Biodegradation of weathered crude oil by microbial communities in solid and melted sea ice.

Oil spilled in the Arctic may drift into ice-covered areas and become trapped until the ice melts. To determine if exposure to oil during freezing may have a priming effect on degradation of the oil, weathered dispersed oil (2-3 mg/L) was frozen into solid ice for 200 days at -10 °C, then melted and incubated for 64 days at 4 °C. No degradation was measured in oil frozen into ice prior to melting. Both total amount of oil and target compounds were biotransformed by the microbial community from the melted ice. However, oil released from melted ice was degraded at a slower rate than oil incubated in fresh seawater at the same temperature (4 °C), and by a different microbial community. These data suggest negligible biodegradation of oil frozen in sea ice, while oil-degrading bacteria surviving in the ice may contribute to biodegradation when the ice melts.

The influences of phytoplankton species, mineral particles and concentrations of dispersed oil on the formation and fate of marine oil-related aggregates.

The formation and fallout of oil-related marine snow have been associated with interactions between dispersed oil and small marine particles, like phytoplankton and mineral particles. In these studies, the influences of phytoplankton species, mineral particle concentration, and oil concentration on the aggregation of oil in seawater (SW) were investigated. The experiments were performed in a low-turbidity carousel incubation system, using natural SW at 13 °C. Aggregation was measured by silhouette camera analyses, and oil compound group distribution and depletion by gas chromatography (GC-FID or GC-MS). Aggregates with median sizes larger than 500 µm in diameter were measured in the presence of dispersed oil and the phytoplankton species Thalassiosira rotula, Phaeocystis globosa, Skeletonema pseudocostatum, but not with the microalgae Micromonas pusilla. When mineral particles (diatomaceous earth) were incubated at different concentrations (5-30 mg/L) with dispersed oil and S. pseudocostatum, the largest aggregates were measured at the lower mineral particle concentration (5 mg/L). Since dispersed oil rapidly dilutes in the marine water column, experiments were performed with oil concentrations of from 10 mg/L to 0.01 mg/L in the presence of S. pseudocostatum and diatomaceous earth. Aggregates larger than 500 µm was measured only at the highest oil concentrations (10 mg/L). However, oil attachment to the marine particles were also measured at low oil concentrations (≤1 mg/L). Depletion of oil compound groups (n-alkanes, naphthalenes, PAHs, decalins) were measured at all oil concentrations, both in aggregate and water phases, with biodegradation as the expected main depletion process. These results showed that oil concentration may be important for oil-related marine snow formation, but that even oil droplets at low concentrations may attach to the particles and be transported by prevailing currents.

Formation and fate of oil-related aggregates (ORAs) in seawater at different temperatures.

In this study, the formation and fate of oil-related aggregates (ORAs) from chemically dispersed oil in seawater (SW) were investigated at different temperatures (5 °C, 13 °C, 20 °C). Experiments in natural SW alone, and in SW amended with typical marine snow constituents (phytoplankton and mineral particles), showed that the presence of algae stimulated the formation of large ORAs, while high SW temperature resulted in faster aggregate formation. The ORAs formed at 5 °C and 13 °C required mineral particles for sinking, while the aggregates also sank in the absence of mineral particles at 20°. Early in the experimental periods, oil compound accumulation in ORAs was faster than biodegradation, particularly in aggregates with algae, followed by rapid biodegradation. High abundances of bacteria associated with hydrocarbon biodegradation were determined in the ORAs, together with algae-associated bacteria, while clustering analyses showed separation between bacterial communities in experiments with oil alone and oil with algae/mineral particles.

Modelling biodegradation of crude oil components at low temperatures.

For oil spilled at sea, the main weathering processes are evaporation, emulsification, photo-oxidation, dispersion and biodegradation. Of these, only biodegradation may completely remove hydrocarbons from the environment in the long term, as the other processes only serve to transform and dilute the oil components. As petroleum development is moving north, the probability of Arctic oil spills increases. Hence, it is imperative to develop methods for comprehensive risk assessment of oil spills in cold and ice-covered waters. Accurate biodegradation rates are an essential part of this, as they are required to predict the long-term effects of marine oil spills. In this paper, we present experimentally determined biodegradation rates for the component groups which are used to represent oil in the OSCAR oil spill model. The experiments have been carried out at seawater temperatures of -2∘C, 0∘C, 5∘C, and 13∘C. We show that for the lighter and more soluble oil components, the changes in degradation rates between 0∘C and 13∘C are well captured by a constant Q10 scaling law. At lower temperatures, and for heavier and less soluble components, the rates are not well described by a constant Q10, probably indicating that oil properties become important for the biodegradation rate.

