Microbial Alkane Biodegradation Genes In Chronically Polluted Sub-Antarctic Coastal Sediments

Petroleum is a very complex chemical containging thousands of hydrocarbon molecules. In the ocean, sediments are the natural sinks of these chemicals. However, in cold environments, petrol can be very persistent causing both short and long-term ecological effects. As oil exploration is becoming more common at higher latitudes it is important to increase our knowledge of cold-environment bioremediation microbes. Bioredemdiation is complex process in itself, involving various microbial organisms. However, particularly in sediments, our understanding of this pathway is disadvantaged as the biodiversity and heterogeneity of the habitat increases its complexity. Most bioremediation work has made significant advances in the both the  tropical and temperate environments. Saturated hydrocarbons include alkanes, branched alkanes and cycloalkanes; and pose the highest proportion in crude oil.  Some previous work has suggested that adding alkanes to environments polluted with other, less degradable chemicals my increase the break-down of these persistent substances. There are a surprising number of microbes that use alkanes as a carbon-source including Alcanivorax, Thalassiolituus, Oleiphilus and Oleispira. These organisms contain various alkane-activating enzymes known as alkB. It has been suggeste that alkB be used as biomarkers for the characterization of aerobic alkane-degrading bacterial populations.

This work studies the alkane-degrading genes and bacterial communities of Ushuaia Bay, South America; described as a chronically polluted environment where off-loading of petroleum is a daily occurrence.

Sediment samples were collected via coring along the low-tide line and at a depth of 11m. These were then stored at -80°C. There were three different treatments of the original sample (OR08). These were the sediment with added 0.46ml of crude oil (expOR08-O), the sediment with added crude oil, ammonium chloride and sodium phosphate (expOR08-ON) and finally, the control sediment (expOR08-c). In order to fully analyse the hydrocarbons, samples were extracted and treated with activated copper and evaporated until 0.2ml was reached. This was loaded into a alumina column to recover the aliphatic and aromatic fractions. Individual alkane (from n-C10 to n-C35) concentrations, Pristine and Phytane isoprenoid levels, total resolved alkanes and total aliphatic hydrocarbons were calculated for each sample.

DNA was also extracted from each sample using a FastDNA SPIN kit. Two DNA extractions were conducted per sample. The alkB gene fragments were then amplified using specific primers, these were cleaned, cloned and sequenced. The V4 hypervaribale region of the 16S rRNA was amplified using pyrosequencing and used to construct alkB gene libraries. The alkB sequences were screened against the GenBank database using BLAST. Alpha-diversity was calculated using OTU (operational taxanomic unit) analysis.

All sample collected from the bay were seen to contain alkane homologous series, isoprenoids pristine, isoprenoid phytane and unresolved complex mixtures. Biodegradation indices showed that biodegradation was an on-going process. A total of 30 OTUs were found and were estimated to cover 95% of the overall alkB gene diversity. Richness was found to be 39-48 OTUs but this was described as a conserved estimate. Groups found relating to alkB were mainly Proteobacteria. The most closely related (and the most common OUT) to alkB were deemed as uncultured microorganisms from cold marine sediments.

After 20 days expOR08-O showed a decease in the concentration of alkanes lower than C20. However expOR08-ON showed further degradation of alkanes.

5 alkB gene OTUs were present in expOR08-O, 4 of which were also present in the environmental samples taken; the most abundant in this treatment was most closely related to Actinobacteria however there were also Gammaproteobacteria. expOR08-ON showed only two OTUs; Kordiimonas gwangyangensis and Rhodococcus spp.

A total of 44,380 reads were obtained from the V hypervariblae region that could be amplified from the three treatments expOR08-O, expOR08-ON, expOR08-c and the environmental samples. All OR slurries showed high richness and low dominance values. Although the expOR08-c was 38% lower than the environmental sediments (attributed to the laboratory conditions), expOR08-O had a lower richness value still and the expOR08-ON was the most simplified.

The authors goes on to explain that this study indicates a high diversity of alkB genes in highly polluted areas. They say that this was consistent over time and space suggesting an ecological relevance to the microbial communities of the sub-Antarctic sediments. The closest matches to the alkB gene sequences were from uncultured microbes suggesting further study in this area. It is explained that different components such as the type of pollution present factors into the communities present at a site. It is hinted that more studies need to be conducted into various environmental factors and as to how these may affect the population dynamics. As the addition of crude oil to the samples resulted in faster degradation of the alkanes. Although these results are still considered controversial. Previous studies have before suggested that the alkB-carrying microbes are positively correlated to alkane concentration however, other studies have debunked this. However this paper the presence of oil results in a strong amplification of alkB genes. This helps to take the first steps toward using alkB genes as biomarkers

Guibert L.M, Loviso C.L, Marcos M.S, Commendatore M.G, Dionisi H.M & Lozada M. 2012. Alkane Biodegradation Genes from Chronically Polluted Subantarctic Coastal Sediments and Their Shifts in Response to Oil Exposure, Microb. Ecol. 64: 605-616.