Metagenomic and metatranscriptomic responses of natural oil degrading bacteria in the presence of dispersants.

Oil biodegradation has been extensively studied in the wake of the deepwater horizon spill, but the application of dispersant to oil spills in marine environments remains controversial. Here, we report metagenomic (MG) and metatranscriptomic (MT) data mining from microcosm experiments investigating the oil degrading potential of Canadian west and east coasts to estimate the gene abundance and activity of oil degrading bacteria in the presence of dispersant. We found that the addition of dispersant to crude oil mainly favours the abundance of Thalassolituus in the summer and Oleispira in the winter, two key natural oil degrading bacteria. We found a high abundance of genes related not only to n-alkane and aromatics degradation but also associated with transporters, two-component systems, bacterial motility, secretion systems and bacterial chemotaxis.

Submersible optical sensors exposed to chemically dispersed crude oil: wave tank simulations for improved oil spill monitoring.

In situ fluorometers were deployed during the Deepwater Horizon (DWH) Gulf of Mexico oil spill to track the subsea oil plume. Uncertainties regarding instrument specifications and capabilities necessitated performance testing of sensors exposed to simulated, dispersed oil plumes. Dynamic ranges of the Chelsea Technologies Group AQUAtracka, Turner Designs Cyclops, Satlantic SUNA and WET Labs, Inc. ECO, exposed to fresh and artificially weathered crude oil, were determined. Sensors were standardized against known oil volumes and total petroleum hydrocarbons and benzene-toluene-ethylbenzene-xylene measurements-both collected during spills, providing oil estimates during wave tank dilution experiments. All sensors estimated oil concentrations down to 300 ppb oil, refuting previous reports. Sensor performance results assist interpretation of DWH oil spill data and formulating future protocols.

Factors that affect water column hydrocarbon concentrations have minor impacts on microbial responses following simulated diesel fuel spills.

Effects of season and mixing on hydrocarbon concentrations and the microbial community response was explored in a series of mesocosm experiments simulating surface spills of diesel into coastal waters. Mixing of any amount contributed to hydrocarbons entering the water column, but diesel fuel composition had a significant effect on hydrocarbon concentrations. Higher initial concentrations of aromatic hydrocarbons resulted in higher water column concentrations, with minimal differences among seasons due to high variability. Regardless of the concentrations of hydrocarbons, prokaryotes increased and there were higher relative abundances of hydrocarbon affiliated bacteria with indications of biodegradation within 4 d of exposure. As concentrations decreased over time, the eukaryote community shifted from the initial community to one which appeared to be composed of organisms with some resilience to hydrocarbons. This series of experiments demonstrates the wide range of conditions under which natural attenuation of diesel fuel is an effective response.

DBWM: A diluted bitumen weathering model.

To help better assist the management of Diluted bitumen (DilBit) spills in marine environment, a model named as DilBit Weathering Model (DBWM) was developed in this study to simulate DilBits weathering in marine environment. The DBWM was developed based on specific algorithms for evaporation, dispersion, biodegradation, as well as density and viscosity changes for DilBit weathering and other widely used algorithms for conventional oil weathering in marine environment. To validate the model, a series of DilBit weathering simulation were conducted and compared with the experimental data. Furthermore, the performance of DBWM was compared with a widely used oil weathering model (Automated Data Inquiry for Oil Spills, ADIOS2). The results demonstrated the feasibility and advantages of the developed DBWM in simulating the weathering of marine DilBit spills. Thus, the proposed DBWM can provide effective decision support to marine DilBit spill management.

Microbial community response to simulated diluted bitumen spills in coastal seawater and implications for oil spill response.

Oil spills in coastal waters can have devastating impacts on local ecosystems, from the microscopic base through to mammals and seabirds. Increasing transport of diluted bitumen has led to concerns about how this novel product might impact coastal ecosystems. A mesocosm study determined that the type of diluent and the season can affect the concentrations of hydrocarbons entering the water column from a surface spill. Those same mesocosms were sampled to determine whether diluent type and season also affected the microbial response to a surface spill. Overall, there were no differences in impacts among the three types of diluted bitumen, but there were consistent responses to all products within each season. Although microbial abundances with diluted bitumen rarely differed from unoiled controls, community structure in these organisms shifted in response to hydrocarbons, with hydrocarbon-degrading bacteria becoming more abundant. The relative abundance of heterotrophic eukaryotes also increased with diluted bitumen, with few photosynthetic organisms responding positively to oil. Overall shifts in the microbial communities were minimal relative to spills of conventional oil products, with low concentrations of hydrocarbons in the water column. Oil spill response should focus on addressing the surface slick to prevent sinking or stranding to minimize ecosystem impacts.

Canadian bitumen is engineered for transport, but the type of product produced can affect spill contingency planning.

