ITGA01 18/7/06 18:06 Page iii

Introduction to Geomicrobiology

Kurt Konhauser ITGC03 18/7/06 18:11 Page 93

3 Cell surface reactivity and metal sorption

One of the consequences of being extremely 3.1 The cell envelope small is that most microorganisms cannot out swim their surrounding aqueous environment. Instead they are subject to viscous forces that 3.1.1 Bacterial cell walls cause them to drag around a thin film of bound water molecules at all times. The im- Bacterial surfaces are highly variable, but one plication of having a watery shell is that micro- common constituent amongst them is a unique organisms must rely on diffusional processes material called peptidoglycan, a polymer con- to extract essential solutes from their local sisting of a network of linear polysaccharide milieu and discard metabolic wastes. As a (or glycan) strands linked together by proteins result, there is a prime necessity for those cells (Schleifer and Kandler, 1972). The backbone to maintain a reactive hydrophilic interface. of the molecule is composed of two amine sugar To a large extent this is facilitated by having derivatives, N-acetylglucosamine and N-acetyl- outer surfaces with anionic organic ligands and muramic acid, that form an alternating, and high surface area:volume ratios that provide repeating, strand. Short peptide chains, with four a large contact area for chemical exchange. or five amino acids, are covalently bound to some Most microorganisms further enhance their of the N-acetylmuramic acid groups (Fig. 3.1). chances for survival by growing attached to They serve to enhance the stability of the submerged solids. There, they may adopt a entire structure by forming direct or interchain more hydrophobic nature to take advantage of cross-links between adjacent glycan strands. The the inorganic and organic molecules that pre- peptide chains are rich in carboxyl (COOH)

ferentially accumulate. Accordingly, through- groups, with lesser amounts of amino (NH2) groups out a microorganism’s life, there is a constant (Beveridge and Murray, 1980). interplay with the external environment, in Despite the enormous variety of bacterial which the surface macromolecules are modified species, most can be classified into two broad in response to changing fluid compositions categories: Gram-positive and Gram-negative. and newly available colonizing surfaces. In this This terminology has its basis in the cell’s response chapter we focus on how cellular design can to the differential staining technique developed facilitate the accumulation of metals onto by Christian Gram in 1884. The Gram stain microbial surfaces, often in excess of mineral involves using four chemicals on dried smears of saturation states. We then examine how model- bacteria in the following sequence: crystal violet, ing their chemical reactivity can be applied to iodine, ethanol, and safranin. Bacteria that are the environmental issues of contaminant bio- able to retain the crystal violet–iodine complex, remediation and biorecovery of economically even after decolorization with ethanol are called valuable metals. Gram-positive. Those that lose their purple ITGC03 18/7/06 18:11 Page 94

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N-acetylmuramic acid (M) N-acetylglucosamine (G)

CH2OH O 1 Glycosidic bond CH2OH 4 O O 2 Glycan CH OH 2 4 strand O O 3 CH2OH O O O NH O OH NH CO M M M M M O H C CH O NH CO 3 CH3 G G G G G CH C M M M M M OH NH CO 3 HCCH3 CO CH3 G G G G G C M M M M CH3

L-alanine L-alanine 1 4 D-glutamine D-glutamine 4 2 Peptide Peptide cross-linkages chain L-lysine L-lysine 4 4 D-alanine D-alanine 3

(M) (G) (M)

Figure 3.1 Structure of peptidoglycan. It is composed of strands of repeating units of N-acetylglucosamine and N-acetylmuramic acid sugar derivatives. The sugars are connected by glycosidic bonds, but the overall resilience comes from the cross-linking of the glycan strands by peptide chains.

coloration and are counterstained with safranin abundances, most bacteria are Gram-negative, to become red are Gram-negative. It is now while the Gram-positive cells are distinguished recognized that these staining characteristics on the universal phylogenetic tree as two sister highlight some fundamental differences in the phyla (the Firmicutes and the Actinobacteria), chemical and structural organization of the cell united by their common cell wall structure. wall (Beveridge and Davies, 1983). In both cell types, the crystal violet–iodine complex pene- (a) Gram-positive bacteria trates the cell wall and stains the cytoplasm. Then during the decolorization step, the ethanol A large proportion of the work conducted on solubilizes some of the membranous material. This the ultrastructure and metal binding properties is where the inherent differences lie. In Gram- of Gram-positive cells has been done using a positive cells, their thick peptidoglycan walls common soil constituent, Bacillus subtilis. Under become dehydrated by the alcohol, the pores the transmission electron microscope (TEM), a in the wall close, and the crystal violet–iodine technique that permits resolution of objects as complex is prevented from escaping. By contrast, small as a few nanometers, these species are Gram-negative cell walls have thin peptido- observed having a single wall layer averaging glycan walls that cannot retain the stain when 25–30 nm thick, which consists of 30–90% the membranes are dissolved. In terms of global peptidoglycan. The remaining materials are ITGC03 18/7/06 18:11 Page 95

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secondary polymers that are covalently attached while the other half is embedded in the peptido- to the peptidoglycan (Fig. 3.2). For instance, when glycan matrix by penetrating through its interstices B. subtilis is grown in the presence of phosphate, (Doyle et al., 1975). Some teichoic acids are also its wall has essentially two chemical compon- bound to membrane lipids, and they are called ents of roughly equal proportion; peptidoglycan lipoteichoic acids. and teichoic acid (Beveridge, 1989a). Teichoic When growth of B. subtilis is limited by the acids are either glycerol- or ribitol-based poly- availability of phosphate, teichoic acid synthesis

saccharides, with a terminal (H2PO3) phosphoryl ceases and it is totally replaced by teichuronic group and glucose or amino acid residues (Ward, acid, a polymer made up of alternating sequences 1981). A phosphodiester group links the teichoic of N-acetylgalactosamine and carboxyl-rich glu- acid chain to N-acetylmuramic acid of the curonic acid, but lacking phosphate. Variations peptidoglycan. Teichoic acids provide a distinct in the type and quantity of secondary polymer asymmetry in composition between the wall’s indicate that the wall composition, at least for inner and outer surfaces because half extends B. subtilis, may be a phenotypic expression of the perpendicularly outwards into the external milieu, environment (Ellwood and Tempest, 1972). Stated

A Peptidoglycan Plasma membrane

50 nm

B Wall-associated protein Teichoic acid Lipoteichoic Chemoreceptor acid

Peptidoglycan (~30 nm thick)

Plasma membrane (not to scale)

Electron transport enzyme Electron transport enzyme Permease ATPase

Figure 3.2 (A) A TEM image of a Bacillus subtilis cell wall (courtesy of Terry Beveridge). (B) Representation of the overall structure of a Gram-positive bacterium. ITGC03 18/7/06 18:11 Page 96

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simply, the bacterium has the means to adapt bound to the lipid A is the “core”, consisting of the its biochemistry to compensate for geochemical unique sugars 3-deoxy-D-mannooctulosonate (also changes in its environment, and as will be dis- known as KDO) and L-glycero-D-mannoheptose cussed below, this has implications for it retain- (or heptose), along with N-acetylglucosamine, ing high surface reactivity. galactose, and a number of other sugars whose exact combination varies between species. Chemically (b) Gram-negative bacteria the core contains carboxyl and cationic amino + groups (NH4 ) that are cross-linked, usually with Much as Bacillus subtilis is the archetypal Gram- carboxyl groups present in excess. The outermost positive bacterium, Escherichia coli has largely region of the LPS is the “O-antigen.” It is made become the model Gram-negative bacterium. up of repeating carbohydrate units that are inter- The walls of E. coli are structurally and chemic- spersed with uronic acids and/or organic phos- ally complex (Beveridge, 1989a). External to the phate groups, the latter comprising 75% of the total plasma membrane is a very thin (3 nm thick) phosphorous associated with the outer membrane, peptidoglycan layer that makes up a mere 10% while the remainder is in the phospholipid. of the cell wall. This, in turn, is overlain by The remaining fraction of the outer mem- another bilayered structure, the outer membrane, brane contains two major types of proteins. that serves as a barrier to the passage of many Lipoproteins are confined to the inner face of unwanted molecules from the external environ- the outer membrane, and they serve to anchor ment into the cell (Fig. 3.3). The narrow region the outer membrane to the peptidoglycan (Di separating the plasma and outer membranes, Rienzo et al., 1978). The other proteins are called the periplasm, contains a hydrated, gel-like porins. They puncture the bilayer and func- form of peptidoglycan. In E. coli it is 12–15 nm tion as small-diameter (up to a few nanometers), thick and occupies approximately 10–20% of water-filled channels that completely span the the total cell volume. Within the periplasm is the outer membrane and regulate the exchange of peptidoglycan layer itself, a number of dissolved low-molecular-weight hydrophilic solutes into components such as amino acids, sugars, vitamins and out of the periplasm along a concentration and ions, and various macromolecules that are gradient (Hancock, 1987). Some porins con- attached to the boundary surfaces (Hobot et al., tain specific binding sites for one or a group of 1984). As discussed in Chapter 2, the periplasm structurally related solutes that they allow in. also houses a number of enzymes involved in Other porins are nonspecific, in that the width of catabolism, e.g., the hydrolytic enzymes and those their channel largely determines the exclusion employed in electron transport. limit for dissolved compounds. Therefore, porins The outer membrane has an asymmetric lipid with restrictive channel widths can both sieve distribution, with phospholipids limited to the out potentially harmful enzymes and other large inner face. The outer face (exposed to the external hydrophilic molecules, while preventing internal environment) contains a uniquely prokaryotic enzymes that are present in the periplasm from molecule, lipopolysaccharide (LPS). Typically, the diffusing out of the cell. There are also a number outer membrane contains 20–25% phospholipid of other proteins located on the exterior surface and 30% LPS. The LPS possesses three distinct of the outer membrane, including those that func- chemical regions (Ferris, 1989). The innermost tion as: (i) mediators for the cellular adsorption, hydrophobic region, called “lipid A,” is the seg- processing, and transport of essential ions into the ment of the LPS that shows the least chemical cytoplasm; (ii) as receptors for bacteriophages, variation between different species. It has a dis- the viruses that infect bacteria; and (iii) chemo- accharide of glucosamine that is acetylated and receptors, helping direct the cell towards or away attached to short-chain fatty acids. Covalently from specific chemicals (Beveridge, 1981). ITGC03 18/7/06 18:11 Page 97

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A Plasma membrane Outer membrane Peptidoglycan

50 nm

B Chemoreceptor

LPS

O-antigen Lipid A

Core Outer membrane

Wall-associated Porin (non-specific) protein Phospholipid Porin (specific) Peptidoglycan (~3 nm thick) Periplasm Plasma membrane (not to scale) Lipoprotein

Electron transport enzyme Electron transport Permease ATPase enzyme

Figure 3.3 (A) A TEM image of a Synechococcus PCC7942 cell wall (courtesy of Maria Dittrich and Martin Obst). (B) Representation of the overall structure of a Gram-negative bacterium.

3.1.2 Bacterial surface layers layers located external to the cell wall (Beveridge and Graham, 1991). These layers are Direct examination of bacterial cells under the defined by both their composition and physical TEM reveals that most possess supplementary characteristics. ITGC03 18/7/06 18:11 Page 98

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(a) Extracellular polymers (EPS)

Extracellular polymers, also known as exopolymers, extracellular polysaccharides, or glycocalyces, are highly hydrated structures (up to 99% water) that are produced inside the cell and excreted to the cell surface. Their consistency is often thixo- trophic, that is, they are able to alternate between a gel and a liquid state. The solid material is pre- dominately a heteropolysaccharide, composed of repeating units of several types of sugar monomers, as well as various carboxyl-rich uronic acids that may make up to 25% of the solid capsular material (Sutherland, 1972). Other cells have EPS domin- Cell ated by proteins (Nielsen et al., 1997). In general, the chemical composition of EPS is extremely diverse, reflecting the different microorganisms that produce them. In fact, even a single strain of bacterium may secrete several types of EPS, each having different physical and chemical properties Capsule depending on nutrient availability, their growth stage, and other environmental parameters. EPS also range in their complexity. Capsules are structured and stable forms firmly attached 600 nm to the cell (e.g., Fig. 3.4). Their thickness can extend several micrometers from the cell surface, and in many instances, the production of capsu- Figure 3.4 TEM image of a capsule surrounding lar material is so extensive that entire colonies Rhizobium trifolii. (Courtesy of Frank Dazzo.) are encapsulated. By contrast, slime layers range from those materials loosely attached to the cell surface to those that are shed into the environ- ment. The latter forms when the bacterium over- 2 They help the bacteria adhere to surfaces and main- produces its capsular material or, for some reason, tain the overall stability of biofilm/mat communities fails to anchor them securely to their surfaces. (see section 6.1.1). This is important because Subsequently, the slime layers are sloughed off organic and inorganic compounds are preferenti- ally concentrated at interfaces, hence surfaces are into their surroundings, where they float freely desirable locations for growth. until they become associated with other solid surfaces (Whitfield, 1988). 3 They provide microorganisms with a reserve of The production of EPS involves a significant carbon and energy. expenditure in energy and carbon by the micro- organism. Accordingly, its formation must have 4 They bind metals, form minerals and serve as chem- benefits to those cells that produce it (Wolfaardt ical buffers at the cell’s periphery, where essential ions are accumulated and toxic substances immobi- et al., 1999). Some of those benefits are: lized. Consequently, EPS can be considered as an additional design strategy by which bacteria control 1 They protect cells from periodic desiccation, extreme the concentration of metals actually reaching the pH values, elevated temperatures, or freezing. vital constituents within the cell. ITGC03 18/7/06 18:11 Page 99

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(b) Sheaths independently) filaments that in response to light can penetrate the viscous extracellular Several filamentous bacteria are completely layers to disperse into the environment, and sub- encased in a structure that resembles a hollow sequently develop back into mature filaments cylinder when devoid of cells. These are known as once they colonize a new substratum (Hoiczyk, sheaths, and for some species they represent the 1998). outermost surface layer (e.g., Fig. 3.5). Sheaths One common feature seems to exist amongst come in two varieties: (i) highly ordered and made the broad group of ensheathed microorganisms, up of proteins, such as those associated with that is, their sheaths have minute particle spacing several species of methanogens (e.g., Patel et al., that makes them impervious to large molecules. 1986); and (ii) fibrillar and predominantly made It is thus likely that they serve as an additional up of neutral sugars, along with variable quantities permeability layer or chemical sieve that filters of uronic and amino acids. They form in associa- out harmful macromolecules (e.g., Phoenix et al., tion with several iron-and manganese-depositing 1999). Moreover, the sheath material in cyano- bacteria, as well as a number of cyanobacteria bacteria has a different surface charge from that (e.g., Weckesser et al., 1988). It is interesting to of the underlying wall material (see section 3.2.3). note that in filamentous cyanobacteria mature This implies that the sheath may mask the cells are fixed within the sheath, yet binding charge characteristics of the wall, possibly by of the sheath to the underlying cell wall can exposing a hydrophobic or uncharged surface be temporarily disrupted to form hormogonia. to the external milieu. This has the effect ofme- These are short, motile (i.e., they can move diating physicochemical reactions between the cell and ions/solids in the external environment (Phoenix et al., 2002).

(c) S-layers

Regularly structured layers, also known as S- layers, are more highly organized than both EPS and sheaths (Koval, 1988). They consist of proteinaceous layers, with carbohydrates occa- sionally present as a minor component. The Cell regularity of their ordering is so great that the S-layers can be considered paracrystalline. S- layers are ubiquitous in nature and are found as part of the cell envelope in virtually every taxonomic group of both Bacteria and Archaea (e.g., Fig. 3.6). Although compositionally and structurally Sheath different from sheaths, both structures have 1 µm similar roles. Many S-layers are arranged so as to form aqueous channels 2–3 nm in diameter, just large enough to allow essential nutrients Figure 3.5 TEM image of Calothrix sp.. These to enter and metabolic wastes to exit, but cyanobacteria produce extremely thick sheaths small enough to exclude some external enzymes that can often double to triple the size of the cell. (e.g., lyzosymes that degrade peptidoglycan) (Courtesy of Vernon Phoenix.) from passing through to the underlying fabric. ITGC03 18/7/06 18:11 Page 100

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The Euryarchaeota (another archaeal phylum) S-layer show a wider range of wall types. Extreme halophiles (e.g., Halococcus sp.) have cell walls composed of complex heteropolysaccharides consisting of several sugars, uronic acids, and amino acids. Within the methanogens, there are a number of wall variations, with each genus having invented its own cell envelope. Some methanogenic genera, such as Methanospirillum, are surrounded by a proteinaceous sheath, while Methanococcus has a S-layer. Others have walls composed of a material similar to peptidoglycan, called pseudomurein, that instead contains N- acetylglucosamine and N-acetyltalosaminuronic 400 nm acid (e.g., Methanobacterium sp.). Interestingly, of all the Archaea subjected to the Gram stain, only the Methanobacterium genus stained Gram- Figure 3.6 TEM image of the cyanobacterium Synechococcus strain GL24 showing the S-layer positive since its pseudomurein wall remained as its outmost surface layer. (Courtesy of Susanne intact after treatment with ethanol (Beveridge Douglas.) and Schultze-Lam, 1996). Another wall variety, possessed by species of Methanosarcina, has a thick layer (up to 200 nm) containing a polymer (Stewart and Beveridge, 1980). S-layers can also called methanochondroitin that is made up of have different surface charges than that of the uronic acid, N-acetylgalactosamine, and minor underlying wall. At times this may lead to an amounts of glucose and mannose. uncharged surface that is unreactive to metal cations, while at other times the S-layers may 3.1.4 Eukaryotic cell walls bind considerable amounts of metals, even to the point where they nucleate mineral phases. The main structural components of all eukaryotic Once mineralized, S-layers can be shed from the cells are polysaccharides. Most algae have walls cell surface, allowing the cells to rid themselves consisting of a skeletal layer and an encompassing of minerals when the burden becomes excessive amorphous matrix (Fig. 3.7). The main skeletal (e.g., Schultze-Lam et al., 1992). material is cellulose, but it can be modified by the addition of other types of polysaccharides that 3.1.3 Archaeal cell walls give an individual species a unique chemical composition (Hunt, 1986). Three such polysac- The cell envelopes of Archaea are much more charides are mannans, pectins, and xylans. The variable than those in Bacteria (König, 1988). amorphous matrix typically consists of alginate, a In Crenarchaeota (an archaeal phylum), the most linear polymer of repeating units of carboxyl-rich common cell envelope is represented by a single uronic acids that can constitute a large propor- S-layer that is closely associated with the plasma tion of the dry weight of both brown and green membrane. There is no external cell wall, and all algae. Other amorphous components include extreme thermophiles rely entirely on this layer sulfated heteropolysaccharides called fucoidan. for maintaining cell viability. Other thermophiles, Accessory amorphous compounds include sulfated such as Thermoplasma sp., are supported just by galactans (e.g., agar, carrageenan, and porphyran). the plasma membrane. In some algae, the wall is additionally strengthened ITGC03 18/7/06 18:11 Page 101

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Amorphous matrix (alginate or fucoidan – algae) (chitosan – fungi)

Skeletal layer (cellulose – algae) (chitin and glucans – fungi) Cell wall

Plasma membrane (not to scale)

Figure 3.7 The main organic components comprising algal and fungal walls.

by the precipitation of calcium carbonate (as in on the solution pH. To chemically describe the coralline algae and coccolithophores) or silica acid–base properties of a microorganism, let us (as in diatoms). begin our examination with the straightforward The fungal wall is also bilayered, with an release (or dissociation) of a proton from a hypo- inner skeletal layer of chitin (a highly crystalline thetical surface functional group on a bacterium’s polymer of N-acetylglucosamine) and glucans, wall. This deprotonation process, which accur- while the outer layer is made up of amorphous ately describes the behavior of a number of compounds such as chitosan (a deacetylated functional groups associated with cell surfaces chitin). The secondary components of the wall (e.g., hydroxyl, carboxyl, sulfhydryl, and phos- include proteins, lipids, polyphosphates, phenols phate), leads to the formation of an organic (a compound with an −OH group attached to an anion, or ligand, and the concomitant release of aromatic ring), and melanin pigments, as well as H+. On the other hand, amino and amide groups various inorganic ions that make up part of the are neutral when deprotonated and positively wall-cementing matrix (Gadd, 1993). charged when protonated. The combined pro- tonation states of the functional groups on a cell surface largely determines its hydrophilic/ 3.2 Microbial surface charge hydrophobic characteristics at any given pH. In its most simplistic form, deprotonation can be expressed by the following equilibrium 3.2.1 Acid–base chemistry of reaction: microbial surfaces R–AH ←→ R–A− + H+ (3.1) One of the characteristic properties of many organic functional groups is that they are where R denotes the parent organic compound amphoteric, that is, they can each either bind to which each protonated ligand type, A, is or release protons (H+) into solution depending attached. The distribution of protonated and ITGC03 18/7/06 18:11 Page 102

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deprotonated ligands can be quantified with the are often used interchangeably. The larger the

corresponding mass action equation: value of Ka, the more dissociation of protons into solution (i.e., the stronger the acid). Import- − + = [R–A ][H ] antly, each functional group has its own Ka, and Keq (3.2) − [R–AH] based on equation (3.2), the pH at which [R–A ] and [R–AH] are equivalent is known as the =− < where Keq is the equilibrium constant for the pKa value, where pKa log10Ka. At pH pKa a > reversible reaction. Equilibrium constants for functional group is protonated and at pH pKa it ionization reactions are also called dissociation is deprotonated (Fig. 3.8a). − or acidity constants (Ka). [R–A ] and [R–AH] The ionization of functional groups in the represent the concentration of exposed deproton- cell wall provides an electrical charge at the ated and protonated species on the bacterium, bacterium’s surface that results in the formation respectively (in mol L−1), and [H+] represents the of an electric field surrounding the entire cell. activity of protons in solution. The term activity In dilute solutions the surface charge is estab- reflects the “effective concentration” of the lished solely by H+ exchange with the organic chemical species, and it is calculated by multi- ligands, whereas in more concentrated solutions plying the molar concentration by an activity the inherent surface charge can be modified coefficient based on ionic concentration (see by the adsorption of ions. For any given con- Langmuir, 1997 for details). In freshwater, the dition, the mean charge excess of a microbial −1 activity coefficient approaches 1, so for simpli- surface, [L]T (mol mg ), can be calculated as a city, the two terms, activity and concentration, function of pH from the difference between total

A 100% > + NH3 Figure 3.8 (A) Estimated speciation profile of the major functional groups

) − associated with bacterial cell walls + 3 >COOH >COO e.g., Shewanella putrefaciens (modified from Haas et al., 2001). (B) A hypothetical titration profile 50% illustrating charge excess (i.e., net surface charge) resulting from the > > − PO4H2 PO4H deprotonation of functional groups Percent speciation (in S. putrefaciens) and calculated as (relative to 100% NH the difference in proton concentration >NH 2 between the bacterial and blank titrations. The blank titrations are free 0% 46810 of functional groups and correspond to the dissociation of water. The site B 0.25 0.4 densities of distinct functional groups Carboxyl ( µ mol mg deprotonation (drawn as solid gray bars) are Blank titration modeled from charge excess and 0.3 0 Site density Phosphate plotted according to the pH at which − 1

deprotonation dry bacteria) half are deprotonated, the pKa value. − 0.2 dry bacteria) 0.25 Amino The various methods of modeling − 1 deprotonation site density and pKa distribution Charge excess −0.50 0.1 account for the fact that a single type of functional group deprotonates ( µ mol mg Carboxyl pKa Phosphate pKa Amino pKa 0 over a pH range. 46810 ITGC03 18/7/06 18:11 Page 103

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base or acid added to a microbial suspension base titration curves are generated regardless and the equilibrium H+ and OH− ion activities: of whether the experiments were initiated from acidic or alkaline conditions, and apparently − + − − + these extremes in pH do not cause changes in = Ca Cb [OH ] [H ] [L]T (3.3) the cell wall structure through saponification B of lipids or destruction of peptide bonds (e.g., Daughney and Fein, 1998). The components, Ca and Cb, are the concentra- − The charge difference between the microbial tions of acid and base added, respectively, [OH ] + − + surface and the proximal aqueous solution (at and [H ] are the number of moles of OH and H any pH) gives rise to an electrical potential that in the solution at the measured pH, respectively, strongly affects the concentration and spatial and B is the quantity of bacterial biomass (mg). − distribution of ions at the cell–water interface. If the density of cells, ρ (cells mg 1), and the − The electrical potential can be modeled using cell wall volume, ν (m3 cell 1), are known, then either electric double layer (EDL) theory, ana- the corresponding cell wall surface charge q − logous to the classical representation of mineral (mol m 3) can be calculated: surfaces (see Dzombak and Morel, 1990 for details) or Donnan exchange, which has been [L] F q = T (3.4) used to characterize the charge associated with ρν ion-penetrable cell walls (e.g., Yee et al., 2004). The EDL model describes the distribution In this reaction, F is the Faraday constant (the of charge on a surface, with an inner electrical amount of electric charge carried by one mole layer consisting of the surface proper (the surface ψ of electrons). When such calculations are done potential, 0), and an outer layer of oppositely over an entire pH range, a so-called acid–base charged ions, or counter-ions, fixed both directly titration curve is created that shows the pH to the surface (referred to as the Stern layer) or range over which some functional groups are more diffusely (referred to as the Gouy layer). chemically active and how the net surface charge Most of the surface charge is neutralized by of the cell varies with pH (Fig. 3.8b). the tightly bound (usually covalently) counter- The calculations above can also yield import- ions in the Stern layer, forming what is known ant information about the number of moles of as the inner-sphere complex. The remaining reactive surface sites, which reflects the buffer- charge is balanced by the Gouy layer whose con- ing capacity of the cell over a given pH range centration of counter-ions declines rapidly away (e.g., Fein et al., 1997). A large difference in from the solid surface. Outer-sphere complexes the total base or acid added and the free H+ ion are those in which the solute ions and surface activity indicates significant pH buffering and a species are attracted by electrostatic forces alone high concentration of surface functional groups. (Fig. 3.9). On a plot of charge excess this is shown by a The Donnan model describes the distribution steep slope. A small difference in the total base or of electrical potential within the wall matrix + ψ acid added and the free H ion activity indicates (the Donnan potential, D), which it treats as weak pH buffering and a low concentration of a porous structure with homogeneous cross- surface functional groups. This translates into a linked ionizable functional groups. It further gentle slope on the charge excess plot. Crucially, assumes that the transition between cell wall and the proton–bacteria reactions are fully reversible, solution is very thin compared to the thickness with the adsorption or desorption of protons of the wall, and that exchange reactions are reaching the same equilibrium concentration at strictly electrostatic, controlled by differences in any give pH value. Consequently, similar acid– the valency of the ions, not their size. ITGC03 18/7/06 18:11 Page 104

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A Cell wall the overall surface charge. This process is termed + electrophoresis, and the electrophoretic mobility + + + + + of a microbial suspension can be quantified by + + + Cation measuring their velocity in an electric field. This + + + Anion measurement can, in turn, be used to calculate + + + the zeta potential (ξ), using the Smoluchowski + + + equation below (Hunter, 2001):

Anionic ligands + + + + + + + ηµ Shear ξ = (3.5) plane ε ε 0

B The component “µ” is the electrophoretic mobility of a particle, ε and ε are the relative Cations 0 dielectric constants of the vacuum and solution, respectively, and η is the viscosity of the solu- tion. The zeta potential reflects the electrical

Concentration potential at the interfacial region (viewed as a Anions shear plane) separating the Stern layer, where cations are held tightly in place and move with Inner the bacterium, and the Gouy layer, where ions Diffuse Bulk layer are mobile (Wilson et al., 2001). A negative ξ layer (Guoy) solution (Stern) potential indicates that the bacterium is nega- C Donnan tively charged and migrates towards the positively ψΨ potential ( D) charged electrode in an electrical field, while a Surface positive ξ potential indicates the opposite. The Ψψ potential ( 0) greater the absolute value of the ξ potential, Zeta the greater is the charge density on the surface Potential potential (Blake et al., 1994). Electrophoretic mobility (ξ) measurements, however, underestimate the true surface potential because they actually measure Distance the adsorbed cations in the Stern layer as well as the cell’s anionic ligands. Unfortunately, tech- Figure 3.9 (A) Representation of the electrical niques are not presently available to measure double layer, with anions and cations surrounding the surface potential itself, so the closest we can the cell surface. (B) Change in concentration of measure is at the shear plane. cations and anions away from the negatively The electrophoretic mobility of different charged bacterial cell wall. (C) The electrical potential across the cell wall. (Modified from Blake species varies significantly with the elemental et al., 1994.) composition of the cell wall or extracellular layers. For instance, there is a direct correlation between the cell surface N/P atomic concentra- tion ratio and the cell’s electrostatic charge. By 3.2.2 Electrophoretic mobility studying various bacteria and fungi, Mozes et al. (1988) showed that the presence of phosphate When subjected to an electric field, micro- groups played a major role in determining the organisms move in a direction, and at a rate, anionic surface charges, while nitrogenous groups commensurate with the polarity and density of were linked to increased positive charge. ITGC03 18/7/06 18:11 Page 105

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Electrophoretic mobility is also pH depend- one microbial species versus another could yield ent because the activity of protons in solution widely different values simply because of subtle controls the ionization reactions of functional conformational variations within the wall macro- groups at the microbial surface (Ahimou et al., molecules. Furthermore, titration experiments 2001). This leads to the concept of isoelectric are only able to resolve those groups that con- point, which is defined as the pH value where tribute significant amounts of protons to solution. net surface charge equals zero. The isoelectric Minor groups are simply undetectable with the point can be estimated from acid–base titrations, resolution of current techniques. Therefore, it but it can also be directly measured with elec- is important to keep in mind that the model- trophoretic mobility experiments because at the derived binding sites do not directly represent isoelectric point microorganisms do not exhibit the functional groups of the cell surface; their motion in an electric field. The isoelectric point identity can only be inferred by comparison of the

of bacterial walls is typically between pH 2 functional group pKa values with pKa values of and 4, and no fundamental differences exist in model compounds. Unequivocal identification of the isoelectric behavior of Gram-positive and the types of functional groups responsible for acid– Gram-negative cells (Harden and Harris, 1953). base buffering can be obtained by spectroscopic This means that at low pH, when the surface techniques, such as Fourier transform infrared functional groups are fully protonated, bacteria spectroscopy (FTIR), or gas/liquid chromato- are either neutral or positively charged, the graphy of cell wall extracts. latter being the result of a cell possessing Despite the inherent variability, acid–base abundant amino groups. Meanwhile, at the modeling of bacteria has clearly shown that growth pH of most bacteria, cells inherently dis- they have a quantifiable and characteristic geo- play a net negative charge and the magnitude chemical reactivity that reflects a suite of func- of negativity increases with higher pH values tional groups in their outermost structures. (e.g., reaction (3.1)). Therefore, under low pH One method of modeling is to constrain the

conditions, most bacterial surfaces behave hydro- number of pKa values to fit the titration data. phobically, and become increasingly hydrophilic In experiments with intact B. subtilis cells, with increasing pH. Fein et al. (1997) demonstrated that a three-

pKa model could effectively quantify the buffer- 3.2.3 Chemical equilibrium models ing effect provided by the cell wall surfaces. For instance, at low pH the deprotonation of One of the major challenges facing researchers carboxyl groups could accurately predict the today is how to interpret acid–base titration buffering capacity of the biomass from pH 2 to 6

data in terms of cell wall biochemistry. Ascribing (reaction (3.6)). A two-pKa model, including pKa values from a titration curve to specific func- phosphate groups, accurately mimicked the titra- tional groups is not so clear-cut because there tion curves up to pH 7.5 (reaction (3.7)). At pH

can be considerable variation in pKa values for values above 7.5, a three-pKa model yielded an the same functional group. This occurs because excellent fit to experimental data. Although the magnitude of the dissociation constant is the authors inferred that the third deprotona- controlled by the structure of the molecule to tion reaction involved the loss of protons by which it is attached (see Martell and Smith, hydroxyl groups (reaction (3.8)), those sites were + 1977 for details). Consequently, a single carboxyl more likely to be cationic amino groups NH3 group in two different organic acids will have because they deprotonate at pH values above

different pKa values, as will an organic acid with 8–11 (reaction (3.9)), while hydroxyl groups multiple carboxylic groups. As might then be tend to deprotonate at pH values above 10

expected, the pKa for a carboxyl group on (Hunt, 1986). ITGC03 18/7/06 18:11 Page 106

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Table 3.1 Relative total concentration of reactive ligands amongst various bacteria.

Species Total ligand Ionic strength Reference concentration (mol L−1) (µmol mg−1 dry bacteria)

Rhodococcus erythropolis 0.93 0.01–1.0 Plette et al., 1995 Bacillus subtilis 0.22 0.3 Fein et al., 1997 Bacillus subtilis 0.50 0.01–0.025 Cox et al., 1999 Bacillus subtilis 1.60 0.001–0.1 Yee et al., 2004 Bacillus cereus 2.29 0.01 He and Tebo, 1998 Bacillus licheniformis 0.29 0.1 Daughney et al., 1998 Shewanella putrefaciens 1.77 0.1 Sokolov et al., 2001 Shewanella putrefaciens 0.08 0.1 Haas et al., 2001 Calothrix sp. 0.80 0.01 Phoenix et al., 2002

+ − → − + R-COOH OH R-COO H2O (3.6) phosphodiesters, and very basic sites, such as the hydroxyl groups, could not be observed in + − → − + R-PO4H2 OH R-PO4H H2O (3.7) the titration range of the experiment. Their − − results instead yielded a total ligand density of R-OH + OH → R-O + H O (3.8) 2 0.50 µmol mg−1 of bacteria. + + − → + R-NH3 OH R-NH2 H2O (3.9) Titration studies have been performed on a wide range of bacteria. What their collective When the total site densities were calculated results demonstrate is that the relative total con- over the entire pH range of their experiments, centrations of surface functional groups can vary the distribution of proposed functional groups by over an order of magnitude amongst a range were as follows: 0.12 µmol of carboxyl groups/mg of bacteria, and even within the same species bacteria; 0.04 µmol phosphate groups/mg bac- (Table 3.1), and that the acid–base behavior teria; and 0.06 µmol hydroxyl groups/mg bacteria, of the surface ligands are weakly affected by making a total concentration of 0.22 µmol mg−1 solution ionic strength. So, the question is, aside

of bacteria (dry weight). from pKa variations due to coordination of any Other models fix the acidity constants and given functional group, what else might be caus- determine the minimum number of ligand sites ing variations in the magnitude of the surface required to achieve a good fit to the titration charge? As discussed above, differences in surface data. For instance, Cox et al. (1999) used this charge between different species can be ascribed technique to resolve five proton binding sites to the types and densities of exposed functional on the cell walls of B. subtilis: two types of groups, i.e., the Gram-positive wall of B. subtilis

carboxyl sites at low pKa values, phosphoryl versus the Gram-negative wall of E. coli. Vari- sites at circumneutral pKa values, and two sites ations amongst the same species, however, are with high pKa values, which were attributed to most likely a function of either subtle changes either amino or phenol (pKa 8–12) groups. Very in: (i) nutrient conditions; or (ii) population acidic sites, such as some carboxylic acids and growth phase: ITGC03 18/7/06 18:11 Page 107

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1 An individual bacterium has the means to alter its extracellular layers. Microorganisms with EPS, surface chemistry to compensate for the chemical sheaths, or S-layers will have a more complex composition of the aqueous environment. Take for charge distribution because those layers differ example B. subtilis, it can change the secondary compositionally from that of the underlying polymers associated with the peptidoglycan in cell wall. This was highlighted in a recent study response to the levels of dissolved inorganic phos- of isolated sheaths and intact filaments of the phate. When teichoic acids are produced, phos- phoryl groups (pK between 5.6 and 7.2) and cyanobacterium, Calothrix sp. (Phoenix et al., a 2002). Electrophoretic mobility measurements phosphodiester groups (pKa between 3.2 and 3.5) are most abundant, while a cell loaded with of cell walls showed completely different profiles teichuronic acids has instead an abundant supply of to those of isolated sheath material (Fig. 3.10).

carboxyl groups (pKa between 4 and 6). At other While the wall was characterized by a net nega- times, when the cells may need to behave more tive surface charge, the sheath’s charge was found hydrophobically, they can strategically place to be near neutral, indicating that the domin- positively charged functional groups into the wall ant electronegative carboxyl and electropositive that markedly reduce the net negative surface amino groups must occur in approximately equal charge (Beveridge et al., 1982). Similarly, Gram- negative bacteria, such as Shewanella putrefaciens, proportions. Significantly, this study confirmed have variable sugar arrangements in their LPS which that under normal growth conditions, some spe- not only impact the carboxyl:amino ratios, but also cies possess a dual-layered surface charge, i.e., a affect the cross-linking of functional groups and highly electronegative cell wall surrounded by limit which remain unoccupied, and hence may an electroneutral sheath. be ionizable (Moule and Wilkinson, 1989). 50 Isolated 2 Daughney et al. (2001) have observed that expon- sheath entially growing cells of B. subtilis possess four times more carboxyl sites, twice as many phosphate sites, 40 and 1.5 times as many amino sites (per unit weight) as cells in either the stationary or sporulated phase. It would appear that the higher nutrient availability 30 prompts exponential phase cells to modify their Sheath-wall cell wall to be more efficient at metal sequestra- composite tion (see next section), whereas diminished nutrient 20 availability causes the cells to return to perhaps Wall their “default” setting. During starvation, the effects

Number of occurrences (%) 10 on cell surface reactivity become even more pro- nounced. Frequently a large reduction in cell volume and a tendency towards increased hydrophobicity 0 occurs, i.e., a reduction in carboxyl and phos- –4 –3.5–3 –2.5 –2 –1.501 –0.5 0.5 1 1.5 phate groups (Kjelleberg and Hermansson, 1984). µ –1 –1 –1 Associated with growth phase is the cell’s meta- Electrophoretic mobility ( m s V cm ) bolic state. Actively respiring cells pump protons into their wall matrix during respiration. These, in Figure 3.10 Electrophoretic mobility turn, can protonate the surface functional groups, measurements performed on Calothrix sp. rendering them electrically neutral (Koch, 1986). By at pH 5.5. The very electronegative peak at − µ −1 −1 −1 contrast, dead cells no longer produce a proton 2.5 ms V cm is characteristic of exposed gradient, and for that reason, they are likely to be cell wall material with deprotonated carboxyl and + more anionic (Urrutia et al.,1992). phosphate groups. The peak around 0.1 is isolated sheath material, made up predominantly One other point needs emphasizing – all those of polysaccharides. The broader peak at around − studies on cell wall material have not taken into 0.3 is likely a composite of wall and sheath account the fact that most benthic bacteria possess material. (Modified from Phoenix et al., 2002.) ITGC03 18/7/06 18:11 Page 108

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addressed where metal cations bind to bacterial 3.3 Passive metal adsorption cell surfaces and then examine how cell surface composition can influence the partitioning of As the chemical equilibrium models show, certain metals from multi-elemental solutions, as bacteria are not inert objects immune to the encountered in the natural environment. physicochemical conditions in the environ- ment surrounding them. On the contrary, all 3.3.1 Metal adsorption to bacteria bacteria have low isoelectric points, meaning that they should interact with metal cations In their pioneering work on metal binding to and have them intimately associated with B. subtilis, Beveridge and Murray (1976) showed their surfaces. Considering their ubiquity in that when the cell walls were chemically separ- the surface environment, their high population ated and suspended in a supersaturated salt-rich densities wherever suitable sources of nutrition solution, so much metal was bound that they exist, and their characteristically large surface formed dense aggregates. Transition metals, in area to volume ratios, it is easy to understand particular, impart such strong electron-scattering why they are very important agents in metal power that some of them have subsequently sequestration. been used as contrasting agents for electron Some bound metals serve the purpose of microscopy. Indeed, many of the advances made stabilizing the negative charges of the anionic on bacterial ultrastructure over the past three functional groups, and thus are relatively “fixed” decades were made possible by metal staining into place (see section 3.4.1). Others metals are and visualization of biological thin sections much more exchangeable and merely provide a under the TEM (e.g., Fig. 3.11). Alkali and alka- temporary positive charge to counter the nega- line earth metals that were freely soluble in water tive charge induced by the deprotonation of (e.g., Na+, Mg2+, etc.) can also be sequestered the cell’s surface functional groups (Carstensen from solution, but then tend to yield diffusely and Marquis, 1968). The strength of the metal– stained walls. Interestingly, alteration of the ligand bond is given by the surface complexation/ charge density within the wall fabric due to the

binding constant (KM), where M refers to the introduction of different metal cations elicits specific metal of interest. The greater its surface a dimensional response in the peptidoglycan complex formation constant, the less likely a strands. Thus, the cell wall can be made to shrink metal cation will be desorbed into solution. Metal or swell according to the metal staining agent cation sorption is also directly affected by pH, used to give it contrast (Beveridge and Murray, which dictates metal partitioning (or speciation) 1979). within, and between, soluble and solid phases, To reveal the functional groups in the wall to and hence controls its mobility, reactivity, and which metal cations react, a variety of chemical toxicity in aquatic environments. Of particu- treatments can be used to modify or remove elec- lar importance here is the hydrolysis constant, tronegative and electropositive groups. Anionic which measures the tendency of a metal cation carboxyl groups can be neutralized or converted to react with water and form a hydroxide phase, into electropositive sites by treatment with

e.g., Fe(OH)3. water-soluble carbodiimides or ethylenediamide, There have been literally hundreds of studies respectively; teichoic acids can be removed by discussing the metal sorption properties of micro- dilute base; while amino groups can be made organisms (see Ledin, 1999 for review). It would anionic by replacing them with succinyl groups be impossible to cover more than a fraction of or removed by deamination using nitrous acid them here, so instead, the goals of the following (Doyle, 1989). When carboxyl groups in the section are to highlight a few studies that have peptidoglycan of B. subtilis are chemically ITGC03 18/7/06 18:12 Page 109

CELL SURFACE REACTIVITY AND METAL SORPTION 109

number of sites that can bind metals, while decreasing the number of cationic amino groups leads to an increase in metals bound (Doyle et al., 1980). Collectively, these experiments demonstrate that with B. subtilis walls, carboxyl groups are the most electronegative sites, and that the bulk of the binding capacity in Gram- positive bacteria remains associated with the peptidoglycan. Another important feature governing how much metal is bound is the degree of interstrand cross-linking in peptidoglycan. Cross-linking occurs by means of peptide bond formation, which involves the loss of two charged groups for every bond formed. The more cross-linking, the greater the physical compactness of the cell wall, and hence the fewer cations that are required to neutralize the excess anionic ligands in the wall matrix (Marquis et al., 1976). What’s more, metal probes, such as polycationic ferritin (PCF), have shown an inherent heterogeneity in charge distribution, with PCF binding pre- 600 nm ferentially to the negatively charged peptide chains and teichoic acids on the outer wall sur- face of B. subtilis (Sonnenfeld et al., 1985a). The Figure 3.11 The routine use of transition positioning of these polymers likely represents metals to stain cell tissue for transmission electron a favored orientation that exposes carboxyl and microscopy works because the cell surface phosphate groups to the external aqueous envir- functional groups are anionic and they react onment where cations can be scavenged. PCF electrostatically with multivalent metal cations. also tends to label the polar ends of the walls, This is an unidentified species growing at a hot spring in Kenya. Notice how staining with uranyl further implying that the tips of the cells are acetate and lead citrate has revealed many of more electronegative than other sites on the wall the structural details of the cells, including the (Sonnenfeld et al., 1985b). ribosomes (arrow). Bacillus licheniformis walls are unlike those of B. subtilis, in that they contain up to 26% teichuronic acid and 52% teichoic acid, thus neutralized, substantial reduction in the amounts having much less peptidoglycan. In similar metal of metals sorbed occurs (e.g., Beveridge and binding studies as above, the walls bind an order Murray, 1980). Similarly, when teichoic acids of magnitude less metal (e.g., Beveridge et al., are extracted to ascertain how much metal is 1982). However, unlike B. subtilis, the phosphate bound to the peptidoglycan only, metal binding groups in the teichoic acid and carboxyl groups decreases in all instances, yet for most metals in teichuronic acid play a greater role in metal not to the same extent as by the loss of carboxyl adsorption. Therefore, the overall metal binding groups (Matthews et al., 1979). The introduc- ability of these particular Gram-positive cells is tion of positive charges into the B. subtilis wall determined by the amount and type of secondary also results in a marked decrease in the total polymers present. The fact that both secondary ITGC03 18/7/06 18:12 Page 110

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polymers bind metals implies that the bacterium maintains a tight control on the negative:positive charge ratio, regardless of the aqueous conditions in which they grow. When the wall material of Gram-negative cells, such as E. coli, are subjected to metal-rich solutions, it becomes readily evident that they do not adsorb as much metal from solution as do their Gram-positive counterparts. Typically the quantities are less than 10% (e.g., Beveridge and Koval, 1981). Unlike the Gram-positive bacteria, there is a bilayered distribution of 600 nm metals associated with the outer membrane. This pattern stems from the higher phosphate:lipid ratio of the LPS compared to the phospholipids, Figure 3.12 TEM image of two encapsulated ensuring that the outer face is more electro- bacteria that are naturally iron-stained from growing in Fe-rich hydrothermal fluids. negative and subsequently can bind more metal (Ferris and Beveridge, 1984). Although carboxyl groups are also present in the LPS core, only one-third of the groups are available for metal (e.g., Rudd et al., 1983). In fact, species pro- binding; the others being cross-linked to the ducing capsules can tolerate higher metal con- cationic amino groups (Ferris and Beveridge, centrations than those that do not, and it has 1986a). The peptidoglycan in E. coli is chemic- been shown that the proportion of encapsulated ally similar to B. subtilis, and even though it is bacteria increases in metal-polluted sediment, only a monolayer, it reacts more strongly to some whereas mutants that cannot produce capsules metals than the outer membrane. As in the die off (Aislabie and Loutit, 1986). Interestingly, Gram-positive bacteria, this feature is attributed many isolates from metal-contaminated sediment to the availability of carboxyl groups (e.g., Hoyle lose their capsules upon subculture in metal-free and Beveridge, 1984). media, suggesting that the role of capsular pro- Many Gram-positive and Gram-negative duction may be linked to protection against metal bacteria also produce EPS. Due to their hydrated toxicity. nature, dissolved metals can freely diffuse Irrespective of the bacteria studied, what throughout the extracellular layers, binding to has repeatedly been shown in metal binding the anionic carboxyl groups of uronic acids and studies is that there is no apparent stoichiometry the neutrally charged hydroxyl groups of sugars between the quantity of metals that bind to (Geesey and Jang, 1989). Under metal-deficient cell walls and the amount of anionic ligands. In conditions in the growth media or in naturally some instances, large metallic deposits line the dilute solutions, capsules can appear diffuse and wall, while at other times so much metal may be extensive, whereas higher metal concentrations fixed to the cell surface that it forms a distinct can lead to flocculation, and even the precipita- mineral phase. This led Beveridge and Murray tion of metal–capsule composites (e.g., Fig. 3.12). (1976) to originally propose a two-step mech- By possessing a large and reactive surface area, it anism for the metal adsorption process; the is thus not unexpected that a number of studies first step in time is an electrostatic interaction have also documented that encapsulated bacteria between the metal cations and the anionic ligands bind more metals than nonencapsulated varieties in the cell wall. This interaction then acts as ITGC03 18/7/06 18:12 Page 111

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a nucleation site for the deposition of more 107 ] O metal cations from solution, potentially leading 2 H to biomineralization (see Chapter 4). The size 106 of the deposit depends on a number of variables, ]/[M Solute-rich including the concentration of the metals in algae 105 solution and the amount of time through which the reactions proceed. If sufficient time exists, the 104 metal/mineral product grows in size within the Solute-poor intermolecular spaces of the wall fabric or on 103 the outer surface until it is either physically con- Enrichment factor [M strained by the wall polymers or the saturation 102 state diminishes. The end result is a bacterial Ti Zr Cr V AgMnCo Sn As Ni Fe U Pb MoCu Zn CdHg wall that contains copious amounts of metal, often approaching the mass of the bacterial cell itself Figure 3.13 Comparison of metal enrichments (Beveridge, 1984). in filamentous green algae from two contrasting rivers, one solute-rich and the other solute-deficient. (Modified from Konhauser et al., 1993.) 3.3.2 Metal adsorption to eukaryotes from a solute-deficient river (Konhauser and Algae possess a number of functional groups that Fyfe, 1993). Plotting the concentration of metals deprotonate under normal growth conditions. As sorbed to algae versus their dissolved concentra- a result, algal populations can sequester a wide tions further showed algal biomass characterized range of metals, commonly with uptake values in by enrichments of between 102 and 107 for the excess of 100 mg of metal g−1 biomass dry weight metals studied (Fig. 3.13). These patterns reflect (Volesky and Holan, 1995). The functional the strong complexing ability of the reactive groups of greatest importance are the carboxyls ligands, leading to the natural conclusion that associated with uronic acids in alginate because metal concentrations within the algae are a they are appropriately spaced to cross-link and direct reflection of availability. For that reason, neutralize a number of multivalent metals (e.g., algal populations in metal-rich rivers will have Majidi et al., 1990). Consequently, the removal correspondingly high metal accumulations, an of alginate from algal biomass can cause signific- observation that might have great merit when ant decreases in metal binding capacity. Many prospecting for mineral deposits. green algae also contain sulfate esters in cellulose, The capacity of fungi and yeasts to bind

that, because of their low pKa values (between 2.5 metals has also been extensively explored (see and 1), can facilitate metal sorption under very Gadd and Sayer, 2000 for review). The import- acidic conditions (Crist et al., 1992). ance of chitin and chitosan in metal binding For a number of algae, biosorption is clearly has been demonstrated by the observation that a passive process, accumulating available metals their removal from biomass results in a signific- irrespective of whether they serve a physiolog- ant decrease in metal sorption (Galun et al., ical role or not. This statement is supported by a 1983). Within these polymers, protonated amino study comparing biosorption by green benthic groups are strongly linked to the adsorption of algae in two chemically dissimilar river systems, anionic species (e.g., Tsezos, 1986), while phos- where it was observed that metal sorption by phate and carboxyl groups are important in the biomass in a solute-rich river was often greater adsorption of cationic species (e.g., Tobin et al., by an order of magnitude relative to biomass 1990). The carboxyl groups in particular appear ITGC03 18/7/06 18:12 Page 112

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to be responsible for metal accumulation in Despite the different ligand-metal affinities, the the biomass from Aspergillus niger, since 90% cell surface can still be viewed as being largely of the metal binding capacity was irretrievably “non-specific”, in that adsorbed metals can be lost when these groups were chemically modified desorbed as geochemical conditions change (Akthar et al., 1996). The secondary com- (Ledin et al., 1997). In this regard, metal-ligand ponents of fungal cells, such as the phenols and interactions are reliant upon thermodynamics, melanins, are also effective at metal sorption just the same as inorganic systems. A particularly (e.g., Saiz-Jimenez and Shafizadeh, 1984; Caesar- compelling example of nonspecificity comes Tonthat et al., 1995). from Fowle and Fein (1999) who demonstrated that in mixed metal experiments with B. subtilis 3.3.3 Metal cation partitioning and B. licheniformis, the cell walls consistently had a higher affinity for Cd2+, even though Ca2+ As the studies above show, binding of cations concentrations were two orders of magnitude to a microbial surface is largely an electrostatic higher. Moreover, Ca is an important element phenomenon. However, the structural and com- for cell structure and Cd is toxic, but the sorp- positional variability of the wall or extracellular tion behaviors of these two elements were not layers, as well as the unique physicochemical manifestations of the different effects they had properties of each element, adds a level of com- on the cell. Instead, they reflected the chemical plexity to the overall process. In other words, properties of the metal cations (in this context protons and each different cation should be also known as Lewis acids) and those of the capable of interacting in a distinctive way with oxygen-, nitrogen-, and sulfur-containing ligands the reactive ligands on a cell’s surface. that reside within the cell wall (known as Lewis This realization has led to a number of metal bases). These properties are largely understood, binding studies that have compared the relative and given sufficient information about the affinities of protons and various cations for the environment in which a microorganism is grow- exposed functional groups of different micro- ing, it may be possible to extrapolate and predict organisms, by techniques that involve displacing metal binding patterns on a cell surface. one by another. Those studies have highlighted The supply of cations to the cell depends on two very important points, the first being that several external factors, such as their aqueous metals and protons compete for the same surface concentrations or the presence of co-ions and sites. As solution pH decreases, the functional other organic anions that can complex the groups become protonated, displacing loosely metals in solution and affect their bioavail- bound metal cations. Conversely, at circum- ability. Once at the cell periphery, competition neutral pH, the functional groups deprotonate between those metals for organic ligands will and electrostatic interactions with metal cations ensue. To fully understand metal adsorption pro- increasingly takes place (e.g., Crist et al., 1981). cesses, we must consider both the ionic forces, The second finding is that some metals pre- which are the initial electrostatic attractions ferentially bind to different ligands in the cell between a metal cation and the organic ligands, wall, but most importantly, they are not equally and the subsequent covalent forces that arise exchangeable. For instance, trivalent (e.g., La3+, from electron sharing across a metal cation– Fe3+) and divalent metal cations (e.g., Ca2+, Mg2+) ligand molecular orbital (see Williams, 1981; are strongly bound to the wall of B. subtilis, while Hughes and Poole, 1989; Stone, 1997 for monovalent cations (e.g., Na+, K+) are easily lost details). Some of the most important factors in competition with those metals for binding that influence metal binding to cells are briefly sites (Beveridge and Murray, 1976). examined below: ITGC03 18/7/06 18:12 Page 113

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1 Ionic potential – Cations in solution vary in their also most effective when either the highest occupied propensity to coordinate water molecules as a molecular orbital or the lowest unoccupied mole- function of their charge density, that being the cular orbital is a d-orbital. These factors account relationship between the charge of the nucleus for why transition metals (e.g., V, Cr, Mn, Fe, Co, (z) and its radius (r). Most monovalent, and Ni, Cu, Zn, and Mo) exhibit greater covalent bond- many divalent, cations remain unhydrated or form ing than lighter metals (e.g., Al) and metal cations to aquoanions. Contrastingly, trivalent metals displace the left on the periodic table (e.g., the alkali and protons from coordinating water molecules. They alkaline earth metals). Generally, cations that are also displace protons from functional groups with bound only weakly through electrostatic attraction, O− ligands. Thus, the affinity of a metal cation for an like Na+, are effective in competing only with other organic ligand increases dramatically in going from weakly bound ions. This also explains why protons, a +I metal cation (e.g., Na+) to a +III metal cation which are mainly covalently bound, are only dis- (e.g., Fe3+). placed during transition metal uptake, and not during light metal uptake (Crist et al., 1981). 2 Ligand spacing/stereochemistry – Monovalent cations are generally preferred by isolated or widely spaced sites, where they replace single pro- When we consider the log K values for divalent tons on individual ligand sites to neutralize the metal cations and a particular organic ligand, negative charge (e.g., phosphates in phospholipids). we note that as we move from left to right in Under such conditions, a divalent cation may not the periodic table, the values increase, reaching be able to satisfy the two distant sites of negative a maximum with Cu2+ (Fig. 3.14). This trend, charge. By contrast, multivalent cations are pre- called the “Irving–Williams Series,” is observed ferred by closely opposed sites (e.g., carboxyl groups of LPS), where either the ligands are unable with practically all oxygen- and nitrogen-bearing to accommodate two monovalent cations or where ligands (Williams, 1953). Because of their greater greater steric stability is achieved by increased charge and smaller radii, the trivalent metals coordination between the metal and two ligand form even stronger bonds with organic ligands sites. In EPS, the ability of the macromolecules (Stone, 1997). to accommodate intra- and intermolecular cation Although trivalent cations typically have bridging will dictate which metals are prefer- higher affinities for wall material, in solutions entially sorbed. The latter is the principle behind 2+ 2+ chelation, the binding of a metal cation to two where divalent cations, such as Ca and Mg , or more coordinating anionic sites in the same are several orders of magnitude more abundant, biomolecule (known as bidentate or multidentate the outer face of the cell wall would be pre- ligand bonding, respectively). dominantly in the Ca-Mg form. The ubiquitous association of Fe with cell walls also stems from 3 Ligand type – Different metals are favored by its greater concentration in natural waters com- different ligands. Oxygen atoms have a distinct pared to other trace metals (e.g., Cu). It thus affinity for the alkali and alkaline earth metals (e.g., K+, Mg2+, Ca2+) and some transition metals appears that cell wall material can show different (e.g., Fe3+), while nitrogen and sulfur atoms pre- metal binding patterns under different geochem- ferentially bind a number of transition metals ical conditions. On the one hand, it displays dis- (e.g., Ni2+, Co2+, Cu2+, Zn2+, Cd2+, Fe2+). tinct preferentiality in binding one metal from a range of competing cations. On the other hand, 4 Covalent bonding – Once a metal cation is it reacts to soluble ions as if it were an open ion adsorbed, the ability to form covalent bonds with exchange resin. In this regard, microorganisms the ligand is important for complex stability. In bind the cations that are in highest concentration general, cations increasingly form inner-sphere (and stronger) complexes with a given ligand as and, accordingly, it is not surprising that micro- the difference in electronegativity between the two organisms are considered ideal metal scavengers decreases (see Faure, 1988). Covalent bonding is for bioremediation purposes (see section 3.7.1). ITGC03 18/7/06 18:12 Page 114

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12 (?)

