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Chapter 20. Biogenesis and Biodegradation of Sulfide Minerals at Earth's Surface

Chapter 20. Biogenesis and Biodegradation of Sulfide Minerals at Earth's Surface

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492



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TABLE 20.1

Metal Sulfides of Geomicrobial Interest

Mineral or Synthetic

Compound



Formula



Antimony trisulfide



Sb2S3



Argentite

Arsenopyrite

Bornite



Ag2S

FeAsS

Cu5FeS4



Chalcocite



Cu2S



Chalcopyrite

Cobalt sulfide

Covellite



CuFeS2

CoS

CuS



Digenite



CU9S5



Enargite

Galena

Gallium sulfide

Marcasite, pyrite



3Cu2S · As2S5

PbS

Ga2S3

FeS2



Millerite

Molybdenite



NiS

MoS2



Orpiment

Nickel sulfide

Pyrrhotite

Sphalerite



As2S3

NiS

Fe4S5

ZnS



Tetrahedrite



Cu8Sb2S7



References

Silver and Torma (1974), Torma

and Gabra (1977)

Baas Becking and Moore (1961)

Ehrlich (1964)

Cuthbert (1962), Bryner et al.

(1954)

Bryner et al. (1954), Ivanov

(1962), Razzell and Trussell

(1963), Sutton and Corrick

(1963, 1964), Fox (1967),

Nielsen and Beck (1972)

Bryner and Anderson (1957)

Torma (1971)

Bryner et al. (1954), Razzell and

Trussell (1963)

Baas Becking and Moore (1961),

Nielsen and Beck (1972)

Ehrlich (1964)

Silver and Torma (1974)

Torma (1978)

Leathen et al. (1953), Silverman

et al. (1961)

Razzell and Trussell (1963)

Bryner and Anderson (1957),

Bryner and Jameson (1958),

Brierley and Murr (1973)

Ehrlich (1963a)

Torma (1971)

Freke and Tate (1961)

Ivanov et al. (1961), Ivanov

(1962), Malouf and Prater (1961)

Bryner et al. (1954)



The enrichment of the surficial deposits of metal sulfide in and on the oceanic crust has occurred

and is occurring in hydrothermally active regions at seafloor spreading centers (mid-ocean ridges)

at depths of 2500–2699 m. Examples of such sites are the eastern Pacific Ocean at the Galapagos

Rift and the East Pacific Rise (Ballard and Grassle, 1979; Corliss et al., 1979) and the Atlantic

Ocean at the Mid-Atlantic Ridge (Klinkhammer et al., 1985). Metal sulfide deposits are evident on

the seafloor where some hydrothermal vents (black smokers; see Chapters 2 and 17) discharge brine

solution that has a temperature ∼350°C and is metal-laden and charged with H2S. Metal sulfides

such as chalcopyrite (CuFeS2) and sphalerite (ZnS) precipitate around the mouth of these vents as

the brine meets cold seawater and are often deposited in the form of hollow tubes (chimneys). The

hydrothermal solution discharged by these vents originated from seawater that penetrated deep



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into porous volcanic rock (basalt) at the mid-ocean spreading centers to depths as great as 10 km

below the seafloor (Bonatti, 1978). As this water penetrated ever deeper into the rock, it absorbed

heat diffusing away from underlying magma chambers and was subjected to increasing hydrostatic

pressure. This caused the seawater to react with the basalt and pick up various metal species and

hydrogen sulfide. The reactions responsible for these seawater modifications include, among others,

the interaction of magnesium in the seawater with the rock to form new minerals with an accompanying release of acid (H+) (Seyfried and Mottl, 1982). The acid leaches metals from the basalt

(Edmond et al., 1982; Marchig and Grundlach, 1982). H2S is formed by the reduction of the sulfate

in seawater and sulfur in basalt by ferrous iron released from the basalt (Shanks et al., 1981; Mottl

et al., 1979; Styrt et al., 1981). As long as the hydrothermal solution is subjected to high temperature

and pressure in the basalt, metal sulfides are prevented from precipitating.

A quantitatively more significant deposition of metal sulfides occurs within the upper oceanic

crust associated with white smokers. Here hot, metal-charged hydrothermal brine rising from the

lower crust meets and mixes with cold seawater that penetrated the upper crust. The mixing of the

two solutions in the upper crust results in partial cooling of the solution and consequent precipitation of metal sulfides in the upper crust. This contrasts with the precipitation of metal sulfides

associated with black smokers, which occurs external to the crust around the mouth of the vents and

becomes deposited mostly in the walls of the vent chimneys. The brine emerging from the vents of

white smokers is depleted in some base metals but still contains major quantities of iron, manganese, and hydrogen sulfide. It is much cooler than the hydrothermal solution issuing from the vents

of black smokers. Figure 17.17 shows diagrammatically the origin of the hydrothermal solution and

metal sulfides associated with black and white smokers at mid-ocean spreading centers.

A study of bioalteration of sulfur and mineral sulfide samples deployed and incubated under

conditions prevailing in the vicinity of a seafloor hydrothermal vent systems (main Endeavor segment of the Juan de Fuca Ridge axis, Pacific Ocean) revealed ready colonization by Bacteria but not

Archaea (Edwards et al., 2003). Elemental sulfur appeared to be most readily attacked. Extensive

Fe-oxide accumulation on Fe-containing minerals suggested activity of neutrophilic iron-oxidizing

bacteria to the investigators.



