Odd Gunnar Brakstad

Microbial diversity during biodegradation of crude oil in seawater from the North Sea.

Microbial communities were characterized during biodegradation of immobilized oil in seawater from the Statfjord field and the German Bight in the North Sea. Seawater samples were collected at different distances from pollution sources at the two locations. A Statfjord oil was immobilized on hydrophobic synthetic Fluortex fabrics and submerged in closed flasks (no headspace) with natural or sterile seawater and incubated at 13°C for 56 days. Biodegradation of immobilized n-alkanes was measured by gas chromatography, total microbes were enumerated by epifluorescence microscopy, and culturable heterotrophic and oil-degrading microorganisms were quantified by most probable number (MPN) analysis. Polymerase chain reaction (PCR) amplification of bacterial 16S rDNA in water samples was conducted during biodegradation experiments. The amplified 16S rDNA fragments were characterized by denaturing gradient gel electrophoresis (DGGE), and by sequence analysis of cloned inserts. Biodegradation rates of alkanes in seawater collected at different distances from the pollution sources did not differ significantly (P > 0.05). Concentrations of oil-degrading microorganisms showed a temporary peak after 7 days of degradation, with a subsequent decline later in the period. DGGE analysis of 16S rRNA genes showed that community diversity decreased during the first 2–3 weeks of biodegradation, with the emergence of a few dominant bands. Cloning, restriction analysis, and sequence analysis of the 16S rDNA fragments revealed >30 different phylotypes. Abundant types during biodegradation belonged to the α-Proteobacteria, in waters from both Statfjord and the German Bight. Cloning and sequencing studies indicated that the most abundant bacteria during biodegradation belonged to the family Rhodobacteraceae, with the closest relationship to the genera Sulfitobacter and Roseobacter.

Biodegradation of dispersed Macondo oil in seawater at low temperature and different oil droplet sizes

During the Deepwater Horizon (DWH) accident in 2010 a dispersant (Corexit 9500) was applied at the wellhead to disperse the Macondo oil and reduce the formation of surface slicks. A subsurface plume of small oil droplets was generated near the leaking well at 900-1300 m depth. A novel laboratory system was established to investigate biodegradation of small droplet oil dispersions (10 μm or 30 μm droplet sizes) of the Macondo oil premixed with Corexit 9500, using coastal Norwegian seawater at a temperature similar to the DWH plume (4-5°C). Biotransformation of volatile and semivolatile hydrocarbons and oil compound groups was generally faster in the 10 μm than in the 30 μm dispersions, showing the importance of oil droplet size for biodegradation. These data therefore indicated that dispersant treatment to reduce the oil droplet size may increase the biodegradation rates of oil compounds in the deepwater oil droplets.

Modulation of steroidogenesis and xenobiotic biotransformation responses in zebrafish (Danio rerio) exposed to water-soluble fraction of crude oil

