Environ. Sci. Technol. 2008, 42, 3194–3200
Bacillus subtilis Bacteria Hinder the Oxidation and Hydrolysis of Fe2+ Ions MOHAMAD FAKIH, XAVIER CHÂTELLIER,* MÉLANIE DAVRANCHE, AND ALINE DIA University of Rennes 1, CNRS, Géosciences Rennes, UMR 6118, Avenue du Général Leclerc, 35042 Rennes Cedex, France
Received October 4, 2007. Revised manuscript received January 30, 2008. Accepted February 4, 2008.
Bacteria are known to associate closely with secondary iron oxides in natural environments, but it is still unclear whether they catalyze their precipitation. Here, Fe2+ ions were progressively added to various concentrations of Bacillus subtilis bacteria in permanently oxic conditions while maintaining the pH at 6.5 by adding a NaOH solution at a monitored rate. The iron/ bacteria precipitates were characterized by wet chemistry, SEM, and XRD. Abiotic syntheses produced nanolepidocrocite, and their kinetics displayed a strong autocatalytic effect. Biotic syntheses led to the formation of tiny and poorly crystallized particles at intermediate bacterial concentrations and to a complete inhibition of particle formation at high bacterial concentrations. The occurrence of the autocatalytic effect was delayed and its intensity was reduced. Both the oxidation and the hydrolysis of Fe2+ ions were hindered.
Introduction Bacteria affect the cycling of metals by sorption processes (1–4). They also influence the formation and dissolution of minerals, including iron oxides, which can themselves adsorb many organic and inorganic molecules (5–9). Specific bacteria thrive in environments where iron oxides massively precipitate, such as iron springs or acid mine drainage (10–15). More generally, secondary iron oxides are commonly found in close association with all sorts of microorganisms through nonmetabolic processes (16–19). It has been suggested that bacterial cell walls or exopolymers act as templates, which catalyze the nucleation and growth of iron oxide crystals with specific properties (18–25). A few laboratory studies investigated the exposition of bacterial cells to Fe3+ ions (25–27). A continuum between sorption and precipitation of various bacterial cells exposed to Fe3+ ions was observed, with mixed conclusions as to whether the cells were favoring the precipitation of the Fe oxides. The study of the effect of natural organic matter on the kinetics of oxidation of Fe(II) has also led to different conclusions, depending on the type of ligands considered (28, 29). Here, we exposed increasing concentrations of Bacillus subtilis, a model Gram-positive bacterium, which has been used in several studies investigating the interactions between Fe and bacterial surfaces (25–27, 30–33), to progressive additions of Fe2+ ions at constant pH. In addition to the characterization of the precipitates by SEM and XRD, we measured here over time several parameters related to the kinetics of the reaction, such as the rate of proton release and the redox potential. * Corresponding author e-mail:
[email protected]. 3194
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 9, 2008
The dissolved organic carbon (DOC) and Fe(II)/Fe(III) concentrations were determined at selected times. Although metabolic effects were not the focus of this study, we also monitored cell death through enumerations.
Materials and Methods Synthesis of the Fe/Bacteria Composite Suspensions. Bacillus subtilis cells were grown to exponential phase and washed 6 times in 0.01 M NaCl (see section S1 of the Supporting Information for more details). The C and N weight contents of the dry bacteria were determined by pyrolysis at 1800 °C and gas chromatography, using a CN Flash EA 1112 analyzer, and they were found to be equal to 43.7 and 12.1%, respectively. This was used to estimate the total C concentration in the bacterial suspensions (e.g., 568 mg/L of C for 1.30 g/L of bacteria). A Fe(II) stock solution was prepared in 2.5 × 10-3 M HCl ([FeCl2] ) 1.25 × 10-2 M, or 0 for the blank experiments). The bacterial stock suspension was diluted to the desired concentration (cb ) 0, 0.135, 0.350, or 1.30 dry g/L) in 500 mL of 0.01 M NaCl solution, and introduced in a thermostatted beaker set at 25 °C. Starting at the time t ) 0, 20 mL of the