10 Cysteine (N/S)

8

log K Figure 3.14 Complexation of divalent metals to a number 6 of organic compounds. The Ethylenediamine adsorption of the metals follows Oxalate (O) the “Irving–Williams Series.” 4 Note the preferential adsorption Malonate (O) + of Cu2 , irrespective of the Inferred donor ligands (in brackets). 2 (Data from Williams, 1953; + + + + + + Stone, 1997.) Mn2 Fe2 Co2 Ni2 Cu2 Zn2

3.3.4 Competition with anions have proven to be so effective as gold scavengers that they have been employed in the elution Studies comparing the biosorption of metals onto (re-solubilization) of the metal from biomass (see microbial surfaces have generally been limited to section 3.7.2). Other organic compounds used to systems involving dissolved cations only. How- bind gold (in order of their complexation capa- ever, in nature dissolved inorganic and organic city) are thiourea > cyanide > mercaptoethanol; anions balance the positive charges of metal each has been shown more effective than the cations, often leading to dissolved complexes or inorganic anions above (Greene et al., 1986). mineral precipitation. Such reactions impact metal Many microorganisms also produce extracellular biosorption in two ways. First, the metal–anion organic exudates (see below), with metal com- complexes typically have lower affinity for the bio- plexation constants equal to, or stronger, than mass than the free cation does, and subsequently, the adsorption constants associated with the cell they bind less strongly (e.g., Shuttleworth and surface functional groups (e.g., Santana-Casiano Unz, 1993). Second, some anions bind metals et al., 1995). more strongly than biomass, making the metals completely unavailable for biosorption. For example, in studies with dead biomass of Sargassum 3.4 Active metal adsorption natans, a brown marine alga, the presence of − 3− NO3 and PO4 suppressed the amount of gold It would be mistaken to view bacterial metal uptake capacity at acid pH values (Kuyucak and uptake simply as a passive process in which Volesky, 1989a), while high concentrations of sorption occurs as a consequence of cells grow- halide ions (e.g., Cl−, Br−, and I−) diminished gold ing in concentrated solutions where metals adsorption to the green alga Chlorella vulgaris in abound. Instead, microorganisms require a vari- the order consistent with their reactivity towards ety of metals to fulfill internal and external cell Au(III) (Greene et al., 1986). functions (see Silver, 1996, for details). It is, Organic ligands can have an even more pro- therefore, not unusual for them to manipulate nounced effect. In the case of gold, commercially the type and abundance of their organic func- produced organic compounds, such as EDTA, tional groups to retain those metals specifically ITGC03 18/7/06 18:12 Page 115

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required for structural or operative integrity, and ionic strength the conductivity of the bacterial to subsequently regulate their uptake rates to cell wall is dominated by ions confined to the maintain intracellular concentrations at optimal Stern layer, but at high ionic strength, the walls levels for growth and metabolism. At other become saturated with exogenous salts and they times, microorganisms may need to immobilize obtain a conductivity that is roughly propor- toxic metals away from the cell periphery, and tional to the surrounding aqueous environment as such, they produce and expel specific metal (Carstensen et al., 1965). binding chelates into the bulk fluid phase. In The presence of metal cations in the wall matrix either situation, the metal–ligand complexes influences its stability in a number of ways: formed can often be so tenacious that those bound metals are not easily displaced by other 1 Surface wettability – In Gram-negative cells, such as metal ions (e.g., Hoyle and Beveridge, 1983). E. coli, the addition of Mg2+ makes the surfaces Quite clearly, the cell surface is a dynamic layer much more hydrophobic than if they only bound + + that continuously interacts with those metal Ca2 or Na (Ferris, 1989). Along similar lines, if the cations in its immediate vicinity. LPS is removed from the surface, there is an overall increase in hydrophobicity since the outermost layer is inherently electronegative. The degree of 3.4.1 Surface stability requirements hydrophobicity is also related to: (i) the quantity of metals bound to the outer surface of the cell; Metal adsorption onto a cell’s surface has an and (ii) the charge density, and hence hydration, important bearing on the its dielectric properties of the cations. If the bacterium can control surface because those metal cations have an effect on the wettability, then numerous benefits ensue. For conduction of low frequency electric currents. instance, a high degree of hydrophobicity can help Their conductivity can actually be measured by the bacterium contact unwettable surfaces and then stick to them. This is particularly useful in micro- the following equation: colony formation on inert solid surfaces, in the utilization of apolar hydrocarbons for nutrition, σ = w w + 0 w 2 1/2 w cf u [1 2c /cf ) ] (3.10) and in the exclusion of solvated particles such as bacteriophages from adhering onto the cell σ w (Beveridge, 1989b). where w is the cell wall conductivity, cf is w the fixed charge concentration in the wall, u 2 Surface stability – In Gram-positive bacteria, a 0 is the mobility of ions in the wall, and c is the single Mg2+ cation can cross-link the anionic ligands environmental ion concentration (Carstensen between two teichoic acid molecules. In this regard, and Marquis, 1968). This equation indicates it eliminates the repulsive anionic charges between which metals serve as counter-ions for fixed adjacent molecules, giving rise to more dense anionic charges (i.e., those likely to have some but stable structures (Doyle et al., 1974). Under sufficiently alkaline conditions (where the phos- requirement), and which metals remain mobile. + phoryl group is completely deprotonated), Mg2 By measuring the electrical conductance of may even stabilize both O− ligands on the same various wall–cation combinations, it appears, teichoic acid molecule. The peptidoglycan also + for example, that K is less free to move around binds Mg2+ cations, with the peptide chains bending + + in the wall than Na , Mg2 appears to be still in such a way that the carboxyl group of one more tightly bound, while protons are essentially peptide is only the diameter of one Mg2+ cation immobile (Marquis et al., 1976). These results away from the carboxyl group of another peptide correlate well with the affinity series discussed (Fig. 3.15). Studies have also shown that decreas- + ing the number of cationic amino groups in peptido- earlier where it was pointed out that H is bound glycan, through chemical treatment, leads to an more strongly than alkali and alkaline earth increase in metals bound (Doyle et al., 1980). metals at low pH values. The conductivity is also This suggests that the cationic amino groups must directly related to solution ionic strength. At low function normally as competitive counter-ions, and ITGC03 18/7/06 18:12 Page 116

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to some extent neutralize the negative charges wall (Doyle and Koch, 1987). Thus, if the proton associated with the carboxyl or phosphate groups motive force becomes dissipated by the death of of the cell wall (Marquis, 1968). In Gram-negative the cell, then the bound protons in the wall would bacteria, Ca2+ functions in a similar manner to systematically be lost and the autolytic enzymes neutralize the numerous electronegative charges would become activated. This results in the of the LPS, thereby bridging adjacent molecules of uncontrolled breakdown of cell wall material, and LPS together and anchoring the outer membrane to eventually the exposure of cytoplasmic material to the underlying peptidoglycan layer. In experiments the external aqueous environment (Jollife et al., where calcium is removed from the membrane, an 1981). Experiments, however, have documented increase in the electrostatic repulsion between the that the addition of metal cations, such as Fe(III), to constituent anionic ligands occurs, thereby limit- lysed cells limits cellular degradation. This increases ing how close the individual components of the the preservation potential of cellular remains, and membrane can approach one another (Ferris and might explain why some organic remains are Beveridge, 1986b). Subsequently, the LPS is forced retained in the geological rock record (e.g., Ferris to adopt a tighter curvature, causing it to bleb and et al., 1988). become sloughed off. Calcium is also required for the proper assembly of S-layers in a number of species, and in calcium-deficient growth media, no 3.4.2 Metal binding to microbial surface layers are formed (Smit, 1987). exudates

3 De-activation of autolysins – Autolysins are enzymes The production of microbial exudates is of global that break down the cross-links in peptidoglycan significance in terms of trace metal cycling. Al- so that the cell wall can be restructured during growth and cell division. At the growth pH of most though many of these organic ligands are poorly cells autolytic activity is controlled by the active characterized, recent studies have shown that a extrusion, and retention, of protons in the cell number of dissolved metals, such as Cu(II), Fe(III),

++ 2 ++ Mg O– Mg O– O– P O O– P O O O HHC HHC RHC RHC HHC HHC O 1 O +Mg+ OOP – –OOP O O Teichoic acid Figure 3.15 Representation of a Bacillus subtilis cell wall, showing how magnesium possibly functions to cross-link (1) the phosphodiester groups of two teichoic acid molecules, (2) the terminal phosphoryl groups within a single teichoic acid molecule, 3 and (3) between two carboxyl groups M O O M C +Mg+ C associated with the peptide stems in O– –O M M peptidoglycan. M, N-acetylmuramic M G M acid; G, N-acetylglucosamine. ITGC03 18/7/06 18:12 Page 117

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Zn(II), and Cd(II), exist in nature predominantly oxygen atoms, with the hydroxyl proton being as organo-metallic complexes (Sunda, 2000). displaced. Frequently, three such groups are found The exudates can have both detrimental and on a single siderophore molecule, and hence beneficial properties for microbial communities. it requires six oxygen ligands to satisfy the pre- Sometimes the exudates are in direct competi- ferred octahedral geometry of ferric iron, each tion with cell surfaces for metals, limiting their having partial double-bonded characteristics availability. At other times, organic exudates can (Fig. 3.16). Hydroxamate siderophores are pro- be utilized by microorganisms as either a means duced by many types of fungi, and they are of sequestering metals from the external environ- the most effective Fe chelators at mildly acidic ment to supplement their nutritional needs or to neutral pH. Like hydroxamates, catecholates to immobilize some metals extracellularly as a also occur in triplicate so that they can facilitate method of detoxification. An example of each is tridentate bonding with ferric iron. They are given in the next section. produced by all classes of bacteria, and they tend to be the more important Fe chelators at alka- (a) Siderophores line pH (Hersman, 2000). Irrespective of which siderophore is used, once the iron is bound, Iron is a key element for all microorganisms, the Fe(III)–siderophore complex reacts with a yet the insolubility of Fe(III) at circumneutral receptor site on the cell’s surface and is then pH means that it is often the limiting nutrient transported to the plasma membrane. There it is for growth. Many bacteria and fungi get around dismantled, and Fe(III) is released and reduced this impasse by excreting low molecular weight, to Fe(II). Fe(III)-specific ligands known as siderophores The biosynthesis of siderophores is tightly (Neilands, 1989). Siderophores, and their break- controlled by iron levels, such that they only down products, make up a large component of become activated when dissolved Fe(III) con- the strong Fe(III)-binding ligands that regulate centrations are negligible. Interestingly, higher Fe(III) species in surface ocean water (e.g., levels of siderophores are produced in response Wilhelm and Trick, 1994). to increasingly insoluble iron sources, such as Siderophores have several properties that hematite (e.g., Hersman et al., 2000). Production make them ideal Fe(III) chelators, namely a high of siderophores is also related to cell growth phase. solubility, an abundance of oxygen ligands, and a Siderophores are produced most abundantly tendency towards bi- and multidentate ligation during exponential phase, they then level out that forms coordinative positions around the during stationary phase, and with time, decrease central Fe(III) cation. Significantly, they form in concentration as bacteria run out of nutri- especially strong 1:1 surface complexes, and their ents and begin to lyse (Kalinowski et al., 2000a). stability constants for Fe(III) can be as high as On a much larger scale, recent experiments in 1052. This important property maintains iron in the equatorial Pacific have demonstrated that a “soluble” form that minimizes its loss from the with the addition of iron, a threefold increase in aqueous environment by the precipitation of the concentration of Fe-binding organic ligands solid-phase ferric hydroxide (Hider, 1984). occurred, leading to a concomitant increase So far over 200 siderophores have been iden- in microbial biomass production (Hutchins and tified, with most broadly divided into two classes Bruland, 1998). Interestingly, many species pro- based on their metal chelating properties, either duce siderophores in great excess of their require- hydroxamates or catecholates (Winklemann, ments (because many are lost via diffusion and 1991). The most common siderophores contain advection), yet when levels of iron become suf- the hydroxamic functional group, which forms ficiently high, i.e., an order of magnitude above a five-member ring with Fe3+ between the two micromolar levels, their production is repressed ITGC03 18/7/06 18:12 Page 118

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R2

N R1 O

R1 O R1 O O + 3+ 3+ + + (3x) Fe (aq) Fe 3H N N O R2 OH R2 O Hydroxamate O N

R2

R1

R (–1)

O (–1) OH O O + 3+ 3+ + (3x) Fe (aq) Fe + 3H OH O O O R R R Figure 3.16 Fe(III) Catecholate complexation reactions with hydroxamate and catecholate siderophores. (–1)

and the cells meet their iron needs via low-affinity benefit the binding of Mg2+ because exposing Fe uptake systems (Page, 1993). such ligands could effectively cleanse the waters of such transition metals, preventing them from (b) Metal binding ligands interfering with binding to the O-donor ligands. As an adaptation to repeated exposure to The preference of a given ligand for certain toxic metal concentrations, cells have evolved metals provides the cell with an opportunity to inducible detoxification mechanisms, such as specifically sequester individual essential elements. the intracellular production of thiol-containing Consider the competition between Mg2+, which is ligands that complex undesirable metals, and biologically required, with a strong Lewis acid such in doing so, mask their presence. One such as Cu2+, which can be toxic to microorganisms example is the synthesis of metallothionein when found in high concentrations. A cell surface proteins in response to the presence of high studded with oxygen ligand-containing functional intracellular copper levels (Williams, 1953). groups would facilitate the accumulation of the Microorganisms can also produce and excrete alkaline earth metal because copper has low affin- extracellular ligands that have extremely high ity for such sites. Conversely, if the same metals surface complexation constants for a number were competing for a ligand that included nitrogen of toxic metals, including copper, cadmium, and or sulfur, then copper would prevail (Hughes and lead. This is desirable for the microbial com- Poole, 1989). In some ways possessing sufficiently munity as a whole because, in most cases, the high amounts of N- and S-ligands on extracellu- toxicity of a free hydrated ion is greater than that lar layers or in microbial exudates can actually of metals complexed with other ligands. One ITGC03 18/7/06 18:12 Page 119

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prevalent example of this is in lakes and oceans, systems. By contrast, the SCM takes into account where greater than 99% of copper is bound to the effects of changing pH, solution composition organic ligands (e.g., Coale and Bruland, 1988; and ionic strength, the acid–base properties of Xue et al., 1996). Two lines of evidence suggest surface functional groups, competitive sorption that the copper-binding ligands are produced with other solutes, and solid-phase mineralogy. by phytoplankton (the microbial portion of the It then draws upon that information to extra- plankton community, versus the animal com- polate to conditions beyond those tested in the ponent, the zooplankton) in order to regulate laboratory. Cu2+ levels in their environment.

3.5.1 Kd coefficients 1 Their distribution varies with biological productivity, such that the ligands occur at a maximum con- When a solute adsorbs onto a surface, the surface centration in the illuminated euphotic zone during is termed the sorbent and the solute is termed seasonal blooms. the sorbate. A plot that quantifies the amount 2 Several species of the cyanobacterial genus, of sorbate sorbed to a solid surface versus the Synechococcus, produce extracellular ligands with concentration of solute in solution is known as stability constants similar to those ligands identified a sorption isotherm. Several models have been in seawater. They can reduce the free Cu2+ concen- proposed to quantify metal adsorption (and tration in seawater by 1000-fold, to levels within conversely desorption) associated with micro- their tolerance limits (Moffett and Brand, 1996). bial surfaces. The simplest is when there is a linear relationship between the amount of metal adsorbed onto the microorganism and the con- 3.5 Bacterial metal centration of metal in solution (Fig. 3.17). Under

sorption models these conditions a distribution coefficient (Kd), that predicts the quantity of metal sorbed to the Many of the early studies described above were biomass, can be used to model the adsorption carried out in conditions supersaturated with reaction (see Langmuir, 1997 for details): respect to the metal of interest. Today, metal sorp- = tion experiments are placing greater emphasis M B Kd M D (3.11) on developing geochemical speciation models

that describe how microorganisms interact In the equation, MB is the mass of metal adsorbed µ −1 with metals and mineral surfaces under natural, per dry unit mass of bacteria ( gg ) and MD is and more realistic, undersaturated geochemical the concentration of dissolved metals in equilib- conditions (see Fein, 2000; Warren and Haack, rium with the bacterial surface (µgml−1). 2001 for reviews). Metal sorption reactions can Distribution coefficients are simple to apply. be quantified using two different approaches: They do not require a detailed knowledge of the (i) bulk partitioning relationships; or (ii) sur- surface or sorption mechanisms, and as such, face complexation models (SCM). In the first they are an uncomplicated means to model the

instance, partitioning relationships, such as Kd, distribution of metals at low concentrations Freundlich and Langmuir isotherms, can easily (e.g., Hsieh et al., 1985). However, linear sorp- be applied to complex systems because they do tion isotherms do not describe sorption in terms not require a detailed understanding of the nature of binding sites, and as the concentration of metal

of the surfaces or the adsorption/desorption increases, the relationship between MB and MD mechanisms involved. However, they are system- eventually becomes nonlinear and Kd coefficients specific, meaning that the results from a set become inapplicable. This occurs because at higher of experiments are not applicable to different metal concentrations the available reactive ligands ITGC03 18/7/06 18:12 Page 120

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sorption isotherm (a Kd isotherm). If N is greater than 1, then the extent of sorption increases with

) increasing metal concentrations, and if N is less B than 1, sorption decreases with increasing metal = MB > < A Kd concentrations. For both cases, N 1 and N 1, MD Site saturation a curvilinear line is obtained when MB is plotted versus MD (Fig. 3.17). The isotherm plot can be = MB linearized by taking the logarithm of the Freund- B K N MD lich equation, such that N becomes essentially the slope of the isotherm (equation (3.13)).

= + log M B log K Nlog M D (3.13)

The Freundlich equation has been widely

Mass of metal sorbed to biomass (M applied to quantify metal adsorption onto micro- bial surfaces. One typical observation is that the Dissolved metal concentration (M ) D highest fraction of metal adsorption occurs at the lowest dissolved concentrations, correspond- Figure 3.17 Typical K (A) and Freundlich d ing to the steepest part of the isotherm plot. (B) isotherms. This indicates that the propensity for cation binding progressively diminishes in the presence become occupied and the affinity between the of increasing concentrations as all available surface and metal gradually decreases (e.g., sorption sites become occupied. At this stage, the curve plateaus out and no more cations Gonçlaves et al., 1987). Since Kd coefficients cannot account for site saturation, they cannot are adsorbed (e.g., Small et al., 1999). Another be applied to define an upper adsorption limit. observation with bacterial biomass is that K values can also decrease at higher cell densities Another limitation with Kd isotherms is that they are specific to each experiment, and as a because the production of significant amounts of organic exudates competes directly with wall result, Kd values for the same sorbate–sorbent combination can vary by orders of magnitude ligands for available cations (Harvey and Leckie, depending on aqueous conditions. 1985). This has similarly been reported in fungi where uptake of metals was lower at higher cell 3.5.2 Freundlich isotherms densities because of (i) reduced cell surface area due to cell–cell attachments and (ii) diminished A more flexible sorption model is the Freundlich mixing of metals with surface ligands (Junghans isotherm. The sorption relationship is ex- and Straube, 1991). pressed as: The Freundlich isotherm more effectively describes metal distribution in complex systems = N M B KM D (3.12) and surfaces with heterogeneous properties, such as bacterial communities, as long as the where N is a fitting parameter. The Freundlich conditions can be directly simulated in the lab. isotherm can generally describe sorption over a However, the Freundlich isotherm is obtained wider range of metal concentrations (from trace by an empirical fit to experimental data, and

to saturation) than Kd coefficients, and it can similar to Kd coefficients, the sorption constants account for nonlinear sorption behavior. If N can vary by many orders of magnitude as a func- equals 1 then the equation becomes a linear tion of solution and system parameters. ITGC03 18/7/06 18:12 Page 121

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3.5.3 Langmuir isotherms In natural solutions the cation concentra- tion may exceed the solubility product of a The Langmuir sorption isotherm was developed mineral phase before the ligand sites are filled. with the concept that a sorbent contains a finite On the isotherm plot this is shown by a vertical number of reactive sites, and once all the sites are upward line. A relevant example of the con- occupied by a monolayer of cations, the surface tinuum between adsorption and precipitation is will no longer adsorb the solute from solution. given with ferric iron (e.g., Warren and Ferris, It also assumes that all sorbed species interact 1998). The relationship describing the hydro- only with the ligand and not with each other. lysis of Fe(III), and its adsorption to cell surface The metal sorption reaction can be expressed by ligands is: a site-specific equilibrium reaction: + 3+ + ←→ 0 + + R-AH Fe 2H2O R-AFe(OH)2 3H (3.19) A− + M + ←→ AM (3.14)

A− is the ligand on the surface and M+ is the In the above equation R-AH represents a pro- tonated functional group and R-AFe(OH) 0 is dissolved metal. An equilibrium constant can 2 be determined from the law of mass action: Fe associated with the ligand. The mass action equation is represented by: [AM] K = (3.15) + − + [R-AFe(OH) 0][H ]3 [A ][M ] K = 2 (3.20) [R-AH][Fe3+] Surface species concentrations can be expressed in terms of moles per liter of solution, per gram In double logarithmic plots of experimental of solid or per cubic centimeter of solid surface. equilibrium data, the initial portion of the curve The upper limit of sorption is defined by the is linear, indicating that adsorption is directly concentration of the ligands on the surface. The proportional to the number of available organic maximum concentration of surface sites, Amax, ligands, i.e., Langmuir-type behavior (Fig. 3.18). is given by: In the Warren and Ferris study, the amount

− of iron adsorbed at this stage approached the [A ] = [A ] + [AM] (3.16) max micromole per milligram range when normalized From equations (3.15) and (3.16), we can derive to cell dry weight, an amount within an order of the Langmuir equation: magnitude of the total surface ligand concentra- tions determined independently by the acid– K[A ][M +] [AM] = max (3.17) base titrations discussed previously. The second 1 + K[M +] stage of surface site saturation and the onset of

+ supersaturation is evidenced where the curves If [AM] is plotted versus [M ], then the line would + plateau as [Fe ]/[H ]3 values increase. The third be a curve that reaches a plateau at the maximum D stage, that being nucleation, begins when [FeD]/ sorption value. The Langmuir isotherm can also + [H ]3 values exceed the recognized equilibrium be expressed in a linearized form: solubility product of poorly ordered ferric hydro- 1 1 1 xide. The curve then undergoes a reversal as the =+(3.18) + dissolved Fe(III) concentration ratio decreases [AM] K[A ][M ] [A ] max max during mineral precipitation, and any additional If 1/[AM] is plotted versus 1/[M], then the inter- Fe added to the system goes directly towards

cept, [1/Amax], represents the maximum sorption mineralization. capacity, while the slope of the line, 1/K[Amax], An advantage of the Langmuir equation is can be used to determine the sorption constant. that it can be expanded to model the sorption ITGC03 18/7/06 18:12 Page 122

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–2 determined. They do not explicitly account for all of the changing parameters in a natural setting, and subsequently cannot be used to estimate the Precipitation –3 extent of sorption in systems not directly studied in the laboratory (Davis and Kent, 1990).

–4

(M) 3.5.4 Surface complexation S Supersaturation Site saturation (nucleation) models (SCM)

log Fe –5 In order to better account for all of the environ- mental variability that underpins the adsorption –6 Adsorption and desorption reactions of metals onto micro- bial surfaces, a number of studies have since turned to the surface complexation model. Unlike –7 the bulk partitioning models, the SCM treats 02468 10 surface complexes formed on minerals and micro- log ([Fe ]/[H+]3) D organisms in a similar manner to aqueous com- plexes, deriving for them equilibrium constants Figure 3.18 Modified Langmuir isotherm that describe their thermodynamic stability. This describing the amount of Fe(III) bound (FeS) to Bacillus subtilis as a function of the amount of means that the SCM must take into account all

dissolved Fe(III) added to the solution (FeD). the changes in aqueous composition, as well as the The graph shows a continuum of three stages acid–base properties of microbial and mineral beginning with adsorption, followed by site surface functional groups (Fein, 2000). Such a pro- saturation, and then nucleation/precipitation. cess has the potential of being extremely useful (Adapted from Warren and Ferris, 1998.) in extrapolating results from select experiments to conditions beyond those directly studied in the laboratory; clearly it is not possible to conduct of two competing solutes onto a surface, and/or experiments using every combination of micro- a surface with two sorption sites. The Langmuir bial species and every type of fluid composition. equation can also be employed to evaluate Importantly, the equilibrium constants obtained nonideal competitive adsorption (termed the from such isolated metal–bacteria or metal– NICA model). The NICA model was originally mineral laboratory experiments can be combined developed to describe proton and metal com- with others to ultimately model and accurately plexation with humic substances, but it has predict the extent of sorption that occurs in more been extended to describe competitive metal complex, multicomponent systems. sorption onto bacterial surfaces (e.g., Plette The pH dependence of metal sorption is et al., 1996). This approach can take into account depicted in Figure 3.19. This feature is com- component heterogeneity or nonideality through monly known as a sorption edge. Under acidic experimentally calibrated fit parameters that are conditions, the cell wall functional groups are system specific and must be determined for each fully protonated and no adsorption of cationic system composition of interest. metal species occurs. Only metals present as Similar to the Freundlich isotherms, Langmuir oxyanions adsorb at low pH. As pH increases, isotherms can accurately quantify metal–bacteria the functional groups systematically deproton- sorption as a function of metal concentration, but ate, forming discrete anionic metal binding their system specificity means that they are only ligands. At low pH those ligands are provided applicable to the conditions at which they were solely by carboxyl groups, at circumneutral pH ITGC03 18/7/06 18:12 Page 123

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A with multiple reactive ligands, the adsorption 100 of aqueous ions can effectively be likened to an abiological process, controlled predominantly by the acid–base properties of the exposed ligands 80 Sorption edge and by the affinity of each type of ligand for a specific ion. Indeed, as discussed earlier, most 60 binding sites on the cell are not tailored to cap- ture specific ions from solution, and subsequently B they can easily exchange them for others in the 40 bulk fluid phase. This point is driven home by

% Metal adsorbed the observation that intact bacteria and their isolated cell wall material exhibit reasonably 20 similar affinities for particular cations, yet that affinity differs between different microorganisms (e.g., Mullen et al., 1989). 0 0246810 Interactions between aqueous metal cations z+ pH (M ) and the most common functional groups in bacterial cell walls (i.e., carboxyl, phosphate, Figure 3.19 Isotherms showing the adsorption of hydroxyl) can be represented by reactions (3.21) any given metal cation to bacterial biomass when to (3.23), respectively: (A) there are more ligands than metals, leading to 100% metal adsorption and a steep sorption edge Mz+ + R-COOH ←→ R-COO(M)(z−1)+ + H+ (3.21) and (B) when there are more metals than biomass and the curve plateaus due to the lack of available z+ + ←→ (z−1)+ + + M R-PO4H2 R-PO4H(M) H (3.22) ligands. On average, carboxyl groups deprotonate

over the pH range 2–6, phosphates pH 5–8, and z+ (z−1)+ + M + R-OH ←→ R-O(M) + H (3.23) amino groups pH 8–11. The release of protons and the adsorption of metal cations to form a charged complex (e.g., (z−1)+ phosphate groups additionally deprotonate, while R-COO(M) ) is quantifiable with the cor- at more alkaline pH, amino (pH 8–11) and responding mass action equation: hydroxyl (pH > 12) groups become increasingly [R-COO(M)(z−1)+][H+] important. Progressive deprotonation reactions K = (3.24) result in increasing metal adsorption, up to a [Mz+][R-COOH] point where potentially all the metal is bound. (curve A, Figure 3.19). In addition, the adsorp- The K value is the experimentally observed tion and desorption of metals reaches the same metal sorption constant that is related to a true

equilibrium concentration at any given pH thermodynamic constant (Kintrinsic), via activity value (Fowle and Fein, 2000). Therefore, it does coefficients, that take into account ionic not matter whether steady-state conditions are strength, surface charge and electrical double approached from site undersaturation (no metal layers (see below). The equilibrium expression associated with bacterial surface) or from site above emphasizes that adsorption of metal cations saturation (virtually all metal associated with by microorganisms depends not only on pH and bacterial surfaces). ionic strength, but also on the number and type Because surface complexation models describe of functional groups per cell. Since ultrastructural bacterial cell walls as heterogeneous surfaces variations exist between different bacterial species, ITGC03 18/7/06 18:12 Page 124

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and even within a single strain, observable differ- 1 Biomass:metal ratios – When abundant biomass ences in metal binding capacity are theorized. An exists, there are excess anionic ligands present additional assumption not immediately obvious on the bacterial surface compared to the dilute from the equilibrium equation, is that all surface concentration of dissolved metals. This means that functional groups are treated as being structurally equilibration between the metals and the surface complexes they form can easily be achieved at and chemically fully equivalent (Buffle, 1990). circumneutral pH. The adsorption edge under In order to consider microbial surfaces as these conditions is steep, and 100% adsorption thermodynamic chemical components, the elec- is potentially attained using whichever ligand trostatic interactions between the surface elec- provides the greatest stability for the newly formed tric field and metal cations can be accounted surface complex (recall Fig. 3.19). At lower for using the following equation (Stumm and biomass:metal ratios, the available ligands may Morgan, 1996): become fully saturated with metals and the rate with which further adsorption takes place diminishes. Accordingly, the adsorption edge is less steep, = −∆ Ψ Kintrinsic K( ZF 0/RT) (3.25) and excess metals will remain in solution unless precipitation occurs. What all this implies is that

Kintrinsic represents the equilibrium constant ref- in solute-rich solutions, several ligands may be erenced to zero surface charge, F is the Faraday required to bind available metals, and that the constant, R is a gas constant, T is absolute tem- quantity of metals adsorbed at a particular pH perature, ∆Z is the change in the charge of the increases as the ratio between the total concentra- tion of microbial surface ligands to the total metal surface species for the reaction of interest, and Ψ concentration increases (e.g., Fein et al., 1997). 0 is the surface potential of the cell. Several Clearly, displaying several types of organic ligands different SC models (e.g., constant capacitance, on a cell wall is how bacteria ensure metal uptake diffuse double layer, and triple layer models) over a wide range of pH. were initially proposed to describe the surface electrical field associated with mineral surfaces 2 Charge modification – While metal accumulation (see Dzombak and Morel, 1990 for details), and onto a microorganism is influenced by the sur- they have now been adapted to microbial sur- face charge characteristics of the exposed ligands, one of the outcomes of metal binding is that faces. Such electrostatic sorption models define a the cell surface progressively becomes less anionic mathematical relationship between surface charge due to charge neutralization within the electric and surface potential, but differ in the assump- double layer (e.g., Plette et al., 1996). Indeed, tions they make about where the adsorbed species with high cation coverage, the surface may even are positioned in the double layer. The constant become positively charged. Some studies have capacitance and diffuse-layer models assume revealed that the charge reversal usually occurs in the pH range where the concentration of un- that all cations are specifically adsorbed at the + complexed divalent cations (e.g., Cu2 ) decreases shear plane, while the triple-layer model assigns and the concentration of their monovalent hydro- adsorbed species to either the shear plane or a xylated cations (e.g., Cu(OH)+) correspondingly more distant plane (Langmuir, 1997). increases (Fig. 3.20). Over this same pH range, maximum metal adsorption takes place, suggest- 3.5.5 Does a generalized sorption ing that the OH- groups of the hydrolyzed model exist? metals play an important role in hydrogen bonding between O-ligands on the bacterial surface and those metals in solution. At more alkaline pH, the Despite the apparent simplicity in using SC cations are neutrally (e.g., Cu(OH)2) or negatively models to determine microorganism–metal inter- 2− (e.g., Cu(OH)2 ) charged, and the overall charge actions, there are a number of factors that obscure on the cells once again becomes negative (Collins the patterns of metal sorption: and Stotzky, 1992). At this stage it is also common ITGC03 18/7/06 18:12 Page 125

CELL SURFACE REACTIVITY AND METAL SORPTION 125 )

–1 3.0 layer between the cell and the ions in the bulk

cm solution. Higher ion availability satisfies the surface

–1 2.0 + charge excesses and results in decreased cell wall

V Cu(OH) –1 1.0 Cu(OH)2 surface potentials. By contrast, dilute solutions allow the electric fields to expand outwards from the cell 0 surface. Saturating the cell surface with cations also –1.0 changes the corresponding isotherms, from those Isoelectric + − Cu2 Cu(OH)3 point with distinct sorption edges at low ionic strength to a –2.0 poorly defined sorption edge at high ionic strength –3.0 (Yee et al., 2004). (iii) Increased ionic strengths lead to competition amongst the various cations –4.0 for the cell’s anionic sites. This, in turn, depends on

Electrophoretic mobility ( µ m s whether a particular cation bonds to the microbial 0123456789 10 pH surface electrostatically as a hydrated, outer-sphere complex or covalently as an inner-sphere complex (Small et al., 2001). Figure 3.20 The relationship between dissolved copper species and the electrophoretic 2+ 4 Kinetics – Metal sorption by microbial biomass mobility of bacteria. In the presence of Cu , cells often involves two distinct stages. The first, which remained negatively charged at pH values between is passive adsorption to the cell surface, is a their isoelectric points and pH 6. At higher pH rapid process occurring within seconds to minutes values, despite continuing deprotonation of after the microorganism comes into contact with cell wall functional groups, bacteria become the metal (e.g., Hu et al., 1996). When the con- positively charged with continued addition of centration of cell ligands exceeds dissolved metal metal. Simultaneously, there is a corresponding concentrations, partitioning can be satisfactorily change in dissolved copper speciation, from an described by a linear relationship (K isotherm) and uncomplexed divalent cation to a monovalent d equilibrium is generally reached within a few hours. hydroxylated cation (Cu(OH)+ ), around pH 6. At During this stage, the contact time of adsorption pH values above 8, the cations become neutrally also exhibits no affect on the kinetics of desorption charged (Cu(OH) ) and then negatively charged 2 or on the concentration of the metals bound to (Cu(OH) −). These species no longer adsorb to the 3 the cell (Fowle et al., 2000). The second stage is cell. The cell surface then reverts back to an overall slower and commonly involves diffusion-controlled, net negative charge, whilst the amino groups intracellular accumulation. This process occurs deprotonate. (Modified from Collins and Stotzky, over several hours, and if sufficient metals are avail- 1992.) able to the cell, it can lead to much higher metal accumulations than that of the first stage (e.g., Khummongkol et al., 1982). to observe a reversal in metal adsorption behavior. Therefore, the pH of the solution not only affects 5 Growth phase – The surface characteristics of any the degree of deprotonation of the cell’s functional given microorganism, and therefore its capacity to groups, but it also influences metal speciation, so sorb metals, can vary with growth conditions. For that different metals present at similar concentra- example, Chang et al. (1997) reported that Pb2+ tions will be differently adsorbed. was most extensively adsorbed by Pseudomonas aeruginosa during stationary phase, Cd2+ was pre- 3 Ionic strength – It influences metal adsorption by three ferentially adsorbed at exponential phase, while principal mechanisms. (i) It affects the activities of Cu2+ was not affected by growth phase. Mean- ions in solution, with higher ionic strengths leading while, B. subtilis cells growing in exponential phase to decreased availability of “free” metal cations in adsorb 5–10% more Cd2+ and Fe3+ than cells at solution: under these conditions, concentration and stationary phase, which, in turn, is 10–20% more activity can no longer be considered equivalent. than that adsorbed by sporulated cells (Daughney (ii) It governs the thickness of the electrical double et al., 2001). ITGC03 18/7/06 18:12 Page 126

126 CHAPTER 3

With all these environmental variables to be 100 considered, is it truly possible to apply chemical 90 equilibrium thermodynamics to quantify metal 80 sorption and desorption on bacterial surfaces? The answer remains to be tested in “natural 70 systems”, but one thing is likely, certain simplify- 60 Bacillus megaturium ing assumptions will need to be made. Fortuit- 50 Staphyloccocs aureus ously, it is now becoming apparent that metal Sporosarcina ureae 40 Bacillus cereus

cations display a similar affinity series for a % Cd adsorbed Streptococcus faecalis 30 Escherichia coli given group of ligands, regardless of whether the Pseudomonas aeruginosa ligands exist on the surface of a microorganism 20 Bacillus subtilis (Fowle and Fein, 2000) or as an aqueous organic species. This means 10 Generalized model that common complexes, such as metal–oxalate 0 or metal–acetate, can be used to predict metal– 5432 109876 pH carboxyl surface stabilities of bacteria for those

metals whose bacterial adsorption behavior has 2+ not yet been measured directly (e.g., Fein et al., Figure 3.21 Plot showing Cd adsorption onto pure cultures of various bacteria. Each 2001). Significantly, this greatly expands the point represents individual batch experiments with number of aqueous metal cations for which 10 −4.1 mol L−1 Cd and 1.0 g L−1 (dry weight) bacteria. adsorption onto bacteria can be modeled. Recent The dotted curve represents the modeled adsorption findings have additionally shown that when behavior. Notice how all the bacterial species biomass:metal ratios are high (i.e., an order of exhibit nearly identical Cd adsorption behavior magnitude more ligand sites than dissolved as a function of pH. (Reprinted from Yee and Fein, metal on a molar basis), metal adsorption onto 2001 with permission from Elsevier.) the walls of various bacterial species all display very similar sorption edges (Yee and Fein, 2001). determined whether it is possible to derive a Each species can adsorb nearly 100% of the generalized model that can actually be applied metal cations at similar pH values, suggesting to quantify the distribution and concentration that patterns of metal adsorption may not be of metals in bacteria-bearing water–rock systems. too species-specific when abundant biomass is present (Fig. 3.21). Although the rationale for SCM is that it is 3.6 The microbial role in possible to describe multiple metals adsorbing onto contaminant mobility multiple surface sites by combining equilibrium constants for each specific chemical reaction One of the main motivations for researching that occurs, the models are not yet sufficiently microbial–metal interactions is that it has wide- developed to predict how lab-based sorption ranging implications for accurately modeling reactions with monocultures compare in an envir- contaminant transport in the environment, and onmental setting with mixed mineral assemblages ultimately the design of effective bioremediation (metal oxides, clays), multiple organic phases strategies (e.g., Bethke and Brady, 2000). The (humic compounds, microbial exudates etc.), significance of microorganisms in contaminant multi-elemental pore waters, and a complex mixed mobility lies in the fact that they comprise a microbial community with species in different significant component of the organic fraction in growth phases held together by EPS and various the subsurface, and they possess highly reactive other extracellular layers of widely different surfaces that allow them to partition metals from compositions. So, at present it remains to be solution into their biomass. In sediment and soils, ITGC03 18/7/06 18:12 Page 127

CELL SURFACE REACTIVITY AND METAL SORPTION 127

this partitioning capacity is of similar, or even driving force for bacterial transport is advec- greater, magnitude to some clays and other organic tion, the process generated by the hydraulic components (e.g., Ledin et al., 1999). gradients that induce groundwater flow. Under When microorganisms become immobilized conditions of limited groundwater movement, onto a solid substratum, or if their movement many bacteria move freely from one location to through an aquifer is inhibited by some form of another by some form of motility, including those permeability barrier, they are likely to reduce the that depend on the propulsive action of flagella transport of contaminants. They do so because (swarming and swimming), and those that depend coating the original mineral surface with bio- partly on cell to cell interactions (gliding). mass frequently increases the metal binding These modes of transport can be quite fast, with properties of the substratum, and immobilized some bacteria showing velocities greater than cells provide additional surface area to which 10 −4 cm s−1 (Characklis, 1981). Many bacteria metals are retained (e.g., Yee and Fein, 2002). At can also move chemotactically in response to a other times, they remain as free-moving particles chemical gradient (Carlile, 1980). through the porous media, enhancing the trans- port and dispersion of sorbed contaminants (e.g., (b) Initial adhesion – effects of solution Lindqvist and Enfield, 1992). chemistry

3.6.1 Microbial sorption to The initial interaction between a bacterium to solid surfaces a mineral, referred to as reversible adhesion, is an instantaneous attraction by long-range forces Within minutes of a solid being submerged holding a bacterium at a small, but finite distance in an aqueous environment, a thin film will some 5–10 nm from a surface. At this stage there collect at the solid–liquid interface due to is no direct physical contact between the cell simple sedimentation and from electrostatic and the solid, and they can readily be removed interactions between the solid and the dis- from the surface by shear forces or the rotational solved ions/suspended materials from the bulk movements of their flagella (Marshall et al., aqueous phase (Neihof and Loeb, 1972). This 1971). The extent of interaction can be pre- is known as “conditioning” the surface with dicted by colloid chemical theories such as the inorganic and organic compounds for the growth Derjaguin–Landau–Verwey–Overbeek (DLVO). of microorganisms. The actual colonization of It describes the magnitude and variation of this surface involves three steps; (i) transport of the potential energy of interaction between a the bacteria to the submerged surface, (ii) their bacterium and a mineral surface as a function of initial adhesion via electrostatic interactions, separation distance (see Shaw, 1966 for details). and (iii) their irreversible attachment to the The interaction arises because there is a tend- substratum through the excretion of EPS or ency for surfaces to obtain a minimum Gibbs free utilization of surface appendages (van Loosdrecht energy by satisfying their charges, and one way et al., 1990). this can be done is through bacterial adsorption (Absolom et al., 1983). (a) Transport to the surface In its simplest form, if steric effects do not play a role, the total Gibbs free energy is obtained In quiescent bodies of water, relatively large from the difference between the van der Waals cells or aggregates settle to the bottom by sedi- attractive energies and the electrostatic repulsive mentation, whereas smaller cells (radii <1 µm) energies (Fig. 3.22). The former are intermole- exhibit a certain degree of diffusive transport cular forces that result from the formation of due to Brownian motion. In aquifers, the primary temporary dipoles created by fluctuating electron ITGC03 18/7/06 18:12 Page 128

128 CHAPTER 3

A (5–10 nm) B (~1 nm) 3 4 4 2 4 4 1 + + + + + + + + + +

+ + +

+ + + + + + + + Bacterium +

+ +

+ + + + + +

mineral surface +

+ + Negatively charged + + + + + +

Low ionic strength High ionic strength G G

GR

GR

GT

GT

GA GA Attraction Repulsion Attraction Repulsion

Figure 3.22 Electrochemical interactions between mineral and cell surfaces of like charge according to the DLVO theory. In low ionic strength solutions (A) minimum total free energy is obtained at a long distance (a few nanometers) from the mineral surface where attractive and repulsive forces are equivalent. This means

that the cell does not closely approach the mineral surface because GT constitutes a barrier to adhesion. In high ionic strength solutions (B) attractive forces dominate at all distances due to increasing ion availability and

decreased electrostatic repulsion. Since GT is negative, cation bridging thus brings the cell closer to the mineral

surface. GA, van der Waals attraction; GR, electrostatic repulsion; GT, total interaction. (Adapted from van Loosdrecht et al., 1990.)

distributions around atoms in each solid. The electrostatic repulsion between the two solids. latter are due to the overlapping double layers At longer distances (several nanometers), the surrounding the mineral surface and cell, which attractive and repulsive forces become balanced.

becomes important only when the cell and solid Then, as ionic strength is increased, GT is lowered are sufficiently close to one another. According to to a minimum value due to a reduction in repul- DLVO theory, the thickness of the double layer sion resulting from increased ion availability and is inversely proportional to the square of the decreased electrostatic interactions (see Stumm ionic strength (van Loosdrecht et al., 1989). Con- and Morgan, 1996). The end result is that cells sequently, as the ionic strength is increased, the can approach the mineral surface to shorter double layers are compressed, and the surface separation distances. The type of cation is import- potential is reduced sufficiently to allow the forces ant here because divalent species (e.g., Mg2+) of attraction to exceed repulsion. In terms of total are more effective at removing bacteria from

Gibbs free energy (GT), at low ionic strength, solutions of identical ionic strength than those + GT has a positive maximum at short separation fluids containing Na (Simoni et al., 2000). distances (~1 nm) that represents an activa- Once the bacterium overcomes the repulsive tion energy barrier for adhesion due to the large forces and gets close to the surface, short-ranged ITGC03 18/7/06 18:12 Page 129

CELL SURFACE REACTIVITY AND METAL SORPTION 129

forces, such as hydrogen bonding, then ultimately 100 determine the strength of adhesion. The relation- ship between ionic strength and bacterial attach- 80 0 ment is borne out in a number of experimental >AI-OH + studies that have shown an increased number of 60 >AI-OH2 bacteria on mineral surfaces in high ionic strength

% Sites − solutions, while reduction of ionic strength acts in 40 R-COO − an opposite manner (e.g., Jewett et al., 1995). The

