This study has used STXM at a variety of absorption edges to map metal and organic species in a complex natural microbial biofilm grown in a model system that had been exposed to 10 mg L−1 Ni2+ for 24 h at the end of a 10-week incubation period. The results show that Ni was sorbed only on the sheaths of filamentous bacteria, which was also the location where high levels of Mn, Si, Ca and Fe were found. The Ni was not sorbed on the rod-shaped bacteria, unbound EPS or discrete particles of muscovite, SiO2 and CaCO3. The sorption of the Mn, Si, Fe and Ca (bio)minerals, as discussed below, likely occurred prior to exposing the biofilm to Ni. We interpret these results to indicate that sorption to (bio)minerals already formed on these filamentous sheaths was the major reason for the selective adsorption of Ni in the biofilm. To put the Ni sorption in a proper perspective, the (bio) mineralization within the sheath and the sheath organic sorption sites are discussed prior to discussing possible mechanisms of Ni sorption on to the sheaths and associated minerals.
Biominerals on the sheaths
The lack of a protein signal in the sheaths of the filamentous bacteria (Fig. 2A) indicated that they were devoid of bacterial cells, as cells contain roughly 50% protein (Ingraham et al., 1983). One of the main functions of sheaths is to protect the cell from being encrusted by minerals, through the shedding of mineral-encased sheaths (Hallberg & Ferris, 2004; Phoenix & Konhauser, 2008), which may be the reason for the discarded sheaths in the biofilm. Another function is to protect cells from toxic species, including metals (Benning et al., 2004). Ni is typically toxic at the 10 mg L−1 level towards micro-organisms, thus it is possible that the sheaths were shed in response to the sorption of Ni (Phoenix & Konhauser, 2008). The experimental design employed in this study did not allow us to determine how the sheaths were discarded.
Mineral formation by microbes involves complex interactions between metal ions in solution and reactive components of the biofilm (Drews & Weckesser, 1982; Douglas & Beveridge, 1998; De Yoreo & Vekilov, 2003). Metal ion binding to biological surfaces appears to be at least a two-step process. The first step is passive adsorption, whereby there is an electrostatic interaction between the metal ion and the surface reactive groups (e.g. carboxyl, phosphate) on the cell walls and/or outer external cell layers (e.g. EPS, sheath) (Weckesser et al., 1988; Urrutia et al., 1992). These surface functional groups are highly reactive towards metal ions on account of their amphoteric nature and low isoelectric point. The second step involves further metal deposition on the metal species initially bound to the surface, usually as a result of protonation and deprotonation reactions of surface hydroxyl or water groups (Miyata & Tazaki, 1997; Tazaki, 2000). With time and favorable thermodynamics, the process eventually leads to biomineral formation. Such biominerals are in turn capable of sorbing metal ions (Jackson et al., 1999). On bacterial surfaces, there are a variety of negatively charged functional groups, including amino, carboxylic, hydroxyl and phosphate (Ferris et al., 1987; Beveridge, 1989; Schultze-Lam et al., 1992; Ledin et al., 1999) that can serve as sites for sorption of metal cations. Also, some minerals carry a negative charge due to cation vacancies in oxide layers, as found in Mn(III) and Mn(IV) oxides (Lanson et al., 2002; Saratovsky et al., 2006; Villalobos et al., 2006), or due to isomorphic substitution in, for example, phyllosilicates (Caillerie et al., 1995), providing a driving force for cation sorption and intercalation. In addition to the negatively charged functional groups, bacterial surfaces also have groups with positive charges such as amino and amide groups that can serve as sorption sites for metal anions (e.g. SiO32−) (Urrutia & Beveridge, 1993, 1994). Also, sorbed cations on bacterial surfaces can sorb anionic species (e.g. CO32−), acting as cation bridges (Konhauser, 2007).