Fe(II) stock solution were added to the suspension at a rate of 0.05 mL/min, using an automated burette (Titrino 794, Metrohm). In parallel, the pH was continuously maintained at 6.5 by adding progressively 4.9 ( 0.7 mL of a 0.1 M NaOH solution, using a second automated burette programmed in a pH stat mode. The total final concentration of Fe was thus equal to approximately 480 µM. After the end of the addition of the Fe(II) solution (t ) 24000 s), the suspension was left in contact with the atmosphere at pH 6.5 for another 56 000 s. Each synthesis was replicated six times, using independently grown bacterial cultures. Three blank experiments were also performed at a bacterial concentration of 1.30 g/L. In one experiment for each type of synthesis, the O2 concentration and the redox potential were recorded. Scanning Electron Microscopy (SEM), X-Ray Diffraction (XRD), Wet Chemistry and Enumerations. In one experiment for each type of synthesis, the suspension was sampled at the times t ) 0, 25 000, and 80 000 s. DOC and dissolved Fe(II)/Fe(III) concentrations were measured using a carbon analyzer and the ferrozine colorimetric method, respectively (34). Total Fe(II) was estimated with the same method, but after an addition of acid to bring the pH down to 2 for one hour first. This was needed to recover the Fe(II) adsorbed onto the bacteria. At the end of the reaction, the Fe/bacteria suspensions were also sampled, washed, observed by SEM, and analyzed by XRD. For one blank experiment and one Fe experiment at 1.30 g/L, which were performed simultaneously using the same bacterial culture, enumerations of the colony forming units (c.f.u.) were realized. The suspensions were also autoclaved at t ) 80 000 s, and the DOC was then measured. More information on the analytical protocols can be found in section S2 of the Supporting Information. Kinetics and Stoichiometry. The normalized rate of proton release v(t), averaged for each period of 1000 s, was calculated as follows: v(t) )
N(OH-) - N(H+) N(Fe2+)
(1)
where N(H+) and N(OH-) were the number of moles of H+ and OH- added during the period of 1000 s considered, and N(Fe2+) ) 1.04 × 10-5 was the number of moles of Fe2+ added per 1000 s for t < 24000 s. The final stoichiometry of the reaction was also calculated by determining how many OH10.1021/es702512n CCC: $40.75
2008 American Chemical Society
Published on Web 04/05/2008
TABLE 1. Chemical and Bacteriological Analyzes of the Suspensions and Final Stoichiometries of the Synthesesa sampling time (seconds)
abiotic
0 25 000 80 000
NA NA NA
0 25 000 80 000 autoclaved
NA NA NA NA
0 25 000 80 000 autoclaved
NA NA NA NA
25 000 80 000
1.0 24 000 s, v(t) decreased much more slowly than in any of the other systems. At t ) 50 000 s, it was larger than 0.1. The measurements of the redox potential in the biotic systems were consistent with the results displayed in Figure 2 for the rate of proton release. Initially the rapid decrease of the redox potential in all systems was suggestive of a build up of the dissolved Fe(II) concentration in the solution. However, sorption processes and the reactions of oxidation and of hydrolysis of the Fe led to a stabilization of the redox potential, which was in all cases comprised between 215 mV and 250 mV for 4000 s < t < 24 000 s. Between t ) 24 000 s and t ) 25 000 s, the redox potential sprang back of 30, 23, and 4 mV, for the syntheses prepared with 0.135, 0.350, and 1.30 g/L, respectively, in comparison with a jump of 42 mV for the abiotic system. In agreement with the chemical analyzes, this suggests that residual Fe(II) lingered for longer periods of time in the biotic 3198
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 9, 2008
