N-221. Growth of Multiple Anaeromyxobacter dehalogenans Populations and a Pseudomonas stutzeri Strain, Using DNA-SIP, in a Single Anaerobic 2,6-Dichlorophenol Soil Enrichment Culture

R. A. Sanford1, J. C. Chee-Sanford2, L. M. Connor2, A. Thorp3, F. E. Loeffler3;
1Univ. of Illinois, Urbana, IL, 2USDA-ARS, Urbana, IL, 3Georgia Inst. of Technology, Atlanta, GA.

The degradation of herbicides under anaerobic conditions in soil has not been extensively studied. Many commonly used herbicides contain halogenated substituents and are structural analogs to compounds known to undergo dehalogenation reactions by bacteria. In this study, we specifically investigated bacterial populations responsible for anaerobic reductive dehalogenation in a highly porous, predominantly sandy soil containing low organic matter with a history of commercial agriculture. Enrichment cultures in butyl rubber-stoppered serum bottles were derived from soil amended with 2,6-dichlorophenol (2,6-DCP) (125 µM) as the electron acceptor and acetate as the primary electron donor. Following successive feedings of 2,6-DCP and monitoring activity by the production of phenol, 13C-DNA stable isotope probing (SIP) coupled to terminal restriction fragment (TRF) analysis was used to identify the metabolically active bacteria. After two feedings of DCP, we detected shifts to higher buoyant densities of DNA from Anaeromyxobacter dehalogenans, a known halorespirer, and Pseudomonas stutzeri. Isolation of eight pure Anaeromyxobacter cultures from the enrichment primarily yielded two distinct clades (4 from each), characterized by a one base difference in 16S rRNA gene sequences. The isolates each demonstrated reductive dehalogenation of 2,6-DCP. We isolated the P. stutzeri strain aerobically, and although it was shown to grow anaerobically using nitrate as an electron acceptor, it did not dehalogenate 2,6-DCP or 2-chlorophenol. The results indicate A. dehalogenans populations mediate reductive dehalogenation processes in the agricultural soil used in this study, and demonstrate a surprising phylogenetic diversity within the ennrichment. The role of P. stutzeri is still unknown in these cultures where no obvious electron acceptor for their growth is available, yet its highly enriched presence along with A. dehalogenans indicates the presence of a linked metabolic food web involving both species.