Microbial transformation and mineralization of hydrocarbons in seawater at low temperatures

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1 Microbial transformation and mineralization of hydrocarbons in seawater at low temperatures Odd Gunnar Brakstad SINTEF, Trondheim 1

2 PROOF project Weathering of marine oil spills in the Arctic Biodegradation Laboratory studies: Biodegradation of petroleum hydrocarbons in cold water 0-5 C Water-soluble fractions Oil phase Weathered oils (e.g. photo-oxidation) Field winter study: Oils in Svalbard fjord ice Interactions between microbes and the oil Possible biotransformation in ice 2

3 PROOF project Weathering of marine oil spills in the Arctic Biodegradation Methodology Laboratory studies Static system with cold seawater as microbial inocula for degradation of hydrocarbons in water-soluble fractions (WSFs) Static system with cold seawater as microbial inocula for degradation of hydrocarbons in immobilized oil films Flow-through seawater with immobilized oil films Field winter study in Svalbard fjord ice Interactions between ice microbes and oil frozen in the ice 3

4 Laboratory studies with static water conditions Objectives of the study Biodegradation studies of petroleum hydrocarbon in thin oil films at low seawater temperatures Biotransformation: Biodepletion of compounds in thin oil films Comparison of oil film biotransformation and dissolution Mineralization: Complete biodegradation to CO 2 in the test flasks Characterization of microbial communities involved in petroleum hydrocarbon degradation on the oil films Input to the fate segment of numerical exposure models OSCAR

5 Seawater source Trondheimsfjorden Trondheim harbour St. Olavs Pir 5

6 Cold seawater: bacterial enrichment in Bushnell-Haas medium Cultures of oil-degrading bacteria with Statjord crude as carbon source Cells/ml (DAPI) C - Surface 0 C - Surface Day 6

7 Biodegradation of oil in static seawater Experimental setup Oil spiked with 14 C-labelled hydrocarbons for studies of mineralization Naphthalene UL- 14 C Phenanthrene-9-14 C n-hexadecane-1-14 C Oils without 14 C-labelled hydrocarbons for dissolution and biotransformation studies Oils immobilised as thin films (appr. 10 µm thick) on hydrophobic Fluortex fabrics Fabrics submerged in natural or sterile seawater seawater acclimated at 5 og 0 C (5-7 days) Biodegradation period 56 days at 5 or 0 C 7

8 Biodegradation test system Oil film immobilization counts FID1 A, (101000\008F0901.D) Crude oil min mi 8 Area: e Area: e Area: e Immobilized oil counts FID1 A, (I:\HPCHEM\2\DATA\101000\010F1101.D) Area: e+008 Area: e Area:

9 Biodegradation test system Adsorbents with immobilized oil in: Normal seawater acclimated at 0 or 5 C Sterile seawater (abiotic controls) Incubation of bottles Immobilised oil submerged in water phase at 0 and 5 C for 56 days (8 weeks) Tubes with CO 2 -traps (1M KOH) Measurements Depletion if HCs from oil films (unlabelled oil fims): GC-FID and GC-MS Mineralization (radiolabelled oil films): Radiorespirometry (β-counts): 14 CO 2 -accumulation 9

10 Dissolution of HCs from oil films Sterile systems 5 C 0 C Depletion (%) Naphthalene Phenanthrene Hexadecane Depletion (%) Naphthalene Phenanthrene Hexadecane Days Days 10

11 Comparison of dissolution and biotransformation on oil films Depletion (%) Naphthalene 5 C NorSW 5 C SterSW 0 C NorSW 0 C SterSW Days 11

12 Comparison of dissolution and biotransformation on oil films Depletion (%) Phenanthrene 5 C NorSW 5 C SterSW 0 C NorSW 0 C SterSW Days 12

13 Comparison of dissolution and biotransformation on oil films Depletion (%) Hexadecane 5 C NorSW 5 C SterSW 0 C NorSW 0 C SterSW Days 13

14 Distribution between HC dissolution and biodegradation in oil films First-order kinetics - non-linear regression analysis Depletion rates Rate constants (k1) C 5 C 0 C 5 C 0 C 5 C Dissolution Biotransformation Naphthalene Phenanthrene n-hexadecane 14

15 Mineralization of hydrocarbons ( 14 CO 2 -accumulation) Mineralization (%) Naphthalene 5 C 0 C Days 15

16 Mineralization of hydrocarbons ( 14 CO 2 -accumulation) Mineralization (%) Phenanthrene 5 C 0 C Days 16

17 Mineralization of hydrocarbons ( 14 CO 2 -accumulation) Mineralization (%) n-hexadecane 5 C 0 C Days 17

18 Measurements microbial communities Bacterial communities adhering to oil films characterized by 16S rrna phylogenetics DNA extracted from fabrics coated with oil films 16S rdna amplified by PCR with universal bacterial primers Diversity of the PCR products analysed by denaturing gradient gel electrophoresis (DGGE) DGGE bands cut and cloned PCR inserts in clone vectors (plasmider) characterised by sequence analyses Taxonomic characterisation by alignments to known sequences in the GenBank database 18

19 PCR-DGGE-sequencing analysis Microbes attached on the oil surface C 5 C Closest match 0 56 Closest match Arcobacter sp. Arcobacter sp. Marine psychrotrophic bacterium Mst37 Psychromonas arctica Uncultured bacterium clone ARKDMS-13 Uncultured Cytophaga sp. Uncultured bacterium SB- 9/21-CS 19

20 Conclusions Oil immobilization on hydrophobic fabrics enabled determination of both hydrocarbon dissolution and biotransformation processes Biodegradation appeared at low temperatures, but at higher rates at 5 than at 0 C Recent studies (WSFs) have shown that the temperature-dependent biodegradation is caused by reduced microbial metabolism rather than reduced bioavailability (Statfjord oil has a pour point of appr. 8 C) Biodegradation rates will be included in exposure model(s) (e.g. OSCAR 2000) Microbial populations associated with oil films were mainly psychrophilic ( cold-loving ) at 0 C, but not 5 C 20

21 Acknowledgements Kristin Bonaunet: Biodegradation experiments, molecular biology, radiorespirometry Inger K. Almås: Chemical analysis (GC-FID and GC-MS) 21