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