As might be expected from the discussions on adsorbed onto positively charged mineral surface the acid–base properties of cells and metal sites (>Al-OH +): sorption, the attachment of bacteria to mineral 2 substrata is governed by the surface charge char- > + + ←→ > + Al-OH2 R-COOH Al-OH2-RCOOH acteristics of both. The effect of pH was recently (3.26) addressed by Yee et al. (2000), who compared the adsorption of B. subtilis onto the minerals quartz A likely explanation for these particular observa-

(SiO2) and corundum (Al2O3). They showed that tions is that the R-COOH sites behave hydro- the quartz surface exhibited a negligible affinity phobically because they are uncharged and not for the bacterium because the mineral surface is significantly hydrated by water molecules. With negatively charged above approximately pH 2, higher pH, the bacterium becomes progressively as is the cell surface above pH 2.5. In this more anionic, increasing the amount of its hydra- case, electrostatic repulsion between the mineral tion and hydrophilicity. In turn, this causes the and the bacterium’s surface is strong enough to cell to detach until the gradually increasing elec- inhibit adsorption. By contrast, the corundum trostatic attraction between the mineral and its surface is positively charged below pH 9, hence surface becomes high enough that the bacterium B. subtilis had a positive affinity towards it under once again becomes adherent. Therefore, attach- normal growth conditions. At very high pH, ment is promoted when either hydrophobic cell both the bacterial and mineral surfaces are surfaces interact with uncharged interfaces, or sufficiently anionic that they repel one another when hydrophilic cell surfaces come into contact (Fig. 3.23). with oppositely charged interfaces. However, electrostatic interactions do not fully This pattern has an important bearing on account for the observed patterns of bacteria- how some microorganisms behave in nature. For mineral adsorption. In the experiments carried instance, benthic cyanobacteria tend to exhibit out by Yee et al. (2000) there was significant hydrophobic characteristics, while planktonic bacterial adsorption onto corundum even under varieties are more hydrophilic in nature (Fattom low pH conditions, where protonated carboxylic and Shilo, 1984). Cyanobacterial hydrophobicity acid groups on the cell surface (R-COOH) appears to have a genetic basis since some species ITGC03 18/7/06 18:12 Page 130

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that are hydrophilic can be made hydrophobic by area to which cells can attach, with higher cell producing extracellular layers (e.g., sheaths) that densities on grains with surface irregularities are electroneutral, and are thus less likely to inter- (DeFlaun and Mayer, 1983). act with water molecules (Phoenix et al., 2002). The surface charge characteristics of minerals, (d) Irreversible attachment and hence their ability to attach microorganisms under normal growth conditions, can also be In the absence of strong shear forces, a bacterium altered by the adsorption of inorganic or organic held closely to the mineral surface is ideally compounds. With quartz, attachment of cells positioned to make use of other means to secure to the mineral can be dramatically increased by a direct and more permanent attachment. This the presence of iron hydroxide coatings. These irreversible phase comes about mainly due to the coatings have an isoelectric point ~8.5 that production of EPS that physically bridges the gap establishes a positive surface charge at neutral between the cell and the solid (e.g., Fig. 3.24). pH so conducive to bacterial adsorption that Recent genetic studies have shown that the subsequent exposure of the bacteria/Fe(III)- physical adhesion to surfaces triggers the expres- coated-quartz assemblage to sterile, dilute water sion of several genes controlling EPS synthesis does not promote desorption (Mills et al., 1994). (Davies and Geesey, 1995). Correspondingly, By contrast, the addition of anionic phosphate biochemical comparisons between benthic and compounds cause the mineral surfaces to become planktonic cells of the same species shows that at sufficiently negatively charged that they repel least 30% of the membrane proteins are expressed the cells (e.g., Sharma et al., 1985). Similarly, when organic matter adsorbs onto Fe-coated quartz, it causes a charge reversal on the mineral surface, making it anionic, and leading to dimin- b ished bacterial attachment (Scholl and Harvey, 1992). Meanwhile, the adsorption of organic matter to bacterial surfaces increases their over- all negative surface charge and leads to increased attraction to the Fe-coated quartz surface, but a decreased attachment of cells in a quartz-only system (Johnson and Logan, 1996). Since the initial adhesion of bacteria is usu- EPS ally reversible and relatively weak, surface shear forces and fluid turbulence cause desorption and elevated levels of cellular wash-out (van Loosdrecht et al., 1989). As a measure of pro- Feldspar 3 µm tection, microorganisms preferentially colonize easily abraded mineral surfaces with some sur- face microtopology. Limestones are particularly Figure 3.24 Using the technique of amenable to surface colonization because of the cryomicroscopy, the complicated structure of EPS ease with which the constituent calcite grains can be visualized in three dimensions without degrade. This likely explains why the number suffering the effects of dehydration during sample of epilithic bacteria on limestone are 10- to preparation. This image shows a plagioclase 100-fold greater than on harder rock types, such feldspar grain, with several bacteria (b) residing as granite, gabbro, rhyolite, basalt, and quartz within the EPS. (From Barker et al., 1997. sandstone (Ferris et al., 1989). The texture of a Reproduced with permission from the mineral also determines the amount of surface Mineralogical Society of America.) ITGC03 18/7/06 18:12 Page 131

CELL SURFACE REACTIVITY AND METAL SORPTION 131

to different extents by cells in these two different formed produce EPS that enhances their adhe- modes of growth (Costerton et al., 1995). Other sive properties, allowing them to take advantage microorganisms employ specific appendages, such of the organic and inorganic compounds that as pili, fibrils, or holdfasts, to anchor themselves accumulate at solid–liquid interfaces (e.g., Dawson onto the solid. et al., 1981). Such tactics may be particularly A direct correlation exists between the num- important in oligotrophic waters where bacteria ber of attached bacteria and the time allowed are exposed to conditions of extreme nutrient for attachment, with increased time leading limitation. The reduction in size is temporary and to a higher number of bacterial collisions with can later be reversed on provision of adequate the surface (Fletcher, 1977). Nonetheless, it nutrients. Other microorganisms respond to only takes 10–30 minutes to form a continuous adverse conditions by producing spores. Some monolayer of cells under laboratory conditions spores, such as those of Bacillus cereus, have a (Characklis, 1973). Cell motility also enhances neutral surface charge that makes them strongly the likelihood that a bacterium will encounter a hydrophobic compared with that of the vegeta- surface, with the kinetic energy being important tive cell (Rönner et al., 1990). in overcoming the electrical repulsive forces. Once attached, the growth of microorganisms 3.6.2 Microbial transport through on surfaces is an autocatalytic process, whereby porous media initial colonization increases surface irregularity and promotes biofilm formation (Little et al., Although bacteria readily attach onto solids, 1997). a fraction of them remain mobile in sub- surface pore waters. Their ability to move freely (e) Effects of cell growth rates through geological material is dependent upon a number of physical parameters, including The relationship between cell growth rates the system hydrodynamics, permeability, and the and attachment is a complex phenomenon that magnitude of the clay fraction (Lawrence and appears to be species-dependent. For example, a Hendry, 1996). number of bacteria show diminished adhesive- As might be expected, flow rates are an ness during exponential phases (e.g., Gilbert important factor in bacterial dispersion. High et al., 1991), whereas others show the opposite flow rates, or the more rapidly the sediment is effect (e.g., Fletcher, 1977). This discrepancy is flushed with groundwater, lead to higher levels directly related to the specific changes in cell of bacterial elution (e.g., Trevors et al., 1990). surface charge between different species during Under such conditions, bacterial transport rates optimal growth conditions. of over 200 m day−1 have been reported (Keswick In cyanobacteria, the formation of hydrophilic et al., 1982). Even under no flow conditions, hormogonia are important for dispersing the some motile bacteria can move through packed species to new environments, yet, as the hormo- sand cores at rates greater than 0.1 m day−1 gonia contact new surfaces, they develop back (Reynolds et al., 1989). Field measurements of into mature trichomes and concomitantly show bacterial motilities even indicate that bacteria increased hydrophobicity (Fattom and Shilo, can be transported through porous aquifers faster 1984). This agrees with the common findings in than chemical tracers because the preferential the laboratory, that during continuous culture at exclusion of bacteria from smaller, more tortu- high dilution rates, many such microorganisms ous pores between sediment particles results in form flocks or adhere to surfaces in the culture a more direct average path of travel for the vessel. unattenuated bacteria (Harvey et al., 1989). In During starvation, bacteria show increased the water-unsaturated (vadose) zone, bacterial levels of adhesion. The “dwarf cells” that are movement is instead influenced by gas saturation. ITGC03 18/7/06 18:12 Page 132

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On the one hand, bacteria preferentially accu- available pores (e.g., Jang et al., 1983). Cell mulate at the gas–water interface, and thus their shape, as quantified by the ratio of cell width to movement can be impeded (Wan et al., 1994). cell length, also affects the transport of bacterial Conversely, increased rates of cell movement can cells, with spheres moving through porous media arise when localized pressure gradients, generated more effectively than rod-shaped cells (Weiss through processes such as fermentation, act to et al., 1995). Other experiments have clearly push bacteria through the pore network. demonstrated how these two properties, size and The pore size distribution in soils/sediment shape, are related. For example, small spheres or the fracture pattern in rock are also key (<1 µm diameter) passed easily through coarse- features governing the spatial distribution of sub- sized sand (1.0 mm), while less than 1% of the surface bacteria. Straining or filtration occurs in larger cells (2 µm diameter rods) passed through unconsolidated material when bacteria are too the fine-grained sand (<0.3 mm) (Fontes et al., large to pass through the pore throat aperture, 1991). This effect was largely the result of a resulting in clogging. Generally if the diameter significant decrease in the hydraulic conductiv- of the bacterium, or the bacterial aggregates, is ity through the fine-grained columns, suggesting greater than the sizes of 5% of the particles in that the degree of macropore flow influences the medium, straining is considered significant the extent of microbial transport (e.g., Fig. 3.25). (Sharma and McInerney, 1994). In a number In rock, conductive fractures have been shown of studies, bacteria smaller than 1.0 µm in to constitute preferential paths for subsurface diameter showed the greatest potential for bacteria in the water-saturated (phreatic) zone, transport through porous media (e.g., Gannon leading to migrations of several kilometers in et al., 1991). However, even if the cells are distance, whereas small-size fissures dramatic- sufficiently small, aggregation of cells in high ally reduced bacterial mobility (e.g., Malard ionic strength solutions can cause them to plug et al., 1994).

Silt

Clay

Figure 3.25 Hypothetical drawing of microbial transport through an aquifer. Two flow paths are shown. At the top, small cocci and rods move easily through the silts but are strained by clay particles. The bottom flow path represents larger cocci that are forced to take a route through the larger pore spaces between grains Fine sand of sand. Note relationship between bacteria and sediment µ ~50 m grains are not to scale. ITGC03 18/7/06 18:12 Page 133

CELL SURFACE REACTIVITY AND METAL SORPTION 133

In addition to influencing soil/sediment utilized in bioremediation strategies (Fig. 3.26). permeability, clays affect contaminant mobility In terms of metal binding, this includes biosorp- because they comprise a significant fraction of tion and bioaccumulation. Biotransformations the solid phase. They also readily form micro- – the reduction of high valence metals to lower aggregates with organic matter, including micro- valence insoluble species, or the oxidation that organisms. Despite the high chemical reactivity leads to the opposite effect – was discussed in of the individual components, aggregation into Chapter 2, while biomineralization – the forma- clay–bacteria composites reduces overall metal tion of insoluble mineral phases – will be the binding ability because clays mask or neutralize subject of the next chapter. adsorbing ligands on the cell (Walker et al., Biosorption, defined here as the process 1989). Nevertheless, once metals are bound to whereby microbial biomass acts as a surface upon clay–bacteria composites, they are difficult to which metals are passively sorbed, has a major remove. Even strong leaching chemicals, such advantage over similar chemical technologies as nitric acid or EDTA, can remobilize only a in that large quantities of inexpensive and fraction of the bound metals. What is particu- easily regenerable fungal and bacterial biomass larly noteworthy is that some metals are more are available from fermentation industry waste, difficult to remove from the bacteria–clay com- sewage sludge, or the many different types posites than from their individual counterparts of marine macroalgae that make up seaweed (Flemming et al., 1990). (see Volesky and Holan, 1995; Gadd, 2002 for reviews). Unlike conventional methods, bio- sorption involves using a nonhazardous material 3.7 Industrial applications based on whose application is broad-ranging; it can bind microbial surface reactivity a suite of metals or it can be employed based on the selectivity for binding a specific metal of concern. Moreover, it can be used under a wide 3.7.1 Bioremediation range of environmental conditions. Both dead and living biomass can bind metals, but the The increasing societal demands for metals has former is generally preferred because it avoids led to a widescale release of metal pollutants into the problems with toxicity, it can be used the environment. Traditional technologies, such under extreme geochemical conditions, and is as chemical precipitation and sludge separa- cheaper to use (see below). Biosorption is most tion, oxidation-reduction, evaporation, electro- effective as a polishing step where waster-water chemical treatment, sorptive resins, and organic with low to medium metal concentrations (up solvents have typically been employed in the to 100 mg L−1) is purified to drinking-water clean-up. While many of the cheaper processes standard. Treatment of wasterwater with high have become inadequate with progressively metal concentrations can lead to rapid exhaus- stringent regulatory effluent limits, the more tion of the biosorbent material and thus may effective methods are prohibitively expensive. require larger than desired amounts of biomass. The need for more affordable technologies has Therefore, pre-treatment of such effluents using led to the evaluation and design of methods other techniques, such as chemical precipita- by which the metal binding properties of micro- tion (which is currently used for 90% of heavy bial biomass could be utilized (Eccles, 1995). metal removal from industrial wasterwater) or Bioremediation is the application of living or electrolytic recovery, may be more economical. dead organisms to degrade or transform hazardous The metal-laden biosorbent is then dealt with in inorganic and organic contaminants. There are one of two ways: it may be incinerated, with the several ways in which microorganisms can be ash disposed of in landfills, or alternatively, the ITGC03 18/7/06 18:12 Page 134

134 CHAPTER 3

Bioaccumulation + Mz Biomineralization + 3+ Biotransformation 3H2S 2As As2S3

− + 2+ HCO + Pb PbCO UO2 + 3 3 Mz − + 3+ − 2− 3OH Fe Fe(OH)3 HAsO4 UO2 − + HPO 2 + UO 2 HUO PO 2− 4 2 2 4 HAsO4 Anionic 3+ + − Cr OH Cr(OH)3 − ligands 3 2 1 7+ + H2AsO3 Te 2H2S TeS2

+ + + Cs Cd2 Pu4 Biosorption

Figure 3.26 Some of the many ways in which natural microbial activity can be used in bioremediation of toxic metals and radionuclides. (Adapted from Lloyd and Macaskie, 2000.)

biomass is regenerated by desorbing the metals metal is bound to metallothioneins or compart- from the biomass, yielding a reusable biosorb- mentalized into vacuoles, and as such, require ent and a highly concentrated metal solution that the cells be physically destroyed (Macaskie (Schiewer and Volesky, 2000). et al., 1996). Bioaccumulation describes absorption of One area that has received significant atten- metals by metabolically active cells. Often toxic tion is the biological removal of radionuclides metals enter the cell as chemical “surrogates,” from low-level nuclear waste processing sites using the transport systems developed by the (e.g., Cs, Te, U, Pu, Np). The biogeochemical cell for other elements. Such systems are self- behavior of these pollutants has become increas- sustaining due to biomass replenishment, and ingly important due to the issues of their disposal, they have the ability to not only absorb high their long-term containment, and ultimately levels of metals, but their excreted metabolic their movement through the environment. Using

wastes (e.g., H2S) can also contribute to metal uranium as just one example, many studies have removal. Unfortunately, living biomass present shown that several genera of filamentous fungi a number of difficulties. First, the final sludge (e.g., Rhizopus, Aspergillus, Penicillium), yeasts for disposal is of high organic content adding (e.g., Saccharomyces), marine algae (Sargassum, to the cost of transportation to the site of repro- Chlorella), and bacteria (e.g., Bacillus, Pseudomonas, cessing or final burial. Second, special care Streptomyces) are very effective scavengers of (and associated high costs) needs to be taken to the radionuclide (see Macaskie and Lloyd, 2002; ensure that the growing microbial population Kalin et al., 2005 for reviews). Maximum accu- is kept uncontaminated by other species and mulation, with a steep sorption edge, occurs maintained through adequate supply of nutri- under acidic conditions, between pH 4 and 6, ents, ideal temperatures, and pH buffering. through monodentate adsorption of a cationic 2+ Third, metals accumulated intracellularly are uranyl ion (UO2 ) onto a deprotonated carboxyl + not as easily recovered as those adsorbed to group to form the surface complex, R-COO-UO2 the surface during biosorption, especially if the (Figure 3.27). At circumneutral pH, U sorption ITGC03 18/7/06 18:12 Page 135

CELL SURFACE REACTIVITY AND METAL SORPTION 135

100

2+ 90 UO2

80 + UO2OH 70

60 UO2(OH)2(aq)

Figure 3.27 Plot showing U(VI) 50 − UO (OH) sorption onto Shewanella putrefaciens 2 3 40 as a function of pH. Under very acidic % U sorption conditions, uranium is poorly sorbed. 30 4– It then reaches a maximum at pH 4–5 UO2(CO3)3 and remains optimal until pH 6. 20 At higher pH, the extent of sorption diminishes as uranium speciation 10 changes from uranyl cations to anionic 0 hydroxide and carbonate species. 1 234 5 6 7 8 9 10 (Modified from Haas et al., 2001.) pH

is best explained by a neutrally charged sur- studies have documented how the biochem- face complex on a phosphate site, yielding ical composition of E. coli cell walls could be

PO4H-UO2(OH). At higher pH, competition modified by inserting sulfur-containing amino of cell-bound U(VI) with aqueous hydroxyl acids from other organisms to increase the adsorp- and carbonate anions reduces the extent of tion of metals that react favorably with the 2+ adsorption, resulting in a release of UO2 back S-ligands (e.g., Sousa et al., 1998). Other studies into solution (Haas et al., 2001). Dead cells are instead focusing on coupling the biosorptive often absorb more uranium than their live abilities of microbial surfaces to the biomineral-

counterparts, presumably due to an increase ization of uranyl phosphate, HUO2PO4. This in accessible metal binding sites (Volesky and has yielded some promising results, with uranium May-Phillips, 1995). uptake values by Citrobacter sp. as much as Uranium uptake is commonly greater than 900% of the cellular dry weight (Macaskie et al., −1 2+ 200 mg U g biomass dry weight, but has been 1992)! The process involves UO2 adsorbing reported to reach values in excess of 50% (e.g., to anionic phosphate ligands in the LPS and Yang and Volesky, 1999). The efficiency of then reacting with phosphate excreted by the uranium uptake is significant because a total cell as a result of its enzymatic overproduction uranium loading capacity of greater than 15% of phosphatase on the cell’s outer membrane of biomass (dry weight) has been defined as an (Macaskie et al., 2000). economic threshold for practical application One technique of metal sorption by microbial when compared to traditional technologies biomass employs freely suspended particles. This (Macaskie, 1991). Given that the amount of permits a high surface area of binding sites, but uranium removed from solution through bio- it suffers from a number of disadvantages. These sorption is ultimately governed by the type of include small and heterogeneous particle size, available ligands, new techniques in molecular low mechanical strength, susceptibility to micro- biology are being developed to enhance the sorp- bial degradation, and difficult biomass/effluent tion capacity of microbial biomass. For instance, separation. In the case of the latter, flotation by ITGC03 18/7/06 18:12 Page 136

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bubble-generation techniques has been examined the bioreactors or valuable metals from solutions as a possible separation process (e.g., Matis et al., where their concentrations are below standard 1996). More often, however, the biomass is immo- recovery levels. In terms of the latter, one such bilized in the form of biofilms or pellets. Nonliving example is gold recovery, because the traditional microorganisms can also be employed in select methods of zinc dust precipitation, carbon wastewater metal removal-treatment systems adsorption, solvent extraction, or ion-exchange where extreme aqueous conditions (i.e., low pH, resins are either of low selectivity or extremely high metal toxicity) are present. Immobilization expensive. A number of studies have shown that occurs through supports such as agar, cellulose, algal cells effectively accumulate gold into their silica, alginate, polyacrylamide gels, collagen, biomass, up to 90% of the gold from solution and metal precipitates (Brierley et al., 1989). (e.g., Hosea et al., 1986). During the process, Based on the various properties highlighted Au(III) is adsorbed onto the cell surfaces, where above, several types of reactors have been devel- it is then reduced to Au(1) or Au(0) by some oped for use in pilot-scale biosorption projects unknown mechanism. Some of the gold even (Volesky, 1990). Briefly, packed-bed columns makes its way into the cytoplasm where it have the wastewater flowing downwards through forms fine-grained, intracellular colloids (e.g., a column filled with biomass. This type of reactor Southam and Beveridge, 1994). More recently, offers the advantage of very high effluent quality it has been demonstrated that various meso- because the stream exiting the column is in con- philic and hyperthermophilic Fe(III)-reducing tact with fresh sorbent material. Unfortunately, bacteria also have the means to precipitate gold clogging occurs when significant concentrations epicellularly by reducing Au(III) to Au(0) with

of suspended solids are involved. Fluidized-bed H2 as the electron donor (Fig. 3.28). This process reactors have the wastewater passing upwards appears to be enzymatically catalyzed, perhaps through the reactor. These reactors avoid the with specific hydrogenases employed to directly problems of clogging but require more effort to reduce Au(III), although attempts to grow ensure that the flow rate is balanced with the Fe(III)-reducing bacteria with Au(III) as the sole biomass size and density. Stirred tanks contain terminal electron acceptor have so far proven biomass dispersed throughout the reactor. They unsuccessful (Kashefi et al., 2001). provide more contact between the biomass and For biomass to be employed in gold bio- wastewaters, but more biomass is generally used recovery, the microbial sorbent would need to to achieve the same quality of effluent as the feature a high maximum loading curve plateau other techniques. A number of novel strategies (in mg Au g−1 of biomass), as well as a steep for improving the biosorption processes are being initial portion of the isotherm indicating a currently developed, including: the use of pulsed high sorption capacity at low equilibrium con- electrical fields to enhance sorption capacity; centrations. Based on a number of dead marine engineering a spiral bioreactor that minimizes algae tested, Kuyucak and Volesky (1989a) docu- space; chemical or heat pretreatment of the mented that Sargassum natans not only exhibited biomass; and growing select, toxic-resistant a desirable steep biosorption isotherm, but it microorganisms in the form of microbial mats also had a maximum uptake comparable to (Lovley and Coates, 1997). commercial ion-exchange resins and activated carbon (Fig. 3.29). Batch kinetic experiments 3.7.2 Biorecovery further indicate that the time required for full biomass saturation with gold depends on the In contrast to bioremediation, biorecovery is the initial aqueous gold concentration, with dilute process whereby microbial biomass is employed solutions requiring only an hour for equilibrium to extract either toxic metals/radionuclides from to be achieved, while concentrated solutions can ITGC03 18/7/06 18:12 Page 137

CELL SURFACE REACTIVITY AND METAL SORPTION 137

pH 2.5 Activated carbon 23°C

400 S.natans biomass) − 1 IRA 400 300

200 A.niger R.arrhizus Gold uptake (mg Au g 100

0 750 nm 0 100 200 300 400 Final concentration (mg L−1)

Figure 3.28 TEM image of epicellular elemental Figure 3.29 Gold biosorption isotherms for gold precipitation associated with Shewanella several different types of microbial biomass and algae. Notice how the gold nicely outlines the cell industrially-used sorbent materials. Starting wall of the intact cells. (Courtesy of Kazem Kashefi gold chloride solutions contained from 10 to −1 and Derek Lovley.) 1000 mg L Au. The graph shows a steep sorption edge for the brown alga Sargassum natans, comparable to the more expensive ion exchange resins (IRA 400) and activated carbon. take several hours. Maximum sorption occurs (Modified from Kuyucak and Volesky, 1989a.) at pH 2.5, as might be expected from the elec- trostatic interaction of protonated ligands and the − anionic, dissolved gold species used (AuCl4 ). the biosorbent for additional metal treatment. In More recently, gold accumulation has been the gold example, Kuyucak and Volesky (1989b) documented for a wide variety of bacteria, fungi, showed that desorption of gold from S. natans and yeasts (Nakajima, 2003). was achievable through the use of a mixture of In order to use biomass for biorecovery, the ferric ammonium sulfate (to oxidize Au0 to Au+), elution of the metal sequestered has to be reason- and thiourea, which forms soluble complexes ably easy to achieve. The eluting solution should with Au+. The elution efficiency was more than also contain the metal in high concentrations 98% effective and the desorption capacity of and the regenerated biosorbent must be capable the eluted biomass remained the same for addi- of another uptake cycle. Many types of eluants tional gold biosorption experiments. Darnall can be used to desorb metals. Some desorbing et al. (1986) also developed an elution scheme agents, such as acids or metal salts, provide cations for selective gold recovery from Chlorella vulgaris. that outcompete the bound metals for the cell’s Most algal-bound metals could be selectively reactive ligands. Another method is to employ desorbed by lowering the pH to 2. However, strong organic ligands (e.g., EDTA) that can strip to desorb the remaining cell-bound Au(III), the the metals from the biomass, thereby “freeing-up” strong ligand mercaptoethanol had to be used. ITGC03 18/7/06 18:12 Page 138

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and reversible electrostatic phenomenon reliant 3.8 Summary upon thermodynamics, analogous to inorganic systems. In this regard, it is not uncommon for Microorganisms have a variety of surface copious amounts of metals to accumulate onto enveloping layers, including the cell wall and the both living and dead microorganisms, at con- extracellular structures residing above them, that centrations far exceeding that predicted based are directly exposed to diffusible components on ligand availability, simply as a consequence in the external aqueous environment. At typical of them being in a concentrated solution. growth pH, these layers are studded with organic Living microorganisms can even compensate for functional groups that are naturally anionic, this by overproducing protective EPS material wettable and thus highly reactive towards metal that sequesters toxic metals, thereby preventing cations. Carboxyl and phosphate groups are the them from disrupting internal cell functions. most important sites for metal adsorption, and Given their ubiquity at the Earth’s surface, their as chemical equilibrium models show, the total rapid rates of metabolism and growth, and their number of reactive ligands are a direct function high chemical reactivity, it is clear that micro- of the architecture and composition of the macro- organisms must play a fundamental role in metal molecules comprising the outermost surfaces. cycling. Indeed, a significant mass of metals in Since considerable ultrastructural variation exists the aqueous environment are intimately associ- between different bacteria, and can even arise ated with cell biomass or tied up as refractory within single species as growth conditions change, organo-metallic complexes. Moreover, the sur- the overall metal sorption capacity of microbial face charge properties of microorganisms further biomass can show fundamental variability. In facilitate their attachment onto submerged sur- dilute solutions, those metals required for meta- faces. This ability has significant implications bolic activities and structural organization are for contaminant transport because metals sorbed preferentially adsorbed from a range of com- onto attached bacteria show limited dispersion peting cations, a property that results from the through the environment. Importantly, these same cell possessing specific ligands that favor one properties have allowed microorganisms to be metal over another. At other times, metal bind- manipulated for a number of industrial processes, ing to a cell’s surface is largely a nonspecific including bioremediation and biorecovery. ITGC04 18/7/06 18:23 Page 139

4 Biomineralization

Microorganisms are remarkably adept at form- barrier through which ions cannot freely diffuse, ing mineral phases. This process, termed bio- minerals form despite external conditions being mineralization, can occur in two different ways. thermodynamically unfavorable. In this chapter The first involves mineral precipitation in the we will review the different types of biominerals open environment, without any apparent con- formed and examine how the process of bio- trol by the cell over the mineral product. This mineralization has affected the geochemical process was defined by Lowenstam (1981) as cycling of mineral-forming elements throughout “biologically induced biomineralization”, with geological time. minerals forming simply as a byproduct of the cell’s metabolic activity or through its inter- actions with the surrounding aqueous environ- 4.1 Biologically induced ment. Simple perturbations, such as the release − − 2+ mineralization of metabolic wastes (e.g., O2, OH , HCO3 , Fe , + NH4 , H2S), enzymatic mediated changes in redox state (e.g., oxidation of Fe(II) or Mn(II)), 4.1.1 Mineral nucleation and growth or the development of a charged cell surface can all induce the nucleation of amorphous to The thermodynamic principles underpinning poorly crystalline minerals with morphologies biomineralization, irrespective of whether they and chemical compositions similar to those pro- are induced or controlled, are the same as those duced by precipitation from sterile solutions. This involved in abiological mineral formation. In is not too surprising considering that biominer- all cases, before any solid can form a certain alization is governed by the same equilibrium amount of energy has to be invested. This energy principles that control abiological mineralization is required for a number of reasons, including: processes. By contrast, “biologically controlled (i) offsetting the potential repulsive interac- biomineralization” is completely regulated, allow- tions between double layers separating the ing the organism to precipitate minerals that solid and solutes; (ii) eliminating the hydration serve some physiological purpose. This process is shells surrounding dissolved ions, so that a specifically designed to form minerals through chemical bond can form between them and the the development of intracellular (within the surface ligands; (iii) removing organic ligands cytoplasm) or epicellular (on the cell wall) that have chelated metal cations; and (iv) to organic matrices, into which specific ions of choice subsequently form a new interface between the are actively introduced and their concentrations nascent nucleus and both the aqueous solution controlled such that mineral saturation states and the underlying substratum upon which it is are appropriately achieved. Because the mineral- formed. The amount of energy required to do this ization site is isolated from outside the cell by a can be viewed as an activation energy barrier, ITGC04 18/7/06 18:23 Page 140

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must exceed the solubility product of the solid mineral phase. In other words, a certain degree of supersaturation has to be reached (see Stumm and Morgan, 1996 for details). This can be

G1* described in thermodynamic terms, where the ∆ G° free energy of nucleation ( Gn) is constrained

Dissolved ∆ ° G2* by the free energy of the bulk solution (∆G ) G1 ∆G ° bulk silica 3 and the free energy of the developing mineral Amorphous ∆G ° nucleus (∆G ): silica 2 min ∆ =∆ +∆ Quartz Gn Gbulk Gmin (4.1) Chemical state The bulk solution free-energy term is, in turn, a secondary function of the degree to which a Figure 4.1 Relation between the standard free solution is oversaturated (lnΩ) (reaction (4.2)), energy (G°) of dissolved solutes (e.g., silica) and Ω the amorphous and crystalline solid phases they where is a value based on the ion activity form upon supersaturation. The larger the decrease product (IAP) of the solution divided by the in ∆G° upon precipitation of the solid, the more solubility product of the corresponding mineral ∆ ∆ stable it will be (e.g., G 3° versus G1°). Yet, for phase (Ksp): recall activity is the “effective con- the solid to form, a certain amount of activation centration” of the chemical species, which is less energy (G*) has to be invested. Therefore, the lower than the actual concentration due to ion com- the activation energy barrier, the faster the reaction plexation in solution – for dilute solutions, activ- proceeds. This helps explain why amorphous silica nucleates first despite being less stable than quartz. ity and concentration are essentially equivalent (Modified from de Vrind-de Jong and de Vrind, terms. The other terms represent Boltzmann’s 1997.) constant (k), temperature (T, in °K), and the number of ions or molecules in the nucleus (n):

∆G =−nkT lnΩ (4.2) and for mineral formation to proceed, the barrier bulk must be overcome by the energy released as a ∆G is a product of the surface area of the consequence of bond formation in the solid min nucleus (A) and interfacial free energy (also phase. The standard free energy (G°) of a solid known as surface tension) of the solid phase (γ) is lower than that of its constituents in solu- (equation 4.3): tion, so if the activation energy barrier can be overcome, the reaction proceeds spontaneously ∆G = γA (4.3) towards mineral nuclei forming (Fig. 4.1). If min instead the activation energy barrier is pro- Accordingly, the overall free energy of nucleation hibitively high, metastable solutions will per- can be written as: sist until either the barrier is reduced or the ∆ =− Ω + γ concentration of ions are diminished, thereby Gn nkTln A (4.4) reducing the thermodynamic driving force towards precipitation. When considering pure solutions in which only The first step in mineral formation is nucleation. the mineral constituents are present, nuclea- This process involves the spontaneous growth of tion is said to be homogeneous. In homogene- a number of critical nuclei of a certain size that ous reactions, critical nuclei are formed simply are resistant to rapid dissolution. For this to occur, by random collisions of ions in a supersaturated the concentration of ions or atoms in solution solution. Conversely, heterogeneous nucleation ITGC04 18/7/06 18:23 Page 141

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involves the development of critical nuclei on value, new surface area is created mainly by the the surfaces of foreign solids. The surface can be nucleation of many small grains characterized by viewed as a template of ideally spaced ligands that high surface area to mass ratios, a regime referred bind and stabilize the nascent nuclei. It essen- to as nucleation-controlled. At activities below tially acts as a catalyst that reduces the interfacial the critical value, surface area increases by the contributions to the activation energy barrier accretion of additional ions to existing grains and thereby increases the nucleation rate. (i.e., crystal-growth controlled). Now, if the com- After critical nuclei are formed, the continued position of a fluid was to start in the nucleation- adsorption of ions to them is accompanied by controlled regime, the generation of new surfaces a decrease in free energy. This process is known by nucleation would rapidly increase, causing the as mineral growth (if the ions are the same as level of supersaturation to collapse to at least the those of the substratum) or surface precipitation critical value. This means that in nature, a degree (if the ions are different), and it goes on spon- of supersaturation above the critical value will taneously until the decreasing supply of ions not be maintained for lengthy periods of time. becomes prohibitive. The initial mineral phase Let us turn to silica precipitation as an example. formed is usually amorphous, characterized by If a concentrated silica solution (10 −2 mol L−1) its high degree of hydration and solubility, and was emitted from a hot spring vent, it would be its lack of intrinsic structure, compared to more supersaturated with regards to all silica phases, stable, crystalline phases. This pattern arises be- but because amorphous silica has the lower inter- cause even though the surface area of the nucleus facial free energy it nucleates first despite quartz increases during hydration, the interfacial free being the more stable phase with lower solubility. energy between the hydrated surface of an As amorphous silica nucleates rapidly it drives amorphous nucleus and a dissolved ion reduces the dissolved silica activity down to its critical ∆ more rapidly, thereby resulting in lower Gmin, value, which happens to be below that required and hence faster nucleation rates than are pos- to nucleate quartz, i.e., to the left side of quartz’s sible for crystalline analogs (Nielson and Söhnel, critical value (Fig. 4.2). 1971). This means that amorphous phases, such Crystalline minerals that would otherwise as amorphous silica (γ = 46 mJ m−2), are kinetic- be difficult or impossible to directly nucleate at ally favored if the solution composition exceeds low temperatures can circumvent the activation their solubility. By comparison, its crystalline energy barriers by making use of the amorphous equivalent, quartz (γ = 350 mJ m−2), has a higher precursors as templates for their own growth. interfacial free energy, is relatively insoluble, Once it begins to grow, the crystal increases its and it nucleates slowly at ambient temperatures. own surface area and, in doing so, controls the Often the transition between amorphous and proximal free ion activity, driving it down crystalline phases involves the precipitation of towards its solubility product. When this hap- metastable phases. pens, the saturation state of the solution moves The nucleation rate also has an important below the solubility of the precursor, causing the bearing on the size of the critical nuclei formed. latter to dissolve (Steefel and Van Cappellen, If the nucleation rate for an amorphous mineral 1990). phase is plotted against the saturation state, a Although thermodynamics can predict the typical curve is obtained. It is characterized by transformation sequence based on energetics, a critical supersaturation value below which it cannot determine the kinetics. Sometimes the nucleation rate is extremely slow and above the reactions are relatively quick, such as the which the nucleation rate increases very rapidly formation of magnetite on ferric hydroxide in (Steefel and Van Cappellan, 1990). What this sediment (see section 4.1.3). At other times, implies is that at ion activities above the critical the reaction rates are immeasurably slow over ITGC04 18/7/06 18:23 Page 142

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1010 hundreds of meters until it too transforms into AB quartz (see section 6.2.7(c)). Another important factor influencing the dissolution of the precursor phase is its large 108 surface area and high solubility compared to the )

–1 newly generated secondary crystals. This feature y

–3 forms the basis for Ostwald ripening, a process 6 10 involving the spontaneous redistribution of mass from the more numerous precursor grains in the system to fewer stable crystals that are typically 104 larger, and more evenly distributed, in size (see

Quartz saturation Baldan, 2002 for details). It can also describe

Nucleation rate (m Crystal the distribution pattern of dissolved ions into Nucleation- 2 growth- colloids (e.g., Iler, 1979). So, as the smaller 10 controlled controlled precursors act as “seeds” for the larger, secondary phases, the latter grow and the area around them becomes depleted of precursors (e.g., Fig. 4.3). The 100 –4 –3 –2 –1 reason Ostwald ripening takes place is that the 10 10 10 10 larger secondary phases are more energetically Silica activity (mol L–1)

Figure 4.2 Rate of heterogeneous nucleation A Supersaturated B Colloid of quartz and amorphous silica at 25°C, using solution formation interfacial free energies of 350 and 46 mJ m−2, respectively. The solid lines reflect the critical silica activities for nucleation of amorphous silica (A) and quartz (B). The graph shows that in a silica supersaturated solution (10 −2 mol L−1) amorphous Cell Monomers silica will nucleate (open circles) in preference to quartz because of its lower interfacial free energy.

Accordingly, the SiO2 activity decreases to the saturation state of amorphous silica, which is below the critical value for quartz. This prevents quartz Silica Oligomer from nucleating in the short term. (Adapted from oligomers dissolution Steefel and Van Cappellen, 1990.) Figure 4.3 An example of Ostwald ripening of silica species at hot springs. (A) As the silica geological time and the amorphous or metastable supersaturated solution is discharged, monomeric phases persist in sediments supersaturated with silica rapidly polymerizes into silica oligomers the thermodynamically most stable minerals. of various size (dimmers, trimers, etc.). Some of those oligomers then bind to solid surfaces, They can show little discernible alteration for tens including microorganisms growing around the of millions of years until pressure-temperature vent. (B) Over the course of hours to days, some changes associated with burial cause the reation of those oligomers increase in size to colloidal sequence to advance to the next stage (Morse dimensions by the accretion of monomers and and Casey, 1988). For example, amorphous silica oligomers. As the colloids grow, the monomeric shells deposited onto the seafloor slowly dis- fraction decreases. As a result, some oligomers solve at shallow depths and re-precipitate as depolymerize, leading to a bimodal distribution cristobalite, which remains stable to depths of of monomer and colloids. ITGC04 18/7/06 18:23 Page 143

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favored than the smaller precursors. This might may lead to secondary magnetite formation. Other seem a bit contradictory to above, but consider microorganisms enzymatically oxidize reduced that even though the formation of small grains metals (e.g., Fe(II)), and facilitate the precipitation is kinetically favored in supersaturated solutions, of metal oxyhydroxides outside the cell wall. they have large surface area to volume ratios and Certainly, the microenvironment surrounding each microbial cell can be quite different from that of the molecules exposed on the surface are energet- bulk aqueous environment, and as a result the cell ically less stable and more reactive than those surfaces can lead to the development of mineral sheltered at the interior. By contrast, larger crystals phases of a type that might not be predicted based (or colloids), with their greater volume to surface solely on our knowledge of the geochemistry of the area ratios, represent a lower energy state, and are bulk fluid (Little et al., 1997). more thermodynamically favored when activation energy barriers of both are overcome. In nature there exists a wide variety of microbial As will become evident in this chapter, mineral precipitates. This leads to the obvious microorganisms contribute significantly to the question – why so much diversity? Fortunately, development of extremely fine grained (often the answer is simple. Those biominerals formed <1 µm in diameter) mineral precipitates. The passively are dependent upon the chemical com- vast majority are formed passively, and in this position of the fluids in which they are growing, regard microorganisms influence mineralization such that a particular microorganism will form in two significant ways: a mineral phase from the solutes immediately available to it. Conversely, the same micro- 1 Reactive surfaces – The cell walls and extracellular organism in a different environment would likely layers contain an abundance of ionized surface form a different mineral phase altogether. The ligands where sorption reactions take place. These sites subsequently lower the interfacial energies variations can be as subtle as a change in redox for heterogeneous nucleation while simultaneously state. For example, it is well known that the decreasing the surface area of the nucleus that is anionic ligands comprising a cell’s surface can in contact with the bulk solution. Furthermore, the form covalent bonds with dissolved Fe(III) spe- spacing of the ligands affects which cations will cies, which, in turn, can lead to charge reversal at be bound (recall section 3.3.3), and thus, some the cell surface. Invariably, this positive charge microorganisms even have the means to control the will attract anionic counter-ions from solution. structure and orientation of the incipient nucleus. So, in the sediment, iron staining of a bacterium For that reason, microorganisms have been likened to mineralizing templates because the composition may lead to the precipitation of an iron sulfate and structure of their functional groups are ideal for precipitate in the oxic zone, whereas another the passive formation of a number of different types bacterium may instead form an iron sulfide at of mineral nuclei. It should be stressed, however, depth, where conditions are reducing. In the that microorganisms only serve to enhance the following section, a number of passively formed precipitation kinetics in supersaturated solutions; biogenic minerals will be discussed, and what they neither increase the extent of precipitation nor will become apparent is that microorganisms do they facilitate precipitation in undersaturated solutions (e.g., Fowle and Fein, 2001). interact with the solutes in intimate contact with their surface layers, and in doing so they 2 Metabolism – Microbial activity can affect mineral essentially function as reactive surfaces for saturation states immediately outside the cell through mineral precipitation. the excretion of metabolites. For example, denitrify- ing or photosynthetic bacteria promote an increase in solution pH that is supportive of carbonate 4.1.2 Iron hydroxides precipitation; sulfate-reducing bacteria induce the − The most geologically widespread biomineral formation of metal sulfides by generating H2S/HS ; while the release of Fe2+ by Fe(III)-reducing bacteria is ferric hydroxide (also loosely referred to as ITGC04 18/7/06 18:23 Page 144

144 CHAPTER 4

ferrihydrite). Its chemical composition is

5Fe2O3·9H2O, but for chemical simplicity it is usually described as Fe(OH)3. Ferric hydroxide has been shown to form in association with microbial biomass in any environment where Fe(II)-bearing

waters come into contact with O2. This includes springs, sediment/soil pore wasters, aquifers, hydro- thermal systems, mine wates, and water distribu- tion systems, to name just a few (see Konhauser, 1998 for review). Fossil structures that resemble modern iron-depositing bacteria have also been found in laminated black cherts and Precambrian µ banded iron formations (BIFs) (Robbins et al., 4 m 1987). As will be discussed in section 7.3.2, there is even some circumstantial evidence suggesting that microbial activity was directly involved in Figure 4.4 TEM image of a lysed bacterium in the initial deposition of Fe-rich sediment, which which the cytoplasm (arrow) has been completely replaced by ferric hydroxide. (From Konhauser and later consolidated to make BIF. Ferris, 1996. Reproduced with permission from the When Fe-encrusted cells are viewed in detail Geological Society of America.) under the transmission electron microscope (TEM), it is apparent that mineralization occurs through a series of stages, often beginning with the cell’s way of compartmentalizing and isolat- Fe-adsorption to extracellular polymers (EPS) or ing unwanted iron into a localized precipitate wall material (recall Fig. 3.12), followed by the (Brake et al., 2002). nucleation of small (<100 nm in diameter) ferric hydroxide grains, and with sufficient time, the (a) Passive iron mineralization complete encrustation of the cell. These steps have also been demonstrated experimentally and The actual role microorganisms play in ferric described by Langmuir-type isotherms showing hydroxide formation can range from the com- the continuum between metal adsorption and pletely passive to that more facilitated in nature. mineral precipitation (e.g., Warren and Ferris, Yet, by our current definitions, this process is 1998). Not only do bacteria serve as templates not considered biologically controlled because for iron deposition, but their organic remains the microorganisms do not manage all aspects of frequently become incorporated into the mineral the mineralization process. In the most passive precipitates during crystal growth such that the of examples, dissolved Fe(II) transported into sediment ends up with iron–organic composites an oxygenated environment at circumneutral

that may, or may not, retain features of their pH spontaneously reacts with dissolved O2 to microbial origins (e.g. Fig. 4.4). Intracellular precipitate inorganically as ferric hydroxide on mineralization typically occurs when the plasma available nucleation sites. Microorganisms simply membrane has been breached during cell lysis, yet act as such sites, and over a short period of time two recent reports of viable bacteria (Shewanella submerged communities can become completely putrefaciens) and photosynthetic protists (Euglena encrusted in amorphous iron (commonly referred mutabilis) that contain intracellular ferric hydro- to as ochre because of their bright red/brown color) xide granules suggest that the minerals may either as abiological surface catalysis accelerates the rate serve some unrecognized physiological function of mineral precipitation. While initial microscopic (Glasauer et al., 2002) or that they may represent observations of such samples often indicate a ITGC04 18/7/06 18:23 Page 145

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paucity of microorganisms, staining the iron-rich visible inhabitant of many freshwater, low- sediment with fluorescent dyes for nucleic acids oxygenated iron seeps is Leptothrix ochracea. (e.g., acridine orange) often reveals high densities This chemoheterotroph frequently forms thick of microorganisms closely associated with the iron filamentous layers comprising tangled matrices precipitates (e.g., Emerson and Revsbech, 1994a). of tubular sheaths encrusted in iron. In an iron This brings up one important point – it is often seep in Denmark, cell densities range from quite difficult to delineate microbial versus abio- approximately 108–109 cells cm−3 (Emerson and logical contributions to mineral precipitation. So, Revsbech, 1994a). Those high numbers pro- just because a microorganism is associated with a moted Fe(III) accumulation rates of 3 mm day−1. mineral phase does not mean it formed it! One interesting observation made was that it At other time microorganisms are more active was rare to find intact filaments of L. ochracea in the mineralization process in that ferric hydrox- cells inside the sheaths (e.g., Fig. 4.5). This cor- ide forms through the oxidation and hydrolysis of relates well with experiments that have shown cell-bound Fe(II), the binding of ferric ion species Leptothrix continuously abandons its sheath at + 2+ 3+ µ −1 (e.g., Fe(OH)2 ; Fe(OH) ; Fe ) and colloids to a rate of 1–2 m min , leaving behind sheaths negatively charged ligands, or the alteration of of 1–10 cells in length that continue to deposit local pH and redox conditions around the cell ferric hydroxide. This would seem to indicate due to their metabolic activity. Indeed, the iron- that the microorganisms actively prevent them- coatings on cells grown in Fe-rich cultures are selves from becoming permanently fixed into sufficiently dense to visualize the bacteria under the mineral matrix (van Veen et al., 1978). the TEM without the standard use of metal stains (MacRae and Edwards, 1972). Because of the ubiquity of iron biomineralization in nature, it was suggested that under circumneutral condi- tions any microorganism that produces anionic ligands will nonspecifically adsorb iron cations or fine-grained iron oxyhydroxides from the sur- rounding waters (Ghiorse, 1984). This is not unexpected given that the isoelectric point of pure ferric hydroxide is between 8 and 9. Ferric hydroxide also develops on the organic remains of dead cells, implying that iron mineralization can occur independent of cell physiological state. A natural corollary to this is the observation that organic matter commonly adsorbs onto Fe-rich sediment through reactions with surface >Fe- + > 0 OH2 and Fe-OH groups (Tipping, 1981). 800 nm (b) Chemoheterotrophic iron mineralization

There are a number of microorganisms, the so- Figure 4.5 TEM image of two ferric hydroxide- called iron-depositing bacteria, that facilitate encrusted Leptothrix ochracea cells from an iron iron mineralization by having surface ligands seep in Denmark. The cross-section shows one that promote Fe(II) oxidation, although it is ensheathed cell and one abandoned sheath. not believed that they gain energy from the (From Emerson, 2000. Reproduced with permission process (Emerson, 2000). The most common from the American Society of Microbiology.) ITGC04 18/7/06 18:23 Page 146

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Other heterotrophic bacteria, such as filament- (recall section 2.5.5). Although most enzymatic ous species from the genera Sphaerotilus, Crenothrix, oxidation of Fe(II) occurs at extremely low pH, Clonothrix, and Metallogenium, as well as unicellu- such as in acid rock drainage environments, the lar cocci of the Siderocapsaceae family, can induce activity of Acidithiobacillus ferrooxidans or Leptos- ferric hydroxide precipitation through the oxida- pirillum ferrooxidans generally does not promote tion of organic iron chelates. Essentially, they use in situ ferric hydroxide precipitation because the the organic carbon of such ligands as an energy Fe(III) formed remains soluble until more alkaline source, and as a result, the Fe(III) is freed and pH conditions ensue. However, at neutral pH, easily hydrolyzed (Ghiorse and Ehrlich, 1992). and under partially reduced conditions, chemo- lithoautotrophic Fe(II) oxidation by Gallionella (c) Photoautotrophic iron mineralization ferruginea leads to high rates of iron mineralization (e.g., Søgaard et al., 2000). In fact, their extra- As discussed in section 2.2.3, some anoxygenic cellular stalk can become so heavily encrusted photosynthetic bacteria are capable of oxidiz- with amorphous ferric hydroxide that the major- ing Fe(II) to Fe(III), which then hydrolyzes to ity of the dry weight is iron (recall Fig. 2.25).

