FROM SIMPLE TO COMPLEX DEGRADATION OF HYDROCARBONS. A CONCEPT OF METAGENOME SUCCESSION IN OIL-AMENDED MICROCOSMS Deni Ribicic1*, Roman Netzer2, Odd Gunnar Brakstad2 Norwegian University of Science and Technology- Department of Cancer Research and Molecular Medicine1, SINTEF Materials and ChemistryEnvironmental Technology2 *
[email protected] CRUDE OIL & ENZYMES
BACKGROUND AND OBJECTIVE
Aliphatics
Hydrocarbons are occurring either naturally or anthropogenically in marine environment. In our research we focus mainly on studying the fate and effects of crude oil accidentally released into the marine environment by oil spills. Different types of oil spills can occur in the ocean. Exxon Valdez oil spill is an example of an surface oil spill while Deep-Water Horizon spill is an example of a sub-surface oil spill. A diversity of weathering processes will shape the fate of oil in the ocean. Biodegradation as such, has been extensively investigated over the years, often in combination with chemical dispersantsa well-known oil spill response method.
In this study we aimed to characterize microbial community and metagenome dynamics in microcosms with chemically dispersed oil incubated in cold Norwegian seawater (5 ⁰C). Surface release
Aromatics
• Alkanes (paraffins)
• Monocyclic aromatics
• Branched alkanes (isoprenoids)
• Polycyclic aromatics
• Cycloalkanes (naphtenes)
C1-C4- variety of soluble and particulate methane monooxygenases C5-C16- heme-containing cytochrome P450 CYP153 and non-heme di-iron alkane hydroxylases > C17- uknown enzyme systems
Different types of monooxygenases and dioxygenases Naming- compound accordignaly , e.g. Xylene-XMO, Naphtalene-NDO, BiphenylBPDO, Benzoate- BDO, etc..
Sub-surface release
FIG 1: Comparing total degradation rates in NSOD for n-alkanes, PAHs and VOCs after 64 days of incubation (A) with changing abundance of total (DAPI), heterotrophic (HM) and oil degrading (ODM) microbes (B). Core microbiome structure represented on taxonomic level of family (order), obtained by 16S rRNA gene analysis (C).
FIG 5: Contribution of microorganisms to the abundance of selected hydrocarbon degrading genes. A: Contribution in the sample NSOD-9 for alkane 1-monooxygenase gene. B: Contribution in the sample NSOD-31 for cytochrome P450 gene. C: Contribution in the sample NSOD-9 for aldehyde dehydrogenase gene. D: Contribution in the sample NSOD-9 for the alcohol dehydrogenase gene. E: Contributions in the sample NSOD-31 for the biphenyl dioxygenase, xylene monooxygenase and phenol hydroxylase gene. F: Contribution in the sample NSOD-31 for the catechol dioxygenase gene. G: Contribution in the sample NSOD-31 for the benzoate dioxygenase gene. Samples were selected according to where respective genes were found to be most abundant (see FIG 4A).
CONCEPT OF METAGENOME SUCCESSION
Simple
Complex
FIG 3: The concept itself relies on the general principle of ecological succession which includes the development of a simple to a more complex structure. In the case of metagenome succession, genes encoding enzymes relevant for degradation of simple saturated hydrocarbons are successively substituted with genes encoding enzymes potentially involved in degradation of more complex cyclic and substituted hydrocarbon substrate.
FIG 2: PCoA plots of chemical and microbial community analyses. Plot A recaptures dissimilarities between samples obtained by chemical analysis. Plot B indicates weightedUnifrack beta diversity based on 16S rRNA gene analysis showing development of microbial community in anti-clockwise direction. Replicates from the same time-point are connected with a polygon. Oil free samples are annotated in red font with “ctrl” prefix. Oil dispersion samples are annotated in turquoise color. Numbers indicate incubation time in days.
FIG 4: Heatmap representing relative abundance of selected predicted functions (enzymes) across different samples with numbers indicating total abundance of observed encoding genes (A) coupled to degradation pattern of oil compound groups (B) likely to be degraded by predicted enzymes. Comparison of SEED subsystem functional categories for control samples (NS) and oil incubation samples (NSOD) (C). Metagenomes were analyzed using MG-RAST built-in analysis tool and plotted within R program.
Abundance
ACKNOWLEDGMENTS • This project was financed by the Research Council of Norway and the oil companies Statoil, Det norske, ExxonMobil , Total and ConocoPhillips. • We would like to thank the staff at the lab for performing the chemical analyses (Marianne Rønsberg, Kjersti Amås, Inger Steinsvik). • Thanks to Stephen Techtmann for useful suggestions regarding bioinformatic analysis. Time (days)
