N-162. TRFLP Analysis of Anaerobic Debrominating Enrichments from Coastal Sponges, Tunicates and Sediment

N. A. Lopez, Y-B. Ahn, K. Parisi, M. Häggblom, L. Kerkhof;
Rutgers, The State Univ. of New Jersey, Highland Park, NJ.

Recent work has shown that many species of Mediterranean and tropical marine sponges harbor dehalogenating bacteria. To test whether temperate sponges, tunicates, and sediments also harbor anaerobic dehalogenating bacteria, samples of Microciona prolifera, Haliclona bowerbanki and Halichondria loosanoffi, an undescribed tunicate, Botryllus schlosseri, and sediment were incubated in anaerobic media with short chain fatty acids as electron donor and 2,6-dibromophenol as electron acceptor in the presence or absence of sulfate. Dehalogenation was observed in all sponges, tunicates and sediment samples from Long Island Sound. After detection of activity, enrichment cultures were established using serial dilutions of 10-7 to estimate numbers of endosymbiotic dehalogenating bacteria and test whether the same microbial populations exist within these different samples. There was a large difference in dehalogenating numbers, with sulfate enrichments having activity at 10-7 dilution, while no-sulfate enrichments were only active up to 10-2 for most samples. This suggests that the dehalogenator(s) are also capable of using sulfate as an electron acceptor. Bacterial populations were identified by TRFLP profiling of 16S rRNA genes using MnlI. Analysis of the bacterial community detected 82 different OTU's in the dehalogenating enrichments. Sulfate and non-sulfate bacterial enrichments exhibited different microbial fingerprints. The 207 bp peak was most abundant, appearing in 17 of the samples. Peaks 211 and 214 were the 2 most dominant peaks, in the sulfate samples, whereas peaks 179 and 294 where the most dominant peaks in the non-sulfate samples. Any TRFLP peak with an area greater than 10% of the total profile was considered a dominant peak. Overall, debrominating bacteria appear to be ubiquitous. However the community analyses indicates that they represent a small portion of microbes in temperate marine organisms and sediment. Stable isotope analysis, cloning and sequencing are underway to determine the dehalogenating bacterial species associated with these systems.