Fe(OH)3. This process could be described as Similar to L. ochracea, G. ferruginea is a common “facilitated biomineralization” because ferric iron inhabitant of iron springs, and where it is abund- precipitates as a direct result of the metabolic ant, the stalk material appears to form the sub- activity of the microorganisms. These bacteria are stratum upon which subsequent Fe(II) oxidation phylogenetically diverse and include green sulfur occurs. However, actively growing Gallionella and bacteria (e.g., Chlorobium ferrooxidans), purple Leptothrix populations appear to occupy separate nonsulfur bacteria (e.g., Rhodobacter ferrooxidans), microniches, the former preferring areas of sedi- and purple sulfur bacteria (e.g., Thiodictyon sp.). ment with lower oxygen concentrations (Emerson Ferrous iron can be used as an electron donor and Revsbech, 1994a). by these bacteria because the standard electrode The rates of iron precipitation by G. ferruginea potential for Fe2+/Fe3+ (+0.77 V) is applicable are impressive, with cell densities on the order only at very acidic pH, whereas at more neutral of 109 cells cm−3 oxidizing up to 1200 nmol of pH, the potential shifts to less positive values due Fe(II) per hour. This could lead to a hypothetical to the low solubility of ferric iron cations. For oxidation rate of 1.1 × 10 −11 mol Fe(III) per cell instance, the electrode potential of the Fe2+/Fe3+ each year (Emerson and Revsbech, 1994b). In the couple for the bicarbonate–Fe(II) system at pH 7 wells, water pipes, and field drains comprising is approximately +0.20 V, low enough to provide water distribution systems, the large amounts of sufficient reducing power to sustain microbial iron precipitated by G. ferruginea has long been growth (Ehrenreich and Widdel, 1994). Photo- recognized as a causative agent of serious clogging ferrotrophic growth can also be sustained by the problems (e.g., Ivarson and Sojak, 1978). presence of soluble ferrous iron minerals, such

as siderite (FeCO3) and iron monosulfide (FeS), (e) Hydrothermal ferric hydroxide deposits but not insoluble minerals, such as vivianite

(Fe3(PO4)2), magnetite (Fe3O4), or pyrite (FeS2) Arguably the most persuasive example of ferric (Kappler and Newman, 2004). hydroxide biomineralization is at marine hydro- thermal settings. It commonly precipitates directly (d) Chemolithoautotrophic iron mineralization on the seafloor from diffuse, low temperature emissions, where subsurface mixing of hydro- The formation of ferric hydroxide may also stem thermal fluids with infiltrating seawater produces from the ability of some chemolithoautotrophic dilute, partially oxidized solutions that range in bacteria to oxidize Fe(II) as an energy source temperature from near ambient deep sea (~2°C) ITGC04 18/7/06 18:23 Page 147

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A B

EPS Fe(OH)3

Tube 1 µm

Figure 4.6 (A) SEM close-up of a vestimentiferan tube worm from the vent fields on the southern Juan de Fuca Ridge. The worm is colonized by bacteria that precipitate ferric hydroxide on their cell walls and within the EPS that holds the community together. Eventually the ferric hydroxide grains coalesce between cells and a continuous crust forms in both vertical and horizontal dimensions. This then serves as scaffolding for a new generation of bacteria. (B) Over time, continued iron mineralization leads to the formation of substantial iron deposits that rise as spires from the seafloor. (From Juniper and Tebo, 1995. Reproduced with permission from CRC Press.)

to around 50°C. The deposits themselves range some of which have morphologies reminiscent from centimeter-thick oxyhydroxide coatings to of neutrophilic Fe(II)-oxidizing bacteria, e.g., more voluminous mud deposits (Juniper and twisted ribbons like Gallionella ferruginea and Tebo, 1995). A spectacular example of the former straight sheaths similar to Leptothrix ochracea. can be seen associated with the unicellular and Although direct evidence supporting enzymatic filamentous bacteria colonizing vestimentiferan Fe(II) oxidation was never put forth, indica- tube worms at the southern Juan de Fuca Ridge. tions for a microbial role in mineralization (Fig. 4.6). comes from the fact that the hydrothermal Extensive Fe(III)-rich mud deposits have been waters in which these deposits formed were

described from a number of sites: (i) the shallow slightly acidic (pH 5–6) and low in dissolved O2 = waters of the present caldera of the island of (pO2 0.06 atm). These conditions give a half- Santorini; (ii) the Red and Larson Seamounts time for the oxidation of Fe(II) by O2 in seawater near 21°N on the East Pacific Rise; and (iii) the of approximately 30 years. Coupled with the Loihi Seamount, Hawaii. The Santorini site is strong currents on the seamount, it is highly perhaps the best cited example of the forma- unlikely that spontaneous oxidation of Fe(II) to tion of ferric hydroxide resulting from microbial Fe(III) could occur without biological catalysis activity. There, mineralized stalks of Gallionella (Alt, 1988). ferruginea occur in such masses that it is more The Loihi Seamount is the newest shield than probable that the bacteria are responsible volcano that is part of the Hawaiian archipe- 2+ for iron precipitation (Holm, 1987). lago. The impact of high Fe (but low H2S) The Red Seamount is characterized by an emissions is readily apparent in extensive deposits abundance of Fe-encrusted bacterial filaments, of ferric hydroxide that encircle the vent orifices, ITGC04 18/7/06 18:23 Page 148

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and in the peripheral regions where visible mats must influence the global cycling of trace metals are present. The initial microscopic analyses in seawater (Kennedy et al., 2003). revealed that both deposits were rich in Fe- encrusted sheaths, similar in appearance to (f) Formation of iron oxides Leptothrix ochracea (Karl et al., 1988). Since then, a number of studies have shown that the Once the primary ferric hydroxides are pre- iron deposits have abundant microbial popula- cipitated and incorporated into the sediment, tions associated with them, up to 108 cells ml−1 several diagenetic reactions can subsequently (wet weight) of mat material, and that some of alter their surface reactivity, morphological those cells are microaerophilic Fe(II)-oxidizers. characteristics, and even mineralogy. In most It has been estimated that at least 60% of the natural systems, ferric hydroxide serves as a iron deposited at the Loihi vents is directly precursor to more stable iron oxides, such as

or indirectly attributable to bacterial activity. goethite (FeOOH) and hematite (Fe2O3). The This percentage accounts for the amount of transformation into more crystalline minerals Fe(III) generated through direct catalysis by proceeds through: (i) dehydration and internal the bacteria, as well as the proportion of ferric rearrangement leading to hematite; and (ii) hydroxide that results from Fe(II) auto-oxidation dissolution-reprecipitation leading to goethite on bacterially bound ferric hydroxide particles (Schwertmann and Fitzpatrick, 1992). These (e.g., Emerson and Moyer, 2002). reactions typically occur without biological The recurring observations of bacteria in envir- participation, yet ferric hydroxide associated onments where they are covered in iron suggests with microbial surfaces can similarly undergo that these environments must offer the bacteria these transformations, leading to a cell encrusted propitious growth conditions. Quite possibly the in iron oxides (e.g., Fig. 4.7). Experiments have continual supply of trace metals, which adsorb documented that bacterially produced ferric or co-precipitate directly onto iron hydroxides, hydroxide can undergo spontaneous dehydra- may serve as an ideal nutrient source in close tion to hematite in an aqueous medium in a proximity to the cells (e.g., Ferris et al., 1999). Certainly the high surface reactivity of biogenic ferric hydroxide deposits, often more so than their inorganic equivalents, testifies to the fact that they have very high metal partitioning coefficients. The charge and abundance of the iron hydroxide surfaces will be dependent upon a number of factors, such as salinity, fluid com- position, and pH, the latter relating to surface- charge characteristics such as the isoelectric point. The latter, in turn, will be affected by chemical impurities, i.e., silica-containing iron 200 nm hydroxides have a much lower isoelectric point than pure ferric hydroxides (Schwertmann and Fechter, 1982). Therefore, depending on condi- Figure 4.7 Precipitation of acicular grains of tions the amphoteric biogenic iron precipitates goethite on an unidentified bacterium collected can either sorb anions or metal cations. Signific- from a hot spring microbial mat in Iceland. Note antly, given that ferric hydroxides are a common how some of the mineral grains have been shed constituent of mid ocean ridge (MORs) venting (arrow) from the cell surface. (From Konhauser systems, and that MORs span over 55,000 km on and Ferris, 1996. Reproduced with permission the ocean floor, such metal sorptive properties from the Geological Society of America.) ITGC04 18/7/06 18:23 Page 149

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few hours at 80°C, or in 10–14 days at 40°C (Chukhrov et al., 1973). The mechanisms by which these transforma- tions occur has been addressed by Banfield et al. (2000), who suggest that crystal growth is accom- plished by the elimination of water molecules and the re-assembly of Fe–O–Fe bonds at multiple sites, leading to coarser, polycrystalline material. This, however, requires that some of the particles are not physically adsorbed to the organic ligands because they would constrain the movement and aggregation of surface-bound ferric hydroxide nanoparticles during their natural transforma- tion into an iron oxide.

4.1.3 Magnetite

A great deal of research has focused on the potential for bacteria to contribute to the stable remnant magnetism of modern soils and sedi- ments, and whether biogenic magnetite signals exist in the ancient geomagnetic record (e.g., Kirschvink, 1982). This interest has arisen from the recognition that a number of bacteria appear to form magnetite crystals that are single domain, i.e., grains with a high natural magnetic reman- ence. These biogenic minerals are known to 300 nm precipitate under both “biologically controlled” and “biologically induced” conditions. For the moment, we will concentrate only on Figure 4.8 TEM image of epicellular/ those bacteria that “induce” magnetite formation. extracellular, fine-grained magnetite particles Dissimilatory Fe(III) reducers such as Geobacter formed as a byproduct of Fe(III) reduction by Geobacter metallireducens. (Courtesy of Derek metallireducens and Shewanella putrefaciens are the Lovley.) most extensively studied species shown to pro- duce magnetite crystals as a byproduct of their metabolism – they oxidize fermentation products Most particles (over 95%) are usually found at and reduce Fe(III) from ferric hydroxide (recall the lower end of this size range, which means that section 2.4.4). The magnetite forms outside the they fall within the superparamagnetic size range cell and it is not aligned in chains (e.g., Lovley (nonmagnetic behavior), as a diameter greater et al., 1987). As a matter of fact, this process is than 30 nm is required for permanent, single very reminiscent of how G. metallireducens forms magnetic domain behavior (Moskowitz et al., uraninite from the reduction of uranyl ions 1989). Not surprisingly, in wet mounts Geobacter (recall section 2.4.5(a)). does not orient itself in response to an applied Some characteristic features of these magnetite magnetic field. Despite this low percentage of grains are that they are poorly crystalline and they single domain magnetite, G. metallireducens might consist of a mixture of round and oval particles still be a major contributor to that size fraction that range in size from 10 to 50 nm (Fig. 4.8). because on a per cell basis, they generate some ITGC04 18/7/06 18:23 Page 150

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5000 times more magnetite than a magnetotactic activity of Fe(III)-reducing bacteria. In experi- bacterium (see section 4.2.1). The limitation to ments where Fe(II) was added to cultures of the amount of magnetite produced is primarily Dechlorosoma suillum, with nitrate as the terminal determined by how much available ferric iron electron acceptor, the bacteria induced the pre- can be added to the culture (Frankel, 1987). cipitation of a greenish-gray, mixed Fe(II)–Fe(III) At present, the actual role that Fe(III)-reducing hydroxide, known as green rust. This mineral is bacteria play in magnetite formation remains generally unstable in the environment, and further unresolved. On the face of it, the abiological oxidation led to the formation of magnetite within reaction of Fe2+ with ferric hydroxide should be just 2 weeks (Chaudhuri et al., 2001). Meanwhile, sufficient to precipitate magnetite. Nevertheless, other experimental studies have documented experimental studies show that magnetite does not magnetite formation in association with suspended form if the cultures are incubated at temperatures cultures of phototrophic Fe(II)-oxidizing bacteria, too high for growth, if the inoculated medium is through the reaction of Fe2+ with biogenic ferric sterilized prior to incubation, or if nongrowing cells hydroxide precipitates (Jiao et al., 2005). are added to the experimental solution (Lovley In modern marine and freshwater sediments, et al., 1987). These observations suggest that the much of the magnetite forms in the suboxic layers metabolism of the Fe(III)-reducing bacteria must where Fe(III) reduction takes place (e.g., Karlin contribute more than just Fe2+ to magnetogenesis. et al., 1987). It has even been found associated One possibility is that magnetite formation is with gas seeps and solid bitumen, where its forma- favored by high pH; a condition met during tion appears to be linked to the microbial reduc- Fe(III) reduction (reaction (4.5)). The Fe2+ that tion of iron oxyhydroxides with the hydrocarbons forms then adsorbs onto other ferric hydroxide serving as the electron donors (e.g., McCabe et al., grains, transforming the latter into magnetite 1987). This process is supported by experimental (reaction (4.6)). Therefore, the appropriate com- findings of magnetite accumulation during toluene bination of a high Fe2+ concentration and a high oxidation coupled to Fe(III) reduction by G. pH at the contact of the Fe(III) solid might pro- metallireducens (Lovley and Lonergan, 1990). vide the ideal interface for secondary magnetite Similar processes likely played a role in the formation (Lovley, 1990). geological past. For instance, the isotopically light δ13C values in Precambrian BIF carbonate − + → CH3COO 8Fe(OH)3 minerals and the extensive presence of secondary 2+ + − + − + 8Fe 2HCO3 15OH 5H2O (4.5) magnetite in the same sedimentary sequences suggests that Fe(III)-reducing bacteria were im- 2OH− + Fe2+ + 2Fe(OH) → Fe O + 4H O 3 3 4 2 portant in shaping the mineralogical component (4.6) of the Fe-rich marine sediments during diagenesis Magnetite has also been shown to form by micro- (see section 6.2.4(c)). This respiratory pathway bial reduction of lepidocrocite (γ-FeOOH), a has also been used to explain the general paucity polymorph of goethite (Cooper et al., 2000). In of organic matter in BIFs (Walker, 1984). this case, the actual step in magnetite formation proceeds via a ferrous hydroxide intermediate 4.1.4 Manganese oxides (reaction (4.7)): The development of Mn(III) hydroxides and γ + 2+ + → Mn(IV) oxides occurs in the same types of modern ( -FeOOH)2 Fe H2O γ + + + → oxic–anoxic interfacial environments where ferric ( -FeOOH)2·FeOH H + + + Fe3O4 H2O 2H (4.7) hydroxide forms, but because dissolved Mn(II) is not subject to as rapid a chemical oxidation as Recent studies have now additionally shown that Fe(II), it may accumulate to greater concentrations magnetite formation does not strictly require the in oxic waters and sediment/soil pore waters. For ITGC04 18/7/06 18:23 Page 151

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AB

12 cm 1 cm

Figure 4.9 (A) Photograph of Fe-rich manganese nodules on the seafloor north of the Puerto Rico Trench. Depth is 5339 meters (courtesy of the Woods Hole Oceanographic Institute, WHOI). (B) Cross-section of a nodule from the Blake plateau, off South Carolina, at a depth of 800 meters, showing concentric laminations. Scale bars are approximate (courtesy of Frank Manheim).

part of Earth’s history, chemical stratification synthesis, and thus provides some energy to the might have existed in the water column over much cell community (Ehrlich and Salerno, 1990). of the continental shelves, where deep anoxic Manganese (III/IV) oxyhydroxides are also waters with high Fe2+ and Mn2+ content mixed a major component of metalliferous sediments

with shallower, O2-bearing waters. Not only did found on the flanks of ridge crests and where they this lead to Precambrian BIF deposition, but also settle out from hydrothermal plumes. Analyses some of the world’s largest and most valuable of the hydrothermal plumes emanating from the manganese deposits (Force and Cannon, 1988). southern Juan de Fuca Ridge have shown that the particulate fraction is largely composed of (a) Hydrothermal manganese deposits encapsulated bacteria encrusted in iron and manganese, with Fe-rich particles predominat- At circumneutral pH, it is generally accepted ing near the vent and Mn-rich particles further that most Mn(II) oxidation is due to microbial off-axis. These observations imply that bacteria catalysis (recall section 2.5.6). One environment scavenge metals from the plumes, but whether where this is testable is at some deep-sea hydro- they actively oxidize Fe(II) and Mn(II) was left thermal vents, where Fe(II) is precipitated at unresolved (e.g., Cowen et al., 1986). depth as iron sulfides, thus allowing high con- centrations of Mn(II) to be released into oxidiz- (b) Ferromanganese deposits ing seawater (Mandernack and Tebo, 1993). The most studied hydrothermal manganese oxide It is common for manganese to co-precipitate deposits are those at the Galápagos rift zone, with iron, leading to what are referred to as ferro- where actively accreting mounds consisting of manganese oxides. Likely the most recognized

todorokite/birnessite (MnO2) are being formed examples of ferromanganese precipitation are as a result of the hot, metal-laden fluids percolat- the laminated concretions and nodules that ing up through the sediment (Corliss et al., 1979). form in soils, lake sediments, and on the seafloor Bacterial isolates collected from there suggest (Fig. 4.9). The mass of manganese associated with that Mn(II) oxidation may be coupled to ATP the nodules is truly impressive. In the Pacific ITGC04 18/7/06 18:23 Page 152

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Metal-rich

phytoplankton O2 River input pH 8 (metal oxides) Concretion Transport formation of particles Erosion of (currents) Eroded particles concretions Degrading Metal-oxidizing sink biomass bacteria Mn2+ Transport of nodules 2+ (traction) Mn Mn(IV) reduction zone Fe2+ Oxic 2+ Fe Anoxic

Mn2+ Fe(III) reduction zone Not to scale Fe2+

Figure 4.10 Model showing how ferromanganese nodules might form in certain lake sediments. (Adapted from Dean et al., 1981.)

Ocean, it has been estimated that 1012 tons of incorporation of metals onto some form of nucleus, nodules exist, predominantly in pelagic (deep-sea) which can be any solid mineral or organic sub- sediments, with an annual rate of formation of stratum. With continued accretion of iron and 6 × 106 tons (Mero, 1962). In Oneida Lake, manganese to the existing metal surfaces, a New York, for example, within a 20 km2 area nodule forms that may or may not display con- of the bottom sediment, 106 tons of nodules, centric laminations. In stratified water bodies, averaging 15 cm in diameter, have accumulated some nodules may also be physically transported (Dean and Greeson, 1979). to the anoxic zone by currents or traction. Nodule formation in marine and freshwater There the reductive dissolution of the nodules environments involves a series of microbially cat- re-liberates Mn2+ and Fe2+ directly to the water alyzed reactions. Based on lake models (Fig. 4.10), column. Some of the reduced metals make their the process begins with cyanobacterial and algal way back to surface oxygenated waters where plankton concentrating dissolved manganese and the cycle is repeated. Phytoplankton also con- iron. Upon their death, the microbial biomass and tribute to metal oxidation by producing high-pH, metals are transported to the bottom sediment oxygenated surface waters that are conducive to and buried, where anaerobic respiratory processes the re-oxidation and hydrolysis reactions (e.g., in the suboxic layers release the metals into the Richardson et al., 1988). pore waters. Concurrently, reduction of riverine In the oceans, nodules can be described in particulate Mn(III/IV)/Fe(III) oxyhydroxides terms of two end-members: (i) those formed from occurs. Upward diffusion to the sediment–water overlying seawater Mn2+; and (ii) those with interface facilitates microbial re-oxidation and Mn2+ supplied via diagentic processes (similar to ITGC04 18/7/06 18:23 Page 153

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lakes). Depending on the source flux of Mn2+, inhabit desert varnish, and that some of those concretion growth rates can range anywhere from species can contribute to Mn(II) oxidation. 1 mm to tens of centimeters in a million years However, the question of whether desert varnish (Dymond et al., 1984). is indeed a biological phenomenon, and whether Microscopic examination of nodules regularly it can only form due to microbial activity, remains shows the presence of bacteria on both the sur- unclear. faces and within the nodules, with population densities of the order of 107 cells of bacteria per 4.1.5 Clays cubic millimeter of nodule surface (Burnett and Nealson, 1981). Just how important the micro- Within the past two decades studies in the natural organisms are in terms of the mineralization environment have led to the recognition that process itself is unknown, but concretions up to bacteria mediate the formation of clay-like phases. 5 mm in diameter can be produced under ideal- Some clays form as replacement products from ized laboratory conditions within just 2 months the alteration of primary minerals. For instance, by the Mn(II)-oxidizing bacteria Metallogenium Konhauser et al. (2002a) recently documented sp. (Dubina, 1981). that highly altered, glassy tephras within active steam vents at Kilauea Volcano, Hawaii, con- (c) Desert varnish tained subsurface bacteria with small (<500 nm in diameter), epicellular grains of smectite. They Another deposit that incorporates ferromanganese formed from the elements released into the pore oxides are the so-called desert varnishes. These are waters after the primary glass phase dissolved. the black to orange coatings found on rocks in Clays are also significant components of deep- arid and semiarid environments (see Plate 7). They sea hydrothermal deposits, and many of them con- range in thickness from micrometers to millimeters, tain filamentous, organic structures reminiscent and are rich in variable amounts of Mn-Fe oxides of bacteria (e.g., Juniper and Fouquet, 1988). and clays, but interestingly, their mineralogy Close microscopic examination of these “biogenic and chemical composition is generally unrelated minerals” show intense iron accumulation onto to the underlying rock substratum. Instead, the the filaments, upon which silica appears to have main source of Mn and Fe is rainfall or dust. subsequently precipitated. Some hydrothermal The predominant microbiological forms asso- clay deposits (e.g., nontronite) also comprise ciated with desert varnishes are fungi, which are intertwining microtube-like structures, thought well adapted to the hot and dry conditions. Close to be ensheathed, filamentous Fe(II)-oxidizing examination of varnish shows that fungi hyphae bacteria. It is believed that the cells not only are frequently heavily mineralized and physic- served as templates for clay precipitation, but ally embedded in the varnish texture. In media also that they may have been instrumental in simulating conditions assumed to be similar to creating the unique geochemical conditions that those on desert rock, 50% of the fungi studied favored the formation of nontronite over other precipitated Mn(IV) oxides (Grote and Krumbein, minerals, such as ferric hydroxide or amorphous 1992). Also present are heterotrophic bacteria, silica (Köhler et al., 1994). and a large proportion of them are capable of The most frequent observations of biogenic oxidizing Mn(II) to manganese oxyhydroxides clay phases come from biofilms in lakes and (Dorn and Oberlander, 1981). Desert varnish can rivers. Ferris et al. (1987) initially described com- even be artificially made in the laboratory within plex (Fe, Al)-silicates on bacterial cells grow- months using rock chips, a source of Mn2+, and a ing in metal-contaminated lake sediment in mixed inoculum of fungi, heterotrophic bacteria, northern Ontario. These precipitates ranged from and cyanobacteria (e.g., Krumbein and Jens, 1981). poorly ordered and uncharacterized phases to cry- These results demonstrate that microorganisms stalline forms of the Fe-rich chlorite, chamosite ITGC04 18/7/06 18:23 Page 154

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((Fe)3(Si3Al)O10(OH)2). Since then, similar clayey product of ferric hydroxide, then the latter will sorb precipitates have been reported from various rivers more iron from solution, leading to the develop- around the world (e.g., Konhauser et al., 1993). ment of small (~100 nm diameter), dense, mineral What is particularly remarkable about the riverine aggregates on the outer cell surface (Fig. 4.11A). clays, irrespective of the chemical composition of the waters from which they were sampled, is that they share a number of similar properties: A 1 They are generally amorphous to poorly ordered structures; those crystalline grains attached to cells tend to be detrital in origin. 2 All have grains sizes <1 µm, although the majority are <100 nm. 3 They are commonly attached in a tangential orienta- tion around lightly encrusted cells, while those Ferric Fe-rich on heavily encrusted cells have a more random hydroxide EPS orientation. 4 The grains have a composition dominated by iron, 600 nm silicon, and aluminum, in varying amounts. With the exception of potassium, no other metals are Amorphous present in significant amounts. What is particularly B clay interesting is that the most amorphous grains are ferruginous, while the most crystalline phases are highly siliceous, and tend towards illite-like

[(Al)2(Si4-xAlx)O10(OH)2•Kx] compositions (Konhauser et al., 1998). Based on the observations above, a sequence of events leading to clay biomineralization can be adduced (Konhauser and Urrutia, 1999). In the initial stages, a bacterium adsorbs any number of different Fe cations, e.g., Fe2+, Fe3+, Fe(OH)2+, + 700 nm Fe(OH)2 , depending on solution chemistry and redox potential. If the dissolved iron concentra- tion around the cell surface exceeds the solubility C

Figure 4.11 (right) TEM images of bacteria from a sediment sample in the Rio Solimões, Brazil. (A) Formation of ferric hydroxide aggregates in EPS. (B) Partially encrusted cell with amorphous clays forming on cell wall and within EPS, likely from the precursor ferric hydroxide. (C) Heavily encrusted cell with abundant amorphous and crystalline clay minerals extending several hundred nanometers away from the cell wall. Crystalline clay (Reproduced from Konhauser and Urrutia, 1999 500 nm with permission from Elsevier.) ITGC04 18/7/06 18:23 Page 155

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Alternatively, bacteria can attract pre-formed not been ascertained, but we do know that dimeric nanometer-sized ferric hydroxide particles from silica (the species that accounts for more than suspension (Glasauer et al., 2001), thereby 99% of the oligomeric silica in natural waters) is negating the need for the nucleation step. highly reactive towards iron hydroxide surfaces In most rivers, iron is only found in trace (reaction (4.8)), and it exhibits a strong affinity amounts compared to other solutes, particularly for dissolved aluminum, forming aluminosilicate silica. Under these conditions, the adsorbed/ ions (e.g., reaction (4.9)) that subsequently react particulate iron may instead serve as a kinetic- with cell bound iron: ally favorable site for the development of more +> 0 → complex precipitates of variable clay composi- Si2O(OH)6 Fe-OH >FeSi O (OH) + H O (4.8) tion, morphology, and structure. The reason these 2 2 5 2 clays form is as follows. In the pH range of most + + → Si2O(OH)6 Al(OH)2 natural waters, negatively charged counter-ions, + + AlSi2O2(OH)6 H2O (4.9) or those molecules that are neutrally charged but exhibit residual surface electronegativity This arrangement of ions forms an electric double (e.g., monomeric, oligomeric, and colloidal silica layer with iron cations sorbing to the bacterial species), accumulate near the solution–solid inter- surface as an inner sphere complex, while the face to neutralize the net positive charge of iron. silica-aluminosilicate species attach as more Two surface species of iron oxide exist in this pH diffuse outer layers. The surface charge of these > + > 0 range; Fe-OH2 and Fe-OH , but the majority of composites is inevitably dependent upon the the surface charge is positive at circumneutral pH solution pH, the ionic strength of the solution, (Fig. 4.12). The initial (Fe, Al)-silicate phases and the time of reaction, such that it becomes then form via hydrogen bonding between the progressively more negative as the particles age hydroxyl groups associated with the cell-bound and more silica sorbs. Indeed, this mechanism iron and the hydroxyl groups in the dissolved of binding Fe to the bacterial cell surface and silica, aluminum, or aluminosilicate complexes subsequent reaction with silica (and aluminum) (e.g., Taylor et al., 1997; Davis et al., 2002). from solution has been confirmed in experi- Exactly how these reactions occur in nature has mental systems with Bacillus subtilis (e.g., Urrutia and Beveridge, 1994). 100 > + If the microorganism is subject to sufficiently Fe-OH2 − concentrated solutions, then continued reac- > 0 >Fe-O 80 Fe-OH tion between the solutes and the Fe-bearing

60 cell surface eventually results in the formation of amorphous to poorly ordered clay phases 40 (Fig. 4.11B). Often, these reactions lead to the partial and/or complete encrustation of cells as

% Total surface sites 20 abiological surface reactions accelerate the rate of mineral precipitation: on some microorgan- 0 4 5 6 7 8 9 10 isms, the density of clayey material surrounding pH them can be so extensive such as to extend hundreds of nanometers away from the cell sur- Figure 4.12 A speciation diagram for the surface face (Fig. 4.11C). Then with time, these hydrous of ferric hydroxide in water, shown in terms of compounds dehydrate, some converting to more relative percentages of the three dominant surface stable crystalline forms. Similar steps to this bio- site species as a function of pH. (From Fein et al., logical model have been observed in the growth 2002. Reproduced with permission from Elsevier.) of smectite from amorphous Fe-Si-Al precursors ITGC04 18/7/06 18:23 Page 156

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(Sánchez-Navas et al., 1998) and the glauconit- be only a few millimeters at most, yet when one ization of precursor smectite phases (Amouric takes into consideration the large surface area and Parron, 1985). of any given river bed that is colonized by bio- The reactions above are naturally an over- films, the volume of water that falls directly into simplification because a number of inorganic microbial contact is substantial. In this regard, processes are also at play. It is well known that biofilms dominate the reactivity of the sediment– dissolved Al-Si complexes precipitate as poorly water interface, and through the adsorption of ordered Al-silicates when a state of supersatura- solutes from the water column, they facilitate the tion is achieved (Wada and Wada, 1980). More- transfer of metals into the bottom sediment. The over, Fe cations can readily be incorporated into bound metals may then become immobilized those structures, leading to clay-like products as stable mineral phases that collect as sediment (Farmer et al., 1991). All these reactions can con- on the river bed, sections of the metal-laden ceivably take place in the proximity of the cell biofilms may be sloughed off by high flows and wall or within the extensive EPS, particularly transported downstream to be deposited in a since diffusion through the extracellular layers lake or ocean, or the metals may be recycled back is inherently slow, and a microenvironment can into the overlying water column after microbial be established that is conducive to mineraliza- organic matter mineralization. tion. Additionally, colloidal species of (Fe, Al)- silicates, that either form in the water column 4.1.6 Amorphous silica or are products of weathering and soil forma- tion, may react directly with the outermost Silica precipitation is an important geological cell surface. It follows that anything that will process in many modern terrestrial geothermal neutralize or diminish the surface charge of the systems, where venting of supersaturated solu- colloids (e.g., a bacterial wall if colloids are posi- tions leads to the formation of finely laminated tively charged or adsorbed iron if the colloids siliceous sinters around hot spring or geyser vents. are negatively charged) will cause the particles When the sinter deposits are examined under to flocculate out of solution. the electron microscope, they typically show The ubiquity of (Fe, Al)-silicate precipita- an association between the indigenous micro- tion on freshwater bacteria implies that the organisms and spheroidal, amorphous silica grains latter facilitate this form of biomineralization that form epicellularly on the sheaths or walls, with relative ease. Perhaps the fact they readily and intracellularly after the cells have lysed scavenge iron, and that most rivers and lakes (Fig. 4.13). The silicification process likely typically contain high concentrations of silica, begins with the attachment of silica oligomers and to a lesser extent aluminum, is all that is or preformed silica colloids. These silica species required for the formation of authigenic clays. The then grow on the cell surface, often reaching implications for this mode of biomineralization tens to hundreds of nanometers in diameter. If are, however, quite profound. The sediment– silicification is sustained, particles invariably water interface is influenced by: (i) sedimenta- coalesce until the individual precipitates are tion and entrainment of metal-rich particulate no longer distinguishable, and frequently, entire material; (ii) metal adsorption onto clays, colonies are cemented together in a siliceous metal oxyhydroxides, or organic material in matrix several micrometers thick. the bottom sediment; and (iii) precipitation of It was suggested many years ago that the role of various authigenic mineral phases (Hart, 1982). microorganisms in silica precipitation is largely The role of microorganisms, in particular bio- a passive process (e.g., Walter et al., 1972). In films, has seldom been considered an important geothermal systems, waters originating from influencing factor. The thickness of a biofilm may deep, hot reservoirs, at equilibrium with quartz, ITGC04 18/7/06 18:23 Page 157

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A Figure 4.13 (left) TEM images of silicified bacteria from a hot spring sinter in Iceland. (A) Two cells completely encrusted in amorphous silica spheres (tens of nanometers in diameter). In some places (arrow) the silica has begun to coalesce into a dense mineralized matrix. (B) A lysed cell with abundant intracellular silica grains. (C) Silica precipitation is so extensive that the entire community of cyanobacteria (Calothrix sp.) are encrusted in a dense mineralized matrix. Note how the silicification is restricted to the outer sheath surface (arrow), leaving the cells inside mineral-free. (From Konhauser et al., 2004. 1 µm Reproduced with permission from the Royal Swedish Academy of Sciences.) B surface, decompressional degassing, rapid cool- ing to ambient temperatures, evaporation, and changes in pH all conspire together to cause the fluid to suddenly become supersaturated with respect to amorphous silica (Fournier, 1985). Concurrently, the discharged monomeric silica,

Si(OH)4, polymerizes, initially to oligomers (e.g., dimers, trimers, and tetramers), and eventually to polymeric species with spherical diameters of 1 µm 1–5 nm, as the silanol groups (-Si-OH-) of each oligomer condense and dehydrate to produce the siloxane (-Si-O-Si-) cores of larger polymers. C The polymers grow in size through Ostwald ripening such that a bimodal composition of monomers and particles of colloidal dimensions (>5 nm) are generated. These either remain in suspension due to the external silanol groups exhibiting a residual negative surface charge, they coagulate via cation bridging and nucleate SiO2 homogenously, or they precipitate heterogene- ously on a solid substratum (Ihler, 1979). As microorganisms are present in these polymerizing solutions, they inevitably become 1 µm silicified, much the same as other submerged solids, e.g., pollen, wood, leaves, and sinter. Indeed, Walter (1976a) defined geyserite to commonly contain dissolved silica concentrations mean a laminated, amorphous silica sinter that significantly higher than the solubility of amor- formed in the proximity of vents and fissures phous silica at 100°C (approximately 380 mg L−1). where temperatures in excess of 73°C were Therefore, when these fluids are discharged at the deemed sterile except for scattered thermophilic ITGC04 18/7/06 18:23 Page 158

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microorganisms. This temperature exclusion point surprising given that the actual mechanisms of has since been modified because studies have now silicification (in solutions where homogeneous shown that microorganisms silicify over a range nucleation is not possible) rely on the micro- of temperatures, often in excess of 90°C (e.g., organisms providing reactive surface ligands that Jones and Renaut, 1996). Moreover, examination adsorb silica from solution and, accordingly, of geyserite from Yellowstone and New Zealand reduce the activation energy barriers to hetero- indicate that their surfaces are covered with geneous nucleation. This means that cell surface biofilms and that their laminae generally contain charge may have a fundamental control on the silicified microorganisms. Thus, not all geyserite initial silicification process. can be regarded as being abiological, and it At present there appear to be three different appears that most siliceous sinters have been con- mechanisms by which microorganisms become structed, to some degree, around microorganisms silicified (Fig. 4.14): (Cady and Farmer, 1996). Nevertheless, experimental evidence now exists 1 Hydrogen bonding – Many bacteria, such as that appears to corroborate the view that the Calothrix sp., form sheaths composed of neutrally microbial role in silicification is incidental and charged polysaccharides. This can lead to hydrogen not limited to any particular taxa. In particular, bonding between the hydroxyl groups associated bacteria have little affinity for monomeric silica, with the sugars and the hydroxyl ions of the silica (Phoenix et al., 2002). Although the low reactivity even at high bacterial densities and low pH con- of the sheath gives such cells hydrophobic charac- ditions, where most organic functional groups teristics that facilitates their attachment to solid are fully protonated (Fein et al., 2002). Similarly, submerged substrata, this same property makes under highly supersaturated conditions, the rates them less inhibitive to interaction with the polymeric of silica polymerization and the magnitude of silica silica fraction in solution. precipitated are independent of the presence of bacterial biomass (e.g., Benning et al., 2003; Yee 2 Cation bridging – For microorganisms where the cell wall is the outermost layer, such as Bacillus et al., 2003). Presumably, in concentrated silica subtilis, silicification is limited due to electrostatic solutions there is such a strong chemical driv- charge repulsion between the anionic ligands and ing force for silica polymerization, homogeneous the negatively charged silica species. In order for nucleation, and ultimately silica precipitation that silicification to proceed, some form of cation bridge there is no obvious need for microbial catalysis. It is necessitated, whereby metals adsorbed to the has also been observed that silicification occurs cell can act as positively-charged surfaces for silica on dead cells, and continues autocatalytically and deposition (e.g., Phoenix et al., 2003). abiogenically for some time after their death due 3 Direct electrostatic interactions – Some bacteria, to the high reactivity of the newly formed silica. such as Sulfurihydrogenibium azorense, produce Consequently, silica precipitated in the porous protein-rich biofilms that contain an abundance of spaces between filaments has the same basic mor- cationic amino groups that adsorb polymeric silica phology as the silica precipitated on the original (Lalonde et al., 2005). filaments (e.g., Jones et al., 1998). These findings certainly support the notion that biogenic silici- One of the more exciting revelations recently has fication at thermal springs occurs simply because been that silicification may not be detrimental microorganisms grow in a polymerizing solution to the microorganism (Phoenix et al., 2000). where silicification is inevitable. For instance, when Calothrix are grown in silica With that said, there are species-specific supersaturated solutions for weeks at a time, and patterns of silicification, because different micro- many of the filaments develop extensive mineral organisms are certainly capable of being silicified crusts up to 5 µm thick, the cells still fluoresce, with different degrees of fidelity. This is not they continue to generate oxygen, and the ITGC04 18/7/06 18:23 Page 159

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Hydrogen bonding A OH Siloxane bond OH OH OH Silanol OH Si bond OO OH Bacterium OHOH OSiSi OH + OH Si OH

Cell OO OH H2O H2O wall OH Si Monosilicic OH OH acid OH (Silica oligomer)

OH Sheath

Cation bridging – B COO Net (+) charge Net (–) charge

OH OH OH – H2PO4 Fe OH OH Si OSiOH OH OH OH H2O OH OH OH COO– Fe OH OH Si OSiOH OH OH OH H2O

– Figure 4.14 Three H2PO4 mechanisms by which microorganisms silicify: (A) hydrogen bonding Direct electrostatic interactions between dissolved silica C NH + and hydroxyl groups 3 associated with some Net (–) charge + sheaths; (B) cation NH3 OH OH bridging between silica + and negatively charged NH3 OH Si OSiOH cell walls; (C) direct OH OH electrostatic interactions NH + between silica and 3 OH OH positively charged + amino groups in some NH3 OH Si OSiOH biofilms. Note: stippled + OH OH arrows show release NH3

of H2O after bond formation. + EPS NH3 ITGC04 18/7/06 18:23 Page 160

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mineralized colonies exhibit comparable rates (Merz-Preiß, 2000). The overall reaction that of photosynthesis to nonmineralized colonies. best describes the precipitation process is: Intriguingly, silicification of viable cyanobacterial 2+ + − ←→ + + cells only occurs on the outer surface of the sheath, M 2HCO3 MCO3 CH2O O2 (4.10) whereas lysed cells have silica forming within the cytoplasm. This clearly indicates that the where M2+ represents a divalent metal cation and

sheath is necessary for enabling photosynthetic- MCO3 is a solid carbonate phase. As the cations ally active cyanobacteria to survive mineralization, present in solution can vary from location to by both acting as an alternative mineral nuclea- location, so too can the different carbonate tion site that prevents cell wall and/or cytoplasmic phases. Consequently, it is not uncommon to see mineralization, and by providing a physical filter cyanobacteria in close association with a number that restricts colloidal silica to its outer surface of carbonate minerals, including calcite/aragonite

(recall Fig. 4.13C). Of course at some stage silici- (CaCO3), dolomite (CaMg)(CO3)2, strontionite fication will inhibit diffusional processes. Perhaps (SrCO3), and magnesite (MgCO3). Calcite and their ability to grow upwards within the sheath aragonite are by far the more common carbonate towards the sediment–water interface, where the phases, with the concentration of Mg2+ deter- magnitude of silica encrustation will be less pro- mining the more stable form; high Mg2+ promotes nounced than at depth (i.e., where the sinter is aragonite precipitation, while lower Mg2+ favors older and has been exposed to more silica), is a calcite precipitation. means by which the cyanobacteria survive in The role of cyanobacteria in carbonate precipi- a continuously accreting environment? Biofilm tation is twofold: metabolic fixation of inorganic production may be a different version of this carbon tends to increase solution pH and lead to defense mechanism. a state of supersaturation, while cation adsorption to the cell surface promotes heterogeneous nucle- 4.1.7 Carbonates ation (Fig. 4.15). With respect to photosynthesis, in waters with neutral to slightly alkaline pH, − Microorganisms have played an integral role in cyanobacteria use HCO3 instead of, or in addi- carbonate sedimentation since the Archean. The tion to, CO2 as a carbon source for the dark cycle deposits they form are heterogeneous, but the main (reaction (4.11)). A byproduct of this reaction, component is fine-grained, lithified lime mud hydroxyl ions, are then excreted into the external composed of micrite (1–5 µm crystals of calcium environment where they create localized alkalin- carbonate). It forms as a result of a combina- ization around the cell. This, in turn, induces a tion of processes, including mineralization of change in the carbonate speciation towards the 2− microbial surfaces, chemical precipitation from carbonate (CO3 ) anion (reaction (4.12)): supersaturated solutions, and erosion of existing − ←→ + − carbonate layers (Riding, 2000). Microorganisms HCO3 CO2 OH (4.11) can play both a controlled and passive role in HCO − + OH− ←→ CO 2− + H O (4.12) mineral precipitation. The biologically controlled 3 3 2 mechanisms will be discussed later in this chapter Cyanobacteria also provide reactive ligands to- (section 4.2.4). wards metal cations and, once bound, they can 2− then react with the CO3 anions to form a number (a) Calcium carbonate – mechanism of of carbonate phases, such as aragonite or calcite: mineralization 2− + 2+ ←→ CO3 Ca CaCO3 (4.13) Much emphasis on passive carbonate biominer- alization has been placed on the photosynthetic Extracellular layers are particularly favorable activity and surface reactivity of cyanobacteria sites for nucleation, and cyanobacterial species ITGC04 18/7/06 18:23 Page 161

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Figure 4.15 Significant insights into the mechanisms underpinning freshwater 2– 2– SO4 CO3 cyanobacterial calcification have been Gypsum Calcite obtained from experiments replicating the nucleation nucleation activity of Synechococcus sp. communities in Fayetteville Green Lake, New York. Ca2+ Ca2+ When the cyanobacteria were cultured S-layer Anionic ligands in filter-sterilized lake water (pH 7) they began to precipitate gypsum (CaSO ·2H O) on their surfaces within 4 2 Synechococcus 4 hours of the beginning of the experiment. 2+ 2+ Ca – – Ca The biomineralization of gypsum was a HCO3 + H2O CH2O + O2 + OH two-step process initiated by the binding of Ca2+ to the cell’s S-layer, followed by reaction with dissolved sulfate. Within 24 hours, an increase in the alkalinization of the microenvironment around the cells Ca2+ Ca2+ pushed the solid mineral stability field towards the formation of calcite. (Modified from Thompson and Ferris, 1990.) – 2– – – HCO3 H2O + CO3 HCO3 + OH

that produce sheaths or EPS generally pre- ential binding of the former two cations over cipitate more calcium carbonate than those Mg2+. Other studies have documented that cyano- species without such structures (Pentecost, 1978). bacteria can partition of up to 1.0 wt% strontium When calcium carbonate nucleates on the sheath in calcite (Ferris et al., 1995). The ability for solid- surface it grows radially outwards and, in some phase capture of trace metals/radionuclides during cases, this may lead to the complete encrustation biogenic calcification has important implication of the cell. Conversely, when calcium carbon- for bioremediation strategies in calcium carbonate- ate nucleates within the intermolecular spaces hosted aquifers because those contaminants can of the sheath, the latter may become filled be effectively immobilized from the groundwater with mineral material (Verrecchia et al., 1995). flow (Warren et al., 2001). EPS fosters carbonate precipitation by provid- Much of the foregoing discussion has focused ing diffusion-limited sites that create localized on cyanobacteria. However, a number of studies alkalinity gradients in response to metabolic have described how green and brown algae processes, while simultaneously attracting Ca2+ (e.g., the genera Chara and Halimeda), that grow to its organic ligands (e.g., Pentecost, 1985). Fur- as part of marine microbial mats, precipitate − thermore, the type of functional groups in EPS aragonite as a result of HCO3 uptake during affects carbonate morphology and mineralogy, photosynthesis. In most cases, the crystals lack e.g., spherule vs. euhedral calcite or calcite vs. any organizational motif or preferred crystal aragonite (Braissant et al., 2003). orientation, they vary in size, and they are not Cyanobacteria grown in the presence of various associated with any organic material other than combinations of Sr2+, Mg2+, or Ca2+ can pre- the cell wall (e.g., Borowitzka, 1989). For a limited cipitate instead strontionite, magnesite, or mixed number of algae (e.g., Penicillus sp.), mineral pre- calcite-strontionite carbonates (Schultze-Lam and cipitation occurs within a “sheath-like” structure Beveridge, 1994). In general, cyanobacteria are surrounding the cell wall. This sheath appears to equally capable of incorporating Ca2+ or Sr2+ dur- serve primarily as a diffusion barrier, aiding in ing carbonate mineral formation, while magnesite the establishment of a sufficiently large degree of is easily inhibited from forming by the prefer- alkalinization. ITGC04 18/7/06 18:23 Page 162

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(b) Calcium carbonate – deposits whitings contain nearly 11 mg L−1 of suspended

sediment, with CaCO3 settling rates of the order Small cyanobacteria (<2 µm in size), known as of 35 g m−2 h−1. Based on those rates, Robbins picoplankton, have been linked to fine-grained et al. (1997) calculated that 1.4 × 106 metric tons calcium carbonate precipitation in both lacus- of aragonite are suspended each year, and that trine and shallow marine environments during once sedimented, can account for much of the times of seasonal blooms. These “whiting events” late Holocene bank-top lime muds on the Great are believed to be responsible for the bulk of Bahamas Bank. Field studies have suggested the sedimentary carbonate deposition in some that epicellular calcite precipitation, triggered by

well-described sites, such as Fayetteville Green the fixation of CO2 by cyanobacterial blooms, Lake, New York. There, the unicellular cyano- may play a role in these whiting events (Robbins bacterial genus, Synechococcus, is the dominant and Blackwelder, 1992). Evidence in support phytoplankton in the surface waters. Under the of this hypothesis includes the presence of 25% TEM, Synechococcus is frequently shown to be organic matter by weight in the solid whiting completely mineralized, yet the type of miner- material and SEM/TEM images that show indi- alization is seasonally dependent (Thompson vidual whiting spheres embedded in an organic

et al., 1990). During the cold winter months, matrix, along with the presence of CaCO3 crystals when the Synechococcus cells are dormant, on cyanobacteria surfaces. Just how important

gypsum (CaSO4·2H2O) crystals develop on the picoplankton are to whiting processes will con- S-layers of nonmetabolizing cells. However, in tinue to be the subject of examination, but con- the spring, as the lake water warms and light sidering that Synechococcus blooms are typically intensity increases, the cell population becomes around 105 cells ml−1 and under some conditions more active in number, the pH increases, and can be responsible for 30–70% of the primary the gypsum becomes unstable and dissolves. productivity of the open ocean (e.g., Waterbury Simultaneously, the dominant mineral phase et al., 1979), their biomineralizing abilities might precipitated by individual Synechococcus cells very well be of global importance. changes to calcite (recall Fig. 4.15), which dur- Benthic cyanobacterial communities can form ing the warm summer months, falls as a light an even wider variety of calcareous deposits. When rain of mineral-encrusted biomass to the lake cyanobacteria growing in biofilms calcify, they bottom. Stable carbon isotopic analyses of the can form micritic coatings, crusts, and layers on unconsolidated carbonate sediment shows that submerged substrata. For example, “microreefs,” it is enriched in 13C relative to the bulk dis- consisting of 30% cyanobacteria by weight, have solved inorganic carbon species (Thompson et been described forming on submerged limestone al., 1997). This isotopic difference is caused by gravel in a number of alkaline lakes (Schneider the preferential use of the lighter 12C isotope dur- and Le Campion-Alsumard, 1999). Ooids are ing photosynthesis, which leaves the organic another such example. These small (<2 mm), component depleted in 13C, while the dissolved concentrically layered, spherical grains are com- inorganic carbon, which precipitates as a calcite posed of primary calcium carbonate or replace- around the cells, becomes enriched in 13C by as ment phases that form where gentle or periodic much as 4–5‰. wave action in shallow marine waters and along In the oceans, whiting events can lead to lacustrine shores cause equal precipitation on deposits of considerable size and thickness. On all sides of a cortex of sand, shell fragments, or the Great Bahamas Bank, for example, satellite microbial biomass. Filamentous cyanobacteria, imagery has shown some whitings to cover such as Schizothrix species, have in particular been between 35 and 200 km2 during the summer. heavily implicated in the accretionary process Shinn et al. (1989) have estimated that average because they produce EPS that binds Ca2+, and ITGC04 18/7/06 18:23 Page 163

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their metabolism changes the physicochemical serve as an important biological surface upon properties at the ooid–water interface (Davaud which authigenic calcite nucleates or particulate and Girardclos, 2001). Endolithic varieties, such grains are trapped (e.g., Emeis et al., 1987). as Solentia sp., also contribute to calcification, Thrombolites are macroscopically clotted but through a multicyclic process of microboring microbialites that have become increasingly into existing carbonate grains and concurrent important since the end of the Precambrian. infilling of boreholes with aragonite (Macintyre Their formation has been attributed to rapid et al., 2000). Processes similar to those described rates of calcification by coccoid cyanobacteria above may account for the high magnesium and, as such, sediment binding and trapping are calcite peloids (elliptical to spheroidal structures of minor importance in the overall accretionary 20–60 mm in diameter) that are incorporated process. There are a number of modern throm- in many cemented carbonate deposits in shallow bolites examples, the largest possibly being the marine and lagoonal settings. They are often 40 meter high tower-like deposits found in the characterized by having fine-grained nuclei com- highly alkaline waters of Lake Van in eastern posed of fossilized clumps of bacteria (Chafetz, Anatolia, Turkey (Kempe et al., 1991). Throm- 1986). bolites can also be formed by green algae in sub- Lithified carbonate bioherms (also called tidal marine environments. The deepening water, microbialites) are common in many modern and decrease in salinity, and increase in energy and ancient environments (Fig. 4.16). Freshwater nutrient supply favor algal growth over the cyano- tufa deposits develop at springs and waterfalls in bacterially dominated shallow water stromatolites

limestone terrains, where loss of dissolved CO2 that form with them a laterally gradational bio- due to turbulence and evaporation induces super- facies (Feldmann and McKenzie, 1998). saturation and calcite precipitation on available For much of the Precambrian, stromatolites submerged solids. The cyanobacteria, algae, and were widespread in shallow marine waters. plants that grow in these moist environments Although their relative importance has since inevitably become incorporated into the pre- declined, they are still present in some modern cipitating minerals, a process enhanced by the intertidal and subtidal marine environments (e.g., tendency of the carbonates to become trapped Exuma Sound, Bahamas), seasonally hypersaline in the EPS of the microbial mats (Pentecost embayments (e.g., Shark Bay, Western Australia; and Riding, 1986). A similar process describes see Plate 8), carbonate atolls (e.g., French the formation of speleothems in caves, where Polynesia), and shallow coastal lakes (e.g., Lake rapid degassing of calcium and bicarbonate-rich Clifton, Western Australia). Their mechanisms groundwaters induces a state of supersaturation of formation are discussed in section 6.1.4(b) (Dreybrodt, 1980). Travertine deposits (a denser and their relevance to the Precambrian in form of tufa) are characteristic of a number of section 7.4.2. One of the characteristic features thermal spring deposits (see section 6.1.4(a) of stromatolites are their laminations. The bio- for details). The primary causes of supersatura- logical imprint on lamina texture is created by tion in these systems are the cooling and pressure the orientation of the filamentous cyanobacteria, reduction of the hydrothermal effluent during the adhesiveness and abundance of microbial discharge, and as steam separates from the fluid sheath/EPS material, their propensity to facilit-

phase, CO2 is degassed and the pH correspond- ate calcium carbonate precipitation, and their ingly increases. Similar to sinter formation, the growth response to sediment flux and authigenic role of the main microbial constituents, the mineralization (Seong-Joo et al., 2000). Cruci- cyanobacteria, may be purely incidental (e.g., ally, the microbial mats must be lithified early to Renaut and Jones, 1997). However, the produc- strengthen the deposit, and invariably preserve it tion of EPS by the indigenous community can into the rock record as a stromatolite. ITGC04 18/7/06 18:23 Page 164

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AB

Apron Pond

Slope

1 m 1 m

C D

40 cm 40 cm

Figure 4.16 Examples of various carbonate microbialites. (A) Speleothems in the Carlsbad caverns, New Mexico (courtesy of Peter Jones/NPS). (B) Travertine deposit from Angel Terrace, Mammoth Hot Spring, Yellowstone National Park (courtesy of Bruce Fouke). (C) Thrombolite mounds from Lake Salda, Turkey (courtesy of Michael Russell). (D) Stromatolites exposed at low tide, Hamelin Pool, Western Australia (courtesy of Ken McNamara).