Thermodynamic equilibrium calculations using phreeqc indicated that the Saskatchewan River water was super-saturated with respect to the following species; aragonite (CaCO3), calcite (CaCO3), hydroxyapatite [Ca5(PO4)3OH], dolomite [CaMg(CO3)2], birnessite (MnO2), bixbyite (Mn2O3), hausmannite (Mn3O4), manganite (MnOOH), gibbsite [Al(OH)3], diaspore (AlOOH), ferric hydroxides [Fe(OH)3], goethite (FeOOH), hematite (Fe2O3), magnetite (Fe3O4), Ni(OH)2 and Ni2SiO4. Which species actually forms is of course a function of kinetic factors as well as thermodynamics, and the role of the microbes in bioaccumulation is played through their influence on the crystallization kinetics.
The sheaths of the filamentous bacteria in this study were found to bioaccumulate Mn species in the (II), (III) and (IV) oxidation states (Fig. 5). In natural waters, solid Mn(IV) oxides are ubiquitous (Shiller & Stephens, 2005). Toner et al. (2005) added Mn2+(aq) to Pseudomonas putida biofilms and observed that the Mn(II) was oxidized to solid Mn(III) and Mn(IV) oxides, which bioaccumulated on the bacterial surfaces. Many other laboratory studies have shown that Mn2+(aq) is oxidized by bacteria to Mn(III) and Mn(IV) oxides and bioaccumulated by bacterial surfaces (Bargar et al., 2000; Pecher et al., 2003; Jürgensen et al., 2004; Tebo et al., 2004). Generally, in those laboratory studies, there was a near-complete conversion of the Mn2+(aq) species to Mn(III) and Mn(IV) oxides, in contrast to this study, where a large amount of the Mn (40%) occurred in the Mn(II) oxidation state. The Mn concentration in solution was expected to be relatively constant for the entire 10-week growth period because the water was continually renewed (about 20% exchanged per day) as the bioreactor system was set up to simulate the adjacent river. Thus, there was a constant supply of Mn, presumably as Mn2+(aq) species, to the biofilm. Bargar et al. (2005) showed that Mn(II) could act as a reductant towards biogenic Mn(IV) oxides as it is readily sorbed by them (Adams & Ghiorse, 1988; Nelson et al., 1999; Haack & Warren, 2003), resulting in mixed Mn oxidation (III, IV) products. This fact, and the continued exposure of the biofilm to Mn2+(aq) species, may account for our observations. Our results also provide evidence that Mn(III) and Mn(IV) (bio)minerals are formed in nature at low Mn concentrations (0.4 μm) (Fig. 5, Table 1); previous laboratory studies had shown the same phenomenon but Mn concentrations of 10 μm to 12 mm were used (Bargar et al., 2000, 2005; Villalobos et al., 2003; Toner et al., 2005).
Both Fe(II) and Fe(III) species were detected; however, Fe(III) was the dominant Fe species found on the sheaths. Ferric iron is known to bind tenaciously to bacterial surfaces and form insoluble biominerals (Konhauser, 1998; Warren & Ferris, 1998; Konhauser & Urrutia, 1999; Banfield et al., 2000; Chan et al., 2004). Thus, ferric oxyhydroxide formation on bacterial surfaces is widespread in nature, particularly in oxygenated environments (Konhauser & Urrutia, 1999). The ferric iron may originate from cell-bound ferrous iron (Châtellier et al., 2004), where it undergoes oxidation and hydrolysis, a process recently studied using STXM and other techniques by Miot et al. (2009). Other sources of Fe(III) include sorption of soluble ferric or colloidal species, or from the spontaneous oxidation of ferrous species that come into contact with oxygen, with the bacteria serving as the nucleation sites. The relatively large amount of ferric iron sorbed to the sheaths seems to preclude cell-bound ferrous iron as a significant source. The water used in this study was initially well oxygenated and remained as such in the rotating annular reactor. Thus, soluble ferric species should be present, although ferric colloidal species may dominate as the pH of South Saskatchewan River water was 8.5 (Ferris et al., 1989a,b). Previous studies have demonstrated that bacterial surfaces bioaccumulate ferric species under acid conditions (Ferris et al., 1989a,b; Warren & Ferris, 1998). However, at near-neutral pH bioaccumulation of Fe is much higher than at low pH. Under these oxygenated conditions, Fe(II) is expected to be readily oxidized to Fe(III) (Konhauser, 