Pseudomonas putida Biofilm Mediated Chemical Weathering of Biotite

Thesis (Ph.D.), Department of Crop and Soil Sciences, Washington State UniversityA strain of Pseudomonas putida isolated from the rhizosphere of a white pine (Pinus strobus) seedling was grown in a biofilm drip-flow reactor on biotite and glass coupons under Fe-deficient and Fe-replete conditions. Our hypothesis was that Fe-deficiency, defined at concentrations below 10 µM, would enhance biofilm weathering of the mineral. The biofilm biomass, the specific number of viable cells (SNVC), and the biofilm cation concentrations of Fe, Mg, and K were all measured. Using confocal laser scanning microscopy (CLSM) and a fluorescent pH probe, we established the biofilm pH to be c.a. 4.5 whether grown on glass or biotite coupons. Scanning electron microscopy (SEM) revealed that the small, easy-to-access mineral particles present in abundance on the biotite surfaces prior to biofilm formation were dissolved away after biofilm growth, and sharp edges of score marks on the mineral were softened. Acid digestions of the biofilm revealed higher concentrations of Fe, Mg, and K, in addition to increased biofilm biomass, and more viable cells in the matrix of biofilms grown on biotite as compared to those formed on glass. When grown without Fe supplementation, compared to Fe-replete conditions both on biotite, the biofilm became a more effective cation sink for all three weathered nutrients and contained nearly twice as much polysaccharide determined via High Resolution Magic Angle Spinning proton nuclear magnetic resonance (HRMAS 1H NMR) spectroscopy. CLSM and attenuated total reflectance Fourier transformed infrared (ATR FT-IR) spectroscopy revealed the presence of alginate in the matrix of biofilms grown on biotite with or without iron supplementation, but increased amounts of alginate and the formation of alginate capsules were present in biofilms formed under iron depletion. Alginate has a high concentration of carboxylic acid functional groups and when present in greater quantities, it confers increased cation holding capacity upon the biofilm matrix. We conclude that oligotrophic soil environments can trigger a biofilm of Pseudomonas putida cells to produce larger quantities of alginate, resulting in a more effective sink for weathered nutritive cations that can be supplied to plant roots.Department of Crop and Soil Sciences, Washington State Universit