Biodegradation of weathered crude oil in seawater with frazil ice.

As ice extent in the Arctic is declining, oil and gas activities will increase, with higher risk of oil spills to the marine environment. To determine biotransformation of dispersed weathered oil in newly formed ice, oil dispersions (2-3 ppm) were incubated in a mixture of natural seawater and frazil ice for 125 days at -2 °C. Dispersed oil in seawater without frazil ice were included in the experimental setup. Presence or absence of frazil ice was a strong driver for microbial community structures and affected the rate of oil degradation. n-alkanes were degraded faster in the presence of frazil ice, the opposite was the case for naphthalenes and 2-3 ring PAHs. No degradation of 4-6 ring PAHs was observed in any of the treatments. The total petroleum oil was not degraded to any significant degree, suggesting that oil will freeze into the ice matrix and persist throughout the icy season.

Oil type and temperature dependent biodegradation dynamics - Combining chemical and microbial community data through multivariate analysis.

BACKGROUND: This study investigates a comparative multivariate approach for studying the biodegradation of chemically dispersed oil. The rationale for this approach lies in the inherent complexity of the data and challenges associated with comparing multiple experiments with inconsistent sampling points, with respect to inferring correlations and visualizing multiple datasets with numerous variables. We aim to identify novel correlations among microbial community composition, the chemical change of individual petroleum hydrocarbons, oil type and temperature by creating modelled datasets from inconsistent sampling time points. Four different incubation experiments were conducted with freshly collected Norwegian seawater and either Grane and Troll oil dispersed with Corexit 9500. Incubations were conducted at two different temperatures (5 °C and 13 °C) over a period of 64 days. RESULTS: PCA analysis of modelled chemical datasets and calculated half-lives revealed differences in the biodegradation of individual hydrocarbons among temperatures and oil types. At 5 °C, most n-alkanes biodegraded faster in heavy Grane oil compared to light Troll oil. PCA analysis of modelled microbial community datasets reveal differences between temperature and oil type, especially at low temperature. For both oils, Colwelliaceae and Oceanospirillaceae were more prominent in the colder incubation (5 °C) than the warmer (13 °C). Overall, Colwelliaceae, Oceanospirillaceae, Flavobacteriaceae, Rhodobacteraceae, Alteromonadaceae and Piscirickettsiaceae consistently dominated the microbial community at both temperatures and in both oil types. Other families known to include oil-degrading bacteria were also identified, such as Alcanivoracaceae, Methylophilaceae, Sphingomonadaceae and Erythrobacteraceae, but they were all present in dispersed oil incubations at a low abundance (< 1%). CONCLUSIONS: In the current study, our goal was to introduce a comparative multivariate approach for studying the biodegradation of dispersed oil, including curve-fitted models of datasets for a greater data resolution and comparability. By applying these approaches, we have shown how different temperatures and oil types influence the biodegradation of oil in incubations with inconsistent sampling points. Clustering analysis revealed further how temperature and oil type influence single compound depletion and microbial community composition. Finally, correlation analysis of degraders community, with single compound data, revealed complexity beneath usual abundance cut-offs used for microbial community data in biodegradation studies.

Microbial communities in seawater from an Arctic and a temperate Norwegian fjord and their potentials for biodegradation of chemically dispersed oil at low seawater temperatures.

Biodegradation of chemically dispersed oil at low temperature (0-2 °C) was compared in natural seawater from Arctic (Svalbard) and a temperate (Norway) fjords. The oil was premixed with a dispersant (Corexit 9500) and small-droplet oil dispersions prepared. Faster biotransformation of n-alkanes in the Arctic than in the temperate seawater were associated with the initially higher abundance of the alkane-degrading genus Oleispira in the Arctic than the temperate seawater. Comparable transformation of aromatic hydrocarbons was further associated with the late emergences Cycloclasticus in both seawater sources. The results showed that chemically dispersed oil may be rapidly biodegraded by microbial communities in Arctic seawater. Compared to oil biodegradation studies at higher seawater temperatures, longer lag-periods were experienced here, and may be attributed to both microbial and oil properties at these low seawater temperatures.