2007). In natural waters, Fe(II) can be stabilized through complexation with organic compounds (Theis & Singer, 1974), which may account for the presence of Fe(II) species in this region of the biofilm. Note that many rivers, including the South Saskatchewan river, receive anoxic groundwater containing organic chelated ferrous iron. Another possibility is that some of the Fe(II) species are stabilized in lower pH, or anoxic micro-environments, a situation that has been documented to occur in Pseudomonas aeruginosa biofilms (Hunter & Beveridge, 2005; Hunter et al., 2008). In summary, it could not be determined whether the Fe(III) bioaccumulation by the sheath was from sorption of soluble Fe(III) species, colloidal Fe species, or by oxidation of Fe(II) species. Moreover, Fe is often associated with Mn as a ferromanganese [Mn(II), Fe(II)] precipitate in natural systems (Dean et al., 1981; Konhauser, 2007), forming in an aerobic environment from the reduction of Mn(III), Mn(IV) and Fe(III) oxides bioaccumulated by cyanobacteria or algae. Tazaki (2000) examined natural microbial mats from river water using an electron microscope and observed that Fe–Mn oxides were sorbed only after the sorption of layer silicates. They proposed that bioaccumulated Mn(IV) oxides oxidize Fe(II) with a concomitant reduction to Mn(II), and that Fe(III) (ferrihydrate) was sorbed by the bacterial surfaces. Our observations appear to be consistent with this proposal since silicates were the dominant chemical component of the sheaths.
It has been proposed that aqueous silica is heterogeneously sorbed onto bacterial surface functional groups via three mechanisms: (i) hydrogen bonding, (ii) bonding to the positively charged functional groups and (iii) cation bridging (Urrutia & Beveridge, 1993; Schultze-Lam et al., 1995; Westall et al., 1995; Jones et al., 1997; Konhauser, 2007; Peng et al., 2007). Urrutia & Beveridge (1993, 1994) showed that Fe pretreatment of bacterial cells at pH 8 enhanced the binding of silicate, whereas at acidic values silicate was more favorably bound when the cells were not treated with Fe. Similar results have been obtained by other researchers (Scheidegger et al., 1993; Manceau et al., 1995; Fortin et al., 1998; Fein et al., 2002). Under such conditions, Konhauser & Urrutia (1999) suggested that Fe sorbs to the bacterial surfaces followed by Si and Al sorption. In this study, the pH of the river water was 8.5, and Fe(III) was sorbed by the sheath. From the analysis of the Si 1s image sequence (Fig. 8) and the detailed elemental analysis (Fig. 10), it is evident that the organo-silicate component dominated the composition of the sheath of the filamentous bacteria. The presence of the organosilicon spectroscopic signal indicates that a C–O–Si bond was present. Heinen (1965) used IR to study the interaction of Si with the bacteria Proteus mirabilis and also observed C–O–Si signals.
Ca2+ was bioaccumulated by the sheaths (Fig. 7), but there was relatively little carbonate (Fig. 2D). CaCO3 minerals are ubiquitous in nature (Riding, 2000; Braissant et al., 2003; Dittrich & Obst, 2004; Benzerara et al., 2006). It is well known that micro-organisms, particularly cyanobacteria, facilitate carbonate precipitation by the adsorption of Ca2+ cations to their cell surfaces and photosynthetic uptake of HCO3− and the concomitant release of OH− under limited CO2 supply (Obst et al., 2006, 2009; Ercole et al., 2007; Lalonde et al., 2007a). That is, Ca2+ acts as a cation bridge between the negatively charged functional groups on the bacterial surface and the carbonate anion. The amount of Ca2+ on the sheath was much higher than the amount of carbonate. Obst et al. (2009) have shown that Ca2+ is sorbed by planktonic cyanobacteria cells on surface EPS. Aragonite-like CaCO3 was then nucleated and grown from the adsorbed Ca2+, and both coexisted on the cyanobacterial surface. Therefore, in this study, it is likely that both CaCO3 and adsorbed Ca2+ were present on the sheaths. Note that the formation of siderite (FeCO3) and rhodochrosite (MnCO3) was unlikely to have occurred in our system as these minerals usually form in anaerobic environments in the presence of microbes (Konhauser, 2007).
