Biorefining promises the development of efficient processes for the conversion of renewable sources of carbon and energy into large volume commodity chemicals. The US Department of Energy (USDOE) recently released a prioritized list of building block chemicals for future biorefining endeavors, which includes 3-hydroxypropionic acid (3-HP). However, synthesis of organic acids at high titers can be toxic to host microorganisms. The overarching goal of this research is to determine if engineering organic acid tolerance will have a beneficial effect on production characteristics (i.e. productivity, titer). We have developed a new high-resolution, genome-wide approach that can be used to monitor enrichment and dilution of individual clones within a genomic-library population. This method includes creation of representative genomic libraries with varying insert size, growth of clones in selective environments, interrogation of the selected population using microarrays, and a mathematical multi-scale analysis to identify the gene(s) for which increased copy number improves overall fitness. We have utilized this method to select for genes that confer organic acid tolerance in E. coli. Selections using 3-HP were carried out in microaerobic chemostats fed by buffered minimal media. We have identified several loci with putative functions involving transport systems and biofilm formation for which increased copy confers 3-HP tolerance. Confirmations of tolerance improvement were carried out by determining minimum inhibitory concentrations (MICs) and constructing growth curves of selected transformants in the presence of 3-HP and other organic acids. A 20% increase in MIC for 3-HP was observed on E. coli clones overexpressing the yeaP, yeiM, and yli regions of the genome when compared with wild type E. coli. In addition to tolerance genes, we have also identified five different genes conferring biofilm phenotypes under microaerobic conditions in minimal media. The most prominent biofilm phenotypes observed in the chemostats were due to increased copy of the same yli operon and yeaP gene that conferred increased 3HP tolerance, suggesting that increased matrix production may play a role in mediating organic-acid tolerance in minimal media. To investigate further, we next constructed disruptional mutant libraries for the yeaP and yli clones and selected for mutants that displayed defective biofilm phenotypes. Greater than 80% of selected mutants resulted in loss of biofilm phenotypes for the yeaP and yli clones. Finally, the disruptional mutant libraries were utilized to identify mutants that display increased biofilm phenotypes in cultures fed with rich media. Collectively, these results have begun to shed light into organic acid tolerance, overlaps between tolerance and biofilm phenotypes, and how such overlaps might be used to improve metabolic engineering organic-acid production.