Microbial community and metagenome dynamics during biodegradation of dispersed oil reveals potential key-players in cold Norwegian seawater.

Oil biodegradation as a weathering process has been extensively investigated over the years, especially after the Deepwater Horizon blowout. In this study, we performed microcosm experiments at 5 °C with chemically dispersed oil in non-amended seawater. We link biodegradation processes with microbial community and metagenome dynamics and explain the succession based on substrate specialization. Reconstructed genomes and 16S rRNA gene analysis revealed that Bermanella and Zhongshania were the main contributors to initial n-alkane breakdown, while subsequent abundances of Colwellia and microorganisms closely related to Porticoccaceae were involved in secondary n­alkane breakdown and beta­oxidation. Cycloclasticus, Porticoccaceae and Spongiiabcteraceae were associated with degradation of mono- and poly-cyclic aromatics. Successional pattern of genes coding for hydrocarbon degrading enzymes at metagenome level, and reconstructed genomic content, revealed a high differentiation of bacteria involved in hydrocarbon biodegradation. A cooperation among oil degrading microorganisms is thus needed for the complete substrate transformation.

Does Microbial Biodegradation of Water-Soluble Components of Oil Reduce the Toxicity to Early Life Stages of Fish?

Microbial degradation following oil spills results in metabolites from the original oil. Metabolites are expected to display lower bioaccumulation potential and acute toxicity to marine organisms due to microbial-facilitated incorporation of chemical functional groups and a general decrease in lipophilicity. The toxicity and characterization of metabolites are poorly studied. The purpose of the present work was to evaluate the toxicity of degraded (0-21 days) water-soluble oil components. Low-energy water accommodated fraction (LE-WAF) of a weathered crude oil was prepared with nutrient amended seawater at 5 °C, kept in the dark, and sampled at 0, 10, 14, and 21 days. Samples were extracted with dichloromethane and toxicity experiments were conducted with reconstituted extracts. Toxicity experiments were conducted for 4 days on developing cod ( Gadus morhua) embryos during a critical period of their heart development. After exposure, embryos were kept in clean seawater and observed until 5 days post hatch. Survival, hatching, morphometric aberrations, and cardiac function was studied. The expected decrease in sublethal toxicity during the biodegradation period was not found, indicating that metabolites formed during biodegradation likely contributed to larvae toxicity.

Biotransformation of petroleum hydrocarbons and microbial communities in seawater with oil dispersions and copepod feces.

To determine biotransformation of components in crude oil dispersions in the presence of feces from marine copepods, dispersed oil was incubated alone, with the addition of clean or oil-containing feces. We hypothesized that the feces would contribute with nutrients to bacteria, and higher concentrations of oil-degrading bacteria, respectively. Presence of clean feces resulted in higher degradation of aromatic oil compounds, but lower degradation of n-alkanes. Presence of oil-containing feces resulted in higher degradation of n-alkanes. The effect of clean feces on aromatic compounds are suggested to be due to higher concentrations of nutrients in the seawater where aromatic degradation takes place, while the lower degradation of n-alkanes are suggested to be due to a preference by bacteria for feces over these compounds. Large aggregates were observed in oil dispersions with clean feces, which may cause sedimentation of un-weathered lipophilic oil compounds towards the seafloor if formed during oil spills.

Concentrations of viable oil-degrading microorganisms are increased in feces from Calanus finmarchicus feeding in petroleum oil dispersions.

Zooplankton are suggested to be biotic contributors to the transport and weathering of oil in marine environments due to their ingestion of oil. In the present experiment, feeding activity and microbial communities in feces from Calanus finmarchicus feeding in oil dispersions were characterized. Feeding activity was significantly reduced in oil dispersions. The microbial communities in clean and oil-containing copepod feces were dominated by Rhodobacteraceae family bacteria (Lesingera, Phaeobacter, Rugeria, and Sulfitobacter), which were suggested to be indigenous to copepod feces. The results also indicated that these bacteria were metabolizing oil compounds, as a significant increase in the concentrations of viable oil degrading microorganisms was observed in oil-containing feces. This study shows that bacteria in feces from copepods feeding in dilute oil dispersions have capacity for degradation of oil. Zooplankton may therefore contribute to weathering of oil by excreting feces with microbial communities already adapted to degradation of oil.

