An oil spill occurred on Signy Island, South Orkney Islands, Antarctica in 1996. Five soil samples were collected from Signy Island after this event - three were collected from the site of the spill, and two were from pristine soils.
Approximately 300 bacterial colonies were enriched from soil samples at 4°C and at 15°C with benzoate, m-toluate, toluene, phenol, hexadecane, biphenyl, naphthalene, mxylene or p -xylene as the sole carbon and energy source. Bacteria were successfully enriched and isolated from both pristine and oil-contaminated soils.
Three psychrotolerant bacteria were chosen for further study because of their ability to utilize 8 or more of the tested growth compounds (benzoate, m-toluate, toluene, phenol, hexadecane, biphenyl, naphthalene, m-xylene and p-xylene) at 4°C. The bacteria were designated ST41, SB45 and SB204. A 16S rRNA gene was amplified from each bacterium. Analysis of the genes revealed that the 16S rRNA gene from ST41 was 99.2% identical to that of Pseudomonas borealis. The same gene from SB45 was found to be 99% identical to that of Pseudomonas fluorescens, and the 16S rRNA gene amplified from SB204 was 96.4% identical to that of Flavobacteriumjohnsonae.
A gene encoding a catechol 2,3-dioxygenase (C23O) was cloned from ST41 on a
13.5kb fragment. Sequence analysis of the fragment revealed that an almost entire xy/-like meta-pathway operon had been cloned, which was very similar to the xyl metaoperon of the TOL plasmid from the mesophile Pseudomonas putida mt-2.
Escherichia coli was transformed with a plasmid containing the xylE (encoding C23O) from pWW0 and with the gene encoding C23O from ST41. The temperature profiles and thermostabilities of the enzymes were studied. The C23O from ST41 had a lower temperature optimum than XylE, and the former was also less heat stable. A pairwise alignment of the protein sequences revealed a difference of 16 amino acids between the two enzymes (which are both 307 residues), 8 of which were clustered at the beginning of the sequence. A model of the secondary structure of the proteins was predicted using the Garnier-Robson method and this suggested that the difference in the
amino acid sequences results in a delayed onset of an alpha-helix region in the C23O from ST41 compared to XylE. The Karplus method for modelling the backbone chain flexibility of a protein predicted that there was an extra region of flexibility in the region
that lacked the alpha-helix in the C230 from ST41. It has been suggested that an
increase in backbone flexibility leads to the lower temperature optima and decreased
thermostability observed in cold-adapted enzymes.
Having established that there are bacteria capable of utilizing oil and its constituents at
low temperatures, in both pristine and oil-contaminated soils, bioremediation appeared
to be a viable option. Microcosms were set up at 4°C in an attempt to find out how rates
of oil biodegradation could be increased.
The effect of: (1) bioaugmentation with ST41; (2) biostimulation; (3) bioaugmentation
(ST41) + biostimulation; and (4) the addition of water alone, on bioremediation rates
were compared. The decrease in recoverable hydrocarbons was monitored using gas chromatography/mass spectroscopy (GC/MS). Changes in microbial community profiles were tracked using temporal temperature gradient gel electrophoresis (TGGE) with the V3-V4 region of the 16S rRNA gene being analyzed. DNA was purified from a TGGE gel that had been performed using DNA extracted from soil following 12 weeks of treatment. The fragments of DNA were sequenced and analysis of the data indicated the presence of ST41 in the microcosms to which oil had been added, even without bioaugmentation.
The GC/MS results showed that only where bioaugmentation and biostimulation were used together was there the complete degradation of alkanes CwC20.