20.2.2 SEDIMENTARY METAL SULFIDES OF BIOGENIC ORIGIN

Among sedimentary metal sulfides of biogenic origin, iron sulfides are the most common. They are

usually associated with reducing zones in sedimentary deposits in estuarine environments, which

have a plentiful supply of sulfate. The presence of sulfate is important, because the formation of

these metal sulfides is usually the result of an interaction of iron compounds with H2S that originated

from bacterial reduction of the sulfate under anaerobic conditions at these sites. The interaction of

the H2S with the iron compounds leads to the formation of iron pyrite (FeS2). Whether amorphous

sulfide (FeS), mackinawite (FeS), and greigite (Fe3S4) are intermediates in the formation of the

pyrite depends on prevailing environmental conditions (Schoonen and Barnes, 1991a,b; Luther,

1991). In at least one salt marsh (Great Sippewissett Marsh, Massachusetts) where pyrite forms, the

pore waters were found to be undersaturated with respect to these compounds (Jørgensen, 1977;

Fenchel and Blackburn, 1979; Howarth, 1979; Berner, 1984; Giblin and Howarth, 1984; Howarth

and Merkel, 1984). Rapid and extensive microbial pyrite formation has been observed in salt marsh

peat on Cape Cod, Massachusetts (Howarth, 1979). Pyrite formation from biogenic H2S has also

been noted in organic-rich sediments at the Peru Margin of the Pacific Ocean (Mossmann et al.,

1991), in Long Island Sound off the Atlantic coast in Connecticut and New York (Westrich and

Berner, 1984), along the Danish coast (Thode-Andersen and Jørgensen, 1989), and in two seepage

lakes, Gerritsfles and Kliplo, and two moorland ponds in the Netherlands (Marnette et al., 1993).

In many sedimentary environments, pyrite does not represent a permanent sink for iron because

the pyrite may be subject to seasonal reoxidation as conditions in the environment change from

reducing to oxidizing (Luther et al., 1982; Giblin and Howarth, 1984; King et al., 1985; Giblin, 1988).



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Active growth of marsh grass may draw oxygen into the sediment by evapotranspiration (Giblin,

1988). Of all the biogenic sulfide formed in these environments, only a portion is consumed in the

formation of pyrite and other metal sulfides. The rest is reoxidized as it enters the oxidizing zones

(Jørgensen, 1977). This oxidation may be biological or abiological (Fenchel and Blackburn, 1979).

Nonferrous sulfide deposits of sedimentary origin, especially biogenic ones, appear to be relatively rare. They are generally thought to have formed syngenetically. The metals in question were

precipitated by hydrogen sulfide of hydrothermal origin (abiotic formation) or of microbial origin

and then buried in contemporaneously formed sediment. The limiting conditions for sedimentary

sulfide formation by bacteria as calculated by Rickard (1973) require a minimum of 0.1% carbon

(dry weight) and an enriched source of metals such as a hydrothermal solution if more than 1%

metal is to be deposited. More recent studies of microbial sulfate reduction revealed, however, that

a significant amount of reducing power for sulfate reduction may be furnished by hydrogen (H2 ),

which would lower the requirement for organic carbon correspondingly (Nedwell and Banat, 1981;

see also Section 19.9).

Examples of nonferrous sedimentary sulfide deposits, which may have been biogenically formed,

include the Permian Kupferschiefer of Mansfeld in Germany (Love, 1962; Stanton, 1972, p. 1139),

Black Sea sediments (Bonatti, 1972, p. 51), the Roan Antelope Deposit in Zambia and Katanga

(Africa) (Cuthbert, 1962; Stanton, 1972, p. 1139), the Zechstein Deposit in southwestern Poland

(Serkies et al., 1967), and the deposits in Pernatty Lagoon (Australia) (Lambert et al., 1971). By

contrast, the sulfide deposit in the Pine Point Pb–Zn property in Northwest Territories, Canada,

was abiotically formed (Powell and MacQueen, 1984). δ34S analyses of the metal sulfides in this

deposit suggest that the sulfide resulted from a reaction between bitumen and sulfate at elevated

temperature and pressure.

As an example of ongoing nonferrous sulfide biodeposition, the following observation at the

Piquette Pb–Zn deposit in Tennyson, Wisconsin, must be cited. At this site, investigators examined

a flooded tunnel in carbonate rock and found the presence of biofilms in which aerotolerant members of sulfate-reducing bacteria of the family Desulfobacteriaceae were precipitating sphalerite

(ZnS) at a pH between ∼7.2 and 8.6. The sphalerite accumulated in the biofilm in aggregates of

particles that had a diameter of 2–5 nm (Labrenz et al., 2000).

Although most instances of metal sulfide biogenesis in nature are associated with bacterial sulfate

reduction, at least one case of biogenesis of galena has been attributed to the aerobic mineralization

of organic sulfur compounds by Sarcina flava Bary (Dévigne, 1968a,b, 1973). The Sarcina strain

was isolated from earthy concretions between crystals of galena in an accumulation in a karstic

pocket located in the lead–zinc deposit of Djebel Azered, Tunisia. In laboratory experiments, the

organism was shown to produce PbS from Pb2+ bound to sulfhydryl groups of sulfur-containing

amino acids in peptone.



