is more energetically favorable than fermentation without Fe(III) reduction (Lovley and Phillips, 1989a), it has not been demonstrated that the minor transfer of electron equivalents to Fe(III) or Mn(IV) during fermentation causes any increase in cell yield In contrast to these fermentative microorganisms, several microorganisms can partially or completely oxidize fermentable sugars and amino acids with the reduction of Fe(III) and conserve energy from this metabolism, as discussed below Table Organisms known to reduce Fe(III) but not known to conserve energy from Fe (III) reduction Sulfate- reducing Microorganisms Many respiratory microorganisms that grow anaerobically with sulfate serving as the electron acceptor also have the ability to enzymatically reduce iron [Fe(III); Table 1] Electron donors that support Fe(III) reduction are the same ones that support sulfate reduction by sulfate-reducing microorganisms However, none of these sulfate reducers have been shown to grow with Fe(III) serving as the sole electron acceptor (Lovley et al., 1993b) This is true despite the fact that sulfate reducers have a higher affinity for hydrogen, and possibly for other electron donors, than for sulfate when Fe(III) serves as the electron acceptor (Coleman et al., 1993; Lovley et al., 1993c) The advantage to sulfate reducers in reducing Fe(III), if there is one, has not been thoroughly investigated Because it has been found that the intermediate electron carrier, cytochrome c3, can function as an Fe(III) reductase (Lovley et al., 1993), intermediate electron carriers involved in sulfate reduction may inadvertently reduce Fe(III) because it has been found that the intermediate electron carrier, cytochrome c3 can function as an Fe(III) reductase (Lovley et al., 1993b) Alternatively, Fe(III) reduction by sulfate reducers may be a strategy to hasten Fe(III) depletion and enhance conditions for sulfate reduction Furthermore, the possibility that sulfatereducing microorganisms may be able to generate ATP as the result of Fe(III) reduction, even if they can not grow with Fe(III) as the sole electron acceptor, has not been ruled out (Lovley et al., 1993c) In contrast to the sulfate-reducing microorganisms discussed above, which could not be grown with Fe(III) as the sole electron acceptor, it has been suggested (Tebo and Obraztsova, 1998) that the sulfate-reducing microorganism "Desulfotomaculum reducens" could also conserve energy to support growth by reducing Fe(III), Mn(IV), U(VI), and Cr(VI) (Tebo and Obraztsova, 1998) However, the data supporting the claim that energy is gained from electron transport to metals is curious For example, when the culture was grown on 400 μM U(VI), the cell yield was greater than when the culture reduced mmol Fe(III) This occurs despite the fact that the number of electrons transferred to Fe(III) was ten-fold higher than the electron transfer to U(VI) and that Fe(III) reduction is energetically more favorable than U(VI) reduction Cell yields with metals as the electron acceptor were comparable to those during sulfate reduction even though electron transfer to sulfate was at least 250-fold, and in some instances 2500-fold, greater than electron transfer to the metals These results suggest that the presence of the metals had some additional influence on growth other than just serving as an electron acceptor Several sulfate-reducing microorganisms can oxidize S° to sulfate, with Mn(IV) serving as the electron acceptor, but were not found to conserve energy to support growth from this reaction (Lovley and Phillips, 1994a) Enrichment cultures that are established at circumneutral pH with S° as the electron donor and Mn(IV) or Fe(III) as the electron acceptor typically yield microorganisms which that disproportionate S° to sulfate and sulfide (Thamdrup et al., 1993) The Fe(III) or Mn(IV) serve to abiotically reoxidize the sulfide produced Microorganisms that Conserve Energy to Support Growth from Fe(III) and Mn(IV) Reduction The Fe(III)- and Mn(IV)-respiring microorganisms (FMR) which are known to conserve energy to support growth from Fe(III) and Mn(IV) reduction (Table 2) are phylogenetically (Fig 2) and morphologically (Fig 3) diverse Most of the FMR grow by oxidizing organic compounds or hydrogen with the reduction of Fe(III) or Mn(IV), but S° oxidation coupled to Fe(III) reduction also can provide energy to support growth of microorganisms growing at low pH The various types of FMR are briefly described below Fig Phylogenetic tree, based on 16S rDNA sequences, of microorganisms known to conserve energy to support growth from Fe(III) reduction The tree was inferred using the Kimura two-parameter model in TREECON for Windows (Van der Peer and De Wachter, 1994) Bootstrap values at nodes were calculated from one hundred replicates Fig Phase contrast micrographs of various organisms that conserve energy to support growth from Fe(III) reduction Bar equals μm, all micrographs at equivalent magnification CaCl2 · 2H2O 0.1 g PROCEDURE: Salt Stock contains, per 100 ml: MgCl2 · 6H2O CaCl2 · 2H2O 21.2 g 3.04 g PROCEDURE: Vitamin Solution contains, per liter: Biotin Folic acid Pyridoxine HCl Riboflavin Thiamine Nicotinic acid Pantothenic acid B-12 p-Aminobenzoic acid Thioctic acid 2.0 mg 2.0 mg 10.0 mg 5.0 mg 5.0 mg 5.0 mg 5.0 mg 0.1 mg 5.0 mg 5.0 mg PROCEDURE: Mineral Solution grams per liter Trisodium nitrilotriacetic acid MgSO4 MnSO4 · H2O NaCl FeSO4 · 7H2O CaCl2 · 2H2O CoCl2 · 6H2O ZnCl2 CuSO4 · 5H2O AlK(SO4)2 · 12H2O H3BO3 Na2MoO4 NiCl2 · 6H2O Na2WO4 · 2H2O 1.5 g 3.0 g 0.5 g 1.0 g 0.1 g 0.1 g 0.1 g 0.13 g 0.01 g 0.01 g 0.01 g 0.025 g 0.024 g 0.025 g Preparation of Fe(III) and Mn(IV) Forms Poorly Crystalline Fe(III) Oxide Dissolve FeCl3·6H2O in water to provide final concentration of 0.4M Stir continually while SLOWLY adjusting the