The induction of CYP enzyme activities, particularly CYP1A1, through the aryl hydrocarbon receptor (AhR) in most vertebrate species is among the most studied biochemical response to planar and aromatic organic contaminant exposure. Since P450 families play central roles in the oxidative metabolism of a wide range of exogenous and endogenous compounds, interactions between the biotransformation processes and reproductive physiological responses are inevitable. Steroidogenesis is the process by which specialized cells in specific tissues, such as the gonad, brain (neurosteroids) and kidney, synthesize steroid hormones. In the present study, we evaluated the effects of water-soluble fraction (WSF) of crude oil on the xenobiotic biotransformation and steroidogenic processes in the head (brain) and whole-body tissue of a model species by transcript analysis using quantitative (real-time) polymerase chain reaction (qPCR), enzyme activities and steroid hormone (testosterone: T and 17beta-estradiol: E2) levels using enzyme immune assay (EIA). Our data showed that exposure of fish to WSF produced an apparent concentration-specific increase of AhR1, CYP1A1 and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) mRNA levels, and decrease of AhR2. On the activity level, WSF produced concentration-specific increase of ethoxyresorufin O-deethylase (EROD), benzyloxyresorufin (BROD) methoxyresorufin (MROD) and pentoxyresorufin (PROD) activities in whole-body tissue. In the steroidogenic pathway, WSF exposure produced apparent concentration-specific decrease of ER* and ERbeta, steroidogenic acute regulatory (StAR) protein, cytochrome P450 side-chain cleavage (P450scc), P450aromA and P450aromB mRNA expression. For steroid hormones, while T levels decreased, E2 levels increased in an apparent WSF concentration-specific manner. In general, the xenobiotic biotransformation and estrogenic responses showed negative relationship after exposure of zebrafish to WSF, suggesting an interaction between these physiological pathways. The relationship between WSF mediated changes in brain StAR, P450scc, 3beta-HSD, ER*alpha, ERbeta, P450aromA, P450aromB and whole-body steroid hormone levels suggests that the experimental animals might be experiencing altered neurosteroidogenesis probably through increased activity level of the biotransformation system. Thus, these responses might represent sensitive diagnostic tools for short-term and acute exposure of fish or other aquatic organisms to WSF.

Acute toxic effects of produced water in relation to chemical composition and dispersion

Abstract The total discharge of produced water in the Norwegian sector of the North Sea is expected to increase to 90 × 10 6 m 3 within the year 2000, with nearly all of this originating from oil production. Produced water from three oil fields in this sector showed large differences in chemical composition and toxicity towards four test organisms ( Skeletonema costatum, Mytilus edulis, Abra alba, Crassostrea gigas ). Values of EC 50 for these organisms ranged from 0.2 to ca 30% of produced water in the test medium. Biodegradation of the produced water changed the chemical composition and generally reduced the toxicity. Model data for dispersion combined with toxicity estimates indicates that acute toxicity should be expected only in the immediate vicinity of the outlets, while at a distance (i.e. > 2 km) toxic effects are considered negligible.

Characterisation of culture-independent and -dependent microbial communities in a high-temperature offshore chalk petroleum reservoir

Recent studies have indicated that oil reservoirs harbour diverse microbial communities. Culture-dependent and culture-independent methods were used to evaluate the microbial diversity in produced water samples of the Ekofisk oil field, a high temperature, and fractured chalk reservoir in the North Sea. DGGE analyses of 16S rRNA gene fragments were used to assess the microbial diversity of both archaeal and bacterial communities in produced water samples and enrichment cultures from 4 different wells (B-08, X-08, X-18 and X-25). Low diversity communities were found when 16S rDNA libraries of bacterial and archaeal assemblages were generated from total community DNA obtained from produced water samples and enrichment cultures. Sequence analysis of the clones indicated close matches to microbes associated with high-temperature oil reservoirs or other similar environments. Sequences were found to be similar to members of the genera Thermotoga, Caminicella, Thermoanaerobacter, Archaeoglobus, Thermococcus, and Methanobulbus. Enrichment cultures obtained from the produced water samples were dominated by sheathed rods. Sequence analyses of the cultures indicated predominance of the genera Petrotoga, Arcobacter, Archaeoglobus and Thermococcus. The communities of both produced water and enrichment cultures appeared to be dominated by thermophilic fermenters capable of reducing sulphur compounds. These results suggest that the biochemical processes in the Ekofisk chalk reservoir are similar to those observed in high-temperature sandstone reservoirs.

