Marine microbial response to hydrocarbons: an experimental approach

LANFRANCONI, M.P.; CHRISTIE-OLEZA, J.A.; BOSCH, R.; NOGALES B.

Baeza (ESPAÑA)

Workshop; Microbial Diversity in the Biosphere: Trends and new perspectives; 2007

Petroleum hydrocarbons are amongst the most widespread contaminants in the environment. Refined petroleum products accumulate around recreational coastal areas due to human-derived activities such as boating operations in marinas. The frequent exposure to low hydrocarbon concentration results in chronic pollution. Although hydrocarbon degradation by marine microbial communities has been extensively studied, it is usually related to big accidental oil spills. However, the relevance of chronic pollution caused by refined petroleum products, regarding its effect on marine microbial communities, has received little attention.  To evaluate the impact of chronic hydrocarbon pollution on microbial communities we propose to study changes in microbial diversity. In order to avoid the influence of many uncontrollable environmental variables, we have used laboratory experimental microcosms to analyze the effects of hydrocarbon addition on the structure and functioning of marine microbial communities. In this study, microbial microcosms containing 45 l of bay water from Mediterranean Sea (Mallorca, Cala Penyes Rotges) were used. The experiments were performed in duplicate: two control microcosms (developed in absence of diesel oil) and two treated microcosms (containing commercial diesel at low concentration). In marine waters, microbial composition has been shown to change along seasonal cycles. This suggests that temperature can modulate microbial composition in the ocean. In this context we are performing winter and summer experiments. In control experiments, total bacterial counts were performed by DAPI and specific probes were used to detect most important bacterial groups using FISH technique. We found an increase in Eubacteria number, particularly g-Proteobacteria, along time and a decrease in microbial diversity as showed by TRFLP analysis of 16S rRNA genes. No changes in cyanobacteria number was detected, except at the end of the experiment when an increase was observed. These results allowed us to determine the maximal incubation time of microcosms. Final incubation time, where the microbial community still reflected the original marine community, was 5 days. Subsequently, a winter experiment introducing diesel contamination was performed during 5 days. The succession and function of control seawater microbial community was compared with that of diesel-treated seawater, simulating permanent exposure to low hydrocarbon concentration. A significant increase in cyanobacterial numbers was obtained in control experiments in contrast to the results obtained in diesel-treated microcosms where bacterial phytoplankton number was similar along the incubation time. The microbial response to oil pollution was also analyzed at functional level. We considered essential processes to community function such as photosynthesis (psbA and pufM), carbon and nitrogen fixation (rbcL and nifH, respectively), hydrocarbon degradation (nahAc, nahG, nahW and alkB) and the recently discovered proteorhodopsins. We have optimized amplification reactions to test gene expression by RT-PCR in diesel-treated and control microcosms.