Endocrine and AhR-CYP1A pathway responses to the water-soluble fraction of oil in zebrafish (Danio rerio Hamilton).

Crude oil is a complex mixture of compounds of which the water-soluble fraction (WSF) is considered to be bioavailable and potentially toxic to aquatic biota. Containing numerous compounds, WSF becomes a source of multiple chemical stressors to wildlife when introduced into the environment. To study the combined effects of WSF components on aquatic biota, the model species zebrafish (Danio rerio Hamilton) was exposed for 24 or 72 h to 10 or 50% WSF solution of known composition, generated from artificially weathered North Sea crude oil. Hepatic expression of genes involved in the aryl hydrocarbon receptor-cytochrome P-450 1A (AhR-CYP1A) pathway (AhR2, AhRR1, CYP1A1) and steroidogenesis (StAR, CYP11A, 3ß-HSD, CYP19A, CYP19B) was measured, as well as estrogen receptors ERα and ERß1. Induction of CYP1A and particularly of AhRR1 was observed while ERα and steroidogenic enzymes CYP11A and 3ß-HSD were downregulated. Regression analysis demonstrated a negative relationship between AhR-CYP1A pathway and endocrine transcript levels, although causality remains to be established. These findings indicate that exposure to WSF of oil disrupts steroidogenesis and may therefore constitute a potential risk for reproductive ability of aquatic organisms. In addition, it is proposed that hepatic gene expression of AhRR1 may serve as a novel biomarker of WSF exposure.

Estimation of hydrocarbon biodegradation rates in marine environments: a critical review of the Q10 approach.

Offshore oil & gas industry is moving exploration and production activities into Arctic and deep water regions. Governmental regulations require environmental impact assessments before operations to evaluate the possible effects of accidental oil releases. These are often performed by numerical fate models, like the Oil Spill Contingency and Response (OSCAR) model, which has become an industry standard in Norway. In this model, biodegradation rates are adjusted to local conditions by temperature compensation according to a Q10 approach. Q10 is the multiplier by which rates of enzymatic reactions increase at a 10 °C temperature rise. Herein, this Q10 approach implemented in the OSCAR model is investigated based on published data and novel obtained results. Overall, biodegradation rate predictions calculated by temperature compensation are found to be questionable, and choosing one universal Q10 value is considered not feasible. The high variation in Q10 values is herein attributed to indirect effects of temperature.

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.

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.

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.

Responses of microbial communities in Arctic sea ice after contamination by crude petroleum oil.

Microbial communities associated with Arctic fjord ice polluted with petroleum oils were investigated in this study. A winter field experiment was conducted in the Van Mijen Fjord (Svalbard) from February to June 2004, in which the ice was contaminated with a North Sea paraffinic oil. Holes were drilled in the ice and oil samples frozen into the ice at the start of the experiment. Samples, including cores of both oil-contaminated and clean ice, were collected from the field site 33, 74, and 112 days after oil application. The sampled cores were separated into three sections and processed for microbiological and chemical analyses. In the oil-contaminated cores, enumerations of total prokaryotic cells by fluorescence microscopy and colony-forming units (CFU) counts of heterotrophic prokaryotes both showed stimulation of microbial growth, while concentrations of oil-degrading prokaryotes remained at similar levels in contaminated and clean ice. Analysis of polymerase chain reaction (PCR)-amplified bacterial 16S rRNA gene fragments by denaturing gradient gel electrophoresis (DGGE) revealed that bacterial communities in oil-contaminated ice generated fewer bands than communities in clean ice, although banding patterns changed both in contaminated and clean ice during the experimental period. Microbial communities in unpolluted ice and in cores contaminated with the paraffinic oil were examined by cloning and sequence analysis. In the contaminated cores, the communities became predominated by Gammaproteobacteria related to the genera Colwellia, Marinomonas, and Glaciecola, while clean ice included more heterogeneous populations. Chemical analysis of the oil-contaminated ice cores with determinations of n-C17/Pristane and naphthalene/phenanthrene ratios indicated slow oil biodegradation in the ice, primarily in the deeper parts of the ice with low hydrocarbon concentrations.