Chemical and toxicological characterization of an unresolved complex mixture‐rich biodegraded crude oil

Chemical and toxicological characterization of unresolved complex mixtures in the water-soluble fraction of an artificially weathered Norwegian Sea crude oil was determined by a combination of chemical analysis and toxicity testing in fish in vitro bioassays. The water-soluble fraction of the crude oil was separated into 14 increasingly polar fractions by preparative high-pressure liquid chromatography. The in vitro toxicity (7-ethoxyresorufin O-deethylase activity, estrogenicity, and metabolic inhibition) of these fractions was characterized in a primary culture of liver cells (hepatocytes) from rainbow trout (Oncorhynchus mykiss). The main contributor to toxicity was one of the most polar fractions, accounting gravimetrically for more than 70% of the organic material in the water-soluble fraction and dominated by an unresolved complex mixture. Chemical analysis by gas chromatography-mass spectrometry and comprehensive two-dimensional gas chromatography-time of flight-mass spectrometry identified a large number of cyclic and aromatic sulfoxide compounds and low amounts of benzothiophenes (<0.1% of total mass) in this fraction. Commonly monitored toxic components of crude oil (e.g., naphthalenes, polycyclic aromatic hydrocarbons, and alkylated phenols) eluted in less polar fractions, characterized by somewhat lower toxicity. Normalization of in vitro responses to the mass in each fraction demonstrated a more even distribution of toxicity, indicating that toxicity in the individual fractions was related to the amount of material present. Although polar and nonpolar compounds contribute additively to crude oil toxicity, the water-soluble fraction was dominated by polar compounds because of their high aqueous solubility and the high oil-water loading. Under these conditions, the polar unresolved complex mixture-rich fraction might account for a large portion of crude oil toxicity because of its high abundance in the water-soluble fraction.

Molecular effects of diethanolamine exposure on Calanus finmarchicus (Crustacea: Copepoda).

Alkanolamines are surface-active chemicals used in a wide range of industrial, agricultural and pharmaceutical applications and products. Of particular interest is the use of alkanolamines such as diethanolamine (DEA) in the removal of CO(2) from natural gas and for CO(2) capture following fossil fuel combustion. Despite this widespread use, relatively little is known about the ecotoxicological impacts of these compounds. In an attempt to assess the potential effects of alkanolamines in the marine environment, a key species in the North Atlantic, the planktonic copepod Calanus finmarchicus, was studied for molecular effects following sublethal exposure to DEA. DEA-induced alterations in transcriptome and metabolome profiling were assessed using a suppression subtractive hybridization (SSH) gene library method and high resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR), respectively. Effects were observed on transcription of genes reportedly involved in lipid metabolism, antioxidant systems, metal binding, and amino acid and protein catabolism. These effects were accompanied by altered expression of fatty acid derivates, amino acids (threonine, methionine, glutamine, arginine, alanine and leucine) and cholines (choline, phosphocholine and glycerophosphocholine). Together, SSH and HR-MAS NMR offer complementary screening tools for the assessment of molecular responses of C. finmarchicus to DEA and can be used in the study of other chemicals and organisms. Concentration-response and time-response relationships between DEA exposure and single gene transcription were investigated using quantitative PCR. Specific relationships were found between DEA exposure and the transcription of genes involved in protein catabolism (ubiquitin-specific protease-7), metal ion homeostasis (ferritin) and defence against oxidative stress (gamma-glutamylcysteine synthase, glutathione synthase and Cu/Zn-superoxide dismutase). At the lowest alkanolamine concentration used in these experiments, which corresponded to 0.5% of the LC(50) concentration, no transcriptional effects were observed, giving information regarding the lower molecular effect level. Finally, similar transcription patterns were observed for a number of different genes following exposure to DEA, which indicates analogous mechanisms of toxicity and response.

Gross CO2 and CH4 emissions from the Nam Ngum and Nam Leuk sub-tropical reservoirs in Lao PDR.