Fungi are important constituents of lichens, carbonates (Verrecchia, 2000). One environment and not only do they excrete large quantities of where fungi biomineralize is in calcretes, terrestrial organic acids that contribute to rock weather- calcareous hardgrounds that are widely distributed ing (see section 5.1.2(c)), but they also form throughout the arid and semiarid regions of the authigenic mineral phases, mainly oxalates and world. In such deposits, fungi are often covered ITGC04 18/7/06 18:23 Page 165

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et al., 1995; Wright, 1999). Furthermore, SRB have since been shown to experimentally induce the formation of dolomite crystals identical in composition and morphology to those found in the natural systems from which the bacteria were isolated (Warthmann et al., 2000). The role of SRB in dolomite formation is twofold. First, the process of sulfate reduction overcomes the kinetic barrier to dolomite forma- tion by increasing the pH and alkalinity, and by removing sulfate, which is a known inhibitor µ to dolomite formation. Since sulfate occurs in 5 m 2+ 2− seawater as a Mg -SO4 ion pair, its removal also increases the availability of “free” Mg2+ cations in the microenvironment around the cell for Figure 4.17 SEM image of calcium oxalate dolomite precipitation (van Lith et al., 2003a). crystals on a fungal filament from a calcrete collected at Galilee, Israel. (Courtesy of Eric Interestingly, only pure cultures of metabolizing Verrecchia.) SRB form dolomite, and even then some pure strains form high Mg-calcite instead. What this implies is that dolomite formation requires with calcium oxalate crystals that form from the specific environmental conditions, and differ- reaction of cell-released oxalic acid with Ca2+ ences in metabolic activity, salinity or substrate around the cell (Fig. 4.17). The calcium oxalates concentration play a role in the establishment can then transform into calcite, resulting in the of chemical gradients around the cells that some- infilling of any available pore spaces and the times favors dolomite precipitation, whereas at formation of a hard cement. Fungi (and bacteria) other times it favors the precipitation of different also appear to play a significant role in the trans- minerals. Second, the cell surfaces of SRB con- formation of woody tissues in trees to calcite. centrate Ca2+ and Mg2+ cations around the cell. In the Ivory Coast and Cameroon, some of the Because of the relatively large size of the dolomite trees are actually being calcified in situ, and if the grains to the SRB themselves, it is likely that quantity of inorganic carbon per tree is extra- the cell material involved in metal binding is polated to account for similar trees throughout the EPS that holds the aggregates of cells (and tropical Africa, then this biological process could mineral grains) together (Fig. 4.18). Once bound, represent a significant long-term carbon sink these cations subsequently serve as favorable 2− (Braissant et al., 2004). adsorption sites for CO3 ions, in a process reminiscent of that for calcite precipitation (c) Dolomite (recall Fig. 4.15). It would thus appear that the metabolic activity of the SRB and their surface The abiological formation of dolomite has proven reactivity are complementary in removing all difficult at room temperature in the laboratory. kinetic inhibitors to the formation of a mineral This is not unexpected given that in nature that would otherwise be difficult to precipitate dolomite commonly forms as a secondary replace- under normal environmental conditions. In fact, ment mineral of earlier calcite and/or aragonite. the bacteria can be so effective at promoting Therefore, it was of great interest to find that dolomitization, with rates on the order of 500 mg the activity of sulfate-reducing bacteria (SRB) L−1 month−1 (van Lith et al., 2003b), that the could mediate primary dolomite formation under cells themselves can be completely dwarfed by anoxic, hypersaline conditions (e.g., Vasconcelos the product of their labor as the numerous small ITGC04 18/7/06 18:23 Page 166

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ent to achieve siderite stability. As a consequence, siderite tends to precipitate in the suboxic layers Dolomite of freshwater and estuarine sediments, where low dissolved sulfate levels constrain SRB activity (Postma, 1982). Siderite also forms in some anoxic marine sediments, within the zone of methano- genesis, where rapid sedimentation rates lead to subsurface sulfate depletion (Gautier, 1982). One further constraint on siderite formation is SRB that the Fe/Ca ratio of the pore water should 3 µm be high enough to stabilize siderite over calcite, hence a lowering of Fe(III) reduction rates causes siderite precipitation to cease in favor of calcite Figure 4.18 SEM image showing the or dolomite (Curtis et al., 1986). Rhodochrosite relationship between the sulfate-reducing bacteria (MnCO3) is formed in a similar environment Desulfovibrio hydrogenovorans and the dolomite as siderite, but it can also precipitate in sulfidic crystals experimentally precipitated along with sediments because of the high solubility of MnS them in culture. Not evident from the micrograph (Neumann et al., 2002). is the EPS coating all the dolomite grains. Siderite and rhodochrosite can be produced (From van Lith et al., 2003a. Reproduced with permission from Blackwell Publishing Ltd.) experimentally through the reductive dissolu- tion of ferric hydroxide and MnO2, respectively (Roden and Lovley, 1993). Both processes involve crystals grow in size (via Ostwald ripening) to two steps: the first being the reduction of the metal form the large crystals shown in Fig. 4.18. (recall reactions (2.30) and (2.28), respectively), Most recently, dolomite has also been shown and the second the reaction of the reduced − to form in basalt-hosted aquifers, in association metals with excess HCO3 (reactions (4.14) and with methanogens (Roberts et al., 2004). Dis- (4.15), respectively): solution of basalt yields elevated pore-water con- + + 2 2 2+ + − + − → + centrations of dissolved Ca and Mg , which Fe HCO3 OH FeCO3 H2O (4.14) then adsorbs onto the methanogen’s surface. 2+ + − + − → + When coupled with methanogenic consumption Mn HCO3 OH MnCO3 H2O (4.15)

of CO2, leading to alkalinity generation, a state of localized carbonate supersaturation can easily The minerals formed experimentally are very sim- be attained. What is surprising about this work, ilar to those crystals formed naturally, particularly however, is that the dolomite grains, only tens in the case of siderite concretions (e.g. Fig. 4.19). of nanometers in size, form directly on the cell This strengthens the argument that bacterial surface, at times completely encrusting the cells. processes are responsible for early diagenetic siderite precipitation (Mortimer et al., 1997). (d) Siderite and rhodochrosite But, whether the bacteria play a role in their formation beyond supplying the necessary ions The formation of siderite is generally limited remains unresolved. to sedimentary environments where pore water 2+ Fe concentrations exceed dissolved H2S – when 4.1.8 Phosphates sufficient H2S is produced, the precipitation of FeS and pyrite (see section 4.1.10) never allows The formation of phosphate minerals is inti- ferrous iron concentrations to reach levels suffici- mately associated with microbial activity. In ITGC04 18/7/06 18:23 Page 167

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pre-existing substrata, including organic matter derived from the microbial cells themselves. As the more stable phase, calcium fluorapatite

(Ca10(PO4)6−x(CO3)xF2+x), then appears, it causes pore water diffusion of phosphate towards the locus of nucleation, thereby bringing down the bulk degree of supersaturation towards cal- cium fluorapatite solubility (Van Cappellen and Berner, 1988). To support its growth, calcium fluorapatite then either uses the precursor as an epitaxial (chemically matching) template or it causes the precursor to dissolve and reprecipit- 20 µm ate as a more stable phase. At some depth, nucleation and growth of calcium fluorapatite ceases due to rising levels of carbonate alkalinity Figure 4.19 SEM image of rhombohedral accompanying the anaerobic decomposition of siderite produced in culture by Geobacter residual organic matter buried in the sediments metallireducens. (Courtesy of Rob Mortimer.) (Jahnke, 1984). Given sufficient time, even calcium fluorapatite will eventually transform into either francolite, a highly substituted form modern sediment, phosphogenesis arises from a of fluorapatite, or the most stable phosphatic

series of independent biogeochemical reactions phase, that being apatite (Ca10(PO4)6F2), with beginning with the accumulation of dissolved the concomitant loss of CO2 and fluorine. inorganic phosphate (usually in the form of Phosphorites are fine-grained, organic-rich sedi- − 2− H2PO4 or HPO4 , with a pKa of 7.2 for the ments containing more than 10% (by volume) − → 2− + + ionization reaction, H2PO4 HPO4 H ) phosphate minerals in the form of nodules, crusts, by phytoplankton. Upon death of the cells, the coatings, and pelletal grains. The sites of their biomass sinks and thus serves as a vehicle by deposition tend towards coastal and shelf envir- which phosphate is supplied from the water onments, where upwelling of phosphate-rich, column to the sediments, and eventually released deep ocean waters leads to high phytoplanktonic into the interstitial pore waters via heterotrophic productivity, while the shallowness of deposi- degradation (Gulbrandsen, 1969). From there, tion ensures that much of the particulate organic its fate is multifold: some is readily scavenged by matter reaches the seafloor. Upwelling water also other microorganisms that store it as an energy facilitates phosphogenesis because: (i) deep cold source; some is adsorbed to minerals phases, water rising towards the surface is heated and tends

such as ferric hydroxide; while the remainder to lose CO2 due to a decrease in pressure; and diffuses into the overlying water column. Dis- (ii) the phytoplankton fix CO2 during photosyn- similatory Fe(III) reduction or reduction of ferric thesis. Thus, water unusually rich in phosphate oxyhydroxides by reaction with bacterially gener- moves into a region of increasing pH that should ated hydrogen sulfide, also serves as a supple- favor deposition of calcium phosphate until the mentary source of dissolved pore water phosphate levels of alkalinity become inhibitory (Burnett, (Gächter et al., 1988). 1977). This model is supported by the organic- High localized rates of phosphate release can rich nature of recent phosphorite deposits form- promote the rapid nucleation of amorphous ing on the continental shelves off the coasts calcium fluorapatite phases throughout the of Southwest Africa and South America (e.g., sediment pore spaces and on the surfaces of Bremner, 1980; Glenn and Arthur, 1988). Off the ITGC04 18/7/06 18:23 Page 168

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Peruvian coast, nodules with diameters of several centimeters grow at rates approaching 1 mm yr−1. A The resulting phosphatic concretions are resist- ant to transport by currents, and as a result can be mechanically exhumed and concentrated during periods of sediment reworking. Microbial structures make up a major part of the modern phosphorite framework, typically comprising filamentous mats of cyanobacteria (Fig. 4.20A) and sulfur-oxidizing bacteria imme- 50 µm diately capping the zone of calcium fluorapa- tite precipitation. Similarly, a close association of benthic microbial activity with the forma- B tion of calcium fluorapatite can be widely traced in ancient phosphogenic environments (e.g., Krajewski et al., 1994). The organic matter in fossil phosphorites exhibit features indicative of intense biodegradation of organic matter at, or near, the seafloor, while the microfabrics preserved show that an abundant and diverse benthic microbial assemblage existed at the time 100 µm of mineralization. Stromatolitic phosphorites are an excellent example of the close spatial associa- tion between the activities of ancient microbial Figure 4.20 (A) SEM image of an mats and the precipitation of phosphatic minerals experimentally phosphatized microbial mat (e.g. Fig. 4.20B). Furthermore, precipitation of dominated by filamentous cyanobacteria apatitic precursor phases was a common mechan- (Oscillatoria sp.). The filaments are coated with ism of bacterial preservation. This has been well thin layers of carbonate fluorapatite, which formed documented in the Upper Cretaceous–Lower as a result of rapid precipitation of an amorphous calcium phosphate precursor phase. (B) Polished Eocene Mishash Formation in Israel, where section of a phosphatic columnar microstromatolites phosphatized mats are preserved as dense apatite from an Upper Cretaceous sequence in the Polish overgrowths on remnants of filamentous cyano- Jura Chain. The microfabric consists of alternating bacterial sheaths and fungal hyphae, while compact (dark gray) and porous (white to pale coccoid cyanobacteria were preserved as apatite gray) apatitic laminae. The latter contain remnants infillings (Soudry and Champetier, 1983). of unicellular microorganisms. (Courtesy of Microorganisms can also play an active role in Krzysztof Krajewski.) the mineralization process. One way is through anaerobic respiratory pathways that release metal phosphate-rich organic compounds (e.g., RNA), cations into the pore waters, where they then in the presence of a calcium source (e.g., calcite), react with dissolved phosphate. For example, produces calcium fluorapatite (Prévôt et al., 1989), in experimental studies with Fe(III)-reducing while phosphate released through the activity of bacteria, the ferrous phosphate, vivianite, fre- outer membrane-bound phosphatase enzymes, in 2+ quently forms as a secondary product after the solutions containing UO2 , has been shown to metabolic release of ferrous iron into a phospha- induce the precipitation of uranium phosphate terich medium (e.g., Lovley and Phillips, 1988b). minerals (Macaskie et al., 2000). Microbial In other studies, bacterial decomposition of redox processes may further promote chemical ITGC04 18/7/06 18:23 Page 169

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gradients and associated pH shifts that help localize calcium fluorapatite precipitation at certain sites in the sedimentary layers (e.g., Van Cappellen and Berner, 1991). In addition, micro- bial mats may behave as physical barriers by reducing the diffusion of phosphate back into the overlying water column. Empty sheaths and degraded microbial remains would function in a similar manner (Soudry, 2000). Whether or not there is a direct cellular con- trol over phosphate mineralization is much more ambiguous. In most phosphate-rich sediments, 1 µm the formation of calcium fluorapatite precursors is a rapid process that takes advantage of any substratum available, and when abundant bio- Figure 4.21 TEM image of a lichen scraped off mass is present, it can appear as though bacteria the surface of a granodiorite outcrop on Ellesmere are favorable nucleation sites. Yet, experiments Island, Canada. Arrows indicate the numerous specifically designed to test the microbial role Fe-phosphate grains that are associated with the have concluded that there is no evidence to cyanobacterial cell walls and EPS. The large dark suggest that phosphate minerals nucleate pre- objects within the cells are polyphosphate granules ferentially on bacteria; calcium fluorapatite grains that store temporary excess phosphate, while the were noted to develop on or close to the cell, as large electron-translucent granules inside the cells are polyhydroxy butyrate bodies that function as well as on solids devoid of bacteria (Hirschler energy reserves. (From Konhauser et al., 1994. et al., 1990). With that said, detailed microscopic Reproduced with permission from the National examination of lichens, growing on exposed rock Research Council of Canada.) outcrops on Ellesmere Island, in the Canadian Arctic, highlight how ferric iron adsorbed onto cyanobacterial walls and their EPS react with mineral growth reveal that the S-layer contains dissolved phosphate (Konhauser et al., 1994). This small, regularly arranged pores that facilitate the reaction leads to the secondary precipitation of initial nucleation of the gypsum grains (Schultze- iron phosphate grains, compositionally similar Lam et al., 1992). Continued aggregation of the

to strengite (FePO4·2H2O), throughout the bio- gypsum grains eventually enshrouds the entire mass (Fig. 4.21). In this particular instance, the cell surface such that the S-layer becomes com- microbial community concentrated phosphate pletely obscured by the growing gypsum crystals. within the biofilm by taking advantage of the high It is interesting that while the cells are still adsorptive affinity of Fe(III) for phosphate anions. active, the mineralized S-layers are shed from the cell wall into the external environment so that 4.1.9 Sulfates the cells can grow unabated by the biominerals they just formed. Given that the adsorption of (a) Gypsum, celestite, and barite dissolved sulfate to the cell-bound calcium is an abiological process, the sloughed off S-layer We have already examined how some Synech- material then continues to nucleate additional ococcus species directly contribute to the forma- gypsum grains during its descent to, and in, the tion of gypsum deposits during the winter in bottom sediment. Fayetteville Green Lake. Experimental studies Evaporitic environments, where salinities fre- of the cell surface during the initial stages of quently reach the brine stage, are more typical ITGC04 18/7/06 18:23 Page 170

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for gypsum deposition. Modern mats grow abundantly in such concentrated solutions, and A it is not uncommon to find laminated gypsum deposits or columnar to conical stromatolitic structures that result from periodically con- trolled phases of microbial mat development and gypsum precipitation (Fig. 4.22A). There are also a number of ancient stromatolitic gypsum deposits, the most notable being those from the Upper Miocene (Messinian) that circumvent much of the Mediterranean shoreline. Although most of the traces of the ancient microbial communities associated with those deposits 15 cm are poorly preserved, the relation between the mats and gypsum are still recognizable by the B laminations (Fig. 4.22B). Each set of laminae resulted from two superimposed processes con- trolled by seasonal variations in salinity: (i) growth of cyanobacterial mats during periods of low salinity; and (ii) interstitial crystalliza- tion of gypsum when trapped brines reached salinities prohibitively high for the growth of most indigenous microorganisms (Rouchy and Monty, 2000). At times, remains of the micro- organisms even became incorporated into the accreting gypsum crystals. 3 cm Because a seasonality effect appears to con- trol the ratio of gypsum to calcite precipitated in Fayetteville Green Lake, Schultze-Lam and Beveridge (1994) tested whether cyanobacteria Figure 4.22 (A) Modern gypsified columnar stromatolites from the Ojo de Liebre Lagoon in could promote a similar sulfate-to-carbonate Baja California (courtesy of Catherine Pierre). transformation when other alkaline earth metals (B) Ancient gypsified stromatolites from the Upper 2+ were present in solution, namely Sr . In their Miocene (Messinian) Polemi Basin, Cyprus experimental set-up, the authors inoculated displaying similar columnar structures as the Synechococcus cells into artificial lake water with modern counterparts (courtesy of Jean Marie 2− 2+ Rouchy). high concentrations of SO4 and Sr . The precipitates that initially appeared were small grains (tens of nanometers in diameter) com-

posed of the mineral celestite (SrSO4). In time, is that: (i) the cyanobacterial cell wall avidly the celestite grains grew in size until the pre- bound both Ca2+ and Sr2+; and (ii) the minerals cipitates completely covered the cells. Then, the that ultimately formed were simply a consequence mineralogy of the precipitates changed in com- of the available counter-ions. Along similar lines,

position from celestite to strontionite (SrCO3), the heterotrophic bacterium, Myxococcus xanthus, the carbonate anion being derived by the same has been reported to form barite (BaSO4), simply alkalinization process described for calcite pre- by exposing it to a solution rich in Ba2+ (González- cipitation. What is intriguing about these results Munoz et al., 2003). ITGC04 18/7/06 18:23 Page 171

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(b) Iron sulfates were typically encrusted in a dense mineralized matrix in which individual precipitates appear

Schwertmannite (Fe8O8(OH)6SO4) and jarosite to have coalesced. What was unexpected was + + (MFe3(SO4)2(OH)6), where M may be H , Na , that the Fe:S atomic weight ratio decreased + + K , or NH4 , typically occur as ochreous surface from 3.5:1 at 15 cm to 1.9:1 at 30 cm, highlight- precipitates on stream beds receiving iron and ing the continued reactivity of the ferric iron sulfate-rich, acid rock drainage (ARD). When for dissolved sulfate as the grains became pro- ARD comes in contact with fresh water at an gressively buried. off-site location, the oxidation and hydrolysis of Fe(II) results in a voluminous yellow precipitate, 4.1.10 Sulfide minerals characterized by its high reactivity and efficiency at scavenging other ions from the effluent. At The formation of low temperature sulfide low pH, schwertmannite and jarosite precipitate minerals (i.e., <100°C) is indirectly linked to through anion bridging of ferric iron colloids the activity of dissimilatory sulfate reduction. As (reactions (4.16) and (4.17), respectively). At discussed in Chapter 2, SRB couple the oxida- higher alkalinity, and in the absence of appreci- tion of simple organic molecules to the reduction able sulfate, the neutralizing effects of relatively of sulfate, thereby generating dissolved hydrogen unpolluted stream water results instead in the sulfide. It, in turn, reacts abiologically with a precipitation of either ferric hydroxide or number of existing mineral phases within the goethite (Bigham et al., 1996). sediment, including ferric oxyhydroxides. The microbial role is simply to generate the reduct- 3+ + 2− + → 8Fe SO4 14H2O ant, and as experimental studies showed many Fe O (OH) SO + 22H+ (4.16) 8 8 6 4 years ago, there is no crystallographic differences + + 3+ + 2− + → between iron sulfides formed in the presence of, M 3Fe 2SO4 6H2O + + or absence of, microorganisms (Rickard, 1969). MFe3(SO4)2(OH)6 6H (4.17) Not surprisingly, in fine-grained anoxic sedi- Although bacteria are directly involved in the ments, sulfide minerals are commonly found oxidation of sulfidic minerals and the generation to be in close association with organic matter of ARD (see section 5.2.2(c)), their involvement (see section 6.2.5(a) for details). In fact, there in the subsequent precipitation of amorphous is generally a good positive linear correlation iron and sulfur phases is less clear. It is well estab- between the organic carbon and mineral sulfide lished that the metabolic oxidation of ferrous contents in normal marine shales throughout sulfate solutions by Acidithiobacillus ferrooxidans the Phanerozoic (Raiswell and Berner, 1986). experimentally leads to the formation of jarosite Moreover, patterns of sulfur isotopic fractiona- (e.g., Ivarson, 1973). Other experiments with tions in many sedimentary sulfide deposits are Bacillus subtilis similarly generated ferric sulfate consistent with this form of mineralization, and phases of variable stoichiometry depending on the support a biological origin of reduced sulfur

initial Fe(II)/SO4 ratio used (Fortin and Ferris, (see Box 7.3 for details). 1998). However, only one study has confirmed a One of the most prevalent sulfide minerals is

direct bacterial role in mineralization, that being pyrite (FeS2). Although the precise mechanisms of an abandoned coal mine drainage lagoon in by which pyrite forms at temperatures below West Glamorgan, Wales (Clarke et al., 1997). 100°C remains the subject of debate, it is In the shallow subsurface sediments, a number believed to involve a number of Fe sulfide pre- of unidentified bacteria displayed granular, fine- cursors progressively richer in sulfur (Sweeney grained Fe(III)-S precipitates attached to their and Kaplan, 1973). The process begins with outer surfaces, while at greater depths, the cells the local precipitation of an amorphous iron ITGC04 18/7/06 18:23 Page 172

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0 Fe(OH) S atom, usually elemental sulfur (S ), and the 3 > + (Reaction 4.19) concomitant formation of Fe(II)OH2 , after Organic reaction with a water molecule; (iv) detach- carbon ment of Fe2+ through a weakening of the bonds (2) between the reduced iron and the O2− ions of 2+ Fe(III) Fe FeS the crystalline lattice, thereby exposing a new reduction surface site on the ferric hydroxide; and (v) reac- zone + − tion of Fe2 with HS to form FeS (dos Santos Afonso and Stumm, 1992; Poulton et al., 2004a). – − HS– (1) HS The first step is usually quite fast unless HS 2– reacts instead with a dissolved cation or another SO4 mineral phase. In euxinic basins (chemically 2– SO4 stratified bodies of water with anoxic waters below Sulfate (Reaction 4.18) the chemocline), the high availability of HS− reduction 2+ zone and Fe leads to rapid monosulfide nucleation within the water column itself (Wilkin and Barnes, 1997). Figure 4.23 Two modes of iron monosulfide Once formed, iron monosulfide converts formation. rapidly into mackinawite. In turn, mackinawite can react with any number of intermediate sulfur species with oxidation states between sulfate and sulfide. One such pathway is the reaction monosulfide phase, e.g., FeS. In sediments, this with elemental sulfur to form greigite, Fe S initial mineralization stage is driven by two 3 4 (reaction (4.20)). The transformation of greigite separate pathways (Fig. 4.23). One pathway + to pyrite then requires a major crystallographic involves Fe2 , produced during biological Fe(III) reorganization of both the iron and sulfur, likely reduction, diffusing down from the suboxic layers involving a dissolution-reprecipitation pathway into the sulfate reduction zone, where it reacts − (Schoonen and Barnes, 1991b). with pore water sulfide (in the form of HS at marine pH) to form FeS (reaction (4.18)). 3FeS + S0 → Fe S (4.20) The second pathway (reaction (4.19)) involves 3 4 dissolved sulfide, from the underlying anoxic Based on the rapid formation of pyrite in some sediments, diffusing upwards where it is removed sedimentary environments, it has also been pro- more slowly, but in greater amounts, by reaction posed that FeS might react with other partially with ferric oxyhydroxide (Canfield, 1989): oxidized sulfur phases, including polythionates 2− 2− 2+ + − → + + (SxO6 ), thiosulfate (S2O3 ), or polysulfides Fe HS FeS H (4.18) 2− (Sx ), and in doing so, avoid the greigite inter- + − → + 0 + + − mediate step (Luther, 1991): 6Fe(OH)3 9HS 6FeS 3S 9H2O 9OH (4.19) + − → + 2− + + FeS HSx FeS2 Sx−1 H (4.21) Reaction (4.19) is actually much more com- plicated than written because it involves five Most controversially, it has been argued that steps: (i) inner-sphere surface complex formation pyrite formation can also proceed at tempera- between the >Fe(III)-OH ligand with HS− to tures lower than 100°C under strictly anoxic > + − form Fe(III)SH OH ; (ii) electron transfer conditions by reaction of FeS with H2S (reaction from S(−1) to Fe(III); (iii) release of an oxidized (4.22)) (e.g., Rickard, 1997). Despite experiments ITGC04 18/7/06 18:23 Page 173

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demonstrating this reaction pathway (Drobner et al., 1990), they are inconsistent with results obtained from many field and laboratory studies that indicate that the mackinawite to pyrite con-

version requires a weak oxidant, not H2S. Instead, it is widely accepted that the environment for pyritization must be slightly oxidizing (Benning et al., 2000).

+ → + FeS H2S FeS2 H2 (4.22)

The numerous intermediate steps may appear 2 µm needlessly complicated, but the direct precipita- tion of pyrite at temperatures below 100°C is unfavorable since its rate of nucleation is slow Figure 4.24 SEM image showing the spherical compared to its formation via the FeS precursor aggregation of individual pyrite crystals to give a (Schoonen and Barnes, 1991a). This makes sense typical framboidal morphology. Sample collected in light of the fact that the activation energy from recent sediments in the Black Sea. (Courtesy of barrier to pyrite nucleation is so high that in Rick Wilkin.) order for this step to occur at a significant rate, the solution must exceed saturation with respect to iron monosulfide. However, once iron mono- being important if greigite was the precursor sulfide supersaturation is attained, it will nucle- phase; (ii) uniformity in the size and morphology ate considerably faster than pyrite and drive the of their microcrystals, suggesting simultaneous reactant concentrations below the critical value nucleation and similar growth rates for the same for pyrite. It is only when pyrite begins to grow period of time prior to aggregation; and (iii) that it can control the saturation state of the fluid overall spheroidicity that might reflect pseudo- and, therefore, cause the concentrations of Fe2+ morphism of a pre-existing spheroidal body, and S2− to diminish enough that the precursor possibly an organism or a microcolony. Certainly, dissolves. Thus, the precipitation of iron sulfides the latter is a possibility when framboids are follows the Ostwald sequence for consecu- found associated with extant microbial mats tive reactions, i.e., the thermodynamically least (e.g., Popa et al., 2004). Framboids that form stable phase forms first. Consistent with this is within the water column are a completely differ- the near universal observation that sediment ent matter because they tend to be smaller and pore waters are saturated or slightly under- less variable in overall size, features that probably saturated with respect to iron monosulfides, but reflect rapid nucleation in the water column with are always supersaturated with respect to pyrite less time for growth (Wilkin et al., 1996). The (e.g., Howarth, 1979). distinctions in size and morphology between syn- The most common pyrite textures are clusters genetic framboids (formed in the water column) of framboids, densely packed mineral aggregates and diagenetic framboids (formed in sediment) with sizes on the order of tens of micrometers, make it possible to determine the redox con- and possessing an overall raspberry-like appear- ditions, bulk C/S values, and the degree of ance (e.g., Fig. 4.24). Framboids have three pyritization during deposition of ancient shales characteristics: (i) a microcrystalline arrange- (see section 6.2.5(a) for details). ment that might be an indicator of fast crystal In addition to being primarily responsible growth and/or magnetic aggregation, the latter for the production of dissolved sulfide, bacteria ITGC04 18/7/06 18:23 Page 174

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can also serve as surfaces for iron sulfide pre- cipitation. For example, in a metal-contaminated 4.2 Biologically controlled lake sediment in Sudbury, Ontario, mackinawite mineralization was observed directly on the outer surfaces of bacterial cells and their membranous debris What makes biologically controlled biominer- (Ferris et al., 1987). Some bacteria from the same alization different from the processes discussed samples also precipitated millerite (NiS), indi- above is that the microorganism exerts consider- cating that the presence of competing cations able control over all aspects of the nucleation and can alter the final product of sulfide biominer- mineral growth stages (Mann, 1988). Initially, a alization. Indeed, in the absence of iron, other specific site within the cell is sealed off from the metal sulfides, such as galena (PbS) and sphalerite external environment; this will later become the (ZnS), are associated with microbial biomass in locus of mineralization. Two common methods some black shales, strata-bound and stratiform of space delineation occur. The first involves the base-metal sulfide deposits, and oil reservoirs development of intercellular spaces between a (Machel, 2001). These natural deposits cor- number of cells. The second is the formation of roborate experimental studies suggesting that the intracellular deposition vesicles. formation of organometallic complexes plays a Once the cellular compartment is formed, the critical role in the partitioning of metallic ions next step entails the cells sequestering specific into sulfidic phases. In particular, metals chem- ions of choice and transferring them to the ically complexed to bacteria are more reactive mineralization site, where their concentrations towards hydrogen sulfide than when they are in are increased until a state of supersaturation solution (Mohagheghi et al., 1985). is achieved. Levels of supersaturation are then Bacterial sulfate reduction is likely not an regulated by managing the rate at which mineral important process in the formation of hydro- constituents are brought into the cell via spe- thermal massive sulfides because these minerals cific transport enzymes. Meanwhile, nucleation are precipitated from solutions containing high is controlled by exposing organic ligands with

concentrations of geothermally generated H2S. distinct stereochemical and electrochemical pro- However, surface crusts on hydrothermal chim- perties tailored to interact with the mineralizing neys at northern Gorda Ridge, for instance, ions. These same ligands also act as surrogate showed the preservation of bacterial filaments oxyanions that simulate the first layer of the

in fine-grained chalcopyrite (CuFeS2), pearceite incipient nuclei (Mann et al., 1993). The (Ag14.7−xCu1.3+xAs2S11), and proustite (Ag3AsS3) crystals then grow in a highly ordered manner, (Zierenberg and Schiffman, 1990). The bacteria with their orientation and size governed by the likely played two roles in biomineralization. overall ultrastructure of the membrane-bound First, they adsorbed Ag, As, and Cu, causing compartment. local concentrations of these metals to exceed the solubility products of their sulfides, ultim- 4.2.1 Magnetite ately leading to mineral nucleation. Second, the bacterial mats may have influenced the physio- There are a number of microorganisms that chemical conditions around the chimneys to exert significant control over magnetite forma- favor metal sulfide precipitation. tion. The best understood are the so-called It has also been revealed that natural commun- magnetotactic bacteria, originally described by ities of SRB can generate essentially pure ZnS Blakemore (1975). These are a diverse group of deposits from dilute groundwater. This extends the aquatic species (predominantly Proteobacteria), possibility for a biogenic role in low temperature that share three basic features (Bazylinski and metal sulfide ore deposits (Labrenz et al., 2000). Frankel, 2000): ITGC04 18/7/06 18:23 Page 175

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1 They are microaerophilic, meaning that they exhibit poor growth at atmospheric concentrations of oxygen. Flagellum

2 Most have bidirectional motility, being able to pro- pel themselves forwards or backwards by rotating one of their polar flagella.

3 They possess a number of intracellular, linearly arranged membrane-bound structures called mag- netosomes that house the mineral grains (Fig. 4.25). Magnetosome Most magnetotactic bacteria produce on average 20 or so magnetosomes, although a 9 µm diameter, 500 nm coccoid bacterium was identified that possessed up to 1000 magnetosomes (Vali et al., 1987). Figure 4.25 TEM image of a magnetotactic Unlike the magnetite formed via Fe(III) reduc- bacterium, designated strain MV-4, grown in pure tion, the crystals formed by magnetotactic bacteria culture. Cells of this strain produce a single chain of have unique morphologies (always either cubic, magnetite crystals that longitudinally traverse the rectangular, or arrow-shaped), they are free from cell. Inset shows close-up of the magnetosome crystallographic imperfections, and chemically membrane (arrow) that surrounds each individual particle. (Courtesy of Dennis Bazylinski.) they are quite pure Fe3O4 (Bazylinski, 1996). Considering that many other metals will be present in their immediate surroundings, this implies that magnetotactic bacteria have the each hemisphere selecting the predominant means to exclude nonmagnetite-forming ions polarity type amongst the magnetotactic bacteria from the growing magnetite crystals. (Blakemore and Blakemore, 1990). It is, how- Magnetotactic bacteria also precipitate mag- ever, important to stress that the cell is neither netite within a narrow range of crystal sizes, from attracted nor pulled towards the geomagnetic pole, approximately 35 to 120 nm. This establishes but merely aligns itself like a compass needle. stable single magnetic domains. A single 40– Thus, dead cells align similar to living cells. 50 nm magnetosome has a magnetic energy of Magnetotactic bacteria have been recovered 3 × 10 −14 erg. This energy would be sufficient to from a wide variety of environments, where they align it in the Earth’s geomagnetic field were it grow most abundantly at oxic–anoxic interfaces not for the thermal forces (4 × 10 −14 erg) that tend (Fig. 4.26). They are chemoheterotrophic, with to randomize the cell’s orientation in its aqueous oxygen as their usual terminal electron acceptor, environment. However, the magnetosomes are and although cells such as Magnetospirillum arranged in one or more chains that traverse magnetotacticum strain MS-1 produce more the cell along its axis of motility, such that the magnetite when grown with nitrate, they still

magnetic interactions of a single particle cause require at least 1% O2 for magnetite synthesis its magnetic dipole to orient parallel to the other (e.g., Bazylinski and Blakemore, 1983). Other grains. Thus, the total magnetic energy of the magnetotactic bacteria can use ferric iron, cell is the sum of each of the individual par- nitrous oxide, and possibly even sulfate as TEAs ticles; with 20 magnetosomes the cell’s magnetic (Sakaguchi et al., 1993), although the latter has energy is 6 × 10 −13 erg. Significantly, this means not been confirmed by other studies. that the cell is able to align itself passively along The most unresolved issue regarding magneto- geomagnetic field lines while it swims, with the tactic bacteria is what is the purpose of possessing vertical component of the geomagnetic field in magnetic properties? At present we can only ITGC04 18/7/06 18:23 Page 176

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[O2]

Oxic-anoxic interface

[HS–]

Sediment

Figure 4.26 Most magnetite-producing magnetotactic bacteria are found at, or above, the oxic–anoxic interface in sediments and stratified bodies of water, where geochemical conditions are appropriate for magnetite formation. They move freely up and down along the inclined geomagnetic field lines (dotted) in response to changing environmental conditions. (Modified from Bazylinski, 1996.)

speculate on some of the advantages, the most magnetite (Frankel and Blakemore, 1989), espe- probable being that magnetotaxis is a particularly cially since the energy expended would surely useful navigational tool, increasing the cell’s effi- give them a severe competitive disadvantage ciency at locating and maintaining an optimal compared to nonmagnetic species? To com- position in vertical chemical and/or redox gra- plicate matters more, magnetite has also been dients typical of sediments and stratified water found associated with euglenoid algal cells (e.g., bodies. Because the magnetotactic bacteria tend Torres de Araujo et al., 1986) and several types to be microaerophilic, their movement above and of protists, including dinoflagellates and ciliates below the chemocline will have serious repercus- (Bazylinski et al., 2000). The role of magnetite in sions for the health of the cells. So, when a cell these cells is even more of a guess. inadvertently moves too far upwards, and the One thing is clear, intracellular magnetite must concentration of oxygen becomes inhibitory, it serve a purpose because the processes involved in reverses direction (Frankel et al., 1997). Similarly, its formation are complicated and energy inten- if it moves too far down into the sediment where sive (Frankel et al., 1983). Magnetite synthesis hydrogen sulfide concentrations are prohibitively involves a series of geochemical steps that begins high, the cell once again reverses direction and with the uptake of Fe(III) from the surround- moves back upwards. Like most free-swimming ing environment (Fig. 4.27). As discussed in bacteria, magnetotactic bacteria propel themselves section 3.4.2(a), bacteria commonly rely on forward in their aqueous environment by rotat- iron chelators such as siderophores to facilitate ing their helical flagella. However, two questions the solubilization and transport of Fe(III) to the arise: (i) if knowing which way is up versus cell. Once a specific siderophore has sequestered down increases a cell’s efficiency at finding and iron, it then needs to be absorbed by a cell that maintaining an optimal position relative to the requires it. This is accomplished by cell syn- gradient, why then don’t all bacteria inhabiting thesis of specific receptor proteins designed to first suboxic sediments have magnetic properties; and recognize the Fe(III)–siderophore complex and (ii) at the equator, where the geomagnetic field then, with the aid of other transport proteins, lines are horizontal, why would bacteria produce guide the coordinated Fe(III) to the plasma ITGC04 18/7/06 18:23 Page 177

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1

Siderophore Fe(III)

2

Siderophore Fe(III) Preformed 4 3 magnetosomes

5

(1) Fe(II) + O2 Fe(OH)3 Fe O Fe(OH) 3 4 3 (2) Fe(II) + Fe(OH)3 Fe3O4

Figure 4.27 The possible mechanisms by which magnetotactic bacteria form intracellular magnetite. There are a number of steps involved, including (1) sequestration of Fe(III) from the aqueous environment via siderophores; (2) siderophore attachment to a receptor site on the outer membrane; (3) transport of the siderophore through the outer membrane to the plasma membrane, where Fe(III) is reduced to Fe(II); (4) transport of Fe(II) to pre-formed magnetosome; and (5) initial precipitation of ferric hydroxide within the magnetosome, followed by conversion to magnetite. Note: size of siderophore not to scale.

membrane (Neilands, 1989). In some species, as the electron acceptor. The actual crystalliza- the siderophore does not penetrate the plasma tion of magnetite then involves the reaction of membrane, but instead donates the iron to a the ferric hydroxide with more Fe2+: second membrane-bound chelator, while in 2+ + − + → + other species, the entire siderophore is absorbed Fe 2OH 2Fe(OH)3 Fe3O4 4H2O directly into the cytoplasm (Müller and Raymond, (4.23) 1984). In either case, the Fe(III) is reduced to Fe(II), and the latter is then shuttled in some The subsequent adsorption of Fe2+ on to the ferric form through the cytoplasm into the magneto- hydroxide has been suggested as the possible some, which appears to be anchored to the trigger for magnetite formation, with the solid- plasma membrane. Empty magnetosomes have state rearrangement manifest as a growing crystal been observed in iron-starved cells, and recent front of magnetite extending into the precursor molecular work has shown that specific magne- phase (Mann et al., 1984). This mineralization tosome-associated proteins play a role in vesicle scenario is, in part, borne out of the observation formation prior to biomineralization (Komeili that some anaerobes, that are capable of dissimilat- et al., 2004). In the magnetosome, Fe(II) is then ory Fe(III) reduction, produce a large number of

re-oxidized to ferric hydroxide, perhaps with O2 small (30–50 nm in diameter), intracellular grains ITGC04 18/7/06 18:23 Page 178

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of ferric hydroxide, as well as lesser amounts of magnetite (Glasauer et al., 2002). A B One aspect perhaps not readily obvious is that the mineralization process requires spatial segregation of regions differing in Eh (the redox potential, see Box 6.1 for details) and pH because the necessary conditions to precipitate ferric hydroxide are quite different from those needed to subsequently transform it into magnetite. Add to that the constraints over magnetite morpho- logy and size, it seems clear that the magnetosome must function under precise biogeochemical and genetic control (Gorby et al., 1988). The characteristic properties of intracellular magnetite are often clearly recognizable in both recent and ancient sedimentary environments. In fact, it has been proposed that biologically controlled magnetite may persist in deep-sea sedi- ments, and thus contribute to the palaeomagnetic record (e.g., Kirschvink and Chang, 1984). Despite 100 µm the magnetite chains fragmenting upon lysis of the cell, their initial presence can be inferred by observing the morphological/chemical char- Figure 4.28 Comparison of magnetite grains acteristics of magnetically separated fractions from modern and ancient sedimentary environments. of sediment under an electron microscope, and (A) An intact magnetite chain, formed by also by using a magnetometer to measure the magnetotactic bacteria, in recent marine sediments resistance to demagnetization that distinguishes of the Santa Barbara Basin, California. (B) Chain of multidomain from single domain magnetite single-domain magnetite grains extracted from (e.g., Petersen et al., 1986). Fossil magnetotactic limestone within the 2.0 Gyr Gunflint Iron Formation, bacteria may even extend as far back as the Canada. (Adapted from Chang and Kirschvink, 1989. Reproduced with permission from the Precambrian, with magnetofossils extracted from Annual Reviews in Earth and Planetary Sciences.) the 2.0 Gyr Gunflint Iron Formation (Fig. 4.28) possibly representing the oldest evidence of con- trolled biomineralization (Chang et al., 1989). as magnetic. The lower magnetism of greigite, however, is compensated for by the fact that the 4.2.2 Greigite greigite-producing bacteria tend to have many more magnetosome crystals, as many as 100 per

The formation of greigite (Fe3S4) proceeds by cell (Pósfai et al., 1998). Morphologies of greigite the same controlled intracellular mineralization include cuboidal and rectangular prismatic crystals process as described for the magnetite-generating in the size range 30–120 nm. magnetotactic bacteria (e.g., Bazylinski et al., The biomineralization of greigite in magneto- 1993). Individual greigite particles are membrane- tactic bacteria closely resembles the processes bound and organized into chains. They are of sedimentary sulfide formation, whereby also ferromagnetically ordered, providing the amorphous Fe sulfide transforms into cubic bacterium with properties similar to a magnetite- FeS → mackinawite → greigite through a series producing bacterium, although greigite is one-third of solid-state transformations. Similar to above, ITGC04 18/7/06 18:23 Page 179

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the magnetotactic bacteria appear to synthesize and align the nonmagnetic sulfides into chains A prior to the crystals becoming magnetic. Over time, greigite then converts to pyrite under reducing conditions at neutral pH when excess sulfur is present. In one magnetotactic bac- terium, pyrite crystals were observed along with greigite (Mann et al., 1990). Given the lengthy conversion time for greigite to pyrite, it seems unlikely that this process took place during the cell’s lifetime. Instead, greigite and pyrite may be biomineralized separately, indicating that the stoichiometry of the metal (Fe) and nonmetal (S) can vary in some magnetotactic bacteria, result- ing in different mineral assemblages (Heywood µ et al., 1990). 6 m While magnetite-producing bacteria prefer microaerophilic conditions, the greigite pro- B ducers grow below the oxic–anoxic interface, where HS− concentrations are high. Interest- ingly, one bacterium, as described by Bazylinski et al. (1995), could produce both minerals, form- ing magnetite in the oxic zone and greigite in the anoxic zone. This further implies that local oxygen and/or hydrogen sulfide concentrations regulate the type of biomineral formed, but it also 45 µm hints at the possibility that two different sets of genes control the biomineralization of magnetite and greigite. Although none of the greigite- Figure 4.29 SEM micrographs of siliceous producing bacteria have as yet been cultured, eukaryotes. (A) The diatom Mastogloia rRNA analysis has shown that they are associated cocconeiformis retrieved from a lagoon in the Grand Cayman, British West Indies (courtesy of with sulfate-reducing bacteria (DeLong et al., Hilary Corlett). (B) Fossil radiolarian Dictyomitra 1993). Therefore, if these bacteria can reduce andersoni from early Pleistocene sediments of sulfate, it then raises the question of whether Chatham Island, New Zealand (courtesy of Chris the sulfide ions present in greigite originate from Hollis). sulfide present in the aqueous environment, or from sulfate reduction occurring within the cell. to as opaline silica or opal-A) that tend to have 4.2.3 Amorphous silica beautifully ornamented structures (e.g., Fig. 4.29). Together, these microorganisms are the major Unlike the numerous microorganisms that contributors of solid-phase silica fluxes to the passively precipitate amorphous silica from super- seafloor, and it is because of their collective exist- saturated fluids, some eukaryotes, such as radio- ence that modern oceans (and lakes) are very larians and diatoms, exert complete control over undersaturated with respect to amorphous silica the silicification process. The cells are enclosed in (Lowenstam and Weiner, 1989). Indeed, prior to siliceous shells (in this context commonly referred the evolution of the radiolarians (and sponges) ITGC04 18/7/06 18:23 Page 180

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during the Cambrian, the oceans were probably upper ocean mixing was more vigorous. Their at equilibrium with respect to amorphous silica prominence has even been linked to global (110 mg L−1 at less than 25°C). Thereafter, silica cooling episodes during Earth’s history (Pollock, levels began to diminish, and with the evolution 1997). Diatoms similarly affect the microbiology of the diatoms around the late Triassic–early of lakes, and mass balance studies have demon- Jurassic, and their subsequent proliferation by strated that they are responsible for the bulk the middle Cenozoic, seawater silica concentra- of silica sedimentation (e.g., Schelske, 1985). tions progressively declined to modern values of As with all autotrophs, their essential environ- less than 5 mg L−1. mental requirements include sufficient irradi- Radiolarians primarily inhabit surface ocean ance to photosynthesize, and, in order to satisfy waters, and occupy biogeographical zones compar- that demand, continuous residence within the able with other zooplankton (Racki and Cordey, euphotic zone is paramount. In highly produc- 2000). Most species are immotile (i.e., they are tive waters, their shells accumulate in enormous not capable of movement), and they drift along numbers to form a mud known as diatomite. with currents from one water mass to another. One of the most interesting paradoxes about Aside from silica availability, one of the major radiolarians and diatoms, from a geochemical controlling factors in their distribution is tem- perspective, is that they expend considerable perature and salinity, with the highest densities energy in constructing very elaborate shells com- found in warm equatorial waters. Radiolarian posed of a material that is not readily available oozes formed below zones of high productivity to them. So two obvious questions arise: (i) how can contain as many as 100,000 shells per gram of do they form their shells under such seemingly sediment (Armstrong and Brasier, 2005). One unfavorable conditions; and (ii) why do they not interesting growth strategy radiolarians employ is use another mineral, e.g., calcium carbonate, a symbiotic relationship with algae. When food that is easier to form? is scarce, an algal symbiont can provide its host The mechanisms underpinning eukaryote radiolarian with much needed nourishment. silicification are far from resolved (see de Vrind- Diatoms are virtually ubiquitous in the hydro- de Jong and de Vrind, 1997 for details). In the sphere, occupying benthic and planktonic niches case of diatoms, their cell wall is silicified to in both freshwater and seawater. As a group they form a hard shell, or frustule, comprising two tolerate an exceptionally large range of tempera- valves, one overlapping the other. New valves ture, salinity, pH, and nutrient conditions. More are formed within minutes during cell division than 20,000 modern and fossil species of diatoms by the controlled precipitation of silica within are known, 70% of which are marine (Harwood a specialized intracellular, membrane-bound and Nikolaev, 1995). Those marine species, silica deposition vesicle, the SDV. To initiate in particular, play an extremely important eco- the process, the cells actively pump silicic acid logical role, accounting for as much as 40% of from the external aqueous environment across the primary productivity in the oceans (Tréguer the plasma membrane and SDV membrane (the et al., 1995). In addition, diatoms possess intra- silicalemma) with the use of specific transporter cellular storage vesicles that acquire and hoard proteins (Hildebrand et al., 1997). The energy short-term pulses of nutrients while simultane- for this process is driven by photosynthesis (in ously depriving competing photosynthetic micro- the light) and glucose respiration (in the dark). organisms of those essential resources (Tozzi et al., Inside the SDV, the silica concentration is 2004). In this regard, diatoms have periodically increased to a state of supersaturation with respect been the most significant phytoplanktonic species to amorphous silica. At this stage the monomers controlling ocean nutrient cycling, particularly polymerize to form nanoscale colloids that adsorb during glacial periods in Earth’s history when on to the inner face of the silicalemma. It has been ITGC04 18/7/06 18:23 Page 181

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estimated that the rate of silicification within opening their shells serve as effective pre- diatoms is about 106 times higher than abio- dators. As a result, diatoms typically show lower logical formation from supersaturated solutions mortality rates than those of other, smaller algae (Gordon and Drum, 1994). with similar growth rates. It has also recently As the silica is deposited, the SDV increases been speculated that the silica might play a in size, and there is a concomitant creation of role in buffering pH, enabling the enzymatic

a silica concentration gradient from the borders conversion of bicarbonate to CO2 in waters of the vesicle towards the center as a result of where the concentration of the latter is less the polymerization process. During this stage, the than required for photosynthesis (Milligan and diatoms exert additional control over silicifica- Morel, 2002). tion because the silicalemma is lined with a Despite their need for silica, the growth rates mixture of proteins consisting of hydroxyl- and of diatoms remain independent of dissolved polycationic amino-containing amino acids, such silica concentrations until they reach 0.1 mg L−1

as glycine, serine, and tyrosine (Fig. 4.30). They of SiO2 or less. Silica, therefore, seldom becomes provide molecular complementarity with poly- a limiting factor except during intensive diatom meric silica such that it sorbs via hydrogen and blooms, and if this does occur, the diatoms either electrostatic bonding, respectively, to the mem- produce weakly silicified shells or the blooms brane surface (Volcani, 1983). Actually, the silica collapse and they are promptly succeeded by binds so strongly to the proteins that only treat- blooms of other nonsiliceous phytoplankton, ment with hydrogen fluoride re-solubilizes the such as the coccolithophores or cyanobacteria silica. Interestingly, diatoms genetically modify (Schelske and Stoermer, 1971). Furthermore, their SDV proteins by inserting more reactive although ocean surface waters are inherently polycationic amino acids when external silica undersaturated with silica, sufficient quantities concentrations are low (Kröger et al., 1999). are temporarily available at any given time due The SDV can take on a number of shapes, and as to a very effective recycling process in which such, it serves as a template for the manufacture more than 95% of the siliceous shells on their of species-specific shell morphologies. Once a way to, or in, the bottom sediment, are dissolved. completed valve is formed, a new plasma mem- This explains how the estimated total present- brane forms behind it, leaving the old plasma day silica production by siliceous eukaryotes and SDV membranes as an organic casing that (2.5 × 1016 g yr−1) is 25 times the input of silica protects the siliceous valve against dissolution to the oceans from rivers, submarine weathering, in the undersaturated waters. and submarine volcanism (Heath, 1974). The The reason for silica use by diatoms is purely efficient recycling process comes about because speculative, but it may have its answer in the once the cells are dead, the plasma membrane genetic legacy of when these cells evolved in a and silicalemma are degraded by chemohetero- more silica-rich hydrosphere and it was energet- trophic bacteria residing in the water column ically “cheaper” to construct a cell wall with and seafloor, and the amorphous silica that silica rather than with organic carbon (Raven, makes up the shells suddenly finds itself exposed, 1983). In any case, there are number of possible and in acute disequilibrium with the under- benefits to possessing siliceous shells, perhaps saturated waters (Bidle and Azam, 1999). The the most notable being as armour against pre- siliceous shells rapidly dissolve, with the silica dation by zooplankton. For instance, Hamm re-circulated to the surface waters by diffusion et al. (2003) have shown that the shells are or upwelling. Zooplankton grazing additionally remarkably strong by virtue of their architecture, affects silica re-cycling, as the silica is repackaged and only organisms large enough to ingest them into fecal pellets that are transported rapidly to or digest their intracellular contents without the seafloor. ITGC04 18/7/0618:23Page182