Although small, there was some Al detected on the sheaths. The low level of Al was surprising since Al-silicates are ubiquitous in natural environments (Konhauser et al., 1993, 1994; Konhauser & Urrutia, 1999; Peng et al., 2007). The fact that the aqueous Al concentration (5–20 μg L−1) in this study was 1–2 orders of magnitude lower than that of the other natural freshwater systems studied (Konhauser et al., 1993, 1994) may account for the low levels of Al in the biomineralized sheaths. The concentration of Mg (Table 1) in the natural water was significantly larger than most of the other metals, except Ca; however, it was not sorbed by the sheaths or the rod-shaped bacteria. This is attributed to the fact that Mg2+ is very soluble, i.e. it prefers to bind to a water molecule rather than a phosphate or carboxylate oxyanion (Collins, 2006).
Even though all of the biofilm components were exposed simultaneously to Ni under the same environmental conditions, the natural river biofilm selectively sorbed Ni only on the sheaths of the filamentous bacteria. Ni sorption was not apparent on the rod-shaped bacteria, the unbound EPS or on the discrete mineral particles (i.e. muscovite, SiO2, CaCO3). This stark contrast in Ni sorption capabilities by the various components led us to examine and compare the biochemistry and mineralogy of these components to understand the Ni sorption processes taking place in the biofilm. In solution, simple Ni2+ salts immediately dissociate to [Ni(OH2)6]2+ with the original ligands acting as non-coordinating or weakly coordinating counter-anions (Richens, 1997). STXM showed that the Ni species that sorbed to the sheath in this study was in the +2 oxidation state (Fig. 4), confirming that the Ni2+ cation was sorbed without oxidation or reduction. Bacterial surfaces have negatively charged surface functional groups that could serve as sites for adsorbing Ni2+ (Beveridge & Murray, 1980; Sar et al., 1999; Konhauser, 2007). Ni adsorption by the cell wall of a pure culture of the cyanobacteria Anabaena cylindrical has been demonstrated (Campbell & Smith, 1986). Our results (Figs 5–8 and 11) indicate that extensive (bio)mineralization on the sheath had occurred with a strong preference to the sheath surface. Jackson et al. (1999), studying Cu adsorption on benthic communities, not only showed that blockage of cell wall ligands by Si-, Al- and Fe-bearing mineral deposits occurs but also showed extensive sorption of Cu by the cell walls, although the sorption was weaker than that observed for the mineral phases. Other researchers (Beveridge & Murray, 1980; Doyle et al., 1980) studying pure bacterial cultures have shown that the introduction of positive charges into cell walls and/or alteration of the charge on carboxyl groups (making them neutral or electropositive) resulted in a decrease in the number of metal-binding sites, thus severely limiting metal sorption onto bacterial surface anionic groups. Templeton et al. (2001, 2003) used the long-period X-ray standing wave technique to probe the sorption of Pb2+ by Burkholderia cepacia biofilms formed on mineral surfaces (e.g. α-Al2O3, α-Fe2O3). At low-Pb concentrations (∼200 μg L−1), the reactive sites on the metal oxides were not passivated by the formation of the biofilm. That is, Pb was preferentially sorbed by the mineral surfaces rather than the biofilm surfaces. When the Pb concentration was increased tenfold, Pb was also sorbed by the biofilm surfaces. In our system, the sheaths were encrusted by the biominerals, whereas in the Templeton et al.’s system the minerals were covered by the biofilm. Thus, it may be expected that the biominerals were the preferred sorption sites. However, the Ni concentration used in this study was high (10 mg L−1), thus sorption to the surface groups on the sheath probably occurred. Nevertheless, even at this high Ni concentration, Ni sorption by the rod-shaped bacteria was not observed. In the case of the rod-shaped bacteria, the type and abundance of their surface anionic functional groups may have been significantly different than those on the sheath surface, so as to exclude Ni sorption (Konhauser, 2007).
