Geomicrobiology Journal, 22:207–218, 2005 Copyright c  Taylor & Francis Inc. ISSN: 0149-0451 print / 1362-3087 online DOI: 10.1080/01490450590947724 FTIR Spectroscopic Study of Biogenic Mn-Oxide Formation by Pseudomonas putida GB-1 Sanjai J. Parikh and Jon Chorover Department of Soil, Water, and Environmental Science, The University of Arizona, Tucson, Arizona 85721, USA Biomineralization in heterogeneous aqueous systems results from a complex association between pre-existing surfaces, bacte- rial cells, extracellular biomacromolecules, and neoformed precip- itates. Fourier transform infrared (FTIR) spectroscopy was used in several complementary sample introduction modes (attenuated total reflectance [ATR], diffuse reflectance [DRIFT], and transmis- sion) to investigate the processes of cell adhesion, biofilm growth, and biological Mn-oxidation by Pseudomonas putida strain GB-1. Distinct differences in the adhesive properties of GB-1 were ob- served upon Mn oxidation. No adhesion to the ZnSe crystal surface was observed for planktonic GB-1 cells coated with biogenic MnO x , whereas cell adhesion was extensive and a GB-1 biofilm was readily grown on ZnSe, CdTe, and Ge crystals prior to Mn-oxidation. IR peak intensity ratios reveal changes in biomolecular (carbohydrate, phosphate, and protein) composition during biologically catalyzed Mn-oxidation. In situ monitoring via ATR-FTIR of an active GB-1 biofilm and DRIFT data revealed an increase in extracellular pro- tein (amide I and II) during Mn(II) oxidation, whereas transmission mode measurements suggest an overall increase in carbohydrate and phosphate moieties. The FTIR spectrum of biogenic Mn ox- ide comprises Mn-O stretching vibrations characteristic of various known Mn oxides (e.g., “acid” birnessite, romanechite, todorokite), but it is not identical to known synthetic solids, possibly because of solid-phase incorporation of biomolecular constituents. The results suggest that, when biogenic MnO x accumulates on the surfaces of planktonic cells, adhesion of the bacteria to other negatively charged surfaces is hindered via blocking of surficial proteins. Keywords FTIR spectroscopy, biomineralization, Mn oxidizing bac- teria, Pseudomonas putida, Mn(IV) oxides, bacterial adhesion Received 16 September 2004; accepted 18 January 2005. We thank Dr. Bradley M. Tebo and Brian Clement for donation of GB-1 cells and providing information critical for cell growth and Mn-oxidation. We also thank Martha Conklin for useful discussions at early stages of this research and Hanna L. Gilbert for her assistance with SEM-EDS analysis. TEM-EDS assistance and analysis was provided by Sunkyung Choi and David Bentley. This research was supported by the National Science Foundation CRAEMS program (Grant CHE- 0089156). Address correspondence to Jon Chorover, Department of Soil, Water, and Environmental Science, University of Arizona, Tucson, AZ 85721, USA. E-mail: chorover@cals.arizona.edu INTRODUCTION Manganese is the second most abundant transition metal in the earth’s crust behind Fe and, like Fe, its oxidation-reduction reactions are largely mediated by biological activity (Lovley 2000). Microbial catalysis is known to accelerate the kinetics of Mn(II) oxidation and promote the formation of Mn(IV) ox- ide minerals in natural waters (Nealson et al. 1988; Tebo et al. 1997). Manganese oxides (MnO x ) are produced biogenically by numerous species of bacteria, including Pseudomonas putida strain GB-1, a fresh-water, facultative-aerobic, gram-negative bacteria. The formation of MnO x can influence the environmen- tal fate of other metals (e.g., Cu, Co, Cd, Zn, Ni, and Pb) through co-precipitation and adsorption reactions (Nelson et al. 1999, 2002; Tani et al. 2003, 2004; Tebo et al. 2004). The physiologi- cal basis for bacterially-mediated Mn oxidation is not known, but it is thought that biogenic MnO x may serve to protect cells from Mn or other metal toxicity (e.g., heavy metals), UV irradiation, or other potential threats (Brouwers et al. 2000). The identity of biogenic MnOx phases is diverse with appar- ent dependence on the type of microbial catalyst and conditions of formation. For example, Mandernack et al. (1995) reported mixed phase minerals (hausmmannite, Mn 3 O 4 ; feiknechtite, β - MnOOH; manganite, γ -MnOOH, and Na-buserite) following Mn(II) oxidation by a marine Bacillus strain SG-1, whereas a nanocrystalline todorokite-like mineral was produced by Lep- tothrix discophora strain SP-6 (Kim et al. 2003). The MnO x formed by P. putida strain MnB1 was found to be most similar to “acid” birnessite (Villalobos et al. 2003), whereas Mn oxide crusts from Pinal Creek, AZ comprise a mixture of todorokite and birnessite or takanelite/ranciete, possibly deriving from a buserite precursor (Bilinski et al. 2002; Gilbert 2003). The mechanisms of biotic Mn(II) oxidation and the subse- quent binding of MnO x to cell surfaces are not known. One or more enzymes is likely responsible for catalyzing Mn(II) oxida- tion. Recent research indicates that the cumA gene, a multicopper oxidase (Brouwers et al. 1999; Francis and Tebo 2001), and/or a general secretion pathway (xcp in Pseudomonas species) gene (de Vrind et al. 2003) are integrally involved in Mn oxida- tion. However, these genes have not been identified unambigu- ously. Although great progress has been made in identifying 207