20.3



PRINCIPLES OF METAL SULFIDE FORMATION



Metal sulfides in nature result from an interaction between an appropriate metal ion and biogenically or abiogenically formed sulfide ion:

M2+ + S2− → MS



(20.1)



The source of the sulfide in the reaction determines whether a metal sulfide is considered to be of

biogenic or abiogenic origin. In the case of biogenic sulfide, it does not matter whether the sulfide

resulted from bacterial sulfate reduction (Chapter 19) or from bacterial mineralization of organic

sulfur-containing compounds (Dévigne, 1968a,b, 1973). Because of their relative insolubility, the

metal sulfides form readily at ambient temperatures and pressures. Table 20.2 lists solubility products for a few common simple sulfide compounds.

The following calculations will show that relatively low concentrations of metal ions, typical in

some lakes, will form metal sulfides by reacting with low concentrations of H2S. The ionic activities



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TABLE 20.2

Solubility Products for Some Metal Sulfides

CdS

Bi2S3

CoS

Cu2S

CuS



1.4 × 10−28

1.6 × 10−72

7 × 10−23

2.5 × 10−50

8.5 × 10−45



FeS

PbS

MnS

Hg2S

HgS



1 × 10−19

3.4 × 10−28

5.6 × 10−16

1 × 10−45

3 × 10−53



NiS

Ag2S

SnS

ZnS

H2S

HS−



3 × 10−21

1 × 10−51

8 × 10−29

1.2 × 10−23

1.1 × 10−7

1 × 10−15



Source: Latimer WM, Hildebrand JH, Reference Book of Inorganic Chemistry. Rev ed.,

Macmillan, New York, 1942; Weast RC, Astle MJ, CRC Handbook of Chemistry

and Physics. 63rd ed., CRC Press, Boca Raton, FL, 1982.



in these calculations are taken as approximately equal to concentration because of the low concentrations involved. The following examines the case of amorphous iron sulfide (FeS) formation.

The ionization constant for FeS is

[Fe2+][S2−] = 10−19



(20.2)



[S2−] = 10−21.96[H2S]/[H+]2



(20.3)



The ionization constant for H2S is



This relationship is derived from the constant for the dissociation of H2S into HS− and H+,

[HS−][H+]/[H2S] = 10−6.96



(20.4)



and the constant for the dissociation of HS− into S2− and H+,

[S2−][H+] /[HS−] = 10−15



(20.5)



Substituting Equation 20.3 into Equation 20.2, the following relationship is obtained:

[Fe2+] = [H+]2/[H2S] × 10−19/10−21.96 = [H+]2/[H2S] × 1021.96



(20.6)



Assuming that the bottom water of a lake contains ∼34 mg of H2S L−1 (i.e., 10−3 M) at pH 7,

∼5.08 µg Fe2+ L−1 (i.e., 10−7.04 M) will be precipitated as FeS by 3.4 mg of hydrogen sulfide per

liter (i.e., 10−4 M). The remaining H2S will ensure reducing conditions, which will keep the iron in

the ferrous state. Because ferrous sulfide is one of the most soluble sulfides, metals whose sulfides

have even smaller solubility products will require even less sulfide for precipitation. In the excess of

sulfide, the FeS would probably be transformed into FeS2, which is more stable than FeS.



20.4



LABORATORY EVIDENCE IN SUPPORT OF BIOGENESIS

OF METAL SULFIDES



20.4.1



BATCH CULTURES



Metal sulfides have been generated in laboratory experiments using H2S from bacterial sulfate

reduction. Miller (1949, 1950) reported that sulfides of Sb, Bi, Co, Cd, Fe, Pb, Ni, and Zn were

formed in a lactate-containing broth culture of Desulfovibrio desulfuricans to which insoluble salts



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of selected metals had been added. For instance, he found that bismuth sulfide was formed on addition of (BiO2)2CO3 · H2O, cobalt sulfide on addition of 2CoCO3 · 3Co(OH)2, lead sulfide on addition

of 2PbCO3 · Pb(OH)2 or PbSO4, nickel sulfide on addition of NiCO3 or Ni(OH)2, and zinc sulfide

on addition of 2ZnCO3 · 3Zn(OH)2. The metal salt reactants were added as insoluble compounds to

minimize metal toxicity for D. desulfuricans. Metal ion toxicity depends in part on the solubility of

the metal compound from which the ion derives. Obviously, for a metal sulfide to be formed from

another metal compound that is relatively insoluble, the metal sulfide must be even more insoluble

than the source compound of the metal. Miller was not able to demonstrate copper sulfide formation from malachite [CuCO3 · Cu(OH)2], probably because malachite was too insoluble relative to

copper sulfides in the medium. Miller (1949) also showed that with addition of Cd or Zn ions to the

culture medium, the yield of total sulfide produced from sulfate by the bacteria in batch culture was

greater than in the absence of the added metal ions. This was because the uncombined sulfide itself

becomes toxic to sulfate-reducers at high enough concentration.