pH to 7.0 dropwise with 10 M NaOH solution It is extremely important not to let the pH rise above pH even momentarily during the neutralization step because this will result in an Fe(III) oxide that is much less available for microbial reduction Continue to stir for 30 minutes once pH is reached and recheck pH to be sure it has stabilized at pH To remove dissolved chloride, centrifuge the suspension at 5,000 rpm for 15 minutes Discard the supernatant, resuspend the Fe(III) oxide in water, and centrifuge Repeat six times On the last wash, resuspend the Fe(III) oxide to a final volume of approximately 400 ml, and after determining iron content, adjust Fe(III) concentration to approximately mole per liter Typically, Fe(III) oxide is added to individual tubes of media to provide 100 mmol per liter Fe(III)-Citrate Prior to the addition of any of the media constituents, heat 800 ml of water on a stirring hot-plate to near boiling Add Fe(III)-citrate [typically 13.7 g to provide a final concentration of ca 50 mM Fe(III)] Once the ferric citrate is dissolved quickly cool the medium to room temperature in an ice bath Adjust pH to 6.0 using 10N NaOH When the pH approaches 5.0, add the NaOH dropwise Add medium constituents as outlined above Bring to a final volume of liter Do not expose this media to direct sunlight to prevent photoreduction of the Fe(III) Fe(III) Nitrilotriacetic Acid To make a stock of 100 mM Fe(III)-NTA, dissolve 1.64 g of NaHCO in 80 ml water Add 2.56 g C6H6NO6Na3 (sodium nitrilotriacetic acid) and then 2.7 g FeCl 3·6H2O Bring the solution up to 100 ml Sparge the solution with N gas and filter sterilize into a sterile, anaerobic serum bottle Do not autoclave Typically, 100 mM Fe(III)NTA stock is added to individual tubes of media to provide a final concentration of or 10 mmol of Fe(III) Goethite Prepare a 0.4M FeCl3·6H2O solution With continual stirring, adjust the pH to between 11 and 12 with 10 M NaOH solution The suspension will become very thick Ensure continual stirring and rinse the pH electrode frequently The color of this suspension will turn to an ochre color as goethite is formed One week at room temperature followed by 16 hours at 90°C is sufficient to convert the Fe(III) to goethite The suspension should be washed to remove chloride, as described above for poorly crystalline Fe(III) oxide The formation of goethite should be confirmed by X-ray diffraction analysis The Fe(III) oxide also should be tested with extractants (Lovley and Phillips, 1987b; Phillips and Lovley, 1987) to ensure that it does not contain poorly crystalline Fe(III) oxide Hematite Hematite is readily available from chemical supply companies as "Ferric Oxide." Manganese Oxide To l liter of a solution containing 80 mM NaOH and 20 mM KMnO slowly add l liter of 30 mM MnCl2 with mixing Wash the manganese oxide precipitate, as described above for poorly crystalline Fe(III) oxide, to lower the dissolved chloride concentration Enumeration of Fe(III)- and Mn(IV)-reducing Microorganisms The FMR in environments can be enumerated with standard most-probable-number (MPN) culturing techniques using variations of media described above Enumerations typically use Fe(III) or AQDS as the electron acceptor with the understanding that the Fe(III)-reducing microorganisms recovered are likely to have the ability to reduce Mn(IV) as well Poorly crystalline Fe(III) oxide or Fe(III)-NTA is preferred over Fe(III)-citrate and Fe(III)-pyrophosphate, which promote the growth of fermentative microorganisms One successful approach has been to add a combination of poorly crystalline Fe(III) oxide (100 mmol/liter) and mM NTA to provide a supply of chelated Fe(III) FMR also can be counted in plate counts in which Fe(III)-NTA or AQDS has been added as the electron acceptor Clearing zones develop around FMR reducing Fe(III)NTA, and growth with AQDS as the electron acceptor results in the formation or orange colonies or zones When possible, molecular enumeration rather than viable culturing enumeration techniques are the preferred methods because of the potential biases associated with the latter The wide phylogenetic diversity of dissimilatory Fe(III) reducing microorganisms and the lack of an identified conserved gene associated with Fe(III) reduction make it impossible to enumerate Fe(III)-reducing microorganisms with one specific gene sequence (Lonergan et al., 1996) However, target 16S rRNA sequences that are selective for known groups of Fe(III)-reducing microorganisms have been identified and have been used to study the distribution of Fe(III)-reducing microorganisms in sedimentary environments (DiChristina and DeLong, 1993; Anderson et al., 1998; Rooney-Varga et al., 1999; Synoeyenbos-West et al., 1999) SUMMARY Microbial reduction of Fe(III) and Mn(IV) is of environmental significance in a variety of aquatic sediments and the subsurface, influencing both the carbon cycle and the fate of many metals and metalloids, in both pristine and contaminated environments Geological and microbiological evidence suggests that Fe(III) reduction was one of the earliest forms of respiration A wide phylogenetic diversity of Fe(III)- and Mn(IV)-reducing microorganisms have been recovered in pure culture, but with the exception of the recently recognized importance of Geobacter in subsurface environments, little is known about the distribution or relative contributions of the various Fe(III)-reducing microorganisms The study of the mechanisms of Fe(III) and Mn(IV) reduction are also in their infancy However, now that methods for culturing these organisms are well-developed, it seems likely that increased insight into the ecophysiology of Fe(III)- and Mn(IV)-reducing microorganisms is forthcoming Literature Cited Anderson, R T., and D R Lovley; 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