Silicalemma Plasma membrane Si(OH)4 + OH NH 3 HO OH OH NH + HO Si OH 3 Hydrogen OH OH OH OH bonding Silica colloid HO NH + OH + OH OH 3 NH3 + HO Si OH NH3 Electrostatic OH OH bonding + + NH3 NH -containing 3 Diatom Amorphous SDV amino acids silica

(Ksp – 110 ppm) OH-containing amino acids Bulk water

(Si(OH)4 – 5 ppm)

Figure 4.30 Representation of how diatoms form their siliceous shells. Despite living in undersaturated solutions with respect to amorphous silica, diatoms actively extract Si(OH)4 from solution and pump it into an intracellular silica deposition vesicle (SDV) that lines the inside of the plasma membrane. There, the concentration of silica is increased to supersaturation. The SDV also contains hydroxyl and cationic amino-containing amino acids that react with colloidal silica, and thus facilitate the subsequent nucleation and mineralization stages. In this regard, the SDV acts as a template, for silicification. ITGC04 18/7/06 18:23 Page 183

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Notwithstanding the efficient recycling, large areas of the seafloor today (some 15%) are still A covered in amorphous silica sediments because silica is less soluble in the colder temperatures associated with the seafloor and there is a progressive loss of reactivity with aging upon sedimentation (Van Cappellen, 1996). These silica-rich deposits are generally associated with regions of active upwelling, where high phyto- plankton growth in the surface waters leads to high sedimentation and fast burial rates. Globally, sediments rich in diatom debris are concentrated in the northern and equatorial Pacific Ocean, 3 µm and around the Antarctic continent, the latter accounting for the majority of the total silica sink B (DeMaster, 1981). In turn, some of the diatom and radiolarian shells become rapidly insulated from the undersaturated bottom ocean waters. Not only does this allow the pore waters in this relatively closed system to attain equilibrium with amorphous silica, leading to diminished rates of shell dissolution, but the deposited layers may become sufficiently thick and impenetrable that they prevent irrigation of the surface sediments by benthic infauna, i.e., those animals that live in the sediment (Pike and Kemp, 1999). In the µ equatorial Pacific today, deposits some 4–6 meters 30 m thick may contain over 400 million diatoms per gram (Armstrong and Brasier, 2005). In the rock record, there are some very signific- Figure 4.31 SEM micrographs of calcareous ant diatomaceous deposits, such as the several eukaryotes. (A) The coccolithophore Calcidiscus hundred meter thick Belridge Diatomite, within leptoporus collected off the coast of Namibia (courtesy of Markus Geisen). (B) The foraminifera the Monterey Formation, an organic-rich deposit Spirillina vivipara as retrieved from a lagoon in the that formed on a Miocene continental margin off Grand Cayman, British West Indies (courtesy of the coast of California. Biogenic deposits formed Hilary Corlett). between the Cambrian and Cretaceous contain instead abundant radiolarian shells, i.e., radiolarite deposits (Racki and Cordey, 2000). Their preserva- from the coccolithophores and foraminifera. tion was generally better considering that seawater These unicellular algae and protozoa, respectively, had higher silica levels in the early Phanerozoic. remove vast amounts of calcium carbonate (in the form of calcite) from seawater to form their 4.2.4 Calcite shells, and today they are considered to be the most important carbonate-secreting organisms Calcium carbonate constitutes the largest fraction on Earth (Fig. 4.31). of known biologically controlled biominerals. The The coccolithophores have been abundant in most impressive carbonate precipitation results the oceans since the Jurassic, yet a population ITGC04 18/7/06 18:23 Page 184

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Figure 4.32 The present-day distribution of the principal types of marine sediments. Calcareous (From Andrews et al., Deep-sea clay Glacial sediments sediments 2004. Reproduced Terrigenous Continental-margin with permission from Siliceous sediments sediments sediments Blackwell Publishing Ltd.)

explosion in the early Cretaceous saw a massive depth where sufficient light exists to support accumulation of carbonates deposited worldwide. photosynthesis. Blooms of this magnitude can be Near the end of the Cretaceous, the coccolitho- responsible for the deposition of thousands of phores suffered a mass extinction; two-thirds of tons of calcite (Holligan et al., 1983). Such large the 50 genera disappeared at that time, though masses of suspended calcite crystals also affect the many new groups appeared later in the Palaeocene light-scattering properties of the surface waters, (Armstrong and Brasier, 2005). The enormous and as a result, the blooms can even be detected extent to which they have historically precipit- from space with satellite imagery (see Plate 9). ated micritic sediment can best be emphasized Significantly, the abundance of coccolithophores by the fact that the Cretaceous chalk deposits influences not only the transfer of carbon from of north-west Europe are formed almost exclu- the atmosphere to the ocean sediment, as well as sively from them. Furthermore, over the past the marine calcium budget, but the cells also 150 Ma (since their first appearance), carbon- generate large fluxes of dimethyl sulfide (DMS) ate sedimentation in the open ocean accounts that leads to considerable albedo affects over the for 65–80% of the global carbonate inventory open ocean, which ultimately may affect global (Andrews et al., 2004). These deep-sea deposits, climate change (Westbroek et al., 1993). which average 0.5 km in thickness, mantle half The foraminifera play a prominent role in the area of the deep ocean (Fig. 4.32). marine ecosystems as micro-omnivores, feeding Modern coccolithophores are abundant at on bacteria, protozoa, and small invertebrates. mid to high latitudes coastal areas in waters vary- They are found in all marine environments, from ing from 2°C to 28°C. Emiliania huxleyi is one the intertidal to the deepest ocean trenches, of the most abundant coccolithophore species, and from the tropics to the poles. Most of the and when nutrients are sufficiently available, estimated 5,000 extant species live in the world’s blooms with cell densities of 108 cells L−1 can oceans. Of these, 40 species are planktonic, while cover several thousand square kilometers, to a the remaining species live on the bottom of the ITGC04 18/7/06 18:23 Page 185

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ocean, on shells, rock, and seaweeds, or in the tion of OH−. However, unlike the cyanobacteria sand and mud (see Murray, 1991 for details). They that excrete the OH− ions into the surround- can be very abundant, comprising over 90% of ing aqueous environment, it appears that in the deep-sea biomass, with bottom sediments the coccolithophores OH− ions are by protons almost exclusively made up of their shells. The generated by the calcifying vesicle (reaction 4.24). oldest foraminifera are from the earliest Cam- This maintains the pH within the depositional brian. Much the same as the coccolithophores, vesicle at an appropriate level for calcification. they underwent a mass extinction at the end The subtle differences between cyanobacteria of the Cretaceous, but then experienced a rapid and coccolithophore calcification clearly high- radiation in the Palaeocene (Tappan and Loeblich, light their differing activities; the former are 1988). The foraminifera produce ornate calcite simply photosynthesizing and inducing calcite shells that range in size from ~30 µm to 1 mm. precipitation as a byproduct of their metabolism Because of their intricate morphologies, fossil (i.e., they do not apparently use the calcite), foraminifera have been widely employed as while the latter controls the process intracellu- biostratigraphic markers, i.e., they can be used to larly in order to precipitate a crystal with precise establish the relative stratigraphic position of size and orientation. sedimentary rocks between different geographic Some algae that comprise reef-building com- localities. Moreover, the shells themselves have munities also incorporate calcium carbonate into proven extremely useful as palaeoenviron- their structures as strengthening agents. These mental indicators of ancient ocean salinity, water consist of the marine red algae (e.g., Corallina sp.) temperatures, surface productivity, and even global that deposit high-magnesium calcite within their climate (e.g., Waelbroeck et al., 2002). cell walls. They possess sulfated galactans and Similar to the silica-secreting eukaryotes, alginates that preferentially bind Ca2+ over Mg2+, there is still some uncertainty regarding the thereby creating localized microenvironments mechanisms underpinning coccolithophore that favor calcite precipitation over aragonite. In and foraminifera shell formation. In the case of addition, the cells appear to be capable of regu- coccolithophores, they have an internal vesicle lating the crystallography and orientation of the that serves as the locus for calcite formation calcite crystals (Borowitzka, 1989). The import- (de Vrind-de Jong and de Vrind, 1997). Import ance of the coralline algae in the carbon cycle 2+ of Ca from CaCO3-saturated seawater occurs lies in the fact that, unlike the coccolithophore passively through Ca2+ channels into the cyto- shells formed in the open oceans, that upon plasm. However, the Ca2+ then proceeds against sedimentation into deeper waters (3000–5000 m) a concentration gradient to get into the vesicle, re-dissolve back into the water column (due to requiring energy in the form of ATP. The exact increased pressures, decreased temperatures, and 2+ mechanism for transferring Ca from the cyto- increased CO2 concentrations below the calcite plasm into the vesicle is unknown, but it is compensation depth), the biogenic reef carbon- hypothesized that specific transport enzymes are ates are semipermanent features in the Earth’s − involved. To complete calcite formation, HCO3 sedimentary record, and therefore represent an is also introduced into the deposition vesicle: enormous carbonate sink.

− + 2+ ←→ + + HCO3 Ca CaCO3 H (4.24) 4.3 Fossilization

Because the concentration of CO2 in seawater may be limiting for photosynthesis, coccolithophores Most microorganisms lack substantial hard parts − have the ability to utilize HCO3 instead (recall and rarely fossilize. Thus, their soft tissues are Fig. 4.15). The net result of this is the genera- rapidly degraded and evidence of their existence ITGC04 18/7/06 18:23 Page 186

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is wiped away with time. Even among the larger, filaments fragmented and coalesced, intracellu- multicellular groups, the fossil record is nowhere lar components were destroyed, and there was near complete. Despite these inherent short- a preferential preservation of the sheath and comings, the limited microfossil assemblages that wall material. The importance of the sheath in exist have proven to be indispensable to our limiting silicification to the outer surfaces of the understandings about the evolution of life on cell has already been discussed in section 4.1.6, Earth because they provide a physical record that and it is interesting that when some cyano- represents the geological and environmental bacteria, such as Calothrix sp., are grown in silica conditions of the time when the organisms were supersaturated conditions, their sheaths double living. to triple in width, up to 10 µm in diameter As will be discussed in Chapter 7, fossil- (Phoenix et al., 2000). In the case of Calothrix, ized prokaryotes are known from very ancient the findings suggest that they genetically respond Precambrian rocks, some potentially as old as to high silica concentrations by adapting their 3.5 billion years. Considering that there is no surface structure to isolate the cell from the evidence to suggest that those microfossils repre- damaging effects of silicification. Crucially, this sent species that controlled biomineralization, it growth response forms a morphological feature suggests that something unique about the species that may be preservable, thereby giving palae- and/or the conditions under which they grew ontologists a clue for recognizing their ancient allowed for their preservation (Konhauser et al., predecessors in the rock record. 2003). The type of mineralization associated Sheaths are not, however, a prerequisite for with soft-part preservation of ancient life forms survival in silica-saturated geothermal waters. is predominantly in the form of silica. This is Oscillatoria, for example, is a cyanobacterium that unsurprising as the small size of the sorbing silica is either not ensheathed or is thinly sheathed, yet colloids, relative to the cell, allows for the entire it has been isolated from various hot springs. outer surface to be completely enshrouded in Studies on unsheathed bacteria have also shown a protective mineral coating. Other fossilizing that some produce robust and durable crusts after minerals, due to their generally larger crystal size, only a week of silicification, whereas others are of secondary importance in terms of their maintain delicately preserved walls that are only ability to preserve intact cells. lightly mineralized (Westall, 1997). Only at very high silica concentrations does significant loss of 4.3.1 Silicification shape and cellular detail occur (Toporski et al., 2002). Recently, exposure of Sulfurihydrogenibium Considering that so many ancient microorganisms azorense, of the order Aquificales, demonstrated are fossilized in silica, there have been surpris- a new twist on coping with high silica concen- ingly few studies trying to elucidate the phys- trations; it produced protein-rich biofilms that ical changes imposed on microorganisms during facilitated silicification, but away from the cell silicification. Based on the assumption that surface (Lalonde et al., 2005). By regulating Achaean microfossils were cyanobacteria, Oehler biofilm production appropriately, the Aquificales (1976) was one of the first to experimentally sub- could potentially contribute to or accelerate ject various cyanobacterial genera to colloidal silicification, though the cells themselves are silica solutions over different lengths of time. unlikely to be preserved. What he showed was that at temperatures of What these studies have collectively shown is ~100°C several months were required for com- that, although the silicification of biomass is an plete silicification, and only slight alteration to inevitable process in silica-supersaturated solu- the cells occurred, while at higher temperatures tions, there remains species-specific patterns of (165°C) the cells mineralized quickly, but the silicification, and ultimately different preservation ITGC04 18/7/06 18:23 Page 187

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potentials with regards to incorporation into the rock record. Unfortunately, at present only a few microorganisms have been analyzed, and in each study different experimental conditions were used. As a consequence not only do the dif- ferent studies yield conflicting results regarding the rates and magnitude of silicification, but no comprehensive database is presently available with which to confidently assess what is required for a siliceous microfossil to form. From what we presently know about silicifica- tion, there are at least three main factors that Sheath µ lead to the short-term preservation of intact cell 1 m structures.

Figure 4.33 1 The timing and rate of silicification relative to TEM image of a lysed cell, growing as part of a cyanobacterial mat, in silica-rich hot death of the microorganisms is of paramount spring waters at Krisuvik, Iceland. This cell exhibits importance. When silicification is rapid, recently both epicellular and intracellular silicification, with lysed cells may resist decay, thereby retaining intact only the sheath and cell wall remaining intact morphologies within a relatively impermeable (From Konhauser et al., 2004. Reproduced with matrix (e.g., Fig. 4.33). Actually, a limited degree permission from the Royal Swedish Academy of of decomposition may help facilitate silicification Sciences.) by exposing cytoplasmic material for hydrogen bonding. Silicification also limits heterotrophic microorganisms from completely degrading the 1999). Conversely, other cells with different cellular cells prior to their incorporation into the sedi- features degraded and left little evidence of their mentary record. By contrast, experimental studies original organic framework (Horodyski et al., have shown that unmineralized cells begin to 1992). Aside from the actual preservation of the degrade within days after death (Bartley, 1996). cell itself, there is some putative evidence to sug- As a result, the remnants of most cells in nature gest that fossilized EPS is widespread in the rock become progressively obscured. This helps explain record (Westall et al., 2000). Whether the struc- the general rarity of recognizable microfossils in tures interpreted in thin section micrographs are the Archean rock record, except under conditions indeed biological needs to be verified, but the of extremely early lithification (see Box 7.5 for premise of their existence is reasonable consider- details). ing that EPS is volumetrically more important than the cells within the biofilm, and as shown above, 2 In Precambrian cherts there is a preservational EPS does provide abundant sites to facilitate bias towards cells that had thick cell walls and/or silicification. sheaths (Knoll, 1985). This is unsurprising since peptidoglycan and the polymers that comprise 3 Ferris et al. (1988) showed that the binding of the sheath are more resilient to degradation than metallic ions, in particular iron to bacterial cell sur- other cell components, and as long as the con- faces, was an important contributing factor to the stituent autolysins are deactivated, they can per- silicification of Bacillus subtilis: cells not pre-stained sist in the environment long after the cell dies. by Fe suffered extensive lysis after several days of Furthermore, those structures are more amenable aging. This inhibition of cell degradation appears to to silicification. Therefore, in terms of preservation be related to the ability of some metals to deactivate potential, fossil assemblages in the Precambrian the cells’ own autolytic enzymes. Correspondingly, may be biased towards microorganisms, such as Walter et al. (1992) suggested that the fossil record some cyanobacteria, simply because they possessed is also prejudiced towards those cells that tolerate suitable ultrastructures (Golubic and Seong-Jao, elevated salinities. ITGC04 18/7/06 18:23 Page 188

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The subtleties of the silicification process are crit- ical because they may control the appearance of A the preserved microorganisms and the features that are needed to identify them in terms of extant taxa. Many of the taxonomically critical features of microorganisms are lost during silicification or are concealed by mineral precipitate. Thus, a silicified microorganism analyzed under SEM 500 nm and/or TEM may display only a few distinct features (e.g., size, general morphology, presence/ B absence of sheath, septa; and rarely cytoplasmic components) that can be used for identification purposes. Therein lies the problem for micro- organism identification by such techniques. For instance, Castenholz and Waterbury (1989) listed 37 characteristics that have been used in the 500 nm identification of cyanobacteria. Unfortunately, as demonstrated by Jones et al. (2001), silicifica- tion may selectively mask and/or destroy some Figure 4.34 SEM images showing the features while preserving others. This can lead formation of “pseudofossils.” These samples to a silicified microorganism that fails to dis- were collected from a glass slide left for 90 hours play key features that indicate what it looked in a New Zealand hot spring where silica concentrations were 450 mg L−1. (A) Nascent like prior to mineralization. A case in point, glass silica beads forming on a mucus strand (arrows). slides left in a silica-supersaturated hot spring pool (B) The mucus strand is completely covered −1 (with 450 mg L SiO2) at 70°C for only 90 hours with silica grains, potentially giving the false showed abundant silicified microorganisms, but appearance of a silica-encrusted filament. silicification concealed essentially all identifi- (From Jones et al., 2004. Reproduced with able features that allowed for their recognition permission from the Geological Society, London.) (Jones et al., 2004). Simultaneously, silicification can generate artefacts that appear to be micro- organisms. In the same hot spring pool, amorph- than 10 morphologically defined taxa; the most ous silica grains (300–400 nm in diameter), that taxa yet described from a silicified hot spring are centred around a mucus strand that is less sinter is 19 (Jones et al., 2003). than 10 nm thick, are morphologically similar in One fundamental point that seems to have appearance to many of the silicified filamentous escaped close scrutiny is that most of the Pre- microorganisms, yet these “pseudofilaments” are cambrian microfossils (as will be described in not cellular in origin (Fig. 4.34). Even in the most Chapter 7) are in chert, yet the original micro- well-preserved silicified microorganisms only a organisms would have been mineralized by amor- few of the taxonomically important charac- phous silica. Therefore, irrespective of how the teristics can be recognized. It is therefore not microorganisms contributed to silicification and surprising that the silicified biota found in the preservation of intact residues, the transforma- hot-spring sinters are usually characterized by tion from amorphous silica to chert would have low diversities despite the fact that the micro- eliminated most of the morphological evidence organisms seem to be so well preserved; silicified of an organic origin. In fact, experiments show cells from such settings typically contain less that during the transformation from opal-A to ITGC04 18/7/06 18:23 Page 189

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opal-CT (cristobalite), most of the detailed wall structures of diatoms are destroyed (Isaacs, 1981). Furthermore, the subsequent phase change from opal-CT to quartz eliminates any remaining wall details, although gross morphology may yet be preserved (Riech and van Rad, 1979).

4.3.2 Other authigenic minerals

In nonsiliceous environments, cell preservation operates in a fine balance between decay and mineralization. On the one hand heterotrophic microorganisms consume the lysed cells, while on the other hand, the metabolic byproducts 3 µm of their metabolism promote elevated saturation states and mineral encrustation of the cellular remains. As might be expected, given our Figure 4.35 SEM image of the fabric in a discussions above, alkalinity generation and phosphorite layer from the Triassic Bravaisberget phosphate/sulfide release lead to the formation Formation of Spitzbergen, Norway. Aggregates of of a wide variety of authigenic minerals. apatite globules encapsulate remnants of microbial cells. It is presumed that the globules formed as a In experiments with decaying shrimp, where result of rapid phosphate precipitation on coccoid the system was freely open to diffusional-related bacteria that inhabited the surface layers of an processes, calcium carbonate forms in a manner organic and phosphate-rich sediment. (Courtesy of typical of biologically induced biomineraliza- Krzysztof Krajewski.) tion. Although calcite retains the gross morpho- logy of the soft tissue, it completely obliterates the finer detail. Contrastingly, when the sys- (e.g., Martill, 1988). They occur as phosphatized tem is closed and diffusion limited, the organic replicas replacing the soft tissues, often with material is replicated in a more detailed man- detail at the subcellular level. In addition to the ner in calcium phosphate; the source of the presence of characteristic microbial fabrics, in phosphate being the shrimp itself (Briggs and some phosphorites, apatitic molds retain remnants Kear, 1993). The underlying determinant for of the original microorganisms (e.g., Fig. 4.35). which mineral forms appears to be pH; more Examples include the presence of filamentous

alkaline values lead to CaCO3, while more cyanobacteria in the Mishash Formation, Israel; neutral values lead to CaPO4. Paradoxically, the reported fungal mat remains in the Tertiary exceptional preservation of soft tissue in fossils phosphorites of Morocco; and the globule-like requires elevated, rather than restricted, micro- clusters of apatite-encrusted coccoid bacteria in the bial activity as this leads to anaerobically driven Triassic Bravaisberget Formation of Spitzbergen mineral authigenesis (Sageman et al., 1999). It is (Krajewski et al., 1994). also important that mineralization occurs quickly, The preservation of soft parts in pyrite is a as once the morphology is stabilized by initial rarer feature in ancient sediments. At present, mineralization, the potential for preservation of the best described examples are associated with soft tissue record is greatly enhanced. the Hunsrück Slate of Budenbach, Germany, Bacterial communities themselves are often the Beecher’s Trilobite Bed of New York State, involved in the preservation of soft tissues and the Burgess Shale of British Columbia. In ITGC04 18/7/06 18:23 Page 190

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general three modes of pyrite fossilization can Fe2+ + HS− → FeS + H+ (4.26) be recognized (Canfield and Raiswell, 1991): The limited nature of fossil pyritization implies 1 Mineralized tissue – Refractory tissues such as that specific sedimentary conditions must have cellulose and chitin may be preserved by the pre- existed at the site of organic decay during the cipitation of pyrite in their pore spaces. time of burial (Raiswell, 1997). One constraint is that pyritization took place before compaction 2 Mineral coats – The preservation of very degrad- of the soft tissue. Another requirement is that able soft parts most commonly occurs by outline pyritization, but it rarely preserves internal struc- the generation of dissolved sulfide, formed at the tures because the crystals are too coarse and form expense of the decaying organisms, had to pro- too late to replicate the finest details. This typic- ceed at a comparatively slow rate because sulfidic ally occurs as a pyritized layer of bacteria that pore waters contain negligible concentrations pseudomorph the original structure. of iron. In other words, pyritization needs to >> 2+ be limited to the decay site, but if H2S Fe , 3 Mineral casts or molds – This style of preservation then no ferrous iron would likely be proximal to involves the greatest degree of information loss, since only the fossil outline is preserved. The casts the organic material. Based on modern sediment and molds result from diffuse early diagenetic pyrit- studies, such conditions could have been met ization in the surrounding sediments. during the earliest stages of sulfate reduction, at shallow burial depths in the suboxic zone, where Pyritization can also preserve shells when it Fe(II)-rich pore waters (from Fe(III) reduction) replaces or coats the carbonate minerals (see were supersaturated with respect to iron mono- Plate 10). Replacement under these situations sulfides (Fig. 4.36). Sulfur isotope data corre- occurs when the solid-phase carbonate is dis- spondingly suggests that 32S-enriched sulfide, solved by H+ (reaction (4.25)). The loss of produced at the expense of the organic matter, protons can then potentially lead to a state of was unable to diffuse away from the decay site supersaturation with respect to FeS, as reaction and into the surrounding sediments. Consistent (4.26) is driven from left to right: with this, the host sediments must have con- tained relatively high concentrations of ferric + + → 2+ + − CaCO3 H Ca HCO3 (4.25) oxyhydroxides at deposition.

Mass of organic matter 2+ FeS decaying by 2− Fe 2+ SO4 Fe + sulfate reduction Fe2 − 2− HS SO4 2− SO4 − − 2+ − HCO3 Fe + HS Fe2 FeS HCO3

+ Zone of Fe2 2+ 2− 2+ Fe sulfide SO 2+ Fe 4 Fe precipitation

Figure 4.36 Model of organic matter pyritization. Its oxidation by SRB releases HS− into the pore waters immediately surrounding the degrading biomass. Those pore waters, however, must have sufficiently high Fe2+ so − that FeS (and ultimately FeS2) precipitation is confined to the decay site, instead of HS diffusing away into the surrounding sediments. (Adapted from Raiswell, 1997.) ITGC04 18/7/06 18:23 Page 191

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must be of considerable importance to the func- 4.4 Summary tioning of the cell. In either case, the influence biomineralization has on elemental cycling in Microorganisms form an immense variety of aqueous and sedimentary environments cannot authigenic minerals. In the majority of cases, be overstated. Modern Fe, Mn, Si, Ca, P, C, and S bacterial biomineralization is a two-step process, cycles are all affected by biomineralizing processes. where cations are initially bound to the anionic Although individual grains are micrometer in ligands of the cell’s surface, and subsequently scale, if one takes into account the total amount they serve as heterogeneous nucleation sites for of biomineralizing biomass, it is not difficult to mineral precipitation. The biogenic minerals imagine how they can represent a significant geo- appear identical to those produced abiologically logical driver that partitions elements from the because they are governed by the same thermo- hydrosphere into the sediments. In this regard, dynamic principles. As the latter stages of min- the precipitation of carbonate minerals by micro- eralization are inorganically driven, the type of organisms is especially relevant because these biomineral formed is inevitably dependent on the minerals represent the final products in the weather- available counter-ions, and hence the chemical ing of silicate minerals, and a long-term sink for composition of the waters in which the micro- atmospheric carbon dioxide (see section 5.1.6). organisms are growing. For far fewer microorgan- Importantly, biomineralization has been occur- isms, biomineralization is a regulated process. The ring over geological time, as is evident by the formation of these minerals is a significant drain common occurrence of limestone, BIF, chert, sul- on their energy reserves, so the mineral formed fide, and phosphorite deposits in the rock record. ITGC05 18/7/06 18:14 Page 192

5 Microbial weathering

Chemical weathering of the Earth’s upper crust the global climate. This chapter will examine includes two major types of processes, mineral dis- how microorganisms influence weathering of solution and mineral oxidation. Microorganisms the Earth’s crust, and then consider how such play a fundamental role in both. They attach processes can have both environmental and to exposed mineral surfaces, coat them with commercial ramifications. extracellular polymers (EPS), and physically dis- rupt the grains in their attempt to gain access to nutrients and energy in the underlying substrata. 5.1 Mineral dissolution At the same time, they create a complex micro- environment at the mineral–water interface, where metabolically catalyzed redox reactions and 5.1.1 Reactivity at mineral surfaces the generation of acids and complexing agents lead to pH and concentration gradients markedly Chemical weathering rates of minerals are con- different from the bulk solution. This often pro- trolled by their composition, morphology, and motes a state of thermodynamic disequilibrium texture, as well as the geochemistry of the sur- that fosters faster rates of chemical weathering. rounding fluids. The primary determinant under- Microbial EPS further serves as a site for the pinning whether a mineral dissolves or not is precipitation of secondary minerals, with com- the competition between: (i) the strength of the positions and morphologies distinct from those chemical bonds holding the crystal structure inorganically precipitated in the bulk solution. together, i.e., Coulombic interactions; and (ii) the Aside from its localized effect on degrading hydration energy of ions at the mineral’s surface individual mineral grains or even entire rock (Banfield and Hamers, 1997). If the Coulombic outcrops, microbial weathering has profoundly interactions are large but the hydration energy influenced the Earth’s surface environment over is small, the solid is insoluble, whereas in the geological time. Microorganisms have expedited opposite case, the solid dissolves easily. soil formation since their evolution onto land Although these relationships can be used during the Archean, while the solutes released to infer the thermodynamic properties of the through chemical weathering have affected the mineral in a given solution, they say little about composition of the hydrosphere, from micro- the overall rates of dissolution. Furthermore, scale soil pore waters to the enormity of the during the dissolution of any given mineral there oceans. The biochemical weathering of some are a number of intermediate chemical steps that silicate minerals, and the subsequent deposi- take place before an atom or molecule is dis- tion of calcium carbonate on the seafloor, are solved from the surface, and each one of those also linked through a feedback cycle that steps has a specific reaction rate (Morse and

impacts atmospheric CO2 levels, and ultimately Arvidson, 2002). Those steps include: ITGC05 18/7/06 18:14 Page 193

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1 transport of reactants through the solution to the (Berner, 1978). Thus, for most of the minerals mineral surface; discussed in this chapter, steps 2–5 can be viewed as an activation energy barrier that restricts the 2 adsorption of the reactants to the mineral surface; rate of hydrolysis and the subsequent transfer of the hydrated ions into the surrounding solution. 3 migration of the reactants on the surface to an “active” site; This process costs energy that is recovered once the ion is removed completely. 4 the actual chemical reaction between the adsorbed There are a number of important variables reactant and the mineral, which may additionally governing the kinetics of dissolution: involve several intermediate steps where bonds are broken and hydrolysis occurs; 1 Structure of the crystal lattice – Mineral dissolution rates are related to the strength of the metal– 5 migration of the hydrated ions away from the anion bonds. Some minerals (e.g., feldspar) dis- reaction site and desorption into solution; solve slowly because they possess an extensively cross-linked structure of silica tetrahedra. In terms 6 transport of the products away from the mineral of feldspar, the magnitude of dissolution depends surface into the bulk solution. on the relative abundance of Al and Si sites at the mineral surface, with Al sites more susceptible to As in any chemical reaction, one of the above dissolution than the Si sites. As a result, feldspar is steps will be the slowest, the so-called “rate- subject to selective leaching, though there tends limiting step”. Steps 1 and 6 involve the diffusive to be a sufficient amount of unreactive bonds left or advective transport of reactants and products near the mineral surface to maintain integrity once the reactive constituents are removed. Conversely, through the solution, and when either of these minerals with poorly cross-linked fabrics, such as steps are rate-limiting, the reaction is said to be olivine, dissolve rapidly and uniformly (Casey and transport- (or diffusion)-controlled. Steps 2–5 Bunker, 1990). occur on the mineral surface, and when one of them is slowest, the reaction is surface- 2 Orientation of the crystal surface – Atoms at sur- controlled. The dissolution of highly soluble faces always have higher free energy than atoms minerals tends to be transport-controlled, such in a three-dimensional crystal because the former have lower coordination and strongly asymmetric that ions are detached so rapidly from the sur- bonding configurations compared with atoms within face that they build up in concentration to form the bulk crystal. Thus, crystals tend to adopt shapes a saturated solution adjacent to the mineral that minimize their surface free energy (Herring, surface. Dissolution is then regulated by the dis- 1951), that being the surface area and interfacial persal of those ions into the surrounding under- free energy terms in equation (4.3). Similarly, as saturated bulk solution. Crystals dissolved in this the crystal size decreases, its surface reactivity manner will exhibit smooth surfaces because increases because small particles have relatively high surface area:volume ratios. ion detachment occurs over the entire surface so quickly that crystallographically controlled 3 Defects on the crystal surface – Not only do surface features, such as etching, do not occur. particular crystals have different energies, but By contrast, relatively insoluble minerals have different locations on an exposed crystal surface surface-controlled reaction rates until very high have variable energies. For example, most crystal degrees of disequilibrium are achieved. This surfaces are not consistently flat, but instead have means that ion detachment is sufficiently slow a stepped topography (Fig. 5.1). Atoms at these step edges have a lower coordination (but higher that they cannot build up at the minerals surface. energy) than those on the flat portions because Instead, the ions diffuse or advect away from the they have two sides exposed to the solution, and surface rapidly enough that their concentration therefore it is easier to form a complete hydration at the surface is equal to that in bulk solution shell around them so as to reduce the activation ITGC05 18/7/06 18:14 Page 194

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A Step edge Dislocation B

Kink site

Figure 5.1 (A) Diagram illustrating how mineral surfaces are not perfectly flat, but instead have a number of imperfections, such as step edges, kink sites, and dislocations. (B) Scanning tunneling microscope image of the surface of the mineral galena, highlighting some of those defects. (Courtesy of Steve Higgins.)

barrier to hydrolysis. Kink sites, where step edges we covered how the interface between a cell’s turn, are most reactive, with three sides exposed to surface and the surrounding aqueous solution is solution and fewer bonds to adjacent ions. Crystals characterized by an electrical potential that arises also have various types of imperfections. Those from the ionization of surface functional groups. imperfections include dislocations and planar defects The same concepts hold true for mineral sur- (e.g., stacking faults, grain boundaries), whereby faces because most have at least a monolayer rows of atoms in the crystal are slightly out of place, of adsorbed water, and their surface functional and hence more energetic than “perfect” surfaces. groups, commonly containing oxygen or hydroxyl These then become strongly preferred sites of ligands, undergo protonation and deprotonation chemical reactivity, marked by selective etching and reactions (e.g., reactions (5.1) and (5.2)): growth of pits on the underlying mineral surface (Drever, 1988). > + + →> + Al-OH H Al-OH2 low pH (5.1) 4 Adsorbed molecules – Mineral dissolution rates > + − →> − + are often diminished if a particular cation or anion Al-OH OH Al-O H2O high pH (5.2) is adsorbed that can block access of water to a specific reactive site, e.g., adsorption of phosphate 5 Reduction or oxidation – Redox dissolution reactions decreases calcite dissolution rates (Berner and are important since electron exchange alters the Morse, 1974). This is particularly the case at kinks, oxygen–metal bond strengths. Iron and manganese where because of their excess surface energy, oxyhydroxides are subject to reductive dissolution,

they are preferred sites for the adsorption of ions. whereas sulfides and some silicate (e.g., Fe2SiO4)

By contrast, rates of dissolution are enhanced by and metal oxides (e.g., Cu2O) are prone to oxidative the adsorption of protons. They induce a redis- dissolution. In either case, the concentration of the tribution of the overall electron charge on the electron-donating and -accepting species, as well as minerals’ surface, which then fosters the slow the activities of H+ and OH−, are important para- rupture of the oxygen–metal bonds in the crystal meters in determining dissolution rates (Hering and lattice (Casey and Ludwig, 1995). In Chapter 3, Stumm, 1990). ITGC05 18/7/06 18:14 Page 195

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5.1.2 Microbial colonization and fuel hydrolysis and other chemical reactions organic reactions (Welch et al., 1999). EPS also serves as a sub- strate for heterotrophic bacteria, some of which Immediately upon exposure of a rock at the generate acids (see below) that facilitate chem- Earth’s surface, a community of bacteria, algae, ical attack on the underlying minerals (e.g., fungi, and/or lichens attach to the newly avail- Ferris and Lowson, 1997). able solid surfaces (see Plate 11). For the micro- These biological processes work in tandem organisms, the minerals in the rock are a rich with frost wedging, diurnal thermal expansion, source of bioessential elements, available only if and alternate wetting-drying processes to physic- they can extract them from the crystal lattice. ally break the rock down into the smaller lithic Indeed, in oligotrophic (nutrient-poor) terrestrial fragments that are more susceptible to dissolution environments, mineral solubilization and ele- by rain and the effects of organic acids. Then, mental cycling can be requisite for the microbial as the minerals become loosened, macrofauna communities survival (e.g., Konhauser et al., 1994). (e.g., nematodes) accentuate the erosional pro- To attain those nutrients, colonizing microorgan- cess through mechanical abrasion as they graze isms do two things: (i) they physically penetrate (Schneider and Le Campion-Alsumard, 1999). into the rock causing disaggregation of the Eventually, the original rock is transformed into mineral; and (ii) they produce organic acids that the finer-grained mineral component comprising act as dissolving agents. primitive soils.

(a) Physical processes µ As microorganisms colonize rock surfaces, fungal 100 m filaments (called hyphae) exploit cracks, cleav- Fungus ages, and grain boundaries to gain access to new mineral resources. In doing so, they cause several EPS Hyphae alteration features, ranging from simple surface Alga roughing by etching and pitting to extensive sm physical disintegration of the minerals (Barker et al., 1997). The latter includes detachment, separation, and exfoliation of some constituent grains along cleavage planes (e.g., Fig. 5.2). Shattered Minerals without cleavage planes, e.g., quartz, mineral show no such features. Furthermore, grain bound- (sm) ary misfits at the interface between minerals, as well as the new pore spaces created through volume changes associated with the conversion of primary minerals into secondary clay phases, Figure 5.2 SEM image of the interface provide sub-nanometer-scale conduits that are between the crustose lichen Porpidia exploited as weak points by the fungal hyphae albocaerulescens and the rock syenite. Notice (e.g., Barker and Banfield, 1996). the extensive nature of mineral shattering Bacteria also enshroud all exposed mineral and how some of the mineral grains have been surfaces in EPS (recall Fig. 3.24). From a weather- exfoliated from the rock by the fungal hyphae ing perspective, the ability of these compounds (arrow) and subsequently coated in EPS. to retain water helps promote mineral fracturing (Reprinted from Barker and Banfield, 1996 with and it increases the residence time for water to permission from Elsevier.) ITGC05 18/7/06 18:14 Page 196

196 CHAPTER 5

(b) Role of endoliths mountain ranges, albeit over geological time (Büdel et al., 2005). Rocks are regularly colonized by endolithic bac- teria that grow within the natural cavities and (c) Production of organic acids fractures. Those that are photosynthetic, such as the cyanobacteria, are often evident as a distinct Once bacteria and fungi become established on blue-green layer 1–10 mm below the rock sur- a newly exposed mineral surface, they immedi- face, where sufficient light penetrates (Vestal, ately begin to accelerate dissolution through 1988). The role of endolithic microorganisms in the production of organic acids. The majority weathering of limestone and dolomite has been of organic acids they generate are byproducts of well documented (e.g., Pentecost, 1992). Their fermentation and/or various intermediate steps main contribution is to actively bore into the host of the aerobic respiration of glucose, but some rock by solubilizing cementing mineral grains. microorganisms further excrete organic acids This generates more room for their growth, as when growth is limited by the absence of an well as the macrofauna that graze upon their essential nutrient. Many of the fungal acids con- lysed cells. In some carbonate rocks, it has been tain multiple carboxyl groups that dissociate estimated that endolithic communities average at circumneutral pH (Berthelin, 1983). For 2 more than half a million cells per cm (Golubic example, oxalic acid has two pKa values at pH 1.3 et al., 1970). Such high population densities have and 4.2, while citric acid is a tricarboxylic acid

the effect of significantly enhancing erosion rates, with three pKa values at 3.1, 4.7, and 6.4. The and along the Adriatic coast, for example, they low pKa values makes both citric and oxalic acids contribute to an estimated 2 kg m−2 of coastline fairly strong acids. Each lichen also produces a dissolved annually (Schneider and Le Campion- unique suite of compounds, called lichen acids, Alsumard, 1999). that are synthesized by the fungi from carbo- Examination of sandstone outcrops (e.g., in hydrates supplied by the phycobiont (Easton, South Africa) has shown that endoliths also 1997). Some 300 compounds unique to lichens contribute to the onset of chemical weathering have been identified. through the process of substratum alkalinization, The organic acids increase mineral dissolution which involves the cells producing sufficient both directly and indirectly. In the first instance, hydroxyl ions, as a byproduct of photosynthesis the majority of the organic acids dissociate into (recall section 4.1.7), to increase the pH up to 11 organic anions and protons. Some of the protons (Büdel et al., 2005). These values are not only then react with the ligands of the mineral surface high enough to enhance bulk silica dissolution (i.e., protonation reactions), causing a weaken- in the endolithic zone, but the associated shift ing of the metal–oxygen bonds, and ultimately in the carbonate speciation facilitates some the release of a metal cation from the surface. minor precipitation of carbonates. As a result Concurrently, the organic anions react with metal of the dissolution process, the upper portion cations at the mineral surface, similarly destabil- of the rock is loosened and then eroded away izing the metal–oxygen bonds, and promoting by wind and flowing water. These weathering dissolution through the formation of a metal– patterns bear a striking similarity to sandstone chelate complex. Eventual detachment of the outcrops elsewhere, such as the Ross Desert of chelate exposes underlying oxygen atoms to Antarctica (Friedmann and Weed, 1987). Such further protonation reactions. Therefore, sys- exfoliative processes not only modify landscape tems with high concentrations of tridentate

geomorphology, but it has even been proposed (three pKa) or bidentate (two pKa) organic acids that they may be responsible for denuding entire tend to contribute to high levels of ion release, ITGC05 18/7/06 18:14 Page 197

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2− while monofunctional groups, such as acetic acid, the oxalate anion (C2O4 ) is a bidentate ligand have a lesser effect (e.g., Welch and Ullman, that can form a four-member chelate ring when 1993). Of all the acids listed above, oxalic acid is it binds to a divalent metal (Fig. 5.3). Trivalent the most abundant in natural systems (reaching metals that normally form an octahedral six- millimolar concentrations), and it has frequently coordinated complex (e.g., Al3+, Fe3+, Cr3+) can been observed that oxalic acid production is bind three oxalates to form an anionic complex correlated with high solute availability (e.g., Ca2+) (Gadd, 1999). Such chelation process are import- in soils and the reprecipitation of oxalate salts, ant for metal mobility because some, such as such as calcium oxalate (e.g., Braissant et al., Al3+ liberated during silicate dissolution, would

2004). In aquifers, concentrations of tens of naturally precipitate as gibbsite (Al(OH)3) at pH micromolar have been reported, the lower values values >3, were it not complexed into an organic indicative of the fact that the organic acids are form (reaction (5.3)). It is only after the oxalate often not produced in situ, but instead migrated component is microbially oxidized that aluminum in from adjacent organic-rich soils (McMahon is finally precipitated. and Chapelle, 1991). 3+ + 2− → 3− Deprotonated organic anions (e.g., oxalate, Al 3C2O4 Al(C2O4)3 (5.3) citrate) indirectly affect dissolution rates by complexing with metals in solution (compared Along similar lines, chelation of Al3+ and Fe3+ to the mineral’s surface as above), thereby lower- by oxalate anions increases nutrient availability ing the solution’s saturation state (e.g., Bennett to vegetation because inhibiting gibbsite and et al., 1988). EPS acts in a similar manner, par- ferric hydroxide precipitation permits higher ticularly those rich in alginate, which contain an phosphate levels in soil pore waters (Graustein abundance of reactive carboxyl groups (Welch et al., 1977). et al., 1999). Some organic anions are very strong Citrates are also strong metal chelators chelators, and depending on the relative con- (Fig. 5.3). In soils, this has important implications centration of the anions versus metal cations in for Al3+ mobility because aluminum–citrate com- solution, pH, and the stability constants of the plexes render the metal less toxic to plant roots various complexes, they can effectively partition (Jones and Kochian, 1996). However, citrate’s a metal cation that has dissolved from the mineral chelating ability may have an adverse environ- into an organo-metallic complex. As an example, mental impact when it facilitates the leaching

A B – – –OOC – O O O O H2O CH2COO CH2COOH COOH C C 2+ C M2+ HO C COOH M COOH C C H O – OH O O– O– O CH2COOH 2 OOCH2C Oxalic Metal–oxalate complex Citric Metal–citrate complex acid (overall charge –2) acid (overall charge –1)

Figure 5.3 (A) Structure of oxalic acid and a metal–oxalate complex. (B) Structure of citric acid and a metal–citrate complex. ITGC05 18/7/06 18:14 Page 198

198 CHAPTER 5

and subsequent mobilization of toxic metal con- to the mineral’s surface, followed by structural taminants away from soils or waste disposal sites. re-arrangement and dewatering, and eventu- For instance, uranium forms a complex of two ally detachment of a molecule of Fe(III)- 2+ uranyl ions (UO2 ) and two citrate molecules hydroxamate (Holmén and Casey, 1996). Even involving four carboxyl and two hydroxyl groups. when a trihydroxamate siderophore, such as This complex is not easily degraded by bacteria, deferriferrioxamine, is added to goethite, only one and consequently the radionuclides could enter Fe(III) center is coordinated at a time (Cocozza into public water supplies if left unattenuated et al., 2002). Siderophores can similarly dis- (Francis et al., 1992). solve hematite at rates comparable to oxalic and ascorbic acids, or to dissolution induced by pro- (d) Production of siderophores ton adsorption (Hersman et al., 1995). In fact, siderophores likely work in concert with protons Siderophores are another example of multident- and other organic ligands to promote ferric oxy- ate organic ligands that form strong complexes hydroxide mineral dissolution. with metal cations. They are by definition Fe(III) There have also been a number of studies specific, and show higher formation constants on Fe-silicate dissolution promoted by sidero- = 25 50 (log Kf 10 –10 ) than low molecular weight phores or their commercial analogs. Common = 8 organic acids, such as oxalic acid (log Kf 10 ). granitic soil minerals, such as hornblende They are also found in reasonably high abund- (a predominantly Fe(II)-containing amphibole ance, averaging around 10 −6 mol L−1 in soil pore with some substituted Fe(III)), are rapidly dis- water (Hersman, 2000). Taking into account a solved in the presence of catecholate-producing 1:1 binding of siderophore to Fe, and assuming bacteria of the genus Streptomyces. Experiments that each siderophore is only used once (although have shown that within just a matter of days in actuality many are re-used), siderophores alone after inoculation, there was a fivefold increase could remove up to 10 −6 mol L−1 of Fe from solu- in Fe release compared to abiological controls tion. This chelating ability, in turn, will invari- (Fig. 5.4), and a doubling of cell mass in ably affect the dissolution of Fe(III)-bearing hornblende-containing cultures relative to con- oxyhydroxide and silicate minerals. trol cultures with Streptomyces only (Liermann During dissolution of ferric oxyhydroxides, for et al., 2000). Moreover, the bacteria penetrated example, the coordination of the Fe(III) in the so deeply into pits and cracks that neither crystal lattice is altered, such that it exchanges chemical treatment nor extreme heating could its O2− or OH− ligands for water or an organic fully remove them from the mineral’s surface ligand. In proton-promoted dissolution, H+ is (Fig. 5.5). Other catecholate siderophore- adsorbed to the metal surface causing polariza- producing microorganisms, including bacteria of tion of the neighboring >Fe-OH or >Fe-O the genus Arthrobacter, similarly enhanced dis- groups. In either case, this leads to a weakening solution rates (Kalinowski et al., 2000b). In both of the Fe(III)-anion bond and the subsequent sets of experiments, adding more siderophores detachment of Fe3+ into solution (Stumm and only temporarily increased dissolution rates. At Sulzberger, 1992). some stage the hornblende surfaces became Although siderophores are generally better Fe-depleted, and dissolution rates declined non- able to chelate dissolved Fe(III) species because linearly, i.e., in a manner similar to the sorption they can form a complete five-member ring, the isotherms from Chapter 3. It is also interesting to hydroxamate groups of a siderophore can also note that when the commercially available hydrox- bind to Fe atoms on mineral surfaces. In the case amate siderophore, desferrioxamine mesylate of goethite, the dissolution process begins with (DFAM), was used, comparable rates of dissolu- the adsorption of a single hydroxamate group tion occurred, suggesting that it was siderophore ITGC05 18/7/06 18:14 Page 199

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Medium + hornblende + DFAM

1.5 ) –5 Medium + hornblende + Streptomyces sp. × 10 1

1.0

Figure 5.4 Comparison of Fe released during hornblende 0.5

dissolution experiments as a Concentration of Fe (mol L function of time between buffered solutions, with and without the Medium + hornblende soil isolate Streptomyces sp., and with a commercially available hydroxamate siderophore, Medium + Streptomyces sp. desferrioxamine mesylate (DFAM). 0 (Modified from Liermann et al., 0 24681012 14 16 18 20 22 2000.) Time (days)

production, irrespective of type, that was driving mineral dissolution. There is a direct relationship between micro- bial growth, iron availability, and siderophore/ chelate production, as first recognized by Page

and Huyer (1984). Using a common N2-fixing bacterium, Azotobacter vinelandii, it was demon- strated that the cells generated different types and amounts of siderophores and organic 50 µm chelates depending on the iron mineral present: (i) the bacterium solubilizes marcasite (FeS2) by producing dihydroxybenzoic acid (DHBA); (ii) solubilization of vivianite and olivine Figure 5.5 Differential interference contrast microscopy image of dendritic colonies of ((Mg,Fe)2SiO4) occurred due to the production Streptomyces sp. on the surface of hornblende. of the siderophore azotochelin, plus DHBA; The arrow points to one of the residual colonies (iii) hematite, goethite, siderite, and pyrite still adherent on the mineral after vigorous induced production of azotochelin and a second cleaning with acetone and the enzyme lysozyme. siderophore, azotobactin, plus DHBA; and (iv) (Reprinted from Liermann et al., 2000 with ilmenite (FeTiO3) and Fe-rich illite caused excess permission from Elsevier.) production of azotochelin and azotobactin, plus ITGC05 18/7/06 18:14 Page 200

200 CHAPTER 5

DHBA. The sequential production of DHBA and What governs the type of secondary mineral two siderophores would seem to indicate that as formed is: (i) the composition of the primary the availability of Fe decreased (due to increasing mineral phase, i.e., felsic versus mafic; (ii) the insolubility of the Fe minerals), the microorgan- concentration of dissolved ions at the interface isms respond by producing more siderophores. between the leached layer and the intact mineral What is clear from that study, and others like surface, and the extent to which they are it, is that different siderophores are required to removed from the weathering zone; and (iii) the sequester Fe from different minerals, and that kinetics of the weathering reaction, which can changing the iron mineralogy can elicit a specific be affected by the temperature and the amount of response from the same microorganism. water through-flow. In the latter case, notice how Although produced in response to Fe stress, the products in reaction (5.5) are more degraded siderophores can also inadvertently complex a as a consequence of more water on the reactant number of other trivalent metals (e.g., Cr3+) side of the reaction. that have a similar ionic potential to Fe3+ (Birch and Bachofen, 1990). Despite forming lower (a) Felsic mineral dissolution stability complexes, divalent metals can also be sequestered by siderophores. In this case, ionic The mechanisms and rates by which feldspar potential is not an issue because only two-thirds minerals dissolve have received more attention of the ligands (e.g., hydroxamate groups) are than any other minerals. This is because they con- utilized. Yet, when a trivalent metal is available, stitute some 70–80% of the labile minerals in the all three hydroxamate groups are used, and the upper continental crust, so their dissolution has siderophore complex must not only be able to important bearing on freshwater composition wrap around the metal cation, but its ligands and secondary mineral formation. Experimental must also be properly arranged (Hernlem et al., studies consistently show that the rate of feldspar 1996). Given that many different metals can be dissolution is low, and essentially pH-independent chelated, siderophores likely play an important in the range of 5–8, due to their extensively role in accelerating the dissolution of a number cross-linked structure (Brady and Walther, 1989). of minerals in the environment. However, dissolution rates increase as the acidity increases, and below pH 5, feldspar dissolves by a n 5.1.3 Silicate weathering factor of aH+ , where “n” is the fractional depend- ence of mineral dissolution on proton activity. Approximately 30% of all minerals are silicates As discussed above, protons have a tendency and it is estimated that 90% of the Earth’s crust to adsorb onto mineral surfaces, where they is made up of silicate-based material. During induce rearrangement of charge in the silicate weathering, silicates typically undergo incon- lattice, and concomitantly the hydrolysis of gruent dissolution, in which most of the easily surface Al–O–Si bonds. This has the combined exchangable (base) cations, such as Ca2+, Mg2+, effect of releasing charge-balancing cations and K+, and Na+, and variable amounts of aluminum creating a leached zone immediately overlying and silica, are leached out of the crystal lattice, the intact crystal (e.g., Blum and Lasaga, 1988). leaving behind a residual clay phase (e.g., reac- The protons then temporarily substitute for the tion (5.4) or metal oxide (e.g., reaction (5.5)): displaced cations, maintaining a local charge balance. Dissolution rates subsequently reach + + + → 2KAlSi3O8 (K-feldspar) 2H 9H2O + a steady state when cation release is equal to Al Si O (OH) (kaolinite) + 4Si(OH) + 2K + 2 2 5 4 4 the rate by which they are exchanged for by H (5.4) (e.g., Helgeson et al., 1984). + + + → Weak inorganic acids, such as carbonic acid, 2KAlSi3O8 2H 14H2O + + + 2Al(OH)3 6Si(OH)4 2K (5.5) exert minimal effect on silicate dissolution rates ITGC05 18/7/06 18:14 Page 201

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because they do not deprotonate completely under pH changes (e.g., Sand and Bock, 1991). When circumneutral pH values, and hence generate the concentrations of those acids become suffici- insufficient protons for chemical attack; they ent, they may even cause congruent dissolution do, however, afford enough acidity to dissolve of the primary mineral phase (reaction (5.6)). In carbonates (see Box 5.1 and section 5.1.4). Even the reaction below, the dissolution of K-feldspar in soils with high aerobic respiration rates, the converts a strong acid (sulfuric) into a weak carbonic acid generated seldom decreases the acid (silicic), that can move through a soil or pH to values below 4.5. On the other hand, sediment in the undissociated form. production of sulfuric and nitric acids (from oxidation of reduced sulfur and nitrogen com- + + → KAlSi3O8 2H2SO4 4H2O 3+ + + + + 2− pounds, respectively) causes severe but localized Al 3Si(OH)4 K 2SO4 (5.6)

Box 5.1 Inorganic carbon speciation

The combination of CO2 with water forms car- type of hydrolysis reaction subsequently trans-

bonic acid (H2CO3). Despite being a relatively forms the carbonic acid to bicarbonate, which is weak acid (it does not readily give up all its the dominant form of soluble inorganic carbon at hydrogen ions when dissolved in water), it can pH values between 6.5 and 10.3: still accelerate the dissolution of some soluble + → 2+ + − mineral phases, such as calcite or aragonite. This CaCO3 H2CO3 Ca 2HCO3

100 − − H2CO3 HCO3 2 CO3 80

60

40 % of total carbon

20

0 4 5 6 7 8 9 10 11 12 pH

pH-dependent speciation of inorganic carbon in solution.