Baas Becking and Moore (1961) also undertook a study of biogenesis of sulfide minerals. Like

Miller, they worked with batch cultures of sulfate-reducing bacteria. The bacteria they employed

were Desulfovibrio desulfuricans and Desulfotomaculum sp. (which they called Clostridium

desulfuricans).

s They grew them in lactate or acetate medium containing steel wool. The steel wool

in the medium was meant to serve as a source of hydrogen for the bacterial reduction of sulfate. The

hydrogen resulted from corrosion of the steel wool by the spontaneous reaction,

Fe0 + 2H2O → H2 + Fe(OH)2



(20.7)



The H2 was then used by the sulfate-reducers in the formation of hydrogen sulfide,

4H 2 ϩ SO24Ϫ ϩ 2Hϩ → H 2S ϩ 4H 2O



(20.8)



The media were saline to simulate marine (near-shore and estuarine) conditions under which the

investigators thought the reactions are likely to occur in nature. They formed ferrous sulfide from

strengite (FePO4) and from hematite (Fe2O3). They also formed covellite (CuS) from malachite

[CuCO3 · Cu(OH)2]; argentite (Ag2S) from silver chloride (Ag2Cl2) and from silver carbonate (AgCO3);

galena (PbS) from lead carbonate (PbCO3) and from lead hydroxycarbonate [PbCO3·Pb(OH)2]; and

sphalerite (ZnS) from smithsonite (ZnCO3). All mineral products were identified by x-ray powder

diffraction studies. Baas Becking and Moore (1961) were unable to form cinnabar (HgS) from mercuric carbonate (HgCO3), probably owing to the toxicity of the Hg2+ ion. They were also unable to

form alabandite (MnS) from rhodochrosite (MnCO3), or bornite (Cu5FeS4) or chalcopyrite (CuFeS2)

from a mixture of cuprous oxide (Cu2O) or malachite and hematite and lepidochrosite. They succeeded in forming covellite from malachite where Miller (1950) failed, probably because they performed their experiment in a saline medium (3% NaCl) in which Cl− could complex Cu2+, thereby

increasing the solubility of Cu2+. The starting materials that were the source of metal were all relatively insoluble, as in Miller’s experiments. Baas Becking and Moore found that in the formation of

covellite and argentite, native copper and silver were respective intermediates that disappeared with

continued bacterial H2S production.

Leleu et al. (1975) synthesized ZnS by passing H2S produced by unnamed strains of sulfatereducing bacteria through a solution of ZnSO4. In one experiment, biogenic H2S formation and ZnS

precipitation by the biogenic H2S occurred in separate vessels. In a second experiment, biogenesis

of H2S and precipitation of ZnS occurred in the same vessel at an initial ZnSO4 concentration in

the culture medium of 10−2 M. The ZnS formed under either experimental condition was identified

as a sphalerite–wurtzite mixture by powder x-ray diffraction. The presence of Zn directly in the

culture medium caused a lag in H2S production, which was not observed when H2S was generated

in a separate vessel.



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20.4.2 COLUMN EXPERIMENT: MODEL FOR BIOGENESIS OF SEDIMENTARY METAL SULFIDES

The relatively high toxicity of many of the heavy metals for sulfate-reducing bacteria has been used

as an argument that these organisms could not have been responsible for metal sulfide precipitation

in nature (Davidson, 1962a,b). However, in a sedimentary environment, metal ions will be mostly

adsorbed to sediment particles such as clays or complexed by organic matter (Hallberg, 1978), which

lessens their toxicity. Such adsorbed or complexed ions are still capable of reacting with sulfide and

precipitating as metal sulfides, as was shown experimentally by Temple and LeRoux (1964). They

constructed a column in which clay or ferric hydroxide slurry carrying adsorbed Cu2+, Pb2+, and

Zn2+ ions was separated by an agar plug from an underlying liquid culture of sulfate-reducers

actively generating hydrogen sulfide in saline medium. They also tested clay that was carrying Fe3+

in this setup. They found that, in time, bands of precipitate formed in the agar plug separating the

slurry of metal-carrying adsorbent from the culture of sulfate-reducing bacteria (Figure 20.1). The

bands formed as upward-diffusing sulfide ion species and downward diffusing, desorbed metal ion

species encountered each other in the agar. Differential desorption of metal ions from the adsorbent

and differential diffusion in the agar accounted for the discrete banding of the various sulfides.

These results demonstrate that biogenesis of relatively large amounts of sulfides in a sedimentary

environment is possible, even in the presence of relatively large amounts of metal ions. The main

requirement is that the metal ions are in a nontoxic form (e.g., adsorbed or complexed) or combined in the form of insoluble mineral oxide, carbonate, or sulfate. As Temple (1964) pointed out,

syngenetic microbial production of metal sulfide in nature is possible. Restrictions on the process,



Metal

ions



Metal

sulfides



Clay or Fe(OH)3 slurry

with adsorbed Fe2+, Cu2+, Pb2+, Zn2+

(source of metal ions)



Banded

precipitate



Agar gel

(medium in which metal ions

encounter sulfide ions and react)



Diffuse

precipitate



SO42−



S2−



Desulfovibrio culture

(source of sulfide)



FIGURE 20.1 Temple and LeRoux column modeling a sedimentary environment in which sulfate-reducers

can precipitate metal sulfides with sulfide they produce. The adsorbents, clay or Fe(OH)3 slurry, control the

concentration of metal ions in solution, and the plug of agar gel prevents physical contact of the sulfatereducers with metal ions. In nature, sediments can act as adsorbents of metal ions. They keep the metal ion

concentration in the interstitial water at a level that does not poison the sulfate-reducers.



