1. PROTECTION OF MESOPORE-ADSORBED TYROSINE FROM MICROBIAL DEGRADATION BY PSEUDOMONAS AERUGINOSA Benjamin C. Stewart* 1, Susanne Daly 1, Ronna Thomsen 1, Andrew R. Zimmerman 2, and Carolyn F. Mathur 1 ( 1 York College of Pennsylvania, Department of Biological Sciences and 2 University of Florida, Department of Geological Sciences) Introduction Mesopores are nanometer- sized pores found in soils and sedimentary rocks. Organic materials can be adsorbed into mesopores Adsorption of organic material to mesoporous rocks is better than adsorption to non-porous rocks. (Zimmerman) Sequestration of petroleum products into mesopores could be a mechanism to prevent natural microbial degradation of this vital energy source. The bacterium Pseudomonas aeruginosa (Pa) can grow in the presence of a single amino acid (such as tyrosine) as an energy source. (Thompson, Daly) Pa can grow as both planktonic (suspended cells) and biofilm (attached) cultures. Tyrosine has an aromatic structure that can adsorb to mesoporous alumina (Zimmerman). Alumina is a common mineral in soils and rocks. The “Mesopore Exclusion Hypothesis” states that mesopores sequester organic compounds from microbial attack. In order to determine if mesopore-adsorbed organic material can be prevented from supporting microbial growth, we developed a microbe-nutrient model using Pa and tyrosine. Methods 0.2g MP-Al + PA 0.2g MP-Al +tyr +PA 1.0g NP-Al +tyr +PA 1.0g NP-Al +PA 0.2g NP-Al +PA 0.2g NP-Al +tyr +PA PA was grown with different combinations of mesoporous and non-mesoporous alumina with tyrosine (0.6-2.0 mM), and placed within well culture plates containing a calcium chloride buffer (10ml) using the protocol in Fig. 4. Wells were incubated at 30 C, and shaken at 50 rpm 7 days. Planktonic cells were harvested from the wells using a pipette. The remaining, attached biofilm cells were stained with crystal violet, excess stain washed, and the remaining stain eluted with ethanol. The presence of planktonic and biofilm cells was measured spectrophotometrically. Relative absorbances were compared to indicate Pa growth under the different experimental conditions. Results Fig. Biofilm growth was only present in wells containing non-porous minerals and tyrosine (2C and 3C). No biofilm formation occurred in wells containing mesoporous minerals, both with & without tyrosine. * Fig. Planktonic growth occurred in the high concentration(2C), nonporous wells containing Pa and tyrosine, and (2B) containing Pa with no tyrosine. There was also significant growth in the NP low concentration (3C) wells, containing Pa and tyrosine. No planktonic growth occurred in wells containing mesoporous minerals, both with & without tyrosine. * * See Legend in Figure 4 Conclusions Pa can grow as both planktonic and biofilm cultures with tyrosine as the sole energy source. Pa does not grow as planktonic or biofilm cultures when tyrosine is adsorbed into mesoporous alumina. Pa does grow as both planktonic and biofilm cultures when tyrosine is adsorbed to non- mesoporous alumina. These observations support our hypothesis and the mesopore exclusion hypothesis. Literature Cited Lewis, K. 2001. Mini Review: Riddle of Biofilm Resistance. Antimicrobial Agents and Chemotherapy. 45: 999- 1006. Mayer, L., et al. 2004. Organic Matter in Small Mesopores in sediments and Soils. Geochemica et Cosmochimica Acta. 68: 3863-3872. Nam, K. and Alexander, M. 1998. Role of Nanoporosity and Hydrophobicity in Sequestration and Bioavailability: Tests with Model Solids. Environmental Science & Technology. 32: 71-74. Zimmerman, A.R., et al. 2003. The Effect of Mineral Mesopores on Organic Matter Adsorption. For Submission to: Organic Geochemistry Fig. 2 Tyrosine structure Hypothesis Pseudomonas aeruginosa can not utilize the nutrient tyrosine for growth when it is adsorbed into mesoporous alumina. Planktonic Cells Biofilm Cells 1 2 3 ABC Fig. 3 Planktonic cells are free- floating and biofilm cells are attached to surfaces Fig. 1 Molecular view of mesopores Fig. 4 Well plate setup. MP-Al = mesoporous alumina; NP-Al = nonporous alumina; +tyr = with adsorbed tyrosine; +Pa = with Pa Fraunhofer-Gesellschaft