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.

Attachment of APAM to mineral particles in seawater.

Polymer injection is used in enhanced oil recovery (EOR) when an oil field ages and the pressure in the reservoir decreases, or for oil fields with heavy oil. By polymer injection, the viscosity of the water injected for pressure support is increased by mixing with a high concentration of a polymer solution. Polymers used in EOR operations are often high molecular weight polyacrylamides, including anionic polyacrylamide (APAM), which may subsequently enter the marine environment with produced water releases. Since seawater (SW) contains mineral particles (MPs) in low concentrations, and polymers like APAM are known to flocculate MPs, we investigated if APAM at different concentrations (0.5-10 mg/L) would attach and flocculate MPs, when these occurred in concentrations relevant for oceanic SW (1 mg/L). Two types of MPs, diatomaceous earth and kaolin, were exposed to fluorescence-tagged APAM (APAM-TAG). A low-energy carousel system with natural seawater (SW) was used for incubation of MPs and APAM-TAG at a temperature relevant for the Norwegian Continental Shelf (13 °C). Attachment to MPs and aggregates of these were analysed by fluorometry and fluorescence microscopy. Particle analyses showed that only minor fractions of the MPs aggregated. When samples were separated in steel filter with a mesh size of 20 µm, APAM-TAG was mainly measured in the flow-through fraction (<20 µm), and the results therefore showed that the polymer mainly remained in the water-phase, or was attached to small particles (<20 µm). For the small fraction of APAM attaching to aggregated MPs, attraction to kaolin was higher than to diatomaceous earth, and fluorescence microscopy analyses confirmed the presence of fluorescent particles at the higher APAM concentrations. MPs at concentrations relevant for oceanic SW are therefore not expected to significantly contribute to sedimentation of APAM dissolved in the water column.

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.

Interaction between microalgae, marine snow and anionic polyacrylamide APAM at marine conditions.

When an oil field ages and the pressure in the reservoir decreases, or for oil fields with heavy oil, there may be a need for enhanced oil recovery (EOR) technologies. Polymer injection is a water-based EOR method where the viscosity of the water injected for pressure support is increased by mixing with a high concentration polymer solution. In this project, the potential fate of a synthetic anionic polyacrylamide (APAM) in seawater was investigated, since these EOR polymers may enter the marine environment with the produced water (PW). The main objective of the study was to determine if the APAM will interact with cells or aggregates (marine snow) of microalgae, resulting in potential polymer transport from the euphotic zone to the seabed. Three different species of microalgae with different degree of autotrophy (autotroph, mixotroph and heterotroph) were exposed to fluorescence-tagged APAM. Attachment to algal cells or aggregates formed by active or heat-inactivated algae were analysed by fluorescence microscopy and fluorometry. Our results suggested that attachment of APAM to cells of the algal species included in his study was negligible. A carousel system with natural seawater (SW) was used for formation of algal aggregates, one of the key components of marine snow. When aggregates of the diatom Thalassiosira rotula were formed in the presence of the fluorescence-tagged APAM, and at SW temperatures relevant for the Norwegian Continental Shelf, the polymer was nearly exclusively measured in the water phase after separation from the aggregates. The aggregate measurements therefore confirmed the results from the attachment studies, and we found no evidence of accumulation of APAM in aggregates formed from algae. Marine snow from algae is therefore not expected to significantly contribute to sedimentation of APAM dissolved in the water column.

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.

Biodegradation in seawater of PAH and alkylphenols from produced water of a North Sea platform.

Operational planned discharges of produced water (PW) to the marine environment from offshore oil production installations, contain low concentrations of dispersed oil compounds, like polycyclic aromatic hydrocarbons (PAHs) and alkylated phenols (APs). Biotransformation in natural seawater (SW) of naphthalenes/PAHs and phenol/APs in field-collected PW from a North Sea platform was investigated in this biodegradation study. The PW was diluted in SW from a Norwegian fjord, and the biodegradation study was performed in slowly rotating carousels at 13 °C over a period of 62 days. Naphthalenes/PAHs and phenol/APs biotransformation was determined by first-order rate kinetics, after normalization against the recalcitrant biomarker 17α(H),21ß(H)-Hopane. The results from this study showed total biotransformation half-lives ranging from 10 to 19 days for groups of naphthalenes and PAHs, while half-lives for APs (C0- to C9-alkylated) were 10-14 days. Biotransformation half-lives of single compounds ranged from 8 to >100 days for naphthalenes and PAHs (median 16 days), and from 5 to 70 days (median 15 days) for phenols and APs. Four of the tested PAHs (chrysene, benzo(b)fluoranthene, benzo(e)pyrene, benzo(g,h,i)perylene) and one AP (4-tert-butylphenol) showed biotransformation half-lives >50 days. This is one of a few studies that has investigated the potential for biodegradation of PW in natural SW. Methods and data from this study may be used as a part of Risk Based Approaches (RBA) for assessments of environmental fate of PW released to the marine environment and as part of the persistence related to risk.

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.