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according to him, are not metal toxicity but free movement of the bacterially generated sulfide and

a need for metal-enriched zones in the sedimentary environment. On a biochemical basis, Temple

suspected that microbial sulfate reduction evolved in the Precambrian. Subsequent stable isotope

analyses of samples representing the early Precambrian in South Africa indicated that extensive

biogenic sulfate reduction occurred at least 2350 million years ago (Cameron, 1982).



20.5



BIOOXIDATION OF METAL SULFIDES



Regardless of whether they are of abiogenic or biogenic origin, metal sulfides in nature may be subject to microbial oxidation. This may take the form of directt or indirectt interaction (Silverman and

Ehrlich, 1964). In direct interaction, the microbes oxidize a metal sulfide in physical contact with

the mineral surface. In indirect interaction, the microbes usually generate an oxidant (commonly

ferric iron from ferrous iron) in the bulk phase. The oxidant then attacks the metal sulfide. In most

instances, the metal is solubilized as a metal ion by either direct or indirect mode of oxidation. The

biooxidation of galena (PbS) is an exception because the mobilized metal reacts with sulfate ion,

which is generated during the oxidation of the lead sulfide and which is also present in the bulk

phase from other sources, to form insoluble lead sulfate (PbSO4). Some microbes can mobilize

metals in metal sulfides in an indirect mode by generating ligands, which may also be acids. These

mobilize the metals by forming soluble metal complexes.



20.5.1



ORGANISMS INVOLVED IN BIOOXIDATION OF METAL SULFIDES



A number of different acidophilic, iron-oxidizing bacteria have been detected at sites where metal

sulfide oxidation is occurring (Norris, 1990; Rawlings, 1997b; Okibe et al., 2003; Mousavi et al.,

2005). The most important of these have been identified as the mesophiles Acidithiobacillus ferrooxidans, Leptospirillum ferrooxidans, Ferroplasma acidiphilum, and F. acidarmanus; the moderate thermophiles Alicyclobacillus tolerans (formerly Sulfobacillus thermosulfidooxidans),

s and

Acidimicrobium ferrooxidans; and extreme thermophiles Sulfolobus spp., and Acidianus brierleyi

(formerly Sulfolobus brierleyi). All are autotrophs, and all but F. acidarmanus grow best in a pH

range of ∼1.5–2.5. F. acidarmanus, a recent discovery, grows at a pH as low as 0 (optimum pH 1.2)

at a temperature of ∼40°C. It was isolated from pyrite surfaces of the ore body at Iron Mountain,

California, and has been described by Edwards et al. (2000a) as a cell-wall-lacking, iron-oxidizing

autotroph. F. acidiphilum, also a recent discovery, and a close relative of F. acidarmanus, was

isolated from a bioleaching pilot plant (Golyshina et al., 2000). It grows in a pH range of 1.3–2.2

(optimum pH 1.7) in a temperature range of 15–45°C. An organism closely related to F. acidiphilum

was isolated from a bioleaching operation by Mintek in South Africa.

Acidithiobacillus ferrooxidans, Leptospirillum ferrooxidans, Alicyclobacillus tolerans, and

Acidimicrobium ferrooxidans are members of the domain Bacteria (Norris, 1997). Sulfolobus

spp., Acidianus brierleyi, Ferroplasma acidarmanus, and F. acidiphilum are members of the domain

Archaea. Although L. ferrooxidans and Acidimicrobium ferrooxidans oxidize Fe2+ and pyrite, they

do not oxidize reduced forms of sulfur as Acidithiobacillus ferrooxidans and Alicyclobacillus tolerans do. This seems to suggest that L. ferrooxidans and Acidimicrobium ferrooxidans can promote

metal sulfide oxidation only by generating Fe3+ from dissolved Fe2+, which then oxidizes metal

sulfide abiotically. However, because of a structural feature possessed by both Acidithiobacillus ferrooxidans and L. ferrooxidans, both organisms may also be able to oxidize metal sulfides by attacking them directly. The common structural feature is exopolymer (EPS) secreted by the cells that

contains bound iron (Gehrke et al., 1995, 1998; Sand et al., 1997; Harneit et al., 2006). Barreto et al.

(2005) have identified the gene cluster involved in EPS formation in Acidithiobacillus ferrooxidans.

The EPSs enable attachment to sulfide mineral surfaces. In addition, as will be explained later,

the iron in the EPS may serve as an electron shuttle or conductor for conveying electrons from the

oxidative half-reaction of metal sulfides to the electron transport system in the plasma membrane of



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the cells via cytochrome in the outer membrane, such as cytochrome Cyc2 in Acidithiobacillus ferrooxidans, and specific electron carriers in the periplasm, such as rusticyanin and cytochrome Cyc1

in Acidithiobacillus ferrooxidans (see Figure 16.4B). It remains to be determined if Acidimicrobium

ferrooxidans forms EPS with bound iron and transfers electrons from its outer membrane to its

plasma membrane by a mechanism similar to that in Acidithiobacillus ferrooxidans.