Canadian bitumen is too viscous to transport by rail and pipeline to markets. One approach to solve this viscosity issue is to dilute the bitumen with a thinning agent to meet transport specifications, but the addition of diluent underutilizes pipeline capacity and increases production cost. A second approach involves the partial refinement of bitumen to produce synthetic crude, which better utilizes pipeline capacity; however, capital and operational costs are high. A third alternative is a new technology that involves wrapping bitumen in a polymer layer to form a solid "puck" termed Canapux, but transportation of this product to coastal ports is limited to rail. Also, greenhouse gas emissions are greater when oil is transported by rail rather than pipeline. In the end, a variety of bitumen products will be transported in Canada, but not all of these products will respond to remediation equally when spilled. In order to ensure effective spill contingency planning, we recommend that engineered bitumen products have physical properties that are resilient to change, within the range of typical response times, after a spill.

Measuring the fate of different diluted bitumen products in coastal surface waters.

Diluted bitumens are produced by adding lower viscosity diluent to highly viscous bitumen to enable it to flow through pipelines and thus may behave differently than conventional oils when spilled into coastal seawater. Simulated surface spills using three different diluted bitumen products were carried out in May, July and November and water column hydrocarbons were monitored over a 14 day period. Volatile and total petroleum hydrocarbons varied in the water column depending on season and type of diluent. In summer, products diluted with synthetic crude or a mixture of condensate and crude released droplets into the water column. Diluted bitumen did not sink to the bottom of the enclosures with surface slicks showing a range of weathering after 14 d. With most of the diluted bitumen product remaining on the surface for 14 d, a rapid conventional clean up response may be effective in low energy, coastal waters.

Fate of diluted bitumen spilled in the coastal waters of British Columbia, Canada.

There is public concern about the behaviour of spilled diluted bitumen (dilbit) in marine and estuarine waters. We provide a preliminary assessment of the results of laboratory experiments and models, in the context of environmental conditions in the coastal waters of British Columbia. Most dilbit spilled within this region would likely float at the surface and be transported to shore by winds and currents. Fresh dilbit is too light to sink in coastal waters. Highly weathered dilbit could sink where salinity is less than 14, typically only near river mouths and in the top 1-3 m of fjords after heavy rainfall. Subsurface plumes of weathered dilbit could re-emerge at the surface. Sinking oil-particle aggregates are unlikely to form in coastal waters. However, dilbit could be entrained below the surface by wave mixing during storms and to depths of 150 m by coherent mixing in the Haro Strait tidal convergence zone.

Inorganic nutrients have a significant, but minimal, impact on a coastal microbial community's response to fresh diluted bitumen.

Microbes capable of degrading hydrocarbons in oil are present in low abundances in coastal waters, but quickly respond to oil following a spill. When estimating potential biodegradation rates in the laboratory, high concentrations of inorganic nutrients are often added to prevent nutrient limitation. In this study, we tested the short term response of coastal microbes to fresh diluted bitumen under varying nutrient conditions in a cold water regime. Total hydrocarbon concentrations changed minimally over five days; however, oil composition changed over time and the abundance of microbes increased in all treatments. Addition of phosphate, with or without nitrogen, resulted in rapid changes in community composition, but after three days treatments no longer differed. Nutrients were never depleted in any treatment suggesting that, even at low inorganic nutrient concentrations, microbial communities can quickly respond to hydrocarbons following a spill.

Chemical dispersants enhance the activity of oil- and gas condensate-degrading marine bacteria.

Application of chemical dispersants to oil spills in the marine environment is a common practice to disperse oil into the water column and stimulate oil biodegradation by increasing its bioavailability to indigenous bacteria capable of naturally metabolizing hydrocarbons. In the context of a spill event, the biodegradation of crude oil and gas condensate off eastern Canada is an essential component of a response strategy. In laboratory experiments, we simulated conditions similar to an oil spill with and without the addition of chemical dispersant under both winter and summer conditions and evaluated the natural attenuation potential for hydrocarbons in near-surface sea water from the vicinity of crude oil and natural gas production facilities off eastern Canada. Chemical analyses were performed to determine hydrocarbon degradation rates, and metagenome binning combined with metatranscriptomics was used to reconstruct abundant bacterial genomes and estimate their oil degradation gene abundance and activity. Our results show important and rapid structural shifts in microbial populations in all three different oil production sites examined following exposure to oil, oil with dispersant and dispersant alone. We found that the addition of dispersant to crude oil enhanced oil degradation rates and favored the abundance and expression of oil-degrading genes from a Thalassolituus sp. (that is, metagenome bin) that harbors multiple alkane hydroxylase (alkB) gene copies. We propose that this member of the Oceanospirillales group would be an important oil degrader when oil spills are treated with dispersant.