Gross CO2 and CH4 emissions (degassing and diffusion from the reservoir) and the carbon balance were assessed in 2009-2010 in two Southeast Asian sub-tropical reservoirs: the Nam Ngum and Nam Leuk Reservoirs (Lao PDR). These two reservoirs are within the same climatic area but differ mainly in age, size, residence time and initial biomass stock. The Nam Leuk Reservoir was impounded in 1999 after partial vegetation clearance and burning. However, GHG emissions are still significant 10 years after impoundment. CH4 diffusive flux ranged from 0.8 (January 2010) to 11.9 mmol m(-2) d(-1) (April 2009) and CO2 diffusive flux ranged from -10.6 (October 2009) to 38.2 mmol m(-2) d(-1) (April 2009). These values are comparable to other tropical reservoirs. Moreover, degassing fluxes at the outlet of the powerhouse downstream of the turbines were very low. The tentative annual carbon balance calculation indicates that this reservoir was a carbon source with an annual carbon export (atmosphere+downstream river) of about 2.2±1.0 GgC yr(-1). The Nam Ngum Reservoir was impounded in 1971 without any significant biomass removal. Diffusive and degassing CO2 and CH4 fluxes were lower than for other tropical reservoirs. Particularly, CO2 diffusive fluxes were always negative with values ranging from -21.2 (April 2009) to -2.7 mmol m(-2) d(-1) (January 2010). CH4 diffusive flux ranged from 0.1 (October 2009) to 0.6 mmol m(-2) d(-1) (April 2009) and no degassing downstream of the turbines was measured. As a consequence of these low values, the reservoir was a carbon sink with an estimated annual uptake of - 53±35 GgC yr(-1).

Depletion and biodegradation of hydrocarbons in dispersions and emulsions of the Macondo 252 oil generated in an oil-on-seawater mesocosm flume basin.

Physically and chemically (Corexit 9500) generated Macondo 252 oil dispersions, or emulsions (no Corexit), were prepared in an oil-on-seawater mesocosm flume basin at 30-32 °C, and studies of oil compound depletion performed for up to 15 days. The use of Corexit 9500 resulted in smaller median droplet size than in a physically generated dispersion. Rapid evaporation of low boiling point oil compounds (C⩽15) appeared in all the experiments. Biodegradation appeared to be an important depletion process for compounds with higher boiling points in the dispersions, but was negligible in the surface emulsions. While n-alkane biodegradation was faster in chemically than in physically dispersed oil no such differences were determined for 3- and 4-ring PAH compounds. In the oil dispersions prepared by Corexit 9500, increased cell concentrations, reduction in bacterial diversity, and a temporary abundance of bacteria containing an alkB gene were associated with oil biodegradation.

Biotransformation and Dissolution of Petroleum Hydrocarbons in Natural Flowing Seawater at Low Temperature

The objective of this study was to establish methods for controlled studies of hydrocarbon depletion from thin oil films in cold natural seawater, and to determine biotransformation in relation to other essential depletion processes. Mineral oil was immobilized on the surface of hydrophobic Fluortex fabrics and used for studies of microbial biodegradation in an experimental seawater flow-through system at low temperatures (5.9–7.4°C) during a test period of 42 days. The seawater was collected from a depth of 90 m, and microbial characterization by epifluorescence microscopy, fluorescence in situ hybridization, and most-probable number analysis showed relatively larger fractions of archaea and oil-degrading microbes than in the corresponding surface water. Chemical analysis of hydrocarbons attached to the fabrics during the test perweree re(H)-hopane showed that the oil remained adsorbed to the fabrics during the test period.Comparison of depletion analysis with calculation of hydrocarbon dissolution in a flow-through system indicated that naphthalenes and smaller PAH compounds (alkylated 2-ring and 3-ring compounds) were removed from the fabrics by dissolution. The data further implied that depletion of n-alkanes and 4–5 ring PAH hydrocarbons were the result of biotransformation processes. PCR amplification of bacterial 16S rRNA genes from microbes adhering on the immobilized oil surfaces showed the dominance of a few bands when analysed in denaturing gradient gel electrophoresis (DGGE). Sequence analysis of DGGE bands revealed phylogenetic affiliation to the α- and γ-subdivisions of proteobacteria and to the Chloroflexus–Flavobacterium–Bacteroides group.