ATR-FTIR Spectroscopy Reveals Bond Formation During Bacterial Adhesion to Iron Oxide Sanjai J. Parikh and Jon Chorover* Department of Soil, Water and EnVironmental Science, The UniVersity of Arizona, 429 Shantz Building #38, Tucson, Arizona 85721 ReceiVed May 12, 2006. In Final Form: July 26, 2006 The contribution of various bacterial surface functional groups to adhesion at hematite and ZnSe surfaces was examined using attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy. When live Shewanella oneidensis, Pseudomonas aeruginosa, and Bacillus subtilis cells were introduced to a horizontal hematite (R-Fe 2 O 3 )- coated internal reflection element (IRE), FTIR peaks emerged corresponding to bacterial phosphate group binding. These IR peaks were not observed when bacteria were introduced to the uncoated ZnSe IRE. When cells were added to colloidal suspensions of R-Fe 2 O 3 at pH 7, spectra included peaks corresponding to P-OFe and ν(COOH), the latter being attributed to bridging of carboxylate at mineral surface OH groups. Selected model organic compounds with P-containing functionalities (phenylphosphonic acid [PPA], adenosine 5′-monophosphate [AMP], 2′-deoxyadenyl- (3′f5′)-2′-deoxyadenosine [DADA], and deoxyribonucleic acid [DNA]) produce spectra with similar peaks corresponding to P-OFe when adsorbed to R-Fe 2 O 3 . The data indicate that both terminal phosphate/phosphonate and phosphodiester groups, either exuded from the cell or present as surface biomolecules, are involved in bacterial adhesion to Fe-oxides through formation of innersphere Fe-phosphate/phosphonate complexes. 1. Introduction Contamination of soil and water from increased urbanization and industrialization threaten human and environmental health. Understanding the mechanisms controlling bacterial adhesion at mineral surfaces is critical for addressing environmental phe- nomena associated with the fate and transport of bacterial cells. These processes are central to both contamination and remediation of soil and groundwater supplies. 1-4 The chemical properties of bacteria and abiotic environmental surfaces influence their mutual adhesion in soils and aquatic systems. Long-range electrostatic forces can diminish adhesion when bacteria and substrate surfaces are of like charge. 5 In addition to electrostatic effects, short- range interactions are controlled by (1) chemical (covalent, ionic, hydrogen) bonding, (2) van der Waals forces, and (3) hydrophobic effects. 6 Microbial adhesion is also influenced by steric effects, which are particularly important for overlapping regions of polymer segments. 7 Steric effects can promote adhesion via bridging of surface macromolecules or inhibit it when biopolymer overlap is unfavorable. 8 Bacteria and many environmental particles exhibit net negative surface charge at pH values typically encountered in natural aqueous systems. 9 For example, quartz and silica are negatively charged at pH > 2.0-3.0. 10 Adhesion of bacteria to negative- charged (e.g., silicate and natural organic) surfaces is thought to be mediated via interaction with cell-surface proteins 11 or hydrophobic interactions. 12 However, in weathering environ- ments, many silicaceous surfaces become coated with a veneer of hydrous Fe oxide, which can confer net positive charge at circumneutral pH. 10 As a result, bacterial adhesion to Fe-oxides is often greater than that observed for silicates, with the difference being attributed to electrostatic attraction. 13-15 Although favorable electrostatics likely contribute to bacterial adhesion at positively charged surfaces, direct bonding of cell surface macromolecules at mineral surface functional groups may also play a role. 11,16-24 The exterior surface of bacterial cells is comprised of extracellular polymeric substances (EPS), teichoic acids (Gram-positive bacteria), lipopolysaccharides (LPS; Gram-negative bacteria), and membrane-bound proteins that can potentially form coordinative bonds with functional groups at mineral surfaces. Biomolecule-surface complexes have been shown to be important to “conditioning film” formation by siderophores and/or EPS, and covalent bonding interactions appear to affect strong adhesion. 25,26 EPS is itself a heterogeneous mixture of polysaccharides, proteins, lipids, and nucleic acids. 18,27 The presence of nucleic acids in EPS and biofilms results from * Corresonding author. E-mail: chorover@cals.arizona.edu. (1) Matthess, G.; Pekdeger, A. Sci. Total EnViron. 1981, 21, 149. (2) Banning, N.; Toze, S.; Mee, B. J. Microbiology (Reading, U.K.) 2003, 149, 47. (3) Abu-Ashour, J.; Lee, H. EnViron. Toxicol. 2000, 15, 149. (4) Corapcioglu, M. Y.; Haridas, A. J. Hydrol. 1984, 72, 149. (5) Marshall, K. C.; Stout, R.; Mitchell, R. J. Gen. Microbiol. 1971, 68, 337. (6) Marshall, K. C. AdV. Colloid Interface Sci. 1986, 25, 59. (7) Neu, T.; Marshall, K. J. Biomater. Appl. 1990, 5, 107. (8) Rijnaarts, H. H. M.; Norde, W.; Lyklema, J.; Zehnder, A. J. B. Colloids Surf. B 1999, 14, 179. 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