DGGE Analysis of Denitrifying Bacterial Diversity in Response to Nutrients and Oil Contamination in Salt Marshes

Catalina Restrepo, Louisiana State University and Agricultural and Mechanical College


Salt marsh ecosystems located at the Mississippi River delta are exposed to high levels of point and nonpoint sources of nutrients such as nitrate-based fertilizers and to oil spills in the Gulf of Mexico. However, there is little information on the effects of eutrophication and oil contamination on the bacterial diversity of denitrifying bacteria in salt marsh sediments of coastal Louisiana. The effects of excess nutrients on the bacterial diversity of denitrifiers were analyzed by injecting sediment cores with high concentrations of nitrate (100μM NO3- and 20,000μM NO3-) and carbon (0% and 20% C concentration from the original sample). After 7 days of incubation, the diversity of denitrifying bacteria at the top (aerobic) and bottom (anaerobic) samples was analyzed with polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). Denitrifying bacterial diversity was significantly lower (P<0.05) at the top than at the bottom when treated with 100μM NO3- and no carbon, but not on the other treatments. The number of species at the top was also significantly higher (P<0.01) in treatments with a high nitrate concentration (20,000 μM NO3-) and where a carbon source was added along with nitrate. The effects of oil contamination on the bacterial diversity of denitrifiers was analyzed by treating sediment cores from black mangroves (Avicennia germinans) with 2% v/wt. contamination with MC252 oil from the Deepwater Horizon spill. DNA was extracted after 24 hours, 21 days and 110 days from both top and bottom samples and followed by PCR and DGGE analysis. No significant changes in the number of species between top and bottom samples were observed, nor when oil addition alone was analyzed. However, significant changes were observed when exposure time was factored in. While samples exposed for 24 hours and 21 days had no significant changes in the number of species of denitrifiers between oiled and un-oiled samples, un-oiled samples exposed for 110 days had a significantly higher (P<0.001) number of species than oiled samples. The decrease in the number of species was probably the consequence of a decrease in the C:N ratio needed for denitrification and bacterial activity.