Escapin is an L-amino acid oxidase that produces antimicrobial metabolites collectively called “Escapin Intermediate Products” (EIP-K). EIP-K and H2O2 together were previously shown to be bactericidal towards diverse planktonic bacteria. The present work investigates the ability of EIP-K and H2O2 to antagonize bacterial biofilms, using Pseudomonas aeruginosa as a model. The project had three aims: 1) determine the most effective concentrations of EIP-K and H2O2 necessary to break down existing P. aeruginosa biofilms, using a crystal violet assay; 2) examine the ability of EIP-K + H2O2 to inhibit biofilm formation, using triphenyl tetrazolium chloride dye; and 3) determine the effect of EIP-K + H2O2 on the viability, biomass and structure of biofilms cultivated in flow cells using confocal laser scanning microscopy (CLSM). Results showed that EIP-K + H2O2 significantly reduced biofilm biomass relative to controls and that the compounds are effective at nanomolar concentrations.

A stable mixed culture, deposited as ATCC 55644, previously shown to degrade petroleum hydrocarbons at relatively high concentrations was used as the source of inoculum. This culture was grown in Stanier’s minimal media, either in the presence of different concentrations of naphthalene, nitrobenzene and toluene (NNT) or naphthalene and toluene (NT) as the sole source of C and/or N. Results showed that the majority of the strains isolated from the mixed culture were able to grow in the presence of NNT or NT. A total of 20 different isolates were isolated from the mixed culture. Individual isolates were grown in Stanier’s minimal medium containing a single hydrocarbon as the source of carbon or carbon and nitrogen. Only one strain was found to grow solely in the presence of nitrobenzene as the source of C and N. Most of the other isolates were able to grow in the presence of naphthalene, toluene, acenaphthene, anthracene, fluoranthene and phenanthrene, n-dodecane, hexadecane, n-pentadecane, n-tetradecane, and n-octadecane. Planktonic and immobilized cells of the controlled mixed culture (ATCC 55644) were grown in separate Sequential Batch Reactors (SBR) using Stanier's media, to which naphthalene, nitrobenzene and toluene were added as the sole source of C and/or N. Biodegradation was determined by measuring the residual hydrocarbon in the SBR and the amount of trapped volatile organic carbon (VOC) and the evolved CO2. Gas chromatography data showed that immobilized cells were able to degrade NNT faster than the planktonic cells. This observation was confirmed by CO2 evolution. Over time the loading of hydrocarbon was significantly increased from a starting level of 400 ppm (Naphthalene), 100 ppm (Nitrobenzene), and 500 ppm (toluene), to a final level of 3000 ppm (Naphthalene), 400 ppm (Nitrobenzene), and 1600 ppm (toluene). While increasing nutrient loading, the frequency of re-feeding with hydrocarbons was changed from an initial re-feeding every 60 hrs to a final re-feeding frequency of 18 hrs. The experiments clearly showed that the attached, mixed microbial community was able to effectively and rapidly degrade high concentrations of hydrocarbons. This demonstrated the practical advantages of employing attached, mixed microbial cultures in a SBR.