systems and that its oxidation was increasingly slowed by the presence of increasing concentrations of bacteria. Complexation of the Fe2+ ions onto the bacterial reactive sites of the cell walls or of the exuded molecules inhibited their quick build-up as free ions in solution. This explains at least to some extent the retardation of the overshoot in the biotic kinetic curves, as well as the slow ending of the reaction at t > 24 000 s. Complexation is regulated by a reversible thermodynamic equilibrium. Hence, as free Fe cations were reacting irreversibly with the iron oxide nuclei or crystals, the equilibrium was moving in favor of the dissociation of still complexed Fe2+ cations. As the bacterial concentration was increasing, the number of bacterial reactive sites was also increasing, moving the equilibrium in favor of the complexation of the Fe2+ or Fe3+ cations and thereby retarding the reactions of hydrolysis with the iron oxide nuclei or particles. Our study indicates that the complexation of Fe2+ cations onto bacterial reactive sites can drastically affect their kinetics of oxidation and hydrolysis, which can be retarded by many hours. At a bacterial concentration of 0.135 g/L, the kinetic curve was similar to the abiotic curve, except that it was shifted in time by about 5000 s, which corresponded to a Fe addition of about 100 µM. The bacterial surfaces were able to adsorb around 45 µM of Fe at most (30). On the other hand, the measured dissolved Fe concentration at t ) 25 000 s was equal to only 7.3 µM in the system at 0.135 g/L, in comparison with 2.7 µM for the abiotic system. Since the DOC concentrations were higher at t ) 25 000 s than at t ) 0, this suggests that the exuded molecules initially present in the suspensions at 0.135 g/L were able to immobilize at most 5–10 µM of Fe. Hence, Fe2+ complexation was likely able to immobilize at most about 55 µM of Fe and to delay the kinetic curves by at most about 3000 s. Hence, other processes also possibly played a significant retardation role on the reaction kinetics of the biotic syntheses at 0.135 g/L. For instance, newly formed iron oxide nuclei might have adhered onto and interacted with the bacterial cell walls. In this case, their reactive sites were unavailable or less easily accessible to dissolved Fe ions for the reaction of hydrolysis. In addition, the physical immobilization of the Fe(III) monomers or oligomers on the cell walls also reduced their ability to react with each other. These two suggestions are supported by the SEM observations, which indicated that most of the iron oxide particles were in close association with the cell walls (Figure 1). In the case of the biotic syntheses at 1.30 g/L, the inhibiting effect of the cell walls on the growth of the crystal nuclei was particularly spectacular. However, even though the stoichiometry of those syntheses was significantly lower than 2, it was still equal to 1.60. Since Fe2+ complexation onto bacteria is known to be a relatively rapid phenomenon (30), and since Fe(II) oxidation alone consumes protons (43), the addition of a significant fraction of the base over a long period of time after the end of the addition of the Fe(II) suggests that the hydrolysis of the Fe(III) had proceeded to a significant extent at t ) 80 000 s in the systems prepared with cb ) 1.30 g/L. We thus believe that Fe in those systems occurred probably mainly as oligomers of Fe(III) atoms. Further work, using, for instance, EXAFS of Mössbauer spectroscopy, would be interesting to characterize more precisely the speciation of the Fe immobilized by the bacterial cells walls and exuded molecules.
Acknowledgments We thank S. Langley for performing the XRD measurements and Professor D. Fortin for graciously supporting the associated cost. Support in the laboratory is also acknowledged from O. Henin and P. Petitjean. Two anonymous reviewers and the editors are thanked for their suggestions.
Finally, the financial support of the CNRS (program “ECCO”) allowed the realization of this study.
Supporting Information Available Additional details concerning the preparation of the bacterial suspensions (section S1); analytical procedures (section S2); bacterial enumerations (Table S1); v(t) for blank experiments (Figure S1); XRD patterns (section S3 and Figure S2); stoichiometries (Figure S3); dissolved oxygen concentration measurements (Figure S4). This information is available free of charge via the Internet at http://pubs.acs.org.