The ability of carbonic acid to cause carbonate 10−2 atm, characteristic of soil pore waters where mineral weathering can be exemplified with two biological activity greatly enhances the produc-

examples. At atmospheric pCO2 levels (approxim- tion of CO2, the pH at calcium carbonate satura- ately 3 × 10 −4 atm), and at saturation with respect tion decreases to 7.0, and the concentrations 2+ − −2.65 −1 to calcium carbonate, the equilibrium pH is 8.3, of Ca and HCO3 increase to 10 mol L 2+ − −1 −2.36 −1 −1 and the concentrations of Ca and HCO3 are (90 mg L ) and 10 mol L (266 mg L ), 10 −3.30 mol L−1 (20 mg L−1) and 10 −3.00 mol L−1 respectively. −1 × (60 mg L ), respectively. At pCO2 levels of 3 continued ITGC05 18/7/06 18:14 Page 202

202 CHAPTER 5

Box 5.1 continued

What this simple example highlights is that an

increase in the partial pressure of CO2 increases

) 100 the concentration of carbonic acid in solution, –1 pH causing more calcium carbonate to dissolve, 80 8 and thus the concentrations of dissolved Ca2+ and − HCO3 to increase. This pattern continues as long

60 pH as CO2 in water can be replenished by exchange with the atmosphere. Conversely, a decrease

40 7 in CO2 partial pressure (due to photosynthetic concentration (mg L

2 + activity or degassing) or loss of water (due to Ca 20 Ca2+ evaporation) causes the solution to become supersaturated, resulting in the precipitation of 0 6 calcium carbonate until equilibrium is restored. 3 × 10–4 3 × 10–3 3 × 10–2 The latter process is commonly manifest in the precipitation of carbonate cements in arid Partial pressure of CO2 (atm) soils (e.g., caliche) or the formation of calcite Relationship between pH and the concentration speleothems that line limestone caverns, such as 2+ of Ca over various CO2 partial pressures in a stalagmites and stalactites. calcium carbonate buffered solution.

Dissolution of feldspar by organic acids is the size and shape of the bacteria colonizing much more effective. Of importance here are the the mineral surface (e.g., Fig. 5.6). To some citric and oxalic acid-producing strains of fungi extent the preferential orientation of etch pits that have been described in nature as effec- along cleavage planes suggests that dissolution tively degrading feldspar minerals into various was crystallographically controlled, with the secondary mineral products (e.g., Jones et al., microorganisms taking advantage of structural 1981). Experimental studies have further demon- weak points (Hiebert and Bennett, 1992). Once strated that these organic acids can increase rates attached, those microorganisms then create a of feldspar dissolution by orders of magnitude, nanoscale reaction zone where organic acids relative to solutions containing inorganic acids and metabolites are concentrated on the mineral of the same acidity (e.g., Welch and Ullman, surface at discrete sites of high reactivity. Micro- 1996). The maximum rate of dissolution occurs bial colonization, and the extent of etching,

near the pH of the organic acid pKa, when both also depends on mineral composition. Using in protons and organic anions are made available to situ microcosms, where mineral surfaces were react with the mineral surface. Organic acids also directly exposed to indigenous bacteria within influence the composition of the residual silicate an aquifer, Bennett et al. (1996) showed that phase because they preferentially solubilize Al-O after several months, the surfaces of microcline bonds (Al sites are more prone to organic ligand (a K-rich feldspar) were much more deeply attack and protonation than Si sites), while in weathered than those of albite (a Na-rich feld- their absence, secondary minerals remain enriched spar). This pattern of dissolution was attributed in Al (Stillings et al., 1996). to the nutritional requirements of the coloniz- Physical evidence for microbial weathering ing bacteria because they were likely potassium comes from surface etch marks that approximate limited (K was very low in the groundwaters), ITGC05 18/7/06 18:14 Page 203

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environments that indicates preferential desilica- tion relative to aluminum or iron, i.e., in tropical soils. The increased mobility of silica is likely brought about by dissolved organic compounds that form soluble silica chelates. This causes a Bacteria lowering of the silicic acid concentration and a concomitant acceleration of quartz dissolu- tion, as evidenced by quartz grains covered in crystallographically oriented etch pits and solu- tion channels (Bennett and Siegel, 1987). Also, at alkaline pH, citrate forms a bidentate com- 5 µm plex with quartz, involving two anionic oxygen ligands interacting with two hydroxyl protons adsorbed onto the quartz surface (Bennett et al., Figure 5.6 SEM image of the surface of a 1988). The interaction might initially take the potassium feldspar crystal collected from a peat form of a weak electron donor–acceptor com- soil. Note the grooves where etch pits have plex, but then the partial electron charge is coalesced. Several different types of bacteria were transferred from the organic anion to the silica observed colonizing the mineral surface (arrows). molecule, increasing the electron density of (Courtesy of Martin Lee and Ian Parsons.) the terminal Si–O bond and invariably making them more susceptible to hydrolysis. The com- and thus were attacking the K-rich feldspars bined effect of reducing soluble silica levels and preferentially. stripping silica from the quartz surface leads to No discussion on felsic silicate dissolution increased quartz dissolution. would be complete without a brief mention of quartz. It is the most stable solid phase of silica, (b) Mafic mineral dissolution with the highest proportion of unreactive silicate groups. Unlike most other silicate minerals, The mafic minerals make up a smaller proportion quartz dissolution is unaffected by acidity except of the continental crust, and their weathering in- at extremely low pH (<2), where high proton volves both dissolution and oxidation-reduction concentrations disrupt silica bonding, or at pH reactions. Olivine, pyroxene, amphibole, and values higher than 8, when deprotonation of sur- biotite are enriched in magnesium and ferrous face Si–O–H bonds occurs (Brady and Walther, iron, and they weather rapidly in oxic environ- 2+ 1990). As such, it weathers extremely slowly ments as the Fe is initially released through under normal surface conditions, preserving the congruent dissolution (e.g., reaction (5.8)), and Si atom in tetrahedral coordination in solution then oxidized and hydrolyzed to ferric hydroxide (reaction (5.7)). This insolubility yields con- (reaction (5.9)): centrations in most surface waters of ~6 mg L−1 + + → 2+ + at 25°C, although most freshwaters have con- Fe2SiO4 (olivine) 4H 2Fe Si(OH)4 (5.8) siderably higher dissolved silica concentrations 2+ + + → + + that reflect feldspar hydrolysis. 2Fe 0.5O2 5H2O 2Fe(OH)3 4H (5.9)

+ → Unlike feldspar, these minerals display little SiO2 2H2O Si(OH)4 (5.7) resistance to weathering because of the relative Despite quartz’s resistance to dissolution, there is lack of Si–O–Si cross-linking. Instead, the ample evidence from both modern and ancient minerals consist of isolated silicate tetrahedra ITGC05 18/7/06 18:14 Page 204

204 CHAPTER 5

attached by cation bridges. Therefore, in an acid expected at pH values where the ferric iron solution, those silicate groups convert intact to released reprecipitates on the weathering surface. silicic acid as the >Fe–O bonds are protonated; Clearly, the extent of dissolution is governed by no hydrolysis reaction is needed. Any leached a balance between the net release of products layer of olivine, for instance, will be thin as there to solution versus the evolution of the mineral are no bridging oxide bonds to maintain integrity surface morphology and reactivity, both of which once the metal cations are removed (Wogelius can be site specific. and Walther, 1991). Basaltic glass is one of the most abundant and In Chapter 1 it was mentioned that bacteria reactive phases in the oceanic crust. Its alteration reside deep within flood basalts, where they begins on a very localized scale with an initial eke out a chemolithoautotrophic living from loss of cations yielding a leached zone several

available sources of H2, some organically sourced micrometers thick, followed by a variable degree and some possibly via water–rock interactions. of dissolution of the silica-rich residues and re- Direct evidence of microbial involvement in polymerization to form a porous silica network that basalt weathering, however, comes from studies eliminates nonbridging oxygen atoms (Thorseth of terrestrial lava flows and the pillow basalts et al., 1992). Bacteria are believed to play an associated with seafloor volcanism. On land, important role in the initiation of dissolution weathering is facilitated largely by lichen growth, reactions because in experiments they rapidly in which the primary rock-forming minerals formed micrometer-thick biofilms on fresh glass undergo oxidation and dissolution as a con- surfaces (Staudigel et al., 1995). After only weeks

sequence of respiratory CO2 production and to months of colonization, the glass displayed pre- the excretion of organic and lichen acids. ferential dissolution at points along fractures, that This can lead to variable stages of etching and subsequently developed into pronounced etch mineral fragmentation (e.g., Jones et al., 1980). marks in a years time (Fig. 5.7). It is interesting Coincident with oxidative dissolution is cation to note that different bacteria were dominant on release, which, in turn, can lead to authigenic mineralization, generally consisting of fine- grained calcium oxalate crystals, along with the formation of ferric hydroxide and various Fe(II)/Fe(III)–silicate assemblages. Microorganisms are also instrumental in alter- ing the kinetics of dissolution, but with variable results. For instance, chemical weathering rates of recent Hawaiian lava flows colonized by lichens have been reported to be at least 100 times that of bare rock (Jackson and Keller, 1970). At low pH, calculations even suggest that olivine dis- 2 µm solution could support significant populations of Fe(II)-oxidizing bacteria (Santelli et al., 2001). Yet, experimental studies have also shown Figure 5.7 SEM image of a polished glass surface a decrease in long-term dissolution rates of left in continuously flowing seawater for 410 days. Fe-rich olivine (fayalite) that is attributed to the Removal of the overlying biofilm reveals significant precipitation of an unreactive alteration rind corrosion, as evident from the etch grooves on the mineral surface, which eventually limits (arrows), some of which exceed 10 µm in length µ fluid exchange with the bulk weathering solu- and 0.5 m in width. (Reprinted from Staudigel tion (Welch and Banfield, 2002). This is to be et al., 1995 with permission from Elsevier.) ITGC05 18/7/06 18:14 Page 205

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the glass surface at different stages of the experi- being that carbonate dissolution directly affects 2+ − ments, likely a result of the changing pH and redox fluxes of Ca and HCO3 to the hydrosphere. conditions during dissolution. Consequently, It has been estimated that over 90% of all large some of the late-stage microorganisms lived within world rivers have chemical compositions dom- those pits, where they accumulated a range of inated by limestone and dolomite dissolution elements derived from the glass (e.g., Fe, Al, (Meybeck, 1979). As such, oceans are chemic- and Si). Often the concentration of elements ally buffered against extreme pH fluctuations, in was sufficiently high to lead to the secondary that the addition of excess acid or base has little formation of fine-grained, geochemically hetero- impact on seawater pH: geneous material, including palagonite, silicate + + → 2+ + − clays, and (Fe, Al)-hydroxides. Other experi- H CaCO3 Ca HCO3 (5.12) ments showed glass dissolution rates on the µ 2+ + − + − → + order of 1 m annually (Thorseth et al., 1995). Ca HCO3 OH CaCO3 H2O (5.13) If similar rates can be applied throughout the seafloor, then glass dissolution could signi- Despite reactions that show calcite being dis- + ficantly contribute to the chemical budget of solved by H2CO3, it is usually H that serves as the ocean, particularly for those elements that the weathering agent (reaction (5.12)) because

are selectively leached (e.g., Mg, Na, Ca). It the rate of hydration of dissolved CO2 to form + has also been hypothesized that since mafic H2CO3 is too slow to be as effective as H (Berner minerals contain the bulk of transition metals and Morse, 1974). In fact, rates of calcite dis- in the upper crust, some of which are bioessen- solution are transport controlled below pH 4. tial nutrients that have low concentrations in Furthermore, unlike feldspar dissolution, where seawater (e.g., Fe), their increased availability the weathering products might accumulate on through biological weathering may actually have the mineral surface causing the rate of dissolu- a significant impact on the marine food chain tion to decrease, the carbonate ions that detach (Staudigel et al., 1998). from the crystal surface are chemically altered to bicarbonate. Thus, in most cases a state of 5.1.4 Carbonate weathering saturation cannot be achieved at the mineral– solution interface, so calcium carbonate con- Carbonate minerals comprise about 20% of tinues to dissolve at low pH. A similar argument Phanerozoic sedimentary rocks. They are amongst can be made at circumneutral pH, but the avail- the most reactive minerals found in abundance ability of free H+ is diminished, leading instead on the Earth’s surface, dissolving congruently to surface-controlled dissolution rates. Kinks at rates that are orders of magnitude faster than are favored sites for carbonate dissolution, and it that of silicate minerals and at much higher pH has been observed that as a kink ion is removed, values (Morse, 1983). Two important carbonate a new kink is formed adjacent to the old one. weathering reactions are the dissolution of calcite Hence, dissolution can be envisaged as the and dolomite, respectively: formation and migration of kinks and the con- sequent retreat of steps until there is widespread + → 2+ + − CaCO3 H2CO3 Ca 2HCO3 (5.10) dissolution of the entire surface (e.g., Lasaga and Luttge, 2001). + → CaMg(CO3)2 2H2CO3 In nature, prolonged dissolution of limestone 2+ + 2+ + − Ca Mg 4HCO3 (5.11) and dolomite is evidenced by the pockmarked surfaces characteristic of karst topography. The These two reactions have a number of environ- surficial features include extensive pavement net- mental implications, one of the most important works with fissures and solution-widened joints, ITGC05 18/7/06 18:14 Page 206

206 CHAPTER 5

type of environment, with higher rates in the hot and humid tropics versus those in the cold and relatively arid tundra. When fresh organic matter is added to soil, three general sequences of degradation take place (Brady, 2002):

1 Initially, the bulk of the material becomes degraded via the release of hydrolytic enzymes from aerobic bacteria and fungi. As long as there is plenty of 30 m fresh organic material available, the number of soil microorganisms remain high (up to 109 cells g−1); often the microbial biomass accounts for one-third of the organic fraction in soil (Fenchel et al., 2000). Figure 5.8 Spectacular karst topography Through fermentation and respiration, the easily showing sinkholes in Permian limestone along the degradable polysaccharides are converted into Little Colorado River. Scale is approximate. CO2, some of which volatilizes and ultimately (Courtesy of Louis Maher.) escapes into the atmosphere, while the remainder reacts with water to produce carbonic acid. As pinnacles, ridges, canyons, lakes, and sinkholes discussed above, the carbonic acid then contributes (Fig. 5.8). Subsurface features include caves to chemical dissolution of some soil minerals. If and their speleothem deposits. Despite being the quantity of easily degradable organic matter commonly attributed to abiological dissolution, present is high, its decomposition via aerobic respiration can also lead to a temporary increase in numerous studies have described the presence the “biological oxygen demand” (BOD), a term that of biofilms covering limestone and dolomite, as refers to the quantity of oxygen required to oxidize well as epilithic bacteria and fungi that bore into, organic matter. and dissolve the underlying carbonates (e.g., Proteins and polynucleotides are simultaneously Ferris and Lowson, 1997). The extent of karst degraded into their constituent amino acids and nucleic acids, respectively. In turn, these are further development, in turn, is a function of mineral + broken down into simple inorganic ions such as NH4 , hardness, with calcite being more prone to dis- − 2− − NO3 , SO4 , and H2PO4 . Organic compounds also solution and endolithic boring than dolomite + + + release various cations, such as Ca2 , Mg2 , and K . (e.g., Jones, 1989). The process that produces these inorganic forms is called mineralization, not to be confused with the 5.1.5 Soil formation formation of minerals. Of these components, nitrate and sulfate are commonly lost due to leaching; phos- (a) Decomposition of organic matter phate is retained as a calcium fluorapatite phase or other insoluble secondary minerals, while the cations Mineral dissolution and organic matter accumu- enter into the soil solution, where they are either taken up by roots, become adsorbed onto negatively lation eventually conspire to form the first layers charged colloids, clays and microbial surfaces, or of soil. The organic fraction is a rather transitory they are leached from the system, particularly if the constituent, lasting from only a few hours to soil minerals are protonated (i.e., an acid soil). several thousand years. This variation occurs, in part, due to the differences in decomposition rates 2 As soon as the easily degraded carbon is exhausted, amongst different compounds; starches, proteins, cell numbers decline. Cellulose is the most common and polynucleotides degrade very quickly; cellu- type of polysaccharide in land plants, yet it is moder- ately difficult to degrade. Thus, some cellulose tends lose and chitin have intermediate rates; while to remain in the residual organic fraction even after lignins degrade very slowly (Table 5.1). Organic prolonged microbial attack. Chitin behaves in a decomposition rates also vary according to the similar manner. ITGC05 18/7/06 18:14 Page 207

MICROBIAL WEATHERING 207

Table 5.1 Relative degradability of organic compounds. (Data compiled by De Leeuw and Largeau, 1993.)

Organic compounds Occurrence Preservation potential

Starch Vascular plants; some algae; bacteria − Fructans Vascular plants; algae; bacteria − DNA/RNA All organisms − Proteins All organisms − Xylans Vascular plants; some algae −/+ Pectins Vascular plants −/+ Mannans Vascular plants; fungi; algae −/+ Galactans Vascular plants; algae −/+ Alginic acids Brown algae −/+ Cellulose Vascular plants; some fungi + Chitin Arthropods; crustaceans; fungi; algae + Peptidoglycan Bacteria + Teichoic acids Gram-positive bacteria + Sheaths Some bacteria + Cutins, suberins Vascular plants +/++ LPS Gram-negative bacteria ++ Tannins Vascular plants; algae +++/++++ Lignins Vascular plants ++++ Cutans Vascular plants ++++

The preservation potential ranges from easily degradable (−), intermediate (−/+ , +, ++), to refractory (+++, ++++).

3 After the moderately degradable organic compon- soils, swamps, etc.) can also lead to very inefficient ents are reduced, only the complex and refractory mineralization rates: in oxic sediment the decay materials (e.g., lignin, resin, and waxes) remain rate is 2–4% per year versus 0.1–0.000001% in relatively intact. Unlike other polymers, lignins the anoxic zone (Swift et al., 1979). have no regular structure to serve as a target for hydrolytic enzymes, and its degradation requires The poorly degradable residues, collectively the collective efforts of a variety of nonspecific known as humic substances, are characterized enzymes (Kirk and Farrell, 1987). Most of those by their black to brown color and their very

enzymes require O2, and, in its absence, anaerobic fine-grained size. On the basis of resistance to degradation rates of cellulose and lignins are only degradation and solubility, humic substances about 1–30% of aerobic respiration rates (Benner have been classified into three groups (Schnitzer et al., 1984). As a result, these compounds can and Khan, 1972). Fulvic acids are lowest in persist in soils and sediment for many thousands of years, particularly if they are associated with clay molecular weight, lightest in color, and soluble minerals that protect them from microbial decay. in both acid and alkali. Humic acids are medium Rapid burial into anoxic layers (water-logged in molecular weight and color, soluble in alkali, ITGC05 18/7/06 18:14 Page 208

208 CHAPTER 5

but insoluble in acid. Humin is highest in mole- ponents. Humics also contain an abundance cular weight, darkest in color, insoluble in both of reactive functional groups, such as carboxyls, alkali and acid, and most resistant to micro- that dissociate at normal pH ranges in soils, bial attack. In environments with high rates sediment, and natural waters (Perdue, 1978). As of cellulose and lignin burial, and where the a result of deprotonation, these anionic ligands environment quickly becomes anoxic, refract- can efficiently sorb and chelate a variety of metal ory organic materials can accumulate to great cations from solution. thicknesses, resulting initially in peat formation, and, if subjected to increased temperatures and (b) Soil profile development pressures that tend to concentrate carbon, they may ultimately be converted into coal through Soil is the ultimate product of mineral weather- the process of coalification. ing, but even as it accumulates, microorgan- Similar to the organic acids discussed previ- isms continue to shape its mineralogical and ously, humics play a vital role in metal cycling. geochemical characteristics into distinct soil They contain at their core abundant polycyclic horizons (Fig. 5.9). The top of the soil, known as aromatic rings connected by aliphatic chains the O-horizon, consists of an accumulation of of different length to form three-dimensional, organic litter that is in various states of decay, flexible biopolymers that possess voids capable from just recently deposited and intact to highly of trapping other organic and inorganic com- degraded with refractory humins. As rainwater

O-horizon (Topsoil richest in organic matter)

E-horizon + 2+ – Downward flow Dissolved/ Zone of leaching CaCO3 + H Ca + HCO3 2+ – (with O2/low pH) chelated (most extensive Fe2SiO4 + 4H2CO3 2Fe + 4HCO3 + Si(OH)4 2+ 3+ – metals in humid climates) 2Fe + 0.5O2 + H2O 2Fe + 2OH

B-horizon 3+ + Acidity Chelates Zone of accumulation 2Fe + 6H2O 2Fe(OH)3 + 6H 3+ + neutralized oxidized (contains soluble AI + 3H2O AI(OH)3 + 3H 2+ – minerals like calcite Ca + 2HCO3 CaCO3 + H2O + CO2 in arid climates)

C-horizon Coarsely broken up bedrock

Bedrock

Figure 5.9 An idealized soil profile showing the various horizons. Some of the important dissolution and precipitation reactions are given to highlight the translocation of Fe, Al, and Ca in the profile. ITGC05 18/7/06 18:14 Page 209

MICROBIAL WEATHERING 209

percolates through this organic-rich layer, in- to soil forming processes, climate, topography organic and biological processes generate acids of of the site, and the indigenous vegetation and varying strengths that promote intense leaching microbiota (see Brady, 2002). The type of parent and removal of the metal cations from the under- material can range from bedrock to detritus lying primary minerals. Because the infiltrating transported to the site via rivers (alluvium), ice

waters also contain dissolved O2, some of those (till), and wind (loess). The nature of the parent metals are oxidized (e.g., Fe(II) to Fe(III)), and material affects such soil characteristics as com- depending on pore water pH, may or may not position, mineralogy, texture, and weathering remain in solution. Organic chelates produced rates. For young soils (just hundreds of years old), in the O-horizon further aid in the solubilization horizons will clearly be more distinct in soils and transport of metals from the uppermost soil formed on granite or basalts than they would if the horizon downwards with the infiltrating pore underlying lithology was sandstone. However, waters. Collectively, these reactions lead to an over longer periods of time, the different soils upper mineral horizon (E-horizon) that becomes converge to a soil type determined by climate, progressively enriched in resistant minerals, such and once the soil is fully developed, it should be as quartz and some metal oxides. stable indefinitely. The top two horizons tend to form the bulk of Climate is important in terms of precipita- the rhizosphere, the depth to which plant roots tion and temperature: both higher rainfall and extend. This zone has intense microbiological temperatures promote greater rates of organic activity adjacent to the plant roots (heterotrophic productivity, more organic decay, and ultimately

respiration, N2 fixation, etc.), and compared to increased organic and inorganic acid generation. the bulk soil, microbial populations here can be Increased temperature and flushing rates also as high as 5 × 109 cells g−1 of root tissue (Russell, serve to enhance the rates of chemical weather- 1977). In addition to the active microbial com- ing. Not surprisingly, thicker soil profiles tend to munities, the plant roots themselves are import- develop in tropical environments, characterized ant in promoting chemical weathering because by accumulations of kaolinite, hematite/goethite, they continuously extrude organic acids, and root and gibbsite. Organic matter accumulation, pro- hairs and their sheaths are rich in organic ligands file development, nutrient cycling, and structural that sorb metal cations. stability are also intimately tied to the type Many of the more soluble cations from the of vegetation and soil microbiota. Take, as an E-horizon end up in groundwater, but some (e.g., example, soils formed under grassland versus Al3+ and Fe3+) are re-precipitated deeper in the forests. The organic matter content of grassland soil, in what is known as the B-horizon, where soils is generally higher than that of forested either the pH is sufficiently buffered to facilitate areas such that the former are darker in color and mineral hydrolysis or the organic chelates (e.g., have higher moisture-holding capacity, while oxalate, citrate) are oxidized by aerobic micro- the acidity associated with coniferous trees will organisms. In arid soils, calcium carbonate may influence soil pore water composition and limits

form as both the loss of CO2 and H2O causes the many types of secondary minerals from forming. saturation state to increase. This movement of metal cations down the soil profile, and their 5.1.6 Weathering and global climate re-precipitation at lower depths, is one of the main causes for the subsequent differentiation A major feedback mechanism controlling atmo-

of soils into specific horizons. spheric pCO2 levels is the weathering of some The extent of soil profile development depends silicates and the subsequent precipitation of Ca- on a number of variables, including the type of Mg carbonates (Kump et al., 2000). During this parent material at the time that they were subject process carbonic acid reacts with minerals, such ITGC05 18/7/06 18:14 Page 210

210 CHAPTER 5

as plagioclase and amphiboles, generating soluble Organisms certainly play an integral role in the 2+ − Ca and HCO3 , and residual clay phases from the carbon cycle. Although carbonic acid arises from incongruent dissolution reactions. The ions are the oxidation of soil organic matter, the ultimate

eventually transported to the oceans where they source of this carbon is atmospheric CO2 fixed are precipitated either biologically, as calcite or via photosynthesis. Importantly, as Berner et al. aragonite shells, or abiologically, as a micritic mud (1983) suggested in their seminal paper more

when a state of supersaturation is achieved: than 20 years ago, the real impact of high CO2 is that it increases Earth’s surface temperatures and + + → CO2 2H2O CaAl2Si2O8 (anorthite) net precipitation. This, in turn, leads to higher + Al2Si2O5(OH)4 CaCO3 (5.14) terrestrial biomass production, increased soil bio- logical activity, more organic decay/acid genera-

Global changes in atmospheric CO2 levels are thus tion, faster chemical weathering rates, and greater determined by the magnitude of the imbalance solute loads carried by rivers. The increased

between the rate of addition of CO2 to the atmo- supply of nutrients to the oceans promotes greater sphere through tectonically induced metamorphic- primary plankton productivity that will further

magmatic decarbonation of limestone/dolomite reduce atmospheric CO2 through photosynthesis and sedimentary organic carbon versus the rate and the precipitation of calcium carbonate shells. of removal by weathering and the incorporation Of course this ends up having a negative feedback

of inorganic carbon into marine sediments and because atmospheric CO2 drawdown inevitably biota (Fig. 5.10). Increased exposure of landmass cools global temperatures, such that biological to surface conditions (through uplift or lowering activity and weathering rates diminish, thereby of sealevel), or higher levels of acid generation, returning the carbonate–silicate cycle to steady should amplify chemical weathering rates and state (Walker et al., 1981). Similarly, as nutrient

swing the balance in favor of atmospheric CO2 fluxes to the oceans decline, plankton produc- drawdown, and potentially glaciation. tivity decreases, and less CO2 is fixed from the

CO2(+)

Shallow water H2CO3 Photosynthesis (–) calcification (–) River flow – 2+ Magma Silica (HCO3 /Ca ) weathering degassing Volcanism/ metamorphism CO (+) Soil Groundwater Plankton/shells (–) 2 discharge (high pCO2) – (H2CO3 HCO3 ) Subducted CO (+) Uplift Organic (–) 2 carbonates/ respiration organic carbon inorganic (–) carbon burial

Figure 5.10 Simplified carbon cycle, showing the addition (+) and removal (−) processes in terms of

atmospheric CO2. ITGC05 18/7/06 18:14 Page 211

MICROBIAL WEATHERING 211

atmosphere. Such biological effects on weather- and their transformation of atmospheric CO2 to ing are a prime example of the so-called Gaia the production of inorganic and organic acids.

hypothesis, in that life helps regulate the Earth’s Their presence thus led to CO2 drawdown, to climate to meet its own needs (Lovelock and the extent that surface temperatures could have Whitfield, 1982; Lovelock, 2000). dropped enough to have facilitated the evolution Implicit in the discussion above is the direct of mesophilic microorganisms, and ultimately link between the hydrosphere–atmosphere– diversification of the terrestrial microbiota. What biosphere subcycle (HAB) and the sediment- is not obvious, however, is how such microorgan- ary subcycle, that includes both the reservoirs of isms could have been prolific given the high UV carbonate minerals and organic carbon. Marine influxes in the absence of an ozone layer. Perhaps, carbonate precipitation is presently dominated they relied instead on a shallow sub-surface mode by calcareous plankton, and to a lesser extent of existence (i.e., as endoliths), where sufficient carbonate-secreting benthic organisms. As indi- visible light could penetrate, or they were pro- cated above, their net mineralizing capacity, tected by some other form of UV shield, such as and ultimately their net burial rate, is controlled an elemental sulfur smog or a mineral crust (see 2+ 2+ − by the riverine flux of Ca /Mg and HCO3 section 7.3.1)? Irrespective of the mechanism, released by surface weathering, and also by if sheltered, and if continually moistened in a seawater–basalt interactions. For the organic warm and wet climate, those communities would

carbon component, the fixation of CO2 into rapidly have become the primary source of soil biomass is achieved principally by photosyn- CO2. The later evolution of fungi, and their thetic organisms, and is shared almost equally key role in soil formation, was likely a critical in terms of primary productivity between the preliminary step for the eventual colonization marine and terrestrial environments (Des Marais, of land by vascular plants in the Silurian 1997). Of that amount of organic carbon pro- (Schwartzman and Volk, 1991). duced, the actual quantity buried into sediments, after respiratory processes, is usually <0.1%, some five times less than the burial rate of carbon- 5.2 Sulfide oxidation ate carbon. Despite that small percentage, the sedimentary carbon reserve is much larger than A number of metal sulfides have the propensity the carbon reserves of the HAB subcycle, and to undergo chemical oxidation when subjected to periodic imbalances in the former are potenti- surface oxidizing conditions. Of those minerals, ally large and can have significant effects on the pyrite (FeS ) is arguably the most important oxidation state of Earth’s surface environment 2 environmentally because it is an extremely (Holland, 1984). common constituent in coal seams, ore bodies, It has been proposed that microbial weather- and shales. ing also played a fundamental role in defining the initial habitability of Earth’s terrestrial envir- onment (Schwartzman and Volk, 1989). These 5.2.1 Pyrite oxidation mechanisms authors argue that various thermophiles, possibly including anoxygenic photoautotrophs and During pyrite’s exposure to oxygenated waters, as chemolithoautotrophs (e.g., methanogens), may in the cessation of a mining operation, both its have occupied much of the land surface as early reduced sulfur and iron atoms become oxidized. as the Archean. These microorganisms would Three electron acceptors are possible: molecular have been effective weathering agents relative to oxygen, hydrogen peroxide and, under acidic con- sterile conditions, primarily from their ability (as ditions, ferric iron (McKibben and Barnes, 1986). biofilms) to retain water at the mineral surface The overall process describing the initiation ITGC05 18/7/06 18:14 Page 212

212 CHAPTER 5

OO − 2S OH H H H H 2

S S S S H2O H2O − − − − S Fe S + S Fe S + Fe O2 2H2O Pyrite 1 2 3

Figure 5.11 Model, based on Goldhaber (1983), of the initial oxidation of pyrite at circumneutral pH by

reaction with O2.

of pyrite oxidation is commonly given by the Although the progressive oxidation of inter- following incongruent reaction: mediate sulfur compounds to sulfate is predicted to follow a linear pathway, instead there are a + + → + FeS2 3.75O2 3.5H2O Fe(OH)3 2H2SO4 number of variables that make S-cycling much (5.15) more complex and still incompletely understood (e.g., Xu and Schoonen, 1995). Thiosulfate is the The oxidation and hydrolysis steps shown in first sulfoxy anion that forms (reaction (5.17)). the above equation involve the loss of 1 electron It is more stable at circumneutral pH than low by ferrous iron and 14 electrons by disulfide, pH, and slowly disproportionates to elemental with the gain of 7.5 electrons by each oxygen per sulfur and sulfite in weakly acid solutions mole of pyrite. All of these redox changes cannot (reaction (5.18)). In the presence of pyrite, thio-

take place in one step; but instead there are a sulfate also oxidizes quickly (with O2) to form series of electron transfer reactions that need tetrathionate (reaction (5.19)). Tetrathionate is consideration. most stable at low pH, and it does do not appear

to be significantly oxidized by O2. By contrast, (a) Sulfur reactions sulfite is not stable except under alkaline con- ditions, and rapidly oxidizes to sulfate in the The first step in the dissolution of pyrite at presence of oxygen or any other strong oxidiz-

circumneutral pH involves attachment of O2 to ing agent (reaction (5.20)). Similarly, elemental the partially protonated sulfur ligands exposed sulfur is oxidized to sulfate at circumneutral at the mineral’s surface (Goldhaber, 1983). The pH (reaction (5.21)). Therefore, in moderately

next step requires breaking the O2 double bond acidic solutions tetrathionate and sulfate pre- − and displacement of S2OH molecules by H2O dominate as the sulfoxy anions at the expense (Fig. 5.11). As long as the fluids at the mineral of thiosulfate and sulfite. surface are circumneutral, the sulfoxy anions dif- − + → 2− + + fuse into the bulk fluid, where they are oxidized S2OH O2 S2O3 H (5.17) to sulfate (reaction (5.16)), via several sulfur − 2− → 0 + 2− 2 S2O3 S SO3 (5.18) intermediates, including thiosulfate (S2O3 ), 2− polythionates (SnO6 ), such as tetrathionate 2− + + + → 2− + 2− 2− 2S2O3 0.5O2 2H S4O6 H2O (5.19) (S4O6 ) and trithionate (S3O 6 ), and sulfite 2− (SO ): 2− + → 2− 3 SO3 0.5O2 SO4 (5.20)

− + + → 2− + + 0 + + → 2− + + S2OH 3O2 H2O 2SO4 3H (5.16) S 1.5O2 H2O SO4 2H (5.21) ITGC05 18/7/06 18:14 Page 213

MICROBIAL WEATHERING 213

In reaction (5.16) the complete oxidation of rise in pH, the hydrolysis of ferric iron to − S2OH to sulfate causes the pH to drop. Accord- form Fe(OH)3 inevitably led to more acidity. ingly, it is sometimes referred to as the initiator Because this reaction normally occurs in the reaction, because its leads to the onset of acidic presence of sulfate, the ferric hydroxide may conditions. Simultaneously, the stability of ele- convert to the more insoluble minerals, jarosite + + mental sulfur increases because of its greater (MFe3(SO4)2(OH)6), where M may be H , H3O , + + + insolubility at low pH. This results in the pre- Na , K , NH4 (reaction (5.25)), or schwert- cipitation and accumulation of micrometer-thick mannite (Fe8O8SO4(OH)6) (e.g., Lazaroff et al., agglomerates of elemental sulfur on the pyrite 1982): − surface (reaction (5.22)), replacing the S2OH + + + 2− → molecules (e.g., Sasaki et al., 1995): M 3Fe(OH)3 2SO4 + − MFe3(SO4)2(OH)6 3OH (5.25) > 2− + + + → 0 + S2 0.5O2 2H 2S H2O (5.22) Reaction (5.23) necessitates that dissolved O2 Precipitation of elemental sulfur has the poten- serves as the oxidizing agent at circumneutral pH tial to form an inert layer that might inhibit owing to the diminished availability of dissolved the diffusion of oxidants to the surface, thereby Fe(III) at pH values greater than 4.5. However, slowing further dissolution. This means that its at this pH range, solid phase or adsorbed Fe(III) rate of oxidation to sulfate (reaction (5.21)), can still serve as an effective oxidant of pyrite if relative to its formation from S(−1), can deter- it is in direct contact with the mineral surface. mine the overall dissolution rates of pyrite. In This can come about in two ways. First, in the turn, these rates are governed by the transport initial oxidation step, Fe(II) diffuses to the sur- rates (diffusion, advection) of oxidizing agents, face and becomes oxidized/hydrolyzed to ferric 3+ hydroxide. Second, dissolved Fe(II) adsorbs onto such as O2 or Fe , to the pyrite surface, the pH of the proximal solution, and the presence pyrite (which has an isoelectric point of 2.5, of S-oxidizing bacteria, as discussed below and hence is anionic at the pH values of most (Nordstrom, 1982). natural waters), where it reacts with, and gives up its electrons to dissolved O2. In either case, (b) Iron reactions the Fe(III) then rapidly accepts electrons from the pyrite. Adsorbed Fe thus acts as an electron shuttle from Fe(II) in pyrite to dissolved O At first, when the pH is still above 4.5, the Fe(II) 2 (Moses and Herman, 1991): exposed during the initial reactions spontane- ously oxidizes in air to form Fe(III) (reaction Fe(II) + pyrite → Fe(II)-pyrite + 0.25O → (5.23)). Some of that ferric iron dissolves, where red red 2 Fe(III)-pyrite + 0.5H O → Fe(II)-pyrite it is hydrolyzed and reprecipitated as ferric red 2 ox (5.26) hydroxide (reaction (5.24)). The remainder is oxidized at the grain surface without going into The geochemical reactions described above are solution. borne out in the surface textures of weathered sulfide minerals, including pyrite and pyrrhotite 2+ + + + → 3+ + Fe 0.25O2 H Fe 0.5H2O (5.23) (Fe7S8), that show several stages in paragenetic alteration sequences (e.g., Nesbitt and Muir, 1994; 3+ + → + + Fe 3H2O Fe(OH)3 3H (5.24) Pratt et al., 1994). Initially, after the loss of − S2OH , the presence of a thin, featureless ferric Note that although the initial oxidation reaction hydroxide layer (8–10 nm thick) forms on the consumed protons, and thus led to a temporary mineral surface. Over time it thickens (up to ITGC05 21/7/06 15:24 Page 214

214 CHAPTER 5

30 nm) through diffusion of Fe(II) into the surface When that happens, Fe2+ becomes stable in the − precipitate. Concomitantly, S( 1) accumulates presence of O2 and its oxidation becomes very in the subsurface layers. At some critical stage, slow (Fig. 5.12). Furthermore, unlike equation the adhesion between the ferric hydroxide and (2.53), Fe(II) oxidation under acidic condi- the S(−1) underlayer is weakened, leading to tions becomes independent of pH (Singer and spalling of the ferric hydroxide into solution. Stumm, 1970), with its kinetic reaction being Removal of Fe then exposes the reduced sulfur, expressed as: which in turn becomes sequentially oxidized and eventually released from the pyrite surface as one − d[Fe(II)] = of the sulfoxy anions. This process becomes to k[Fe(II)][O2] (5.27) some extent self-sustaining because as the acidity dt near the pyrite surface increases, the Fe(II) is −7 −1 −1 more easily leached from the surface layer prior where k = 1.0 × 10 min atm at 25°C. to oxidation, thus increasing the exposure of Ferric hydroxide also becomes considerably more the S-rich sites in the crystal lattice. Thereafter, soluble at low pH (reaction (5.24) now goes 3+ oxidation of >S(−1) to S0 becomes increasingly from right to left), and as the Fe concentration important as the latter becomes stable as a increases with greater acidity, its role becomes solid phase. much more important as the pyrite oxidizing − agent (Moses et al., 1987): The acid generated by the oxidation of S2OH 0 2− and S to SO4 , as well as Fe(III) hydrolysis, begins low, but given the right conditions, the + 3+ + → 2+ + 2− + + FeS2 14Fe 8H2O 15Fe 2SO4 16H pH of the waters can drop to values below 4.5. (5.28)

3

2 3′ 1 1 0 )

− 1 –1

–2 2

log k (day –3 3 –4 Figure 5.12 Comparisons of rate constants as a function of pH for (1) the –5 oxidation of pyrite by Fe3+, (2) the oxidation 2+ of pyrite by O2, and (3) the oxidation of Fe –6 by O2 (modified from Nordstrom, 1982). The variation in rate constants for reaction 0 1 23 54 6789 10 1 results from different proportions of total pH 3+ Fe and FeS2, as calculated by Singer + 3+ + 2+ + 2− + + 1 FeS2 14Fe 8H2O 15Fe 2SO4 16H and Stumm (1969). Reaction 3′ represents + + 2+ + 2− + + 2 FeS2 3.5O2 H2O Fe 2SO4 2H the reaction rate enhancement by 2+ + + + 3+ + Acidithiobacillus ferrooxidans. 3 Fe 0.25O2 H Fe 0.5H2O (After Lacey and Lawson, 1970.) 3′ biological oxidation of reaction 3 ITGC05 18/7/06 18:14 Page 215

MICROBIAL WEATHERING 215

Indeed, at pH values lower than 3, Fe3+ is the (a) Oxidation rate enhancement only important oxidizer of pyrite (Fig. 5.12). The increased Fe3+ availability also enhances Acidophilic Fe(II)-oxidizing bacteria can gener- the oxidation of intermediate sulfoxy anions, ate Fe3+ some five or six orders of magnitude converting them to sulfate, with the further faster relative to sterile conditions (e.g., Lacey effect of increasing acidity (e.g., Druschel et al., and Lawson, 1970). This increase makes the 2003): Fe(II) oxidation rate slightly higher than the rate of the pyrite oxidation by Fe3+. Of course, 2− + 3+ + + → S4O6 3Fe 2.75O2 4.5H2O microorganisms in the environment are always 2− + 2+ + + 4SO4 3Fe 9H (5.29) growth-limited by bioessential elements, pre- dators, or some hydrologic condition, hence, According to molecular orbital theory, reaction their true environmental oxidation rates prob- (5.28) is initiated by the bridging of Fe3+ cations ably approximate the rate of pyrite oxidation by 2− 3+ 3+ to S2 (Luther, 1987). The sulfide moiety is then Fe (Nordstrom and Southam, 1997). The Fe transformed into more oxidized species, such formed under these conditions, being soluble, is 2− as S2O3 , which may oxidize further in solution chemically reactive and can effectively scavenge to polythionates and sulfate depending on the electrons from S(−1) in pyrite, generating Fe2+ availability of further oxidizing agents. Concom- once again. It is then reoxidized to Fe3+ by the itantly, the bound Fe3+ is reduced to Fe2+, and bacteria. Because of this re-cycling process, the the bridging complex is eliminated. This binding formation of Fe3+ can be viewed as an efficient 3+ 2− of Fe to the S2 ligands (compared to O2 that electron acceptor for sustained lithotrophy, cannot bind as easily because of the arrange- with a progressive, rapidly increasing rate of ment of its outer electron shell) further explains pyrite oxidation (called the propogation cycle) why the rates of pyrite oxidation are an order of owing to biocatalysis (Singer and Stumm, 1970). magnitude faster when Fe3+ is available relative Recall from Chapter 2 that under acidic con-

to dissolved O2. The abundance and reactivity of ditions, very little energy is generated through the S(−1) groups for Fe3+ likely also explains the Fe(II) oxidation. Subsequently, these bacteria different dissolution rates displayed by various must oxidize large amounts of reduced iron in iron sulfides, such as pyrite versus arsenopyrite order to sustain themselves, and even a small (Edwards et al., 2001). number of cells can be responsible for exten- sive pyrite oxidation. Not surprisingly, estimates 5.2.2 Biological role in pyrite made in some acid mine environments suggest oxidation that the acidophilic bacteria can account for the majority of pyrite dissolution (e.g., Edwards In acid waters, pyrite can reduce Fe3+ to Fe2+ et al., 2000b). faster than the latter can be regenerated into The most widely studied and environment- 3+ Fe by O2. Accordingly, the pyrite will simply ally important Fe(II)-oxidizing bacteria include reduce all the ferric cations and the reaction the Gram-negative mesophiles, Acidithiobacillus will stop. Thus, the oxidation of ferrous iron ferrooxidans (formerly known as Thiobacillus is considered the rate-determining step in the ferrooxidans) and Leptospirillum ferrooxidans. The abiological oxidation of pyrite (Singer and former is rod-shaped, 0.5 µm in diameter by Stumm, 1970). However, as introduced in 1–2 µm long, and possesses a flagellum that Chapter 1, acidophilic bacteria use reaction enables it to be motile (Fig. 5.13A). It grows best (5.23) as an energy-generating process, and in within the pH range 1.8–2.5. Generally more doing so foster the acidification of their local abundant in the latter stages of sulfide oxidation, environment (Fig. 5.12). when pH declines to 1.8 or less, is L. ferrooxidans. ITGC05 18/7/06 18:14 Page 216

216 CHAPTER 5

It is easily distinguished from A. ferrooxidans even shown that during growth on pyrite, the by its morphology, ranging from helix to curved EPS surrounding A. ferrooxidans becomes studded rods to vibrios, with dimensions of 0.2–0.4 µm with fine-grained elemental sulfur colloids which in diameter by 1–2 µm in length (Fig. 5.13B). are believed to serve as a temporary energy reserve Growing at even lower pH, below 1, is the (Rojas et al., 1995). Many of the other Fe(II)- chemolithoautotroph, Ferroplasma acidarmanus. oxidizing bacteria are only capable of using There are many other acidophilic Fe(II)-oxidizing ferrous iron as a substrate (e.g., L. ferrooxidans), prokaryotes spanning the phylogenetic tree and in low pH experiments where they are the (Fig. 5.14), ranging from mesophiles to thermo- sole chemolithoautotrophs, a build up of ele- philes (Baker and Banfield, 2003). Heterotrophic mental sulfur develops on the pyrite surface bacteria coexist with the autotrophs within (e.g., McGuire et al., 2001). This, in turn, makes tailings, where several members of the genus life possible for those chemolithoautotrophs that Acidiphilium survive by coupling the reduction of can only oxidize reduced sulfur species (e.g., elemental sulfur to the degradation of their auto- Acidithiobacillus thiooxidans, formerly known as trophic neighbors. Filamentous fungi and protozoa Thiobacillus thiooxidans). In the experiments above, are the most common eukaryotes, where they also A. thiooxidans is able to reduce the quantity of function as heterotrophs or grazers, respectively elemental sulfur on the pyrite surface to less than (Johnson and Roberto, 1997). 1% observed on samples exposed to Fe(II)- Not all acidophiles oxidize Fe(II). In fact, oxidizing cultures only. Notably, the removal considerably more energy is available during of elemental sulfur from pyrite surfaces exposes the oxidation of reduced sulfur compounds, the underlying minerals to increased oxidative and A. ferrooxidans will preferentially consume attack. Furthermore, bacterial catalysis of sulfoxy sulfide rather than ferrous iron. Studies have anion oxidation facilitates recycling of sulfur

A B

2 µm 1 µm

Figure 5.13 (A) SEM image showing a pure culture of Acidithiobacillus ferrooxidans on filter paper (courtesy of Aman Haque and Bugscope Project). (B) TEM image of a biofilm dominated by Leptospirillum ferrooxidans (arrow) (Reprinted from Rojas-Chapana and Tributsch, 2004 with permission from Elsevier.) ITGC05 18/7/0618:14Page217

Actinobacteria Thermoprotei Acidimicrobium ferrooxidans Sulfolobus sp. β/γ Proteobacteria Ferrimicrobium acidiphilum Acidianus sp. Metallosphaera sp. Thermoplasmata Fungi Acidithiobacillus ferrooxidans Bacilli Stygiolobus azoricus Thermoplasma sp. Penicillium chrysogenum Acidithiobacillus thiooxidans Sulfobacillus sp. Acidilobus aceticus Ferroplasma sp. Rhodotorula glutinis Acidithiobacillus caldus Alicyclobacillus sp. Picrophilus sp. Tremella globospora Thiomonas cuprina Cryptococcus albidus Acidobacteria α Proteobacteria Acidobacterium capsulatum Stramenopiles Acidiphilium sp. Cafeteria roenbergensis Acidocella sp. Hibberdia magna Acidisphaera rubrifaciens Alveolates Green Algae/plants Acidomonas methanolica Sarcocystis muris Nitrospira Colpidium campylum Chlorella minutissima Chamydomonas noctigama Leptospirillum ferrooxidans Eukarya Archaea Leptospirillum ferriphilum Bacteria Leptospirillum thermoferrooxidans Amoeboids Red Algae Filamoeba nolandi Aquificae Chondrus crispus Dictyostelium discoideum Gracilaria sp. Hydrogenobacter acidophilus Entamoeba histolytica

Figure 5.14 Universal phylogenetic tree based on 16S and 18S rRNA sequences, illustrating the wide distribution of microbial species that have been identified in highly acidic waters. The Bacteria and Archaea are subdivided by taxonomic class, and the Eukarya are outlined by their familiar groupings. Representative species are also provided. Note, branch lengths are not to scale. ITGC05 18/7/06 18:14 Page 218

