Biofilms are created by colonies of bacteria with polymeric substances produced by the cells. With a high-level of tolerance to a variety of stresses, biofilms cause serious problems in both industrial (biofouling and corrosion) and healthcare (persistent infections) settings. On the other hand, biofilms of environmentally friendly bacteria have promising applications in bioremediation and biofuel production. This project will develop a new method for investigating nutrient transport and enzyme activity of using Escherichia coli as a model organism.<br/><br/>The PIs have extensive research experience in E. coli metabolic analysis and biofilm physiology. This team will develop and test a new biofilm study method by integrating several interdisciplinary approaches: 1) culture synthetic biofilms with controlled morphology to reduce spatial variation in gene expression and enzymatic functions; 2) use microtome sectioning to obtain thin slides of biofilm samples with cells in similar metabolic/nutrient transport status; and, 3) apply a stable isotopic labeling method (i.e., 13C-pulse) to trace nutrient mass transfer within biofilm layers as well as intracellular free metabolite conversions along functional pathways. The transient labeling in cascade metabolites can reveal the speed of substrate diffusion through biofilm layers as well as the rate of biotransformation into downstream intracellular metabolites. It can also determine these active biotransformation pathways important for biofilm survival and growth. Ultimately, the outcome of this exploratory project will be a new method for studying biofilm physiology (i.e., intracellular biotransformation) of diverse environmental microbes. This biofilm study technology will reveal regulatory mechanisms of biofilm growth and persistence under environmental stresses (e.g., antibiotic conditions); and it will improve our understandings of diverse biofilm systems from environmental microbes to pathogens. Specifically, Escherichia coli will be used as a model bacterium in this project with complementary studies outlined in the following Tasks: 1. Create synthetic biofilms with rigorously controlled morphology to reduce structural heterogeneity. 2. Optimize microtome technology to quench and sample free metabolites from different layers of biofilm cells, which can be analyzed via liquid chromatography-mass spectrometry. 3. Validate this new method by conducting 13C-pulse experiments with microtome sectioning and liquid chromatography-mass spectrometry analysis of cascade metabolites to determine glucose transfer and biotransformation rates across different layers of E. coli biofilm cells. This project will result in a new approach to section biofilms to detect metabolite biotransformation and probe the pathway function with desired spatial resolutions. The interdisciplinary nature of this work and the development of new tools outlined herein will build the foundation for advanced research and innovation to address many grand challenges associated with microbial biofilms at molecular level. It will provide new insights into nutrient transport and intracellular enzyme conversions within the biofilm matrix; and help reveal regulatory mechanisms and essential pathways for biofilm survival under diverse environmental conditions. Such insights can help researchers design more effective way to control biofilm physiologies.