Biodegradation of oil spill dispersant surfactants in cold seawater.

While biodegradation of chemically dispersed oil has been well documented, only a few studies have focused on the degradation of the dispersant compounds themselves. The objective of this study was to determine the biodegradation of dispersant surfactants in cold seawater, relevant for deep sea or Arctic conditions. Biotransformation of the surfactants dioctyl-sodium sulfosuccinate (DOSS), Tween 80, Tween 85, and α/ß-ethylhexylsulfosuccinate (EHSS, expected DOSS hydrolysis product) in the commercial dispersants Corexit 9500, Dasic Slickgone NS and Finasol OSR52 were determined. The biotransformation studies of the surfactants were performed in natural seawater at 5 °C over a period of 54 days without oil present. The surfactants were tested at concentrations of 1, 5, and 50 mg/L, the lower concentration being as close as possible to expected field concentrations. Experiments with dispersants concentrations of 1 mg/L resulted in rapid biotransformation of Tween 80 and Tween 85, with depletion after 8 days, while DOSS showed rapid biotransformation after a lag period of 16 days. The degradation half-life of DOSS increased from 4.1 days to >500 days as Corexit 9500 concentrations went from 1 mg/L to 50 mg/L, emphasizing the importance of performing experiments at dispersant concentrations as close as possible to environmentally relevant concentrations. EHSS showed limited degradation compared to other surfactants. This study shows that the surfactants DOSS, Tween 80 and Tween 85 in the three chemical dispersants studied are biodegradable in cold seawater, particularly in environmentally relevant concentrations.

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.

Biodegradation of n-alkanes on oil-seawater interfaces at different temperatures and microbial communities associated with the degradation.

Oil biodegradation studies have mainly focused on microbial processes in dispersions, not specifically on the interfaces between the oil and the seawater in the dispersions. In this study, a hydrophobic adsorbent system, consisting of Fluortex fabrics, was used to investigate biodegradation of n-alkanes and microbial communities on oil-seawater interfaces in natural non-amended seawater. The study was performed over a temperature range from 0 to 20 °C, to determine how temperature affected biodegradation at the oil-seawater interfaces. Biodegradation of n-alkanes were influenced both by seawater temperature and chain-length. Biotransformation rates of n-alkanes decreased by reduced seawater temperature. Low rate coefficients at a seawater temperature of 0 °C were probably associated with changes in physical-chemical properties of alkanes. The primary bacterial colonization of the interfaces was predominated by the family Oceanospirillaceae at all temperatures, demonstrating the wide temperature range of these hydrocarbonoclastic bacteria. The mesophilic genus Oleibacter was predominant at the seawater temperature of 20 °C, and the psychrophilic genus Oleispira at 5 and 0 °C. Upon completion of n-alkane biotransformation, other oil-degrading and heterotrophic bacteria became abundant, including Piscirickettsiaceae (Cycloclasticus), Colwelliaceae (Colwellia), Altermonadaceae (Altermonas), and Rhodobacteraceae. This is one of a few studies that describe the biodegradation of oil, and the microbial communities associated with the degradation, directly at the oil-seawater interfaces over a large temperature interval.

Biotransformation of natural gas and oil compounds associated with marine oil discharges.

Field data from the Deepwater Horizon (DWH) oil spill in the Gulf of Mexico (GoM) suggested that oxidation of gas compounds stimulated biodegradation of oil compounds in the deep sea plume. We performed experiments with local seawater from a Norwegian fjord to examine if the presence of dissolved gas compounds (methane, ethane and propane) affected biodegradation of volatile oil compounds, and if oil compounds likewise affected gas compound oxidation. The results from the experiment showed comparable oil compound biotransformation rates in seawater at 5 °C between seawater with and without soluble gases. Gas oxidation was not affected by the presence of volatile oil compounds. Contrary to DWH deep sea plume data, propane oxidation was not faster than methane oxidation. These data may reflect variations between biodegradation of oil and gas in seawater environments with different history of oil and gas exposure.

Biotransformation of potentially persistent alkylphenols in natural seawater.

Produced water (PW) discharged to the marine environment may contain both natural substances and industrial chemicals that are potentially persistent, bioaccumulating and toxic (PBT). Identification of substances as PBT is dependent upon accurate assessment of biodegradation rates, but these measurements can be impeded where substances exhibit inherently low solubility in water. Examples of substances of this kind include some alkylated phenols (APs). Biotransformation of three APs, suspected to be PBT compounds in PW, was investigated by adopting a new methodology in which they were immobilized to hydrophobic adsorbents submerged in natural seawater. These compounds were not ready biodegradable by conventional screening biochemical oxygen demand (BOD) methods at high concentrations (2 mg/L). However, potential biodegradability for two of the three APs were demonstrated by the immobilization method at low concentrations (appr. 100 µg/L), with biotransformation half-lives <50 days. Thus, standard screening tests should be supplemented by biodegradation methods suited for testing of poorly soluble substances before the persistence of potential PBT substances are defined.

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.