The effect of chemically dispersed oil on marine microbial communities of Norwegian seawater - A molecular and ecological approach
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SAMMENDRAG Produksjon av olje og relaterte undersøkelser har foregått på norsk sokkel i omlag 50 år, og interessen for slik aktivitet i framtida har åpenbart ikke minket. Det finnes tvert imot planer for å utvide aktiviteten inn i arktiske regioner for å utvinne nye ressurser. Som følge av dette, og på grunn av den globale historien rundt oljeutslipp, finnes det både vitenskapelige og samfunnsmessige bekymringer rundt risikoen som er knyttet til slik aktivitet. Oljevern må baseres på eksperimentelle resultater hvor biodegradering av olje spiller en særdeles viktig rolle. Biodegradering av råolje i nordlige og polare farvann er ikke tilstrekkelig studert og mye er fortsatt ukjent. Dette gjelder særlig i kombinasjon med operasjonelle oljevernmetoder som for eksempel bruk av kjemiske dispergeringsmidler i oljekontaminerte arealer. Dette prosjektet er gjennomført for å få en bedre forståelse av dynamikk og kapasitet i mikrobielle samfunn for nedbryting av olje under oljeutslipp i norske farvann hvis utslippene behandles med kjemiske dispergeringsmidler. Arbeidet er strukturert i følgende hovedpunkter: (I) Egenskaper for råolje og kjemiske dispergeringsmidler som påvirker biodegradering; (II) Dynamikk og biodegraderingspotensiale i mikrobielle samfunn ved oljeutslipp under ulike betingelser; (III) Sammenligning av biodegraderingspotensialet for sjøvann fra norske lokaliteter. Vi har påvist at dispergerbarheten av norske råoljer hovedsakelig er avhengig av oljetypen, og at lette råoljer enkelt kan dispergeres i motsetning til tunge og viskose råoljer. Dessuten har vi påvist at effektiviteten av de undersøkte dispergeringsmidlene er forskjellig hvis de brukes for tunge oljer og emulsjoner, mens det ikke ble oppdaget forskjeller for lette oljer. Bruk av kjemiske dispergeringsmidler reduserte ikke biodegradering og påvirket ikke sammensetningen av det mikrobielle samfunnet i særlig grad. Selv om biodegradering er sterkt påvirket og kontrollert av temperaturen i omgivelsene, vil oljetypen, som er karbonkilden, spille en viktig rolle. Selv om de mikrobielle samfunn i det sjøvannet som ble brukt viste forskjeller avhengig av lokalitet og sesong, så ble disse variasjonene redusert til et samfunn dominert av noen få arter som hovedsakelig bidrar til mineralisering av hydrokarbonsubstrater. Våre resultater viser at biodegradering av kjemisk dispergerte oljer er effektiv i både polart og varmere norsk sjøvann, og er sammenlignbart med andre biodegraderingsrater rapportert for havet.SUMMARY Background: Oil exploration and production ventures have been part of the Norwegian continental shelf for about 50 years, and interest in future activities does not seem to have lessened. On the contrary, plans exist to expand the activities into Arctic regions in pursuit of new resources. For this reason, and because of the history of oil spills around the globe, there is a reasonable scientific and public concern regarding risks accompanying such activities. Oil contamination risk assessments must be based on experimental results in which oil biodegradation plays a particular role. Crude oil biodegradation in higher latitude temperate and polar waters is not studied sufficiently and is poorly understood, particularly when combined with operational oil spill methods, like the use of chemical dispersants in oil-affected areas. Aim: This PhD thesis was funded in order to obtain a better understanding of microbial community dynamics and capacity for oil biodegradation during oil spills in Norwegian waters when spilled oil is treated with chemical dispersants. This work is structured into the following thematic objectives: I. Properties of crude oil and chemical dispersant influencing biodegradation II. Microbial community dynamics and biodegradation potential under different oil spill conditions III. Comparison of biodegradation potential of Norwegian seawater sources Results and discussion: The efficacy for dispersibility of characteristic Norwegian crude oils and emulsions using commercially available dispersants was tested. We have used four crude oils with different properties (paraffinic, naphthenic, asphaltenic and a blend of paraffinic and asphaltenic oil) and three available chemical dispersants (Slickgone NS, Corexit 9500A and Finasol OSR-52). Troll emulsions were dispersed with all three dispersants. Corexit and Finasol generated smaller droplets compared to Slickgone; however, all droplet sizes were within the ranges expected to be quickly biodegraded at low temperatures. Dispersibility tests of fresh oils with Slickgone and Corexit showed larger droplet sizes using asphaltenic oil with both dispersants. IFT measurements revealed more rapid dispersant leaching with Slickgone compared to Corexit. We can thus conclude that Corexit 9500A and Finasol OSR-52 proved to be more efficient compared to Slickgone NS when emulsions and asphaltenic oil was dispersed, whereas for light paraffinic and naphthenic oil, the dispersants’ effects were similar. In addition, we investigated whether chemical dispersants negatively influence biotransformation dynamics and microbial community structure during dispersed oil incubations. We found that there were no differences between degradation of mechanically and chemically dispersed oils. Furthermore, seawater enriched only with a dispersant did not significantly differ in microbial community composition compared to seawater controls, whereas mechanically and chemically dispersed oil incubations exhibited substantially different