Settling of dilbit-derived oil-mineral aggregates (OMAs) & transport parameters for oil spill modelling.

The size and settling velocity of oil-mineral aggregates (OMAs) derived from diluted bitumen are primary constituents in predictive models for evaluating the potential fate of oil spilled in the aquatic environment. A series of low sediment concentration (15mg·L-1), colder water (<10°C) wave tank experiments designed to measure variability in these parameters in naturally-formed OMAs in response the presence or absence of chemical dispersant are discussed. Corresponding lab experiments revealed settling velocities of artificially formed OMAs on the order of 0.1-0.4mm·s-1. High-resolution imagery of settling particles were analyzed for particle size, density and settling velocity. In situ formation of OMAs in the wave tank was unsuccessful. Possible effects of chemical dispersant on natural sediment flocculation, the size of suspended oil droplets and clearance rates of suspended particles are discussed.

Metagenomic survey of the taxonomic and functional microbial communities of seawater and sea ice from the Canadian Arctic.

Climate change has resulted in an accelerated decline of Arctic sea ice since 2001 resulting in primary production increases and prolongation of the ice-free season within the Northwest Passage. The taxonomic and functional microbial community composition of the seawater and sea ice of the Canadian Arctic is not very well known. Bacterial communities from the bottom layer of sea ice cores and surface water from 23 locations around Cornwallis Island, NU, Canada, were extensively screened. The bacterial 16S rRNA gene was sequenced for all samples while shotgun metagenomics was performed on selected samples. Bacterial community composition showed large variation throughout the sampling area both for sea ice and seawater. Seawater and sea ice samples harbored significantly distinct microbial communities, both at different taxonomic levels and at the functional level. A key difference between the two sample types was the dominance of algae in sea ice samples, as visualized by the higher relative abundance of algae and photosynthesis-related genes in the metagenomic datasets and the higher chl a concentrations. The relative abundance of various OTUs and functional genes were significantly correlated with multiple environmental parameters, highlighting many potential environmental drivers and ecological strategies.

An oil spill decision matrix in response to surface spills of various bitumen blends.

Canada's production, transport, and sale of diluted bitumen (dilbit) products are expected to increase by a million barrels per day over the next decade. The anticipated growth in oil production and transport increases the risk of oil spills in aquatic areas and places greater demands on oil spill capabilities to respond to spills, which have raised stakeholder concerns. Current oil spill models only predict the transport of bitumen blends that are used in contingency plans and oil spill response strategies, rather than changes in the oil's physical properties that are relevant to spill response. We conducted weathering studies of five oil products (two conventional oils and three bitumen blends) in the Department of Fisheries and Oceans' flume tank. We also considered two initial oil slick thicknesses, 4.0 mm and 7.0 mm. We found that there is a major difference in the time evolution of oil properties (density and viscosity), raising doubts on weathering models that do not consider the thickness of oil. We also developed empirical expressions for the evolution of the density and viscosity of these oil products. The findings from the 4.0 mm results were incorporated with data from the literature to provide an update on the factors to consider during the decision making for spills of diluted bitumen products. The matrix indicated that most response options, including chemical dispersants, work much more effectively within 48 hours of the initiation of weathering. After this window of opportunity closes, natural attenuation or in situ burning is the only option remaining, but containment of oil is a limiting factor for in situ burning.

Hydrocarbon biodegradation by Arctic sea-ice and sub-ice microbial communities during microcosm experiments, Northwest Passage (Nunavut, Canada).

The increasing accessibility to navigation and offshore oil exploration brings risks of hydrocarbon releases in Arctic waters. Bioremediation of hydrocarbons is a promising mitigation strategy but challenges remain, particularly due to low microbial metabolic rates in cold, ice-covered seas. Hydrocarbon degradation potential of ice-associated microbes collected from the Northwest Passage was investigated. Microcosm incubations were run for 15 days at -1.7°C with and without oil to determine the effects of hydrocarbon exposure on microbial abundance, diversity and activity, and to estimate component-specific hydrocarbon loss. Diversity was assessed with automated ribosomal intergenic spacer analysis and Ion Torrent 16S rRNA gene sequencing. Bacterial activity was measured by (3)H-leucine uptake rates. After incubation, sub-ice and sea-ice communities degraded 94% and 48% of the initial hydrocarbons, respectively. Hydrocarbon exposure changed the composition of sea-ice and sub-ice communities; in sea-ice microcosms, Bacteroidetes (mainly Polaribacter) dominated whereas in sub-ice microcosms, the contribution of Epsilonproteobacteria increased, and that of Alphaproteobacteria and Bacteroidetes decreased. Sequencing data revealed a decline in diversity and increases in Colwellia and Moritella in oil-treated microcosms. Low concentration of dissolved organic matter (DOM) in sub-ice seawater may explain higher hydrocarbon degradation when compared to sea ice, where DOM was abundant and composed of labile exopolysaccharides.

