Potential episodic hydrocarbon inputs associated with oil mining and transportation together with chronic introduction of hydrocarbons via urban runoff into the relatively pristine coastal Florida waters poses a significant threat to Florida's fragile marine environment. It is therefore important to understand the extent to which indigenous bacterial populations are able to degrade hydrocarbon compounds and also determine factors that could potentially control and promote the rate at which these compounds are broken down in situ. Previous controlled laboratory experiments carried out by our research group demonstrated that separately both photo-oxidation and cometabolism stimulate bacterial hydrocarbon degradation by natural bacterial assemblages collected from a chronically petroleum contaminated site in Bayboro Bay, Florida. Additionally, we also demonstrated that stable carbon and radiocarbon abundances of respired CO{sub 2} could be used to trace in situ hydrocarbon degradation by indigenous bacterial populations at this same site. This current proposal had two main objectives: (a) to evaluate the cumulative impact of cometabolism and photo-oxidation on hydrocarbon degradation by natural bacterial assemblages collected the same site in Bayboro Bay, Florida and (b) to determine if in situ hydrocarbon degradation by indigenous bacterial populations this site could be traced using natural radiocarbon and stable carbon abundances ofmore » assimilated bacterial carbon. Funds were used for 2 years of full support for one ESI Ph.D. student, April Croxton. To address our first objective a series of closed system bacterial incubations were carried out using photo-oxidized petroleum and pinfish (i.e. cometabolite). Bacterial production of CO{sub 2} was used as the indicator of hydrocarbon degradation and {delta}{sup 13}C analysis of the resultant CO{sub 2} was used to evaluate the source of the respired CO{sub 2} (i.e. petroleum hydrocarbons or the pinfish cometabolite). Results from these time series experiments demonstrated that short-term exposure of petroleum to UV light enhanced hydrocarbon degradation by 48% over that observed for non-photo-oxidized petroleum. Despite the greater bio-availability of the photo-oxidized over the non-photo-oxidized petroleum, an initial lag in CO{sub 2} production was observed indicating potential phototoxicity of the photo- by-products. {delta}{sup 13}C analysis and mass balance calculations reveal that co-metabolism with pinfish resulted in increased hydrocarbon degradation for both photo-oxidized and non-photo-oxidized petroleum each by over 100%. These results demonstrate the cumulative effect of photo-oxidation and co-metabolism on petroleum hydrocarbon degradation by natural bacterial populations indigenous to systems chronically impacted by hydrocarbon input. To address the second objective of this proposal bacterial concentrates were collected from Bayboro Harbor in April 2001 for nucleic acid extraction and subsequent natural radiocarbon abundance analyses. Unfortunately, however, all of these samples were lost due to a faulty compressor in our -70 freezer. The freezer was subsequently repaired and samples were again collected from Bayboro Harbor in June 2002 and again December 2002. Several attempts were made to extract the nucleic acid samples--however, the student was not able to successfully extract and an adequate amount of uncontaminated nucleic acid samples for subsequent natural radiocarbon abundance measurements of the bacterial carbon by accelerator mass spectrometry (i.e. require at least 50 {micro}g carbon for AMS measurement). Consequently, we were not able to address the second objective of this proposed work.« less