Literature Cited (1) Guine, V.; Spadini, L.; Sarret, G.; Muris, M.; Delolme, C.; Gaudet, J. P.; Martins, J. M. F. Zinc sorption to three gram-negative bacteria: combined titration, modeling, and EXAFS study. Environ. Sci. Technol. 2006, 40, 1806–1813. (2) Fein, J. B.; Daughney, C. J.; Yee, N.; Davis, T. A. A chemical equilibrium model for metal adsorption onto bacterial surfaces. Geochim. Cosmochim. Acta 1997, 61, 3319–3328. (3) Daughney, C. J.; Fein, J. B. The effect of ionic strength on the adsorption of H+, Cd2+, Pb2+, and Cu2+ by Bacillus subtilis and Bacillus licheniformis: a surface complexation model. J. Colloid Interface Sci. 1998, 198, 53–77. (4) Burnett, P.-G. G.; Heinrich, H.; Peak, D.; Bremer, P. J.; McQuillan, A. J.; Daughney, C. J. The effect of pH and ionic strength on proton adsorption by the thermophilic bacterium Anoxybacillus flavithermus. Geochim. Cosmochim. Acta 2006, 70, 1914–1927. (5) Dixit, S.; Hering, J. G. Comparison of arsenic(V) and arsenic(III) sorption onto iron oxide minerals: implications for arsenic mobility. Environ. Sci. Technol. 2003, 37, 4182–4189. (6) Gao, Y.; Mucci, A. Acid base reactions, phosphate and arsenate complexation, and their competitive adsorption at the surface of goethite in 0.7 M NaCl solution. Geochim. Cosmochim. Acta 2001, 65, 2361–2378. (7) Randall, S. R.; Sherman, D. M.; Ragnarsdottir, K. V.; Collins, C. R. The mechanism of cadmium surface complexation on iron oxyhydroxide minerals. Geochim. Cosmochim. Acta 1999, 63, 2971–2987. (8) Spadini, L.; Schindler, P. W.; Charlet, L.; Manceau, A.; Ragnarsdottir, K. V. Hydrous ferric oxide: evaluation of Cd-HFO surface complexation models combining Cd-K EXAFS data, potentiometric titration results, and surface site structures identified from mineralogical knowledge. J. Colloid Interface Sci. 2003, 266, 1–18. (9) Ferris, F. G.; Hallberg, R. O.; Lyvén, B.; Pedersen, K. Retention of strontium, cesium, lead and uranium by bacterial iron oxides from a subterranean environment. Appl. Geochem. 2000, 15, 1035–1042. (10) Emerson, D. Microbial oxidation of Fe(II) and Mn(II) at circumneutral pH. In Environmental Microbe-Metal Interactions; Lovley, D. R., Ed.; ASM Press: Washington, DC, 2000. (11) Emerson, D.; Weiss, J. V.; Megonigal, J. P. Iron-oxidizing bacteria are associated with ferric hydroxide precipitates (Fe-plaque) on the roots of wetlands plants. Appl. Environ. Microbiol. 1999, 65, 2758–2761. (12) James, R. E.; Ferris, F. G. Evidence for microbial-mediated iron oxidation at a neutrophilic groundwater spring. Chem. Geol. 2004, 212, 301–311. (13) Anderson, C. R.; Pedersen, K. In situ growth of Gallionella biofilms and partitioning of lanthanides and actinides between biological material and ferric oxyhydroxides. Geobiol. 2003, 1, 169–178. (14) Fortin, D.; Davis, B. S.; Beveridge, T. J. Role of Thiobacillus and sulfate-reducing bacteria in iron biocycling in oxic and acidic mine tailings. FEMS Microbiol. Ecol. 1996, 21, 11–24. (15) Baker, B. J.; Banfield, J. F. Microbial communities in acid mine drainage. FEMS Microbiol. Ecol. 2003, 44, 139–152. (16) Jackson, T. A.; West, M. M.; Leppard, G. G. Accumulation of heavy metals by individually analyzed bacterial cells and associated nonliving material in polluted lake sediments. Environ. Sci. Technol. 1999, 33, 3795–3801. (17) Fortin, D.; Leppard, G. G.; Tessier, A. Characteristics of lacustrine diagenetic iron oxyhydroxides. Geochim. Cosmochim. Acta 1993, 57, 4391–4404. (18) Schultze-Lam, S.; Fortin, D.; Davis, B. S.; Beveridge, T. J. Mineralization of bacterial surfaces. Chem. Geol. 1996, 132, 171– 181.
(19) Fortin, D.; Ferris, F. G. Precipitation of iron, silica, and sulfate on bacterial cell surfaces. Geomicrobiol. J. 1998, 15, 309–324. (20) Fortin, D. What biogenic minerals tell us. Science 2004, 303, 1618–1619. (21) Fortin, D.; Ferris, F. G.; Beveridge, T. J. Surface-mediated mineral development by bacteria. In Geomicrobiology: Interactions between Microbes and Minerals; Banfield, J. F., Nealson, K. H., Eds.; Mineralogical Society of America: Washington DC, 1997, 35, 161–180. (22) Mavrocordatos, D.; Fortin, D. Quantitative characterization of biotic iron oxides by analytical electron microscopy. Am. Mineral. 2002, 87, 940–946. (23) Ferris, F. G.; Schultze, S.; Witten, T. C.; Fyfe, W. S.; Beveridge, T. J. Metal interactions with microbial biofilms in acidic and neutral pH environments. Appl. Environ. Microbiol. 