Although Acidithiobacillus ferrooxidans, Sulfolobus spp., and Acidianus brierleyi are autotrophs, growth of the two archaea in this group of three is stimulated by a trace of yeast extract in laboratory culture. In the absence of dissolved ferrous iron or reduced forms of sulfur in the medium, all

three organisms can use appropriate metal sulfides as energy sources. Depending on the oxidation

state of the metal moiety in the metal sulfide, both the metal and the sulfide may serve as energy

sources. For example, in the oxidation of chalcocite (Cu2S), Acidithiobacillus ferrooxidans can use

the energy from Cu(I) oxidation for CO2 fixation (Nielsen and Beck, 1972) (see also further discussion in Section 20.5.2). Cell extracts from Acidithiobacillus ferrooxidans have been prepared that

catalyze the oxidation of cuprous copper in Cu2S but not of elemental sulfur (Imai et al., 1973). The

oxidation is not inhibited by quinacrine (atebrine). It needs the addition of a trace of iron for proper

activity. The effect of traces of iron on metal sulfide oxidation had been previously noted in experiments in which the addition of 9 mg of ferrous iron per liter of medium stimulated metal sulfide

oxidation by whole cells of Acidithiobacillus ferrooxidans (Ehrlich and Fox, 1967).

Acidithiobacillus ferrooxidans can use NH+4 and some amino acids as nitrogen sources (see

Sugio et al., 1987; see also Chapter 16). At least some strains are able to fix nitrogen (Mackintosh,

1978; Stevens et al., 1986).

Acidithiobacillus ferrooxidans is very versatile in attacking metal sulfides. It has been reported

to oxidize arsenopyrite (FeS2 · FeAs2 or FeAsS), bornite (Cu5FeS4), chalcocite (Cu2S), chalcopyrite

(CuFeS2), covellite (CuS), enargite (3Cu2S· As2S5), galena (PbS), millerite (NiS), orpiment (As2S3),

pyrite (FeS2), marcasite (FeS2), sphalerite (ZnS), stibnite (Sb2S3), and tetrahedrite (Cu8Sb2S7) (see

Silverman and Ehrlich, 1964; Wang et al., 2007). In addition, the oxidation of gallium sulfide, pyrrhotite, and synthetic preparations of CoS, NiS, and ZnS by Acidithiobacillus ferrooxidans has been

reported (Torma, 1971, 1978; Pinka, 1991; Bhatti et al., 1993). The mode of attack of any of these

minerals may be direct, indirect, or both.

Although not as exhaustively tested as Acidithiobacillus ferrooxidans, the archaea Acidianus

brierleyi and Sulfolobus sp. can also oxidize a variety of metal sulfides including pyrite, marcasite,

arsenopyrite, chalcopyrite, NiS, and probably CoS (Brierley, 1978a,b, 1982; Brierley and Murr,

1973; Dew et al., 1999; Wang et al., 2007). Unlike Acidithiobacillus ferrooxidans, Acidianus brierleyi can oxidize molybdenite in the absence of added iron (Brierley and Murr, 1973) because

molybdate ion is less toxic to Acidianus brierleyi than to Acidithiobacillus ferrooxidans (Tuovinen

et al., 1971).



20.5.2



DIRECT OXIDATION



According to the concept of direct oxidation of susceptible metal sulfides as defined by Silverman

and Ehrlich (1964), the crystal lattice of such sulfides is attacked through enzymatic oxidation. To

accomplish this, the microbes have to be in intimate contact with the mineral they attack. Evidence

for rapid attachment of Acidithiobacillus ferrooxidans to mineral surfaces of chalcopyrite particles

(CuFeS2 ) has been presented by McGoran et al. (1969) and Shrihari et al. (1991); to covellite particles by Pogliani et al. (1990); to galena crystals by Tributsch (1976); to pyrite crystals by Bennett

and Tributsch (1978), Rodriguez-Leiva and Tributsch (1988), Mustin et al. (1992), Murthy and

Natarajan (1992), and Edwards et al. (1998); and to pyrite/arsenopyrite-containing auriferous ore by

Norman and Snyman (1988).

Bacterial attachment to mineral sulfide surfaces appears not to be random but to occur at specific

sites and even specific crystal faces. Some evidence suggests that direct microbial attack is initiated

at sites of crystal imperfections. Selective attachment of Acidithiobacillus ferrooxidans or Sulfolobus



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acidocaldarius to newly exposed pyrite crystals in coal is very rapid, that is, ∼90% complete in

2–5 min (Badigian and Myerson, 1986; Chen and Skidmore, 1987, 1988). Although the details of

how microbes attack crystal lattices of metal sulfides once they have attached are in most respects

not yet understood, a collective model is that bacterial cells possessing this ability act as catalytic conductors in transferring electrons from cathodic areas on crystal surfaces of a metal sulfide via an electron transport system in the cell envelope to oxygen (Figure 20.2). The model assumes the existence of

a special electron transport system, which in gram-negative bacteria involves components of the outer

membrane, the periplasm, and the plasma membrane. Indeed, the bacteria can be viewed as cathodic

extensions. They benefit from this process by coupling energy conservation (ATP synthesis) to it.