Effect of dispersant on the composition of the water-accommodated fraction of crude oil and its toxicity to larval marine fish.

Newly hatched mummichog (Fundulus heteroclitus) were exposed in a 96-h static renewal assay to water-accommodated fractions of dispersed crude oil (DWAF) or crude oil (WAF) to evaluate if the dispersant-induced changes in aqueous concentrations of polycyclic aromatic hydrocarbons (PAH) affected larval survival, body length, or ethoxyresorufin-O-deethylase (EROD) activity. Weathered Mesa light crude oil (0.05-1 g/L) and filtered seawater with or without the addition of Corexit 9500 were used to prepare DWAF and WAE At 0.2 g/L, the addition of dispersant caused a two- and fivefold increase in the concentrations of total PAH (sigmaPAH) and high-molecular-weight PAH (HMWPAH) with three or more benzene rings. Highest mortality rates (89%) were observed in larvae exposed to DWAF (0.5 g/L; sigmaPAH, 479 ng/ml). A reduction in body length was correlated with increased levels of sigmaPAH (r2 = 0.65, p = 0.02) and not with HMWPAH. The EROD activity increased linearly with HMWPAH (r2 = 0.99, p = 0.001) and not with sigmaPAH. Thus, chemical dispersion increased both the sigmaPAH concentrations and the proportion of HMWPAH in WAF. Dispersed HMWPAH were bioavailable, as indicated by a significantly increased EROD activity in exposed mummichog larvae, and this may represent a significant hazard for larval fish.

Flume tank studies to elucidate the fate and behavior of diluted bitumen spilled at sea.

An economical alternative to conventional crudes, Canadian bitumen, harvested as a semi-liquid, is diluted with condensate to make it viable to transport by pipeline to coastal areas where it would be shipped by tankers to global markets. Not much is known about the fate of diluted bitumen (dilbit) when spilled at sea. For this purpose, we conducted dilbit (Access Western Blend; AWB and Cold Lake Blend; CLB) weathering studies for 13 days in a flume tank containing seawater. After six days of weathering, droplets detached from the AWB slick and were dense enough to sink in seawater. The density of CLB also increased, but at a slower rate compared to AWB, which was attributed to the high concentration of alkylated polycyclic aromatic hydrocarbons in it, which are more resistant to weathering. An empirical, Monod-type model was introduced and was found to closely simulate the increase in oil density with time. Such a model could be used within oil spill models.

Interfacial film formation: influence on oil spreading rates in lab basin tests and dispersant effectiveness testing in a wave tank.

Test facilities such as lab basins and wave tanks are essential when evaluating the use of chemical dispersants to treat oil spills at sea. However, these test facilities have boundaries (walls) that provide an ideal environment for surface (interfacial) film formation on seawater. Surface films may form from surfactants naturally present in crude oil as well as dispersant drift/overspray when applied to an oil spill. The objective of this study was to examine the impact of surface film formation on oil spreading rates in a small scale lab basin and on dispersant effectiveness conducted in a large scale wave tank. The process of crude oil spreading on the surface of the basin seawater was influenced in the presence of a surface film as shown using a 1st order kinetic model. In addition, interfacial film formation can greatly influence chemically dispersed crude oil in a large scale dynamic wave tank.

The immune response of juvenile Atlantic cod (Gadus morhua L.) to chronic exposure to produced water.

Produced water (PW) is the main discharge from the offshore oil industry and contains oil-derived compounds such as poly-aromatic hydrocarbons, phenols, alkylphenols, and heavy metals. Studies suggest that PW discharges may affect the biota over larger areas from the oil drilling sites at sea than originally predicted. We investigated the effects of chronic exposure to PW on some aspects of juvenile Atlantic cod immunity, stress response and growth by intermittently exposing fish to 0, 100 or 200 ppm of PW for 22 weeks. No significant effects of PW were observed on growth, hepatosomatic index, condition factor or plasma cortisol. The respiratory burst (RB) of circulating leukocytes was significantly elevated in the 100 ppm group only, while the RB of head-kidney leukocytes was significantly decreased in both the 100 and 200 ppm groups. Significant up-regulation of the mRNA expression of beta-2-microglobulin, immunoglobulin-M light chain and interleukins-1beta and -8 was observed in the 200 ppm group, while the down-regulation of interferon stimulated gene 15 was obvious for both the 100 and 200 ppm groups. The results suggest that chronic exposure to environmentally relevant concentrations of PW causes modulations of the immune system of juvenile Atlantic cod with most immune parameters being stimulated, potentially resulting in an energetic cost that may be detrimental to the fish.