Q-077. Novel Raman Microspectroscopy and Stable Isotope Probing to Investigate a Gas Station Site Contaminated by Naphthalene

W. E. Huang1, A. Ferguson2, A. Singer1, I. P. Thompson1, M. J. Bailey1, R. M. Kalin2, M. Larkin2, A. S. Whiteley1;
1CEH-Oxford, Oxford, UNITED KINGDOM, 2Queen's University, Belfast, UNITED KINGDOM.

In a naphthalene contaminated gas station, 14 different naphthalene degraders were isolated from groundwater by growing on a minimal medium with naphthalene as a sole carbon source. These degraders belong to Pseudomonas sp., Comamonas sp., Delftia sp. or Achromobacter sp.. Naphthalene degradation genes of all 14 strains were located in conjugative plamids as they can transfer to P. putida KT2440 by filter mating. Plasmids from 14 strains were extracted and they all have identical nahG gene as pDTG1 from Pseudomonas putida NCIB9816. However, nahR genes in 14 plasmids were variable. Stable isotope probing (SIP) was carried out by introducing 4 ÁM 13C- fully-labelled naphthalene to groundwater and incubated at 13 oC for 72 hrs. During the period, naphthalene was completely degraded whereas the structure of microbial community did not change. In SIP experiment, two key degraders, which were not classified to any of 14 isolates, were identified as beta-proteobacterium. It was revealed that those culturable isolates were only responsive when naphthalene concentration was high (>30 ÁM). FISH (fluorescent in situ hybridization)-Raman Microspectroscopy was employed to confirm that the uncuturable beta-proteobacterium were responsible for naphthalene degradation in situ. Nucleotide probes were designed based on 16S rDNA sequence of the uncuturable beta-proteobacterium and probed to the microbial community after SIP. Raman microspectroscopy, which has capability to distinguish 13C- labelled single bacteria, was used to scan single hybridized cells. The results show that hybridized cells have integrated 13C-carbon and hence confirm two uncuturable beta-proteobacterium were key naphthalene degraders in situ.