1989, 55, 1249–1257. (24) Chan, C. S.; de Stasio, G.; Welch, S. A.; Girasole, M.; Frazer, B.; Nesterova, M.; Fakra, S.; Banfield, J. F. Microbial polysaccharides template assembly of nanocrystal fibers. Science 2004, 303, 1656– 1658. (25) Warren, L. A.; Ferris, F. G. Continuum between sorption and precipitation of Fe(III) on microbial surfaces. Environ. Sci. Technol. 1998, 32, 2331–2337. (26) Wightman, P. G.; Fein, J. B. Iron adsorption by Bacillus subtilis bacterial cell walls. Chem. Geol. 2005, 216, 177–189. (27) Rancourt, D. G.; Thibault, P. J.; Mavrocordatos, D.; Lamarche, G. Hydrous ferric oxide precipitation in the presence of nonmetabolizing bacteria: Constraints on the mechanism of a biotic effect. Geochim. Cosmochim. Acta 2005, 69, 553–577. (28) Rose, A. L.; Waite, T. D. Kinetic model for Fe(II) oxidation in seawater in the absence and presence of natural organic matter. Environ. Sci. Technol. 2002, 36, 433–444. (29) Santana-Casiano, J. M.; Gonzalez-Davila, M.; Rodriguez, M. J.; Millero, F. J. The effect of organic compounds in the oxidation kinetics of Fe(II). Mar. Chem. 2000, 70, 211–222. (30) Chatellier, X.; Fortin, D. Adsorption of ferrous ions onto Bacillus subtilis cells. Chem. Geol. 2004, 212, 209–228. (31) Beveridge, T. J.; Murray, R. G. E. Sites of metal deposition in the cell wall of Bacillus subtilis. J. Bacteriol. 1980, 141, 876–887. (32) Châtellier, X.; Fortin, D.; West, M. M.; Leppard, G. G.; Ferris, F. G. Effect of the presence of bacterial surfaces during the synthesis of Fe oxides by oxidation of ferrous ions. Eur. J. Mineral. 2001, 13, 705–714. (33) Châtellier, X.; West, M. M.; Rose, J.; Fortin, D.; Leppard, G. G.; Ferris, F. G. Characterization of iron-oxides formed by oxidation of ferrous ions in the presence of various bacterial species and inorganic ligands. Geomicrobiol. J. 2004, 21, 99–112. (34) Viollier, E.; Inglett, P. W.; Hunter, K.; Roychoudhury, A. N.; Van Cappellen, P. The ferrozine method revisited: Fe(II)/Fe(III) determination in natural waters. Appl. Geochem. 2000, 15, 785– 790. (35) Cliff, J. B.; Jarman, K. H.; Valentine, N. B.; Golledge, S. L.; Gaspar, D. J.; Wunschel, D. S.; Wahl, K. L. Differentiation of spores of Bacillus subtilis grown in different media by elemental characterization using time-of-flight secondary ion mass spectrometry. Appl. Environ. Microbiol. 2005, 71, 6524–6530. (36) Santo, L. Y.; Doi, R. H. Ultrastructural analysis during germination and outgrowth of Bacillus subtilis spores. J. Bacteriol. 1974, 120, 475–481. (37) Bura, R.; Cheung, M.; Liao, B.; Finlayson, J.; Lee, B. C.; Droppo, I. G.; Leppard, G. G.; Liss, S. N. Composition of extracellular polymeric substances in the activated sludge floc matrix. Water Sci. Technol. 1998, 37, 325–333. (38) Schwertmann, U.; Taylor, R. M. Natural and synthetic poorly crystallized lepidocrocite. Clay Miner. 1979, 14, 285–293. (39) Liu, C.; Zachara, J. M.; Gorby, Y. A.; Szecsody, J. E.; Brown, C. F. Microbial reduction of Fe(III) and sorption/precipitation of Fe(II) on Shewanella putrefaciens strain CN32. Environ. Sci. Technol. 2001, 35, 1385–1393. (40) Roden, E. E.; Urrutia, M. M. Influence of biogenic Fe(II) on bacterial crystalline Fe(III) oxide reduction. Geomicrobiol. J. 2002, 19, 209–251. (41) Urrutia, M. M.; Roden, E. E. Microbial and surface chemistry controls on reduction of synthetic Fe(III) oxide minerals by the dissimilatory iron-reducing bacterium Shewanella alga. Geomicrobiol. J. 1998, 15, 269–291. (42) Daughney, C. J.; Fowle, D. A.; Fortin, D. The effect of growth phase on proton and metal adsorption by Bacillus subtilis. Geochim. Cosmochim. Acta 2001, 65, 1025–1035. (43) Cornell, R. M.; Schwertmann, U. The Iron Oxides; Wiley-VCH: Weinheim, 2003. VOL. 42, NO. 9, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3199
(44) Tamura, H.; Kawamura, S.; Hagayama, M. Acceleration of the oxidation of Fe2+ ions by Fe(III)-oxyhydroxides. Corros. Sci. 1980, 20, 963–971. (45) Tüfekci, N.; Sarikaya, H. Z. Catalytic effects of high Fe(III) concentrations on Fe(II) oxidation. Water Sci. Technol. 1996, 34, 389–396.
3200
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 9, 2008
(46) Williams, A. G. B.; Scherer, M. M. Spectroscopic evidence for Fe(II)-Fe(III) electron transfer at the iron oxide-water interface. Environ. Sci. Technol. 2004, 38, 4782–4790.
ES702512N