The mere spontaneous dissociation of a mineral to yield oxidizable ion species in solution that

Acidithiobacillus ferrooxidans can attack is too small in the case of minerals that are very insoluble

in acid solution. For example, covellite (CuS), in which the only oxidizable constituent is the sulfide,

has a solubility constant of 10−44.07 (Table 20.2). Acidithiobacillus ferrooxidans is able to oxidize

this mineral at pH 2 (see later in this section). Simple calculations show that at equilibrium at pH 2

in water, the dissociation of CuS will only generate a concentration of HS− equal to 10−15.53 M and a

concentration of H2S equal to 10−13.06. This is insufficient for sulfide oxidation by Acidithiobacillus

ferrooxidans because the most recent Ks value for sulfide oxidase in intact cells of this organism has



Cu2S ==> Cu2+ + CuS + 2e



2e



(A)



1/2O2 + 2H+ + 2e ==> H2O

(inner surface of plasma

membrane)

2e



Cu2S ==> Cu2+ + CuS + 2e



Fe3+



Fe2+



(B)



FIGURE 20.2 Schematic representation of direct and indirect oxidation of a particle of Cu2S by

Acidithiobacillus ferrooxidans. (A) Direct oxidation. In this model the bacterial cell, attached to the surface

of a Cu2S particle, acts essentially as a conductor of electrons it removes in the oxidation of Cu(I) of Cu2S

and transfers to oxygen. Not shown is the mechanism by which the electrons cross the interface between the

particle surface and the cell surface. For a possible mechanism, for this electron transfer, see Chapter 16,

Figure 16.4B and discussion in Section 20.5 of this chapter. (B) Indirect oxidation. In this model, planktonic

(unattached) bacterial cells generate and regenerate the oxidant (Fe3+) by oxidizing Fe2+ in the bulk phase.

The iron acts as a shuttle that carries electrons from the oxidation of Cu(I) of Cu2S to the bacterium, which

transfers them to oxygen.



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501



been reported to be 10−5.30 (Pronk et al., 1990). Ks values are a measure of the substrate concentration

at which a reaction velocity catalyzed by intact cells is half-maximal (Michaelis–Menten kinetics).

(See footnote in Section 16.4. Ks for a cell is equivalent to Km for an individual enzyme.) Thus the

mere dissociation of CuS into Cu2+, HS−, and H2S cannot furnish nearly enough sulfide substrate

to sustain its oxidation by Acidithiobacillus ferrooxidans at a reasonable velocity, regardless of

whether HS− or H2S or both are the actual substrate for sulfide oxidase. Because Acidithiobacillus

ferrooxidans can oxidize covellite in the absence of added iron, it must be in direct contact with

a mineral to attack it under that condition. The need for direct contact in covellite oxidation by

Acidithiobacillus ferrooxidans in the absence of added Fe2+ was demonstrated experimentally by

Pogliani et al. (1990). By contrast, Acidithiobacillus thiooxidans was shown by Donati et al. (1995)

to promote covellite oxidation only with the addition to the medium of Fe(III) or by Fe(II) autoxidized to Fe(III). The oxidation of covellite by Fe(III) generates S0 (and possibly small amounts of

other partially reduced and dissolved sulfur species) as first described by Sullivan (1930),

CuS + 2Fe3+ → Cu2+ + S0 + 2Fe2+



(20.9)



As pointed out in Chapter 19, Acidithiobacillus thiooxidans cannot oxidize Fe2+.

On the contrary, if we consider a more soluble sulfide mineral such as ZnS, which has a solubility

constant of 10−22.9, calculations similar to those for CuS show that at pH 2, ZnS dissociation will

yield 10−4.95 M HS− and 10−1.47 H2S (Table 20.2). These concentrations of HS− and H2S are more

than sufficient to satisfy the Ks of 10−5.30 for sulfide oxidase in Acidithiobacillus ferrooxidans and

to permit its growth without direct attack of ZnS at the mineral surface. Indeed, it has been shown

that Acidithiobacillus thiooxidans, which is unable to oxidize Fe2+, will readily promote the dissolution of ZnS at pH 2 (Pistorio et al., 1994). The relative solubility of PbS in acid solution also

explains why Garcia et al. (1995) found that Acidithiobacillus thiooxidans promoted dissolution of

PbS (galena). The solubility constant of this metal sulfide is 10−27.5, a little smaller than that of

ZnS (10−22.9) but significantly larger than that of CuS (10−44.07). At pH 2, PbS dissociates to yield

10−7.25 M HS− and 10−4.77 M H2S (Table 20.2).

The exact nature of the interaction between a sulfide mineral surface and the Acidithiobacillus

ferrooxidans cell surface, on which the enzyme-catalyzed oxidation of the mineral depends, has

become a little clearer in the past few years. Previously, Ingledew proposed that iron bound in the

cell envelope of Acidithiobacillus ferrooxidans served as an electron shuttle that conveys electron

from an external electron donor across the outer membrane to electron carriers in the periplasm of

the cell (see Ingledew, 1986; Ehrlich, 2000). Alternatively, Tributsch (1999) proposed that the ferric

iron bound in the EPS at the cell surface of Acidithiobacillus ferrooxidans in contact with a mineral

surface generated elemental sulfur according to Reaction 20.9. This sulfur was then supposed to

be oxidized by Acidithiobacillus ferrooxidans by a known reaction (see Chapter 19). However, this

proposal assumes that EPS-bound iron(III) is as strong an oxidant as ferric iron in the bulk phase.