We believe that the (bio)mineral(s) that formed on the sheaths prior to the addition of Ni were a major site for Ni sorption. Adsorption of bivalent metal ions on metals oxides has been documented (Tamura & Furuichi, 1997). The sheaths were encrusted by a number of metal species/(bio)minerals (Figs 5–8), all of which may have served as Ni sorption sites on the sheath. Note, however, that co-localization does not necessarily imply chemical interaction between Ni and the metals/(bio)minerals. The Ni–Mn color composite map (Fig. 11A) shows very clearly that the Ni and Mn distributions were very similar. Mn species occurred on the sheath as Mn(II), Mn(III) and Mn(IV) species (Fig. 5). Biogenic manganese oxides (Mn+4) are known to bioaccumulate many metals, including Ni (Tani et al., 2004; Tebo et al., 2004). Villalobos et al. (2005) indicated that organic sorption sites in biofilms do not appear to compete effectively with Mn(IV) oxides in scavenging trace metals such as Pb(II) and Mn(II). This observation may also be applicable to Ni2+. Ni2+ adsorption on MnO2 was shown to occur above pH 4, with maximum adsorption at pH > 7 (Tamura & Furuichi, 1997). In this system, the pH of the water was 8.5; thus, there should have been ample Mn(III) and Mn(IV) oxide sites available for Ni sorption. Besides these pH-dependent sites, Mn(III) and Mn(IV) oxides also carry a negative charge due to cation vacancies in the oxide layers (Saratovsky et al., 2006), and these sites appear to be favorable for Ni sorption. The extent of Ni(II) substitution for Mn(II) in manganese oxides is extremely limited (Kay et al., 2001); thus, it is unlikely that Ni substituted for Mn(II) in the oxides. Rather, we believe that Ni was adsorbed to the Mn(III) and Mn(IV) oxide biominerals on the sheath. Moreover, it was observed that Zn2+ sorption to the organic sites only occurred after depletion of the Mn sorption sites on the Mn(IV) oxides (Toner et al., 2006), supporting the contention that Ni has a preference for Mn over organic sorption sites. In addition, biogenic Mn oxides showed about tenfold higher efficiency than synthetic Mn oxides (γ-MnO2) for sorbing Ni2+, and also had higher irreversibility of Ni sorption on biogenic Mn oxides (Tani et al., 2004). The amount of Ni sorbed appears to be large compared with the amount of Mn(IV) oxides, which may be due to the formation of a Ni biomineral or that Ni was sorbed on other sites.
Fe(III) species were also present on the sheaths of the filamentous bacteria (Fig. 6). Ni2+ sorption by ferric minerals (e.g. hydrous ferric oxides, goethite) was 100% at pH > 7 (Fischer et al., 2007; Xu et al., 2007). At pH 8.5, the ferric iron species on the sheaths are expected to be deprotonated, thus Ni sorption was also likely on Fe(III) species on the sheaths.
The organo-silicate dominates the chemistry of the sheath (Fig. 8). Silica has been shown to adsorb Ni from solution (Xu & Axe, 2005). In the environment silica is often associated with iron oxides. Xu & Axe (2005) also showed that goethite coated with silica adsorbed Ni, and that both the silica and goethite surfaces were available for Ni adsorption. In fact, Ni sorption was greater on the goethite silica-coated material than on the sum of goethite and silica, apparently due to a change in the goethite-coated silica surface properties vs. that of the goethite and silica themselves. Hence, Ni sorption was also likely on organo-silicate species on the sheath.
Muscovite is a non-expandable clay, thus ion exchange is limited to the surface ions, i.e. the potassium and sodium ions in the interlayers are not exchangeable under ambient conditions (Osman et al., 1998). The muscovite in this study sorbed Fe(III), K+ and Ca2+ but the number of surface sites were apparently too low to result in significant Ni adsorption, and/or the sorbed cations were not readily exchangeable, or the effective Ni concentration was too low due to sorption to the other biominerals on the sheaths.