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intermediates, with the net production of increased acidity (e.g., Schippers and Sand, 1999). Recent studies characterizing microbial diver- sity in acidic environments have discovered the presence of numerous other S-oxidizing bacteria that appear to be just as numerically important Pyrite as A. thiooxidans (e.g., Bruneel et al., 2003). In view of the specific role each plays in the overall oxidation process, it is not surprising that we find Fe(II)- and S-oxidizing bacteria growing juxtaposed to one another. This also explains the findings that mixed cultures of chemolitho- A. ferrooxidans autotrophs increase the rates of sulfide mineral dissolution relative to the actions of a single 700 nm species growing in isolation (e.g., Lizama and Suzuki, 1989). Figure 5.15 TEM image of a colony of (b) Importance of attachment Acidithiobacillus ferrooxidans cells growing attached to pyrite. (From Southam and Beveridge, 1992. Reproduced with permission from the Bacterial oxidation of pyrite occurs by two mech- American Society for Microbiology.) anisms: indirect and direct. As discussed above, a number of Fe(II)-oxidizing bacteria generate Fe3+ from Fe2+, which then reacts abiologically 4 Eventually these reconnaissance bacteria multiply, with solid pyrite. This process is considered indi- and form a microcolony directly on the pyrite sur- rect because the bacteria do not directly oxidize face. Given time and sufficient nutrients, the colony expands into a pyrite enshrouding biofilm onto pyrite, and thus can grow attached to nonsulfide which other species may attach. minerals as well. By contrast, most of the bacteria discussed above actually grow on pyrite and Bacterial adsorption onto the pyrite surface is other types of sulfide minerals (e.g., Fig. 5.15), rapid. Experiments have documented that nearly where they directly oxidize and solubilize the 100% of the total population of planktonic reduced iron and sulfur moieties via enzymatic A. ferrooxidans cells can adhere to the pyrite reactions. The attachment of bacteria to pyrite within minutes if sufficient surface area is made can be visualized as occurring in four distinct available (e.g., Bagdigian and Myerson, 1986). steps: The mechanism of attachment is not random, and appears to involve the bacteria colonizing 1 In the first step, bacteria are transported to the fractures or high surface energy sites, such as dis- pyrite surface after it has already been conditioned locations. Aside from free energy gains associ- with inorganic and organic compounds. ated with attachment at dislocations, those sites 2 Once in the vicinity of the surface, electrochemical may afford the acidophiles with a greater flux interactions between the cell and pyrite surface are of reductants – diffusivities along dislocations initiated; the type and strength being governed by can be orders of magnitude greater than through surface properties of the mineral and the bacterium pure crystalline solids (Andrews, 1988). Once (recall section 3.6.1). initiated, the contact sites eventually develop 3 The actual physical attachment of the bacterium to into corrosion pits the size and shape of the the surface occurs by the development of specific bacteria, widening and enlarging until there is structures, such as fibrils or EPS. a pronounced surface roughening (Fig. 5.16). ITGC05 18/7/06 18:14 Page 219

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A. thiooxidans), because their survival hinges on being able to attach to specific sites on the mineral substrate. Interestingly, the species are able to selectively colonize minerals that have low electrochemical stability and are more reac- tive and easier to dissolve. This can be relative to matrix materials (sulfide vs. silicates) or even between different sulfide phases, such as the preferential oxidation of arsenopyrite (FeAsS) over pyrite (Norman and Snyman, 1988). Once attached to pyrite, the production of EPS fixes the cells firmly onto the solid. With time, not 10 µm only does EPS completely enshroud the exposed mineral surfaces, but under the prevailing acidic conditions, it becomes heavily impregnated with Figure 5.16 SEM image showing extensive cationic Fe species. This further facilitates the pyrite dissolution after only 43 days of oxidation by A. ferrooxidans. (From Mustin et al., 1992. bacterium’s electrostatic adsorption onto the Reproduced with permission from the American negatively charged pyrite by lowering the elec- Society for Microbiology.) tronegativity of the bacterium’s surface (at pH values where some of the cell’s functional groups In turn, the corrosion pits serve as conveni- have already deprotonated) and by reducing ent physical recesses and make available newly any double-layer repulsive barriers (Blake et al., altered surfaces for colonization by a second 1994). Moreover, the EPS may actually acceler- wave of bacterial species (e.g., the S-oxidizing ate pyrite oxidation because the Fe adsorbed to it A. thiooxidans). Under ideal growth conditions, can potentially serve as an electron shuttle for this process would continue until the pyrite is conveying electrons from the metal sulfide to the completely degraded (Mustin et al., 1992). cell surface, in a manner reminiscent of reaction In the Gram-negative acidithiobacilli, the (5.26) (Sand et al., 1995). Such a mechanism macromolecule that is responsible for initial might be important because the same EPS that mineral binding is the lipopolysaccharide (LPS). aids in surface adhesion could also present a Although pyrite is negatively charged above potential inhibitor of Fe3+ diffusion away from pH 2.5, the functional groups comprising the the cell. Thus, the iron recycling that takes place LPS are still protonated under acidic conditions, during the propagation cycle can be envisioned thus providing the cells with a neutral to slightly as taking place entirely within the EPS. positive charge that allows them to approach The relative importance of direct versus indi- and attach onto the pyrite surface. It has even rect mechanisms in terms of pyrite dissolution been suggested that variations in LPS chemistry rates remains a subject of dispute. It can be argued afford acidithiobacilli the means by which to that rates of oxidation are faster when Fe3+ reacts distinguish different atoms in the pyrite lattice with pyrite versus bacterial oxidation of Fe(II) (Southam and Beveridge, 1993). This is a useful in the crystal lattice. Furthermore, many of the trait for A. ferrooxidans because it can activate surface weathering features observed on pyrite the appropriate oxidative enzymes depending can be attributed to abiological reactions with on whether Fe or S sites are exposed. The ability bacterially induced Fe3+ (Edwards et al., 2001). to recognize specific electron donors is even Yet, bacteria rapidly attach to pyrite surfaces more crucial for strict Fe(II) oxidizers (e.g., when they become exposed, and species, such as L. ferrooxidans) or reduced sulfur oxidizers (e.g., A. ferrooxidans, do not develop into multiplayer ITGC05 18/7/06 18:14 Page 220

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biofilms, suggesting that each cell needs to be in The effects of pyrite oxidation are often far direct physical contact with the pyrite surface removed from the actual mine site. The reasons in order to grow (e.g., Larrson et al., 1993). for this are simple. The highly acidic and sulfate/ Certainly, by adhering to the solid sulfide they metal-rich effluent, aptly named acid mine drain- ensure that a source of reduced iron and/or sulfur age (a specific type of acid rock drainage), is is in close proximity, thereby minimizing the sometimes initially transported by groundwater time needed for diffusion of reducing equivalents flow before discharging into surface waters. As between the mineral and the bacterium. What long as anoxic effluent is acidic, the Fe2+ is stable is most likely is that both mechanisms work in the absence of bacteria, but once it comes into

concurrently, and that the more reactive sulfides contact with O2 in more alkaline, aerated drain- (e.g., pyrrhotite) are oxidized predominantly age, oxidation and hydrolysis spontaneously occur. by indirect mechanisms, while relatively less Similarly, Fe3+ may be transported away from reactive sulfides (e.g., pyrite) may require more the site of active pyrite oxidation, without ever direct attachment to cause their oxidative dis- having come into chemical contact with any solution. This could clarify why, on a per cell remaining sulfide mineral phases. In the acidic basis, sulfide mineral dissolution rates appear outflow, however, some of it will also precipitate comparable between attached and planktonic as jarosite, or other ferric sulfate minerals, due species (Edwards et al., 1999). to sulfate bridging of ferric iron colloids (recall section 4.1.9(b)). (c) Formation of acid mine drainage (AMD) Mixing of AMD with natural waters in rivers and lakes causes serious degradation in water During coal and metal mining operations, over- quality: burden, waste rock, and mill tailings are dis- posed of in the form of spoil heaps or in tailings 1 both the acid and high dissolved metal content ponds. Those waste materials contain residues (e.g., Fe and trace metals solubilized from the solid- of pyrite and other sulfide minerals that, upon phase sulfides under acidic conditions) are toxic to decommissioning of the mining operation, even- aquatic life; tually become exposed to rain and oxygenated 2 the acidity changes the dissolved inorganic carbon surface waters. This places them into chemical − speciation from HCO3 to H2CO3, thereby diminish- disequilibrium, and subject to the oxidation ing the autotrophic metabolism of a number of transformations discussed above. Within the organisms; vadose zone of the spoil heap, where capillary action on the mineral substrata supplies water 3 the ferric hydroxide/ferric sulfates smother benthic species, inhibiting photosynthesis; for chemolithoautotrophic growth, the sulfide phases provide a source of energy, and the pore 4 the acids have a corroding effect on parts of infras- spaces allow for the influx of CO2 and O2 that tructures along the river course, such as bridges. serve as the carbon source and terminal electron acceptor (TEA), respectively (see Kleinmann If the rate-determining steps are controlled prim- et al., 1981). With all their needs met, sulfide arily by the activity of A. ferrooxidans, then the mineral-oxidizing microorganisms can expediti- oxidation process as a whole depends upon the ously establish themselves and begin the bio- growth conditions of the bacteria. Aside from transformation of fine-grained, pH-neutral, gray the obvious supply of sulfide minerals, one of the sulfide-bearing tailings into the bright yellow most important requirements are that periodic and orange stained, leachate-producing residues rainwater infiltration provides the needed aera- that are a significant environmental concern tion and removal of oxidation products so that (see Plate 12). fresh pyrite surfaces are exposed. It has been ITGC05 18/7/06 18:14 Page 221

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0 + 3+ + → 2− + 2+ + + shown that A. ferrooxidans increases acid produc- S 6Fe 4H2O SO4 6Fe 8H tion for 3–4 days after each rainfall, after which (5.30) acid generation drops back down to ambient conditions (Kleinmann and Crerar, 1979). A Cell yields observed during such anaerobic qualifying statement is, however, needed here growth are comparable to the cell yields in aero- because excessive rainfall will dilute, or remove, bic, Fe(II)-grown cultures, but two times lower

acid build-up, such that the pH may not drop than growth on inorganic sulfur with O2 as the below 4.5, and the onset of the propagation terminal electron acceptor (Pronk et al., 1992). cycle of AMD may never occur. This has been The practical implications of this are that metal shown to happen during heavy rainfalls at Iron solubilizing activity may take place at the center Mountain, California (the most metal-rich and of poorly aerated ore heaps by using ferric iron acidic effluent of any abandoned mine reported that was produced by other bacteria growing at anywhere in the world), where washout has pre- the surface. Indeed, recent findings by Coupland viously been shown to have reduced the micro- and Johnson (2004) have indicated that A. bial populations from 109 cells ml−1 to less than ferrooxidans is the dominant bacterium in both 104 cells ml−1. It also altered the microbial speci- anaerobic and acidic waters from two submerged ation, such that it took a lag time of 6 months mines in Wales. before re-colonization and return to the usual Another major source of controversy regard- mine microbiota was established (Edwards et al., ing acid mine drainage is the origin of the acidity. 2000b). Thus, for AMD to become a significant Although A. ferrooxidans plays an active role problem, acid must be allowed to accumulate in pyrite oxidation once the pH has decreased in the spoil heap pore waters. Such accumula- below 4.5, they were not believed to survive tion can often be a seasonal phenomenon. For at higher pH conditions. Yet, the rather slow example, acid flushing into streams is some- kinetics of abiological oxidation by molecular times observed during spring. The underlying oxygen in air would seem to preclude it as the cause lies with drainage out of the spoil during dominant acidification process. Therefore, it was winter being prevented by the frozen ground, suggested that the initial steps must be micro- yet any unfrozen water within the spoil (kept bially catalyzed, driven perhaps by the activity liquid by the heat generated by the oxidative of neutrophilic chemolithoautotrophs and/or reactions) continues to generate acidity. Then heterotrophs that condition the tailings for sub- when the ground thaws, the acid is discharged. sequent acidophilic populations (e.g., Harrison, In a different scenario, if spoils are allowed to 1978; Blowes et al., 1995). More recently, it has dry out completely during the summer months, become accepted that a succession of neutro- bacterial numbers and AMD also decline as philic and moderate acidophiles microorganisms water becomes limiting for microbial activity are not required to generate the needed acidity. (Olson et al., 1981). In a study of simulated sulfide-rich tailings, it was Due to the relationship between oxygenation observed that A. ferrooxidans not only survives and acid production, it has for many years been at pH values of 7, but it was also able to initi- perceived that as long as oxygen was excluded ate pyrite oxidation and localized acidification from the tailings that pyrite oxidation would within just 2 weeks of colonization (Mielke et al., cease. However, it has now been recognized that 2003). A. ferrooxidans is a facultative anaerobe, capable How the bacteria do this is described as follows.

of surviving in the absence of O2 by using Fe(III) First, the bacterium uses cation bridging to fix as an electron acceptor, provided that H2 or a itself onto high energy sites of the pyrite surface, reduced sulfur species serves as the electron upon which it then excretes EPS to attain a donor (e.g., reaction (5.30)): tenacious bond with the mineral surface. Once ITGC05 18/7/06 18:14 Page 222

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attached, it begins directly oxidizing the Fe(II) and precipitates instead of the intermediate sulfur S(−1) moieties in the crystal lattice, producing anions diffusing away, thereby affording S-oxidizing corrosion pits on the surface into which the cells bacteria with an oxidizable substrate. Simultane- reside. Acidity arises from both the biological ously, ferric hydroxide begins partial solubiliza- oxidation of sulfoxy anions to sulfate and ferric tion. The Fe3+ formed then reacts with pyrite to hydroxide precipitation, of which the latter initiate the propagation cycle. This extends the covers the pyrite surface and becomes embedded acidity to areas away from the immediate surround- within EPS. The ferric hydroxide and EPS then ings, eventually affecting the more neutral bulk act as partial diffusion barriers that maintain H+ pore waters (Fig. 5.17). In essence, the micro- in a nanoenvironment at the mineral surface bial nanoenvironment exhibits physicochemical (a few nm3). Then, as conditions become more conditions conducive to the survival of a particu- favorable, the bacteria multiply to form micro- lar community of species, even though the bulk colonies that are enshrouded in Fe-rich biofilm. pore waters in the tailings sediment is fundament- As the proximal pH drops, elemental sulfur ally different. Nonetheless, this environmental

Stage1 pH 7 Oxygenated pore water

Figure 5.17 Possible model for the colonization of Bacterium EPS pyrite under circumneutral pH conditions. Stage 1: A. ferrooxidans attaches to Pyrite pyrite surface and generates EPS to firmly attach itself to the mineral surface. Stage 2: Stage 2 Dissolution of pyrite causes Fe-rich EPS − release of S2OH , while Fe(II) oxidizes to Fe(III). Oxidation SO 2– 4 of sulfoxy anions and – O S2OH hydrolysis of ferric iron 2 H+ H+ generate acidity that stays confined to the EPS around the cell. Stage 3: Cells Fe(II) Fe(OH)3 forms S(–1) multiply and more acid is created, expanding

Stage 3 + + + + the acid front. The ferric pH 3 H H + + H H H H + + H + hydroxide re-dissolves to H + + H + H H+ Acid expansion front H yield Fe3 that reacts with the H+ + + H+ pyrite, while elemental sulfur H+ H H Fe3+ H+ becomes the stable S-phase upon which species, such as A. thiooxidans, later attach. (Adapted from model originally proposed by Fe(OH)3 dissolves S(–1) Southam and Beveridge, A. thiooxidans 1992, and later Mielke et al., 2003.) S0 layer ITGC05 18/7/06 18:14 Page 223

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modification is transient, and the colonizing micro- tion of metals as sulfide minerals; or adding living/ organisms eventually succumb to the changes dead biomass to adsorb iron and other metals beyond their adaptive capabilities, at which point (recall section 3.7.1). different species begin to predominate and a Wetlands offer perhaps the best approach complete restructuring of the mine waste com- because they represent a potentially long-term, munity ensues. Such changes could include the self-sustaining system in which both the acidity colonization of even more extreme acidophiles is consumed and the metals immobilized prior (e.g., Schrenk et al., 1998) or the advent of to the effluent being discharged into the

Fe(III)- and SO4-reducers, if the tailings become regional waterways (Pulford, 1991). Essentially, O2-depleted (e.g., Fortin and Beveridge, 1997). wetlands remove metals by one of two processes: The exact time associated with the establish- adsorption/absorption by metal-tolerant plants ment of highly acidic effluent is, at present, and FeS precipitation via bacterially mediated 2− still ill defined, but appears to be of the order of sulfate reduction – the SO4 coming from the years to decades. Despite the rapid colonization AMD (Fig. 5.18). The sorption processes tend and onset of localized acid production in tailings, to dominate at the start of wetland construc- modeling predictions and studies at a limited tion, but, over time, mineralization becomes the number of field sites indicate the peak acid load more important process as it converts dissolved occurs 5–10 years after mining, followed by a Fe into an unreactive form, such as pyrite. As a gradual decline over 20–40 years (e.g., Hart result, artificial wetlands are now constructed et al., 1991). The same study projected very long with the view of adding decomposable, organic- decay curves for coal refuse (beyond 50 years) rich substrates that facilitate the growth of sulfate- before acid leachate is depleted. reducing bacteria (SRB) (e.g., Machemer and Today a number of remediation methods are in Wildeman, 1992). Wetlands are also usually low place to curb the spread of AMD away from the maintenance, involving only periodic dredging mine site. They usually develop along two lines: of sediment build-up and addition of limestone (i) prevention of the actual generation of AMD to treat the acid inflow, although wetland effec- at the source; or (ii) treatment of the AMD tiveness has come into question under conditions downstream (see Ledin and Pedersen, 1996 for when high acid loading overwhelms abiological details). In the former case, this may involve: and microbial alkalinity-generating mechanisms chemical treatment by adding alkalinity (via (see Wieder, 1993 for details).

crushed limestone or Ca(OH)2) to the system before the pH drops below values of 4.5; adding 5.2.3 Bioleaching phosphate to inhibit pyrite oxidation; growing

vegetation on spoil heaps to consume O2 and Low grade sulfide ores generally contain a variety diminish water infiltration; capping the tailings of valued metals at concentrations below 0.5%

to prevent O2 diffusion to the sulfides; flooding (wt/wt), and their extraction by smelting after the tailings so that anoxic conditions inhibit the milling and ore enrichment is unfavorable because profusion of A. ferrooxidans; or applying biocides of the high gangue/metal ratio. In order to recover to kill off the Fe(II) and S-oxidizing bacteria. those metals at profit, a number of mining com- The second treatment strategy may involve: panies have utilized technologies that harness diverting AMD to a water treatment plant where the metal-oxidizing or acid-generating activity chemicals are applied to neutralize the acid of microorganisms (see Hackl, 1997 for details). and precipitate iron hydroxide (along with the This process, called bioleaching, places the co-precipitation of the trace metals); adding a metals of value in the solution phase, while the reactive organic substrate to promote bacterial solid residue, if any, is discarded as waste material. sulfate reduction and the subsequent immobiliza- Bioleaching is now also being used in the ITGC05 18/7/06 18:14 Page 224

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Limestone and organic substrate

O2

Oxicoxic zonezone Mine effluent 2– + (metals, SO4 , H ) 1 Fe(III)-Fe(III)-reductionreduction Fe(OH) 3 2 FeS FeS2 SO4--reductionreduction

– 2+ – – 1 8Fe(OH)3 + CH3COO 8Fe + 2HCO3 + 15OH + 5H2O

2– – – – 2 SO4 + CH3COO + H2OH2S + 2HCO3 + OH

Figure 5.18 Model showing how wetlands alleviate acidity and high dissolved metal concentrations from mine drainage. Acid input is initially neutralized by the addition of limestone, causing the hydrolysis of Fe(III) to ferric

hydroxide. Secondary alkalinity is then generated through bacterial Fe(III) and SO4 reduction. Metals are 2+ immobilized during burial through reaction of pore water Fe and H2S, with organic compounds supplied to support the dissimilatory bacterial communities.

bioremediation of municipal wastewaters, where residues. In most cases little effort is made to the organic sludge produced after treatment optimize bacterial activity. This process is a slow, often contains high concentrations of heavy inefficient process, with leach cycles measured metals. Because of the high content of nitrogen, in years and efficiency at most 50% for metals phosphorous, and potassium, sludges have been such as copper. Heap bioleaching is a more used as fertilizers in many areas around the world. sophisticated method. In this process, finely However, to avoid any potential environmental crushed ores are placed on prepared pads, and a contamination, the metal contaminants must dilute sulfuric acid solution (pH ~2) is initially first be removed (e.g., Tyagi et al., 1990). sprayed onto the “heap” to pre-condition the ore for the bacteria. Without the acidification (a) Chemolithoautotrophic oxidation step, bioleaching becomes rapidly ineffectual because waste rocks, made up of silicates, buffer Bioleaching has been used effectively in the the natural acidity generated through pyrite recovery of copper, zinc, lead, arsenic, anti- oxidation before the acidophiles can take hold. mony, nickel, and molybdenum from sulfide ores Simultaneous aeration of the pile is essential (Table 5.2). There are two common methods since the microbial leaching process is an aerobic used, dump bioleaching and heap bioleaching. process. The liquid coming out at the bottom Historically dump leaching has been the most of the pile is collected and transported to a col- widely used method because open-pit mining lection plant where the metal is re-precipitated frequently led to the formation of large piles of and purified. Meanwhile, the Fe(II)-rich liquid, waste rock, some on the order of several million called the lixiviant, is released into an oxidation tons. During dump leaching, acid is added pond to form Fe(III), and then pumped back to to the waste rock, and the indigenous bacteria the top of the pile where the cycle is repeated proliferate naturally by oxidizing sulfide mineral (Fig. 5.19). ITGC05 18/7/06 18:14 Page 225

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Table 5.2 A summary of the reactions involved in the breakdown of various common sulfide minerals. (Data compiled by McIntosh et al., 1997.)

Mineral Reactions

+ + → + + Chalcopyrite 4CuFeS2 17O2 2H2SO4 4CuSO4 2Fe2(SO4)3 2H2O + → Covellite CuS 2O2 CuSO4 + + → + + Chalcocite 5Cu2S 0.5O2 H2SO4 CuSO4 Cu9S5 H2O + + → + + Bornite 4Cu5FeS4 37O2 10H2SO4 20CuSO4 2Fe2(SO4)3 10H2O + → Sphalerite ZnS 2O2 ZnSO4 + → Galena PbS 2O2 PbSO4 + + → + Arsenopyrite 4FeAsS 13O2 6H2O 4FeSO4 4H3AsO4 + + → + + Stibnite 2Sb2S3 13O2 4H2O (SbO)2SO4 (SbO2)2SO4 4H2SO4 + → Millerite NiS 2O2 NiSO4 + + → + Molybdenite 2MoS2 9O2 6H2O 2H2MoO4 4H2SO4

Microorganisms currently used in commercial from chalcopyrite (CuFeS2), followed by Fe. bioleaching operations are exactly the same as By contrast, A. thiooxidans can only oxidize the those found naturally associated with exposed sulfide portion of the ore without preference sulfidic ore, the only difference is that they may for particular metals, and its inability to oxidize have been selected for rapid growth on the ore of Fe(II) reduces the electrochemical effect of interest. The bacteria should also show versatil- having high Fe3+ concentrations (Lizama and ity in attacking different metal sulfides, and they Suzuki, 1988). must be resilient to toxic concentrations of transi- The most suitable copper minerals for heap

tion metals within the lixiviant (Ehrlich, 2002). bioleaching are chalcocite (Cu2S) and covellite Acidithiobacillus ferrooxidans fulfills these criteria (CuS) – the major copper mineral of most mine and, crucially, is ubiquitous in mine tailings. wastes, chalcopyrite, has not been considered Notwithstanding the environmental concerns economically bioleachable due to the long leach posed by growth of A. ferrooxidans and the times required. The overall process of copper other Fe(II)- and S-oxidizing bacteria present bioleaching is predicated on the basis of A. in waste tailings, when those same species are ferrooxidans being capable of directly oxidizing used in the controlled and confined conditions Cu(I) in chalcocite, removing some copper in of a processing plant, the undesirable oxidative the dissolved form (Cu2+), and forming the metabolic processes can serve as catalysts in the mineral covellite (reaction (5.31)): metal extraction process. + + → + 2+ + − The rates of oxidative leaching and the Cu2S 0.5O2 H2O CuS Cu 2OH efficiency of the process can be predicted based (5.31) largely on the electrochemical properties of the metals in a mixed ore and on the metabolic In reaction (5.31), the bacteria utilize Cu(I) abilities of the chemolithoautotrophic species. as an electron donor before the sulfide (Nielsen For example, A. ferrooxidans will solubilize Zn and Beck, 1972), although other studies have from sphalerite (ZnS) much faster than Cu shown the precipitation of the mineral antlerite ITGC05 18/7/06 18:14 Page 226

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Spraying pH 2 lixiviant on copper ore

Nutrients

Aeration Low grade copper ore → 2+ – [1] Cu2S + 0.5O2 + H2O CuS + Cu + 2OH + → 2+ 0 [2] CuS + 0.5O2 + 2H Cu + S + H2O 0 → 2– + [3] S + 1.5O2 + H2O SO4 + 2H Lixiviant pumped back to [4] Cu S + 2Fe3+ → Cu2+ + 2Fe2+ + CuS 2 top of leach dump [5] CuS + 2Fe3+ → Cu2+ + 2Fe2+ + S0

2+ H2SO4 Cu addition

Recovery of copper metal (Cu0) Fe0 + Cu2+ → Cu0 + Fe2+

2+ → 3+ – 4Fe + O2 + 2H2O 4Fe + 4OH Leptospirillium ferrooxidans Acidithiobacillus ferrooxidans Oxidation pond

Copper metal (Cu0)

Figure 5.19 The arrangement of a copper heap leaching plant. In the first step, finely crushed ores are placed on prepared pads, and a dilute sulfuric acid solution (pH 2) is initially sprayed onto the “heap” to pre-condition the ore for Acidithiobacillus ferrooxidans. Once conditions are ideal for the bacteria, they begin oxidizing Cu(I) and S(−1) through a succession of metabolic reactions. Simultaneously, Fe3+, generated from bacterial oxidation processes, reacts abiologically with the same copper minerals. The Cu-rich liquor is then processed, and the Fe2+ from the oxidative and recovery reactions is re-oxidized to Fe3+ by A. ferrooxidans, in an oxidation pond, and pumped back into the heap to be recycled. (Modified from Madigan et al., 2003.) ITGC05 18/7/06 18:14 Page 227

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((Cu3SO4)(OH)4) in their chalcocite bioleach- diffusion of into the heap pile must be applied, ing experiments (Silver and Torma, 1974). The i.e., through the addition of air injection systems. sulfide in covellite is subsequently oxidized by the 3+ same bacteria, initially forming elemental sulfur 2 Acidity – To ensure both sufficient Fe as a lixiviant + and a steady-state population of acidophiles, acidic and Cu2 (reaction (5.32)), but then the sulfur is conditions must be maintained. fully oxidized to sulfate, either by A. ferrooxidans or another bacterial species, such as A. thiooxidans 3 Permeability – The permeability of a heap helps (recall reaction (5.21)): determine the solution distribution and the diffu-

sion of O2 required for bacterial activity. Good + 2+ 0 agglomeration, through mixing ore with acid and CuS + 0.5O + 2H → Cu + S + H O (5.32) 2 2 water to prevent the segregation of fine and coarse material, greatly improves permeability and pre- Because pyrite is a common constituent of metal vents solution channeling. sulfide ores, its oxidation to Fe3+ becomes an additional oxidant of chalcocite and covellite, 4 Nutrients – Leaching bacteria require ammonium, generating more dissolved Cu2+ and Fe2+: phosphate, and potassium, which are supplied as (NH4)2SO4 and KH2PO4, respectively. Typically, these nutrients are added to a heap with a pH < 2, + 3+ → 2+ + 2+ + Cu2S 2Fe Cu 2Fe CuS (5.33) which maximizes bacterial growth and also pre- vents the precipitation of ammonium jarosite. CuS + 2Fe3+ → Cu2+ + 2Fe2+ + S0 (5.34) 5 Heat – A. ferrooxidans grows best at 20–35°C, although activity is evident outside these ranges. This reaction sequence is thermodynamically Temperature is important, and as a general rule predictable considering that the standard elec- of thumb, bacterial activity halves for every 7°C 3+ 2+ trode potential of the Fe /Fe couple is +0.77, temperature drop, so operations in seasonal envir- + + − while that of Cu2 /Cu is +0.15, and S0/S2 is onments need to take into account some pore water −0.27. This means that ferric iron should act as freezing. Furthermore, many of the reactions that an oxidant for both Cu(I) and S(−II) (McIntosh take place within the heap pile are exothermic, and it is not uncommon to record summer temper- et al., 1997). In the presence of O2, and at the + atures in excess of the optimal conditions of the acid pH involved, A. ferrooxidans re-oxidizes Fe2 acidophiles. back to Fe3+, thereby regenerating the lixiviant so that it can oxidize more copper sulfide. (b) Galvanic leaching Thus, copper oxidation is maintained indirectly through bacterial Fe(II) oxidation. An additional reaction mechanism that can There are a number of variables that affect the have a significant affect on bacterial leaching efficiency of bioleaching operations, the most rates is the process of galvanic leaching. Sulfide important being the maintenance of bacterial minerals tend to be electrically conductive, growth rates commensurate with the desired cell thus, when two different sulfide minerals are in densities in the overall system (Schnell, 1997). physical contact, as would be the case in an ore This can be influenced by: deposit, a galvanic couple is created in which the less reactive mineral acts as a cathode, while 1 Oxidants – Ferric iron is considered to be the the more reactive mineral acts as an anode. The primary oxidant in the dissolution of copper, and latter preferentially becomes oxidized and dis- its production is vital for efficient bioleaching. + solved. Minerals can be listed according to their The physical addition of Fe3 is uneconomic, thus bacterially-mediated Fe(II) oxidation is of para- reduction potentials, making it possible to pre- dict how different mineral pairings will interact mount importance. Because O2 is required for A. ferrooxidans, methods to improve its natural (Table 5.3). ITGC05 18/7/06 18:14 Page 228

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chalcopyrite crystal, releasing electrons that Table 5.3 Reduction potentials of some migrate to the surface of the adjacent pyrite Fe- and Cu-bearing sulfide minerals*. crystal (reaction (5.35)). Oxygen that accumu- (After Rossi, 1990.) lates at the pyrite surface is subsequently reduced and hydroxyl ions are produced in accordance Mineral Potential (mV) with the following half reaction (5.36). This process is commonly referred to as an oxygen Chalcocite (Cu S) 350 reactive 2 concentration cell. Chalcopyrite (CuFeS ) 400 d 2 e + + − Stannite (Cu2FeSnS4) 450 e → 2 + 2 + 0 + i CuFeS2 Cu Fe 2S 4e (5.35) Pyrrhotite (FeS) 450 i Tetrahedrite (Cu SbS ) 450 h + + + − → − 3 3 O2 2H 4e 2OH (5.36)

Pyrite (FeS2) 550–600 noble These reactions also cause the formation of metal *H SO solution, pH = 2.5. Open-circuit potential is 2 4 oxyhydroxide and sulfur deposits in the regions measured against a saturated hydrogen electrode. around the zone of chalcopyrite pitting. The latter subsequently provides an ideal substrate for A typical example is where chalcopyrite and S0-oxidizing bacteria, such as A. thiooxidans. This pyrite are in contact. Chalcopyrite, being the increases the sulfate flux to the aqueous phase. more reactive, acts as the anode (Fig. 5.20). The Cu2+ released diffuses away from the surface Thus, oxidation begins at the surface of the into the overlying aqueous phase, while the Fe2+

SO 2– Overlying water 4 O (oxygenated, low pH) 2 2 – + 2OH O2 + 2H Fe3+ Cu2+ Fe2+ Fe(OH)3 S0 1

– CuFeS2 4e (anode)

FeS2 (cathode)

1 S0 oxidizers 2 Fe(II) oxidizers Silicate rock

Figure 5.20 Model of a galvanic cell between pyrite and chalcopyrite. Of the products of chalcopyrite oxidation, Cu2+ diffuses into the water around the sulfide grains; elemental sulfur accumulates on the chalcopyrite surface, where some of it is biologically oxidized by A. thiooxidans; while Fe2+ is oxidized by A. ferrooxidans and then hydrolyzed to ferric hydroxide on the sulfide surfaces. (Modified from McIntosh et al., 1997.) ITGC05 18/7/06 18:14 Page 229

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released is oxidized by A. ferrooxidans (or other fungi were applied at 95°C. The cobalt recovery Fe(II) oxidizers) to Fe3+, and then hydrolyzed was almost 50% (Tzeferis, 1994). Not only is in the more alkaline nanoenvironment above Aspergillus niger able to solubilize a wide range of

the mineral surface created via the O2 reduction insoluble minerals, including phosphates, sulfides, reaction. The net result of this process is the and oxide ores, but it is also able to immobilize preferential and accelerated oxidation of chal- those leached metals (e.g., Cu, Cd, Co, Zn, and copyrite, while pyrite remains relatively unaltered Mn) by the formation of metal oxalate salts. Many until the chalcopyrite has been completely of those metal oxalates are resistant to further depleted (Lawrence et al., 1997). solubilization, suggesting that oxalate formation may be a survival mechanism used by the fungi to (c) Fungal acid production immobilize potentially toxic metal compounds within their immediate surroundings (Sayer and Most biological leaching operations rely on Gadd, 1997). acidophile-assisted oxidative processes because Bioleaching can also be employed to remove the metals of interest are in a reduced oxidation toxic metals from waste materials. This dimin- state, housed within a sulfide framework. How- ishes problems of disposal and opens up new ever, for minerals that contain no redox-active avenues for recycling metal-rich refuse. As dis- sources of energy, simple dissolution may be cussed above, chemolithoautotrophs have been all that is required to liberate those metals. In employed in sewage sludge treatment, and the such situations, fungi tend to be highly effective use of different acidophiles is often advantage- weathering agents because: (i) they tolerate ous because they can induce the mobilization of higher pH levels than the acidophiles; (ii) they metals at low pH. In organic-rich wastes, hetero- can survive high metal exposures; (iii) they can trophic bacteria and fungi are more useful because be more easily manipulated in bioreactors; and they not only degrade the bulk of the organic (iv) they produce high concentrations of organic carbon, thus reducing the volume of waste, but acids that facilitate weathering reactions (Gadd, they also generate acids that effectively leach 1999). Importantly, by altering their growth con- metals from insoluble mineral constituents. For ditions, fungi can be induced to produce acids instance, a strain of Penicillium simplicissimum on an industrial scale. Citric acid production by that was isolated from a metal-contaminated the soil fungus Aspergillus niger is a case in point. site, produced sufficient citric acid to successfully World annual production is estimated at around leach 90% of the zinc from insoluble ZnO- 400,000 tons (Mattey, 1992). Fungal produc- containing industrial filter dust (Schinner and tion can be modified by the concentration and Burgstaller, 1989). type of carbon source, while withholding certain metals from the growth cultures cause the fungi 5.2.4 Biooxidation of refractory gold to increase the amounts of citric acid produced (e.g., Meixner et al., 1985). Many ore bodies contain metals, such as gold, There are many examples of laboratory-scale that are difficult to extract because the metal leaching operations. One is the recovery of Ni is disseminated throughout the host sulfide and Co from low grade laterite ores by species mineral, such as pyrite or arsenopyrite. For of Aspergillus and Penicillium. Nearly 60% of these “refractory” ores, conventional cyanide the available Ni was leached when fungi were or bioleaching methods do not work unless the grown in the presence of the ore and the leach- sulfide minerals can first be destroyed by an ing potential was increased to 70% when the oxidative pre-treatment to liberate the gold. metabolic products obtained from cultivation of Two traditional pre-treatment methods for ITGC05 18/7/06 18:14 Page 230

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refractory gold ores are roasting and pressure constituents of the ores, such as antimony and oxidation. Recently, biooxidation has emerged mercury, can become toxic to the bacteria. as a viable third alternative. Biooxidation uses Other environmental factors that impact on ® similar bacteria as in bioleaching to catalyze the BIOX process are pH, temperature, CO2, the degradation of sulfide minerals, but unlike and nutrients. The optimal temperature for the bioleaching, it leaves the metals of value in the BIOX® bacteria is between 35°C and 45°C; the solid phase. To facilitate the biooxidation pro- bacteria are not killed at 50°C, but their oxida- cess, finely ground ores are separated from the tion rates slow down considerably, and the time gangue and other materials by flotation tech- required for complete conversion of Fe(II) to niques to produce a concentrate that is then added Fe(III) increases from 1 day at 40°C to 3 weeks to a stirred-tank bioreactor, where the chemo- at 50°C. lithoautotrophic bacteria reside (Lindström et al., The BacTech process employs moderate 1992). thermophiles in the biooxidation of refractory There are a number of commercial biooxida- gold-bearing ores (Miller, 1997). Bacteria of the tion processes now in existence. The BIOX® pro- genera Sulfobacillus and Sulfolobus grow optim- cess uses a mixed population of A. ferrooxidans, ally at temperatures of around 50°C, and when A. thiooxidans, and L. ferrooxidans to collectively used in biooxidation experiments, they have oxidize the reduced Fe and S moieties in arseno- been shown to provide higher rates of sulfide pyrite and pyrite (see Dew et al., 1997 for details). mineral dissolution than their mesophilic coun- In biooxidation, the first step in leaching is oxi- terparts. Furthermore, the efficient extraction dation of the sulfide component of the mineral, of copper from chalcopyrite concentrates, which 2+ 3+ and the solubilization of Fe , As (as H3AsO3), cannot be achieved at low temperatures, is a and the sulfoxy species. The reductants enhance notable potential application of thermophiles the growth of free-living chemolithoautotrophic in bioleaching. bacteria, with the concomitant formation of Fe3+, 5+ 2− As (as H3AsO4), and SO4 (see overall reac- tion (5.37)). The Fe3+ subsequently triggers the 5.3 Microbial corrosion abiological oxidation of more arsenopyrite and pyrite (reaction (5.38)): The deterioration of a metal by electrochemical reactions with substances in its environment is + + + + → referred to as corrosion. In most cases the basic 2FeAsS 7O2 2H 2H2O 2H AsO + 2Fe3+ + 2SO 2− (5.37) process underpinning corrosion involves a flow 3 4 4 of electricity between certain areas of a metal surface through a solution that has the ability to FeAsS + Fe3+ + 2.5O + 2H O → 2 2 conduct an electric current. During corrosion, 2Fe2+ + H AsO + SO 2− + H+ (5.38) 3 3 4 metal cations develop at an anodic site and the electrons associated with this dissolution Many bacteria growing naturally on arsenopyrite are accepted at a cathodic site. The metal that may be inhibited by the levels of arsenic released. has received the most attention is elemental The BIOX® bacteria are, however, tolerant to iron (Fe0), although copper, aluminum, lead, As(V) concentrations of 15–20 g L−1. They are and silver also succumb to corrosive reactions. less tolerant to As(III), and become inhibited The corrosion of elemental iron is best known above concentrations of 6 g L−1, although its as rust formation on steel, when in contact rapid oxidation by Fe3+ generally maintains very with oxygen and water. Such reactions are often

low H3AsO3 concentrations. Similarly, other manifest as structural damage to buildings and ITGC05 18/7/06 18:14 Page 231

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deterioration of machinery, ship structures, cars, (Beech and Gaylarde, 1991). Several types of and even airplane fuel tanks. In the absence of microorganisms contribute to metal corrosion, oxygen, any number of other electron accep- but for simplicity, we will consider them in terms tors can be involved in steel corrosion, so that of chemolithoautotrophs, chemoheterotrophs, structures underground are similarly susceptible and fungi. to damage (e.g., pipelines, storage tanks, etc.). Even protons can be reduced because of the very 5.3.1 Chemolithoautotrophs negative electrode potential of Fe0 (reaction (5.39)). Hence, in principle, protons repres- Most chemolithoautotrophs play some role in ent constant potential electron acceptors for metal corrosion. The importance of S- and anaerobic iron corrosion. N-oxidizing bacteria lies in their formation of sulfate and nitrate, and hence sulfuric and nitric 0 + + → 2+ + Fe 2H Fe H2 (5.39) acids, respectively. The degradation of concrete sewers or the deterioration of stone monuments Under normal circumstances Fe0 does not (see below) are the most serious problems corrode completely. Instead, as Fe2+ forms and associated with their growth. dissolves away from the surface, negative charges The role of Fe(II)- and Mn(II)-oxidizing are left on the surface. They are strongly reducing bacteria in steel corrosion is based on their

and, in the presence of O2, rust forms, while ability to form cathodic Fe(OH)3 and MnO2, in anoxic waters, they reduce protons from the respectively. The process is as follows. During dissociation of water to form a protective film the initial stages of steel corrosion, cathodic

of adsorbed H2. These layers limit continued reduction of O2 causes an increase in solution corrosion by serving to some extent as passivity pH above the steel surface, which facilitates layers, or barriers, to further corrosion (Cord- oxidation and hydrolysis of the Fe2+ liberated Ruwisch, 2000). during corrosion. If sufficient amounts of ferric Microorganisms facilitate corrosion in a num- hydroxide form, then the anodic site may even- ber of ways, one of them being through the tually become isolated from the surrounding mechanism of cathodic depolarization, as initi- oxygenated cathode. When that occurs, ferric ally postulated by von Wolzogen Kuehr and hydroxide instead serves the cathode, accepting van der Vlugt (1934). In their theory, reaction electrons directly from the steel (Little et al., (5.39) will continue from left to right as long 1997). The extent of the current, in turn, is 0 as the H2 is continually removed from the Fe governed by the Fe mineralization rate because surface via consumption by various electron the current becomes self-limiting when the

accepting molecules, ranging from O2 to CO2. cathode (Fe(OH)3) is depleted via its reduction 2+ Some microorganisms further facilitate the (to Fe ), and O2 is re-established as the cathode. transfer of electrons from Fe0, by specific catalytic This impasse is overcome by the metabolism enzymes (e.g., hydrogenases), and in the pro- of the metal-oxidizing bacterial community that cess capture energy for growth, while others reside on the steel surface and within the dis- facilitate corrosion simply because they form solution pits. In addition, any soluble Cl− anions biofilms. The EPS primarily contributes to corro- will migrate to the anode to neutralize any build- sion by adsorbing and chelating metals, thereby up of charge, forming heavy metal chlorides that generating localized concentration gradients are extremely corrosive (Fig. 5.21). Given the that affect saturation states. They also promote heterogeneity of the corrosive environment, the establishment of cathodic and anodic sites pitting, rather than the even corrosion of the on the steel surface, that enhances electron flow surface, tends to occur. ITGC05 18/7/06 18:14 Page 232

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Oxygen-containing O2 water CI–

O EPS 0.5O + H+ OH– 2 2 Fe2+ 3+ Initial Fe cathode 1 2+ + 2 Fe H Fe(OH)3 Secondary cathode 2e– 1e–

Bacteria 0 Steel anode (Fe ) Dissolution pit 2+ – 1 4Fe + O2 + 8OH + 2H2O 4Fe(OH)3

– 2+ – 2 Fe(OH)3 + e Fe + 3OH

Figure 5.21 Model illustrating the role of Fe(II)-oxidizing bacteria in metal corrosion. Through their oxidative activity, they generate ferric hydroxide that functions as a cathodic surface, accepting electrons directly from steel. (Adapted from Little et al., 1997.)

5.3.2 Chemoheterotrophs surface (reaction (5.40)): strictly, it is not the sulfur atom that accepts the electrons from the Many studies have investigated the effects of corrosion process but the protons that are part aerobic bacterial activity on the corrosion of of the hydrogen sulfide molecule (Lee et al., iron. However, because the process also occurs 1995). at relatively high rates in the absence of micro- 0 + → + organisms, the microbial effect is not easy to Fe H2S FeS H2 (5.40) monitor or predict. In general, due to the oxygen uptake activity, bacterial biofilms on the metal Mackinawite formation has the effect of acce- surface create localized environments of differ- lerating the corrosion process because once ential aeration that generate cathodic areas electrical contact is established, the steel may 0 (where electrons from Fe reduce O2) spatially behave as an anode, facilitating electron transfer separated from the anodic areas (where ferrous through the cathodic iron sulfide phase, i.e., iron dissolves), resulting in a corrosion current acting as a galvanic cell (Wikjord et al., 1980). and the dissolution of the metal (e.g., Morales If the Fe2+ concentration in solution is low, et al., 1993). the mackinawite alters to greigite, as previously What is likely the most important group covered in section 4.1.10. of anaerobic heterotrophs, in terms of their SRB can directly cause corrosion via their corrosive capabilities, are the SRB (Fig. 5.22). hydrogenase activity, as follows. Hydrogenase

Through their production of H2S, they in- accepts H2 and releases protons. Those protons directly promote corrosion by forming a thin then attack the steel surface, causing its oxida- layer of iron sulfide (mackinawite) on the metal tion and the release of Fe2+, while simultaneously ITGC05 18/7/06 18:14 Page 233

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2– H2SSO4

H S 2 SRB 1 Figure 5.22 Model showing 3 how sulfate-reducing bacteria 4Fe2+ H H S H facilitate the corrosion of steel 2 2 2 2 + through (1) cathodic depolarization, 4 4H2 8H + 2H 2+ FeS Cathode (2) anodic depolarization, (3) H2S Fe production, and (4) indirect supply of H+. (Adapted from Cord-Ruwisch, Steel – Fe0 – 2000.) (anode) 8e

2+ + → + + accepting electrons from the anode to form H2 Fe H2S FeS 2H (5.43) (reaction (5.41)): Although not related to metal corrosion, 0 + + → 2+ + Fe 2H Fe H2 (5.41) bacterial sulfate reduction can lead to the destruction of gypsum deposits. Their activity If an appropriate electron acceptor is then avail- is known to limit the preservation of primary

able to accept H2 (e.g., reaction (5.42)), then gypsum in sediments (Rouchy and Monty, 2000), the hydrogenase once again becomes available to and considering that a number of antiquities

oxidize more H2 generated at the metal surface; are composed of alabaster (a hydrated form of the dissolving process would cease if the H2 was gypsum), this can have important archaeolo- not removed, for reasons discussed above. gical implications. On a different note, industry employs gypsum-degrading SRB to convert + 2− → + + − 4H2 SO4 H2S 2H2O 2OH (5.42) gypsum sludge, produced during flue gas desul- furization, into marketable calcite and elemental By virtue of their resistance to degradation, hydro- sulfur, while simultaneously oxidizing sewage genases may remain viable for months after the sludge as their organic substrate (Kaufman et al., cell is dead, implying that even lysed cells can 1996). continue the corrosion process (Bryant and Under conditions where nitrate serves as the Laishley, 1989). Thus, in terms of the cathodic terminal electron acceptor, some bacteria, such as depolarization theory, SRB trigger the oxidation E. coli, also promote the oxidation of elemental

of metal by removing the protective H2 layer iron through a similar hydrogenase model as and linking the electron flow from the metal to described above (Umbreit, 1976). In recent years,

the metabolic reduction of sulfate, with H2 as methanogens have similarly been added to the the electron donor (Cord-Ruwisch and Widdel, list of microorganisms believed responsible for 1986). Additionally, the hydrogen sulfide gener- corrosion. Like many SRB, methanogens consume ated via sulfate reduction reacts with Fe2+ to form hydrogen and thus are capable of performing FeS and H+ (reaction (5.43)), thereby removing cathodic depolarization-mediated oxidation of Fe2+ from solution and driving reaction (5.41) to elemental iron to produce methane (Boopathy form more products. This is anodic depolarization and Daniels, 1991). Because methanogenesis by Fe2+ removal. The protons, in turn, repeat the involves proton consumption, the overall reac- oxidative attack on the steel surface. tion (5.44) will be affected by the acidity of the ITGC05 18/7/06 18:14 Page 234

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aqueous solution, i.e., at lower pH the reaction becomes more energetically favorable: 5.4 Summary

0 + + + → 2+ + + 4Fe CO2 8H 4Fe CH4 2H2O Microorganisms play an important role in (∆G° =−136 kJ/CH at pH 7 and 37°C) (5.44) 4 accelerating mineral dissolution and oxidation ∆ reactions. Through their production of organic The above reaction has the same G° as previ- + ously shown for methanogens using reaction acids, they supply H ions to attack metal– − oxygen bonds and systematically dissolve the (2.46), that is 136 kJ per mol CH4 at pH 7. As both the SRB and methanogen examples atoms comprising the crystal lattice. Mean- have shown, anodic dissolution of Fe0, when while, the deprotonated organic anions complex with metal cations, thereby affecting mineral coupled only to H2 production, is not energet- ically favorable. This means that the oxidative saturation states, promoting even greater min- reaction must be coupled to cell growth of a micro- eral dissolution. Other microorganisms produce chelates that act in a similar manner to the organism that consumes the H2. In this regard, even Fe(III)-reducing bacteria can accelerate the organic acids. Even upon death, microorganisms rate of corrosion (Iverson, 1987). are important agents in weathering because their decay, via the action of respiring heterotrophs,

5.3.3 Fungi leads to elevated soil CO2 partial pressures, which, in turn, creates carbonic acid. Collectively, these Most fungi are capable of producing organic acids processes have contributed to the erosion of that corrode steel and aluminum, as in the highly exposed outcrops, led to soil formation and publicized corrosion failures of aircraft fuel tanks. influenced global climate since the spread of Another significant corrosion problem is the microbial life onto land. Many microorganisms degradation of cement, and the resulting deteri- have also evolved the capacity to utilize the oration of building materials and nuclear waste energy released by the oxidation of reduced repositories. Minerals in cement, such as hydrated transition metals and sulfur phases. In the deep

calcium silicate and portlandite (Ca(OH)2), sea, the microbial oxidation of Fe(II) in basalts are readily solubilized and decalcified by fungal- likely contributes to the flux of solutes to the generated organic acids. In particular, the fungus bottom waters, while biological sulfide mineral Aspergillus niger seems to promote corrosion oxidation on land functions as the catalyst for through the production of acids within the pore the release of high concentrations of metals, sul- spaces physically created by its hyphae (Perfettini fate and acidity into regional waterways. These et al., 1991). Oxalic acid is also involved in same reactions have industrial implications. On the corrosion of ancient stonework by lichens. the one hand, the oxidative and acid-generating Physical and chemical changes in stonework, properties can be utilized in the biorecovery such as fracturing and encrustation, can lead to of economically valued metals from mine wastes biodegradation, with calcium oxalate being a or in the remediation of toxic metal concentra- significant chemical component in the surface tions in the environment. On the other hand, alteration zone (e.g., Edwards et al., 1994). Con- these same reactions contribute to the corro- comitantly, carbonic acid is generated through sion of various metal structures, necessitating aerobic respiration of lysed lichens, further con- significant financial expenditures on repairs and tributing to chemical weathering. preventative measures.