It has now been demonstrated by Yarzábal et al. (2002a,b) that the outer membrane of

Acidithiobacillus ferrooxidans contains a high-molecular weight c-type cytochrome Cyc2 that

has the capacity to promote the oxidation of Fe2+ to Fe3+ at the outer surface of the outer membrane. Cytochrome Cyc2 in the outer membrane then conveys the electrons it removed from Fe2+

to the multicopper oxidase rusticyanin and from it to a low-molecular weight c-type cytochrome

Cyc1, both located in the periplasm. Cytochrome Cyc1 then passes the electrons it receives to aa3

cytochrome oxidase in the plasma membrane, which passes them to O2 accompanied by energy

conservation via ATP synthase (see also Section 16.4 and Figure 16.4B). Although it remains to

be experimentally demonstrated, it seems reasonable to assume that cytochrome Cyc2 or another

c-type cytochrome in the outer membrane is involved in conveying electrons from the oxidation of

a metal sulfide with which Acidithiobacillus ferrooxidans is in physical contact, to oxygen either

directly or via iron bound in the EPS of the cells acting as an electron shuttle between the mineral

and cytochrome Cyc2 in the outer membrane, as explained later.



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Precedents for electron transfer between an outer cell surface and a mineral surface with which

it is in contact exist among bacteria that are involved in the reduction of insoluble electron acceptors

such as ferric oxide or MnO2 (see Chapters 16 and 17). The electrons in these instances travel in a

direction opposite to that when an oxidation of an insoluble electron donor, like metal sulfide or a

substrate like Fe2+, at the outer surface of the outer membrane is involved.

Gehrke et al. (1995, 1998) and Sand et al. (1997) have proposed that the iron bound in the

EPS around the cells of Acidithiobacillus ferrooxidans mediates attack of the sulfur moiety in

pyrite. They view bulk-phase iron as inducing EPS formation and enabling attachment to pyrite.

However, they do not differentiate unbound, bulk-phase iron and iron bound (complexed) by EPS of

Acidithiobacillus ferrooxidans. Bulk-phase ferric iron when interacting with metal sulfide mineral

behaves as a chemical reactant, which is consumed in the oxidation of the metal sulfide. EPS-bound

iron should be viewed as an electron shuttle, which is reversibly reduced and oxidized during electron transfer from the metal sulfide being oxidized to an acceptor molecule of the cell (e.g., cytochrome Cyc2). This follows from the following consideration. The standard reduction potential

4−

for the Fe3+/Fe2+ couple is +777 mV, whereas that of the Fe(CN)3−

6 / Fe(CN)6 in 0.01 N NaOH

is +460 mV (Weast and Astle, 1982), and that for oxidized cytochrome c/reduced cytochrome c

is +0.245 mmV (Lehninger, 1975, p. 479). Although no reduction potential for EPS-bound iron is

available, its value is likely to lie between that for uncomplexed iron and the porphyrin-bound iron

in cytochrome. EPS-bound iron would therefore be a significantly weaker oxidant and function better

as an electron shuttle.

Although the mechanism by which Acidithiobacillus ferrooxidans promotes direct oxidative

attack of metal sulfides is becoming clearer, that by which other bacteria known to promote such

oxidative attack remains to be clarified. Some may only be able to promote such oxidation by indirect attack, but others are likely to be able to promote it by both direct and indirect attack. Because

Acidithiobacillus ferrooxidans is a gram-negative organism, other gram-negative organisms, such

as Leptospirillum ferrooxidans for instance, may use the same mode of direct attack, but not grampositive organisms or archaea, such as Alicyclobacterium tolerans and Acidianus brierleyi, respectively, that may be capable of it. This is because the envelope structure of gram-positive bacteria and

archaea is very different from that of gram-negative bacteria (see Chapter 6).

Evidence for enzymatic attack of synthetic covellite (CuS) by noting inhibition of oxygen

consumption and Cu2+ and SO42− ion production by Acidithiobacillus ferrooxidans in the presence of the enzyme inhibitor trichloroacetate (8 mM) was obtained by Rickard and Vanselow

(1978). In the case of CuS, only the sulfide moiety of the mineral is attacked because the metal

moiety is already fully oxidized. The oxidation of the mineral probably proceeds in two steps

(Fox, 1967):

CuS ϩ 0.5O2 ϩ 2Hϩ Bacteria



→ Cu2ϩ ϩ S0 ϩ H 2O

S0 ϩ 1.5O2 ϩ H 2O Bacteria



→ SO24Ϫ ϩ 2Hϩ

By contrast, Thiobacillus thioparus promotes covellite oxidation only after autoxidation of the mineral to CuSO4 and S0 (similar to Reaction 20.10 but in the absence of bacterial catalysis; Rickard and

Vanselow, 1978). It is the bacterial catalysis of the oxidation of S0 to sulfate that helps the reaction

by removing a product of the autoxidation of CuS.

In some instances, both an oxidizable metal moiety and the sulfide moiety may be attacked

by separate enzymes, for example, in the case of chalcopyrite (CuFeS2) (assuming the Fe of chalcopyrite to have an oxidation state of +2; Duncan et al., 1967; Shrihari et al., 1991). Although

Duncan and coworkers reported Fe and S to be simultaneously attacked, Shrihari et al. (1991)

found that iron-grown Acidithiobacillus ferrooxidans oxidized the sulfide sulfur of chalcopyrite by

direct attack before oxidizing ferrous iron in solution to ferric iron. When the dissolved ferric iron



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