microbial communities compared to the previous two incubations. Biodegradation dynamics are known to change with altering environmental conditions. Our aim was to inspect how local seawater is influenced by changing temperature and oil type. Seawater temperature played a crucial role in oil degradation dynamics as well as in defining the community composition. Degradation of the dispersed oil proved to be twice as fast at 13°C as at 5°C. Major differences in community composition among two seawater temperatures originated from the contrast in Colwelliaceae abundance, which was more pronounced at 5°C compared to 13°C. Oceanospirillaceae seemed to have preference for cold water as well. The principal aromatics degrader, Cycloclasticus, exhibited a significant increase after only three to six days of incubation at 13°C, while at 5°C, increase was observed only after 13 days to 16 days. Similar trends could be observed for Flavobacteriaceae, Alteromonadaceae and Rhodobacteraceae where the increase in abundance was shown to be more rapid at 13°C. In contrast to temperature, the effects of different crude oil types on biodegradation processes have only been studied to a limited extent. We have found differences in biodegradation dynamics between different oil dispersions occurring for PAH compounds, while there were no significant differences for n-alkanes. However, it was possible to observe that in certain periods, n-alkanes degraded more rapidly in paraffinic oil incubations compared to naphthenic oils. The differences in microbial community composition were found to similarly occur in the early days of incubation, particularly in abundances of principal degraders. Differences in degradation and community composition between distinct oil types interestingly became more prominent at low (compared to temperate) seawater temperatures. One important aim was to identity the genetic potential of the biodegradation key-players in local seawater from a Norwegian fjord (-80m) at low temperature (5°C). For this reason, we reconstructed genomes from metagenomic datasets obtained during biodegradation study. We have found that the Oceanospirillaceae genus Bermanella contributed the most to gene encoding for initial n-alkane degradation. Zhongshania, a Spongiibacteraceae genus, was observed to contain the same genes as well. Porticoccaceae included a high abundance of gene encoding enzymes involved in the utilization of products of initial alkane breakdown. Similar observations were made for one Colwellia and Zhongshania genome. Beta-oxidation genes were found to be abundant in Bermanella and Porticocacceae but were not found as often in Colwellia. Additionally, Porticoccaceae contained genes encoding for aromatics degradation, while Cycloclasticus contained the greatest variety of the respective genes. In the mosaic of genes encoding for different enzyme systems, we found that the genetic profile of different reconstructed genomes shows substrate specificity and a need for cooperation during oil biotransformation. A metagenome succession emerges correspondingly to microbial community succession. This follows a pattern of genes encoding for alkane degradation eventually being substituted with genes encoding for beta oxidation and aromatics degradation. Comparison of biodegradation effectiveness of two Norwegian seawater sources at low seawater temperature (0-2°C) revealed that n-alkanes in dispersed oil were biotransformed faster in an Arctic (Svalbard- SVB) than a temperate (Trondheimsfjord- TRD) seawater at low temperature, and these differences were also reflected in the microbial successions. The n-alkane degradation was primarily associated with the Oceanospirillaceae genus Oleispira, which became more abundant in the SVB than the TRD seawater. However, biotransformation of aromatic hydrocarbons and VOCs were comparable between the two seawater sources, and related to high abundances of the Piscirickettsiaceae genus Cycloclasticus emerging late in the biodegradation period. Larger n-alkanes (nC29+) were not depleted in the dispersions, and longer lag-periods and higher biotransformation rates at the low temperature (0-2°C) were observed when compared to degradation of n-alkanes in corresponding dispersions at higher temperatures (> 5°C). The biotransformation of the aromatic hydrocarbons and VOC at the low seawater temperature was comparable to biodegradation of these groups at higher temperatures. However, the lag-periods were longer at the lower temperature, probably reflecting slower dissolution and the fact that the degradation of these compounds mainly appear in the seawater after dissolution. These data will have implications for the predictions of the fate, as well as the environmental risk, related to oil spill in Arctic and other cold seawater environments after treatment with chemical dispersants. Conclusions: We can conclude that application of chemical dispersant to oil contaminated seawater does not suppress biodegradation, and it does not have a major influence on indigenous microbial community composition. Although biodegradation is largely influenced and controlled by environmental temperature, the crude oil type as a source of carbon also plays an important role. Microbial communities found in selected seawater exhibit seasonal and locational variations; however, these variations converge to a community dominated by a few taxa that have large contribution in mineralization of hydrocarbon substrate. Our findings show that biodegradation of chemically dispersed crude oils occurs effectively in temperate and polar Norwegian seawater, and it is comparable to biodegradation rates found throughout the ocean.