Molecular-based technical adaptation using oil-degrading bacteria for autonomous leakage detection

This project is exploring the application of the Environmental Sample Processor (ESP), a real-time analytical robot enabling advanced measurements at sea and often described as a ?molecular laboratory in a can?.The ESP analytical capabilities are explored particularly towards the environmental monitoring of petroleum activities, with a focus on accidental spill in the high North. The scientific challenges are related to the ability of the ESP to detect specific microbial genes as markers of oil occurrence in the water. Crude oil pollution affects strongly and rapidly the bacterial community composition with the dominance of some oil-degrading bacterial species. Hence, bacterial community changes can be used as an effective biosensor for oil contamination in the marine environment and the ESP is the platform enabling their real-time monitoring at sea. Important project milestones were: identification of molecular markers of crude oil pollution using relevant spill scenario, optimization of assays with protocols mimicking ESP and validation. 1) First, we selected and verified in preliminary experiments the potential bacterial bioindicators of oil biodegradation for this work. After analytical protocol optimization, samples of seawater with relatively high levels of petroleum load (nominal concentration of 0.1%) and low temperature (4ºC) were tested. Our first data analyses confirmed the importance of Gammaproteobacteria (particular role of Oceanospirallaceae family), Alphaproteobacteria and Flavobacteria microbial family as stated in literature. (WP1) 2) In a second series of so-called GRADIENT experiment we performed detailed verification of selected molecular targets using Q-PCR analysis with samples derived from experiments using more realistic loading of contaminants, from 30ppb to 2000 ppb. At that point, the scientific questions were whenever selected bacterial bioindiactors will respond to the petroleum, what is the lowest concentration and timing of the response compared to reference situation with no oil. We identified bacteria, which responded linearly to the dose of petroleum contamination i.e. Alcanivorax sp., Cycloclastius sp., Oleispira antarctica, Marinomonas sp., Thalassolituus oleivorans and others like Colwellia sp. less dose-dependent but sensitive to oil level as low as 30ppb. QPCR results were confirmed by NGS amplicon-based sequencing showing the same compositional shifts in bacterial community. Chemical composition together with the succession of bacterial community during the experiment showed a comprehensive picture. These experiments were performed in controlled conditions with seawater from the Byfjord (Stavanger) used in our facility (WP2). 3) In the field studies performed in Greenland, the observations made in the laboratory were confirmed. Among other, QPCR analysis indicated a significant enrichment of Oleispira antarctica and Colwellia sp. Hence, these two species appear to be robust biosensors of oil for Arctic water. Colwellia bacteria showed the same occurrence independently of season variation and temperature. QPCR assays specific to Oleispira antarctica meets all requirements of a bioindicator of crude oil pollution at low temperatures as well but the occurence of this bacteria is delayed at cold winter temperature compared to Colwellia. Other analyses of field samples (Full City cargo ship, Langesund, Norway) also confirmed the role of Oleispira antarctica, Alcanivorax sp. and Cycloclasticus sp. and bacteria belonging to Colwelliaceae as bioindicators of pollution of crude oil (WP3). 4) Two ESP instruments were shipped to IRIS to test with qPCR and SHA assays implemented during the duration of this project. They were used to analyze control and oil-exposed water samples from a lab mesocosm setup in IRIS mimicking a realistic oil spill in water. After oil addition to the mesocosm, ESP analyzed water for target microbial response each day for 7 days. Bench analyses with protocols mimicking those of the ESP were also performed for comparison. The concentration in the water analyzed by ESP varied from 1.9 mg/l (start) to 0.5 mg/l oil and 56 ?g/l (start) to 4.5 ?g/l sum PAH. The Oleispira assay with qPCR was comparatively good and provided the best response to oil exposure in water after 3 to 4 days. Colwellia increased but showed no difference in control and oil-exposed water. Regarding SHA, the probe arrays tested were weak and did not allow to significantly detect oil in water compared to situation with no oil. A major challenge remained the confounding effect of the incubation itself on the microbial shift in both control and oil-exposed tanks. In conclusion, using ESP q-PCR analytical protocols aiming at quantifying oil-degrading microbes shortly after oil exposure, oil leakage events in the water can be detected. Oleispira is the microbial bioindicator showing best response to crude oil pollution. SHA needs optimization of the probes to oil detection.

Oil and Gas (O&G) industries are moving towards deeper and colder areas requiring new technologies capable of real-time monitoring, and rapid access to operational and environmental information. Regarding subsurface oil leak detection, the Environmental S ampling Processor (ESP) developed by researchers of MBARI (Monterey Bay Aquarium Research Institute) is bearing great potential for underwater sensing related to O&G installations. The ESP is a field-deployable device designed to collect water samples and apply autonomously molecular assay technologies to identify specific marine targets used as markers of water conditions. The objectives of this application are 1) to demonstrate that molecular assays that use oil-degrading microbial targets are suitable for oil leak detection and 2) adapt the ESP device accordingly and validate the performance of this device for near-real time oil leak detection in field-simulated deployment in the laboratory. Three work packages (WP) and several milestones (M) are plann ed: In WP1, we will select oil-degrading microbial species and design the corresponding molecular probes specific to the target species and genes involved in the degradation of petroleum compounds (M1). In WP2, we will proceed with developing the detectio n assays in a fashion that is compatible with the ESP device and in situ operations (M2). After testing the new assays using conventional laboratory equipment, MBARI will assist with testing those assays on the ESP itself (M3). If results of these prelimi nary trials are encouraging, MBARI will bring an ESP to IRIS facility for final laboratory testing using relevant oil leakage scenarios for North Sea and sub-Arctic conditions, and further to explore how the instrument will be deployed in Norwegian waters (M4). This work is in step with MBARIs vision for ESP development within the time frame of this project: deployment of ESP on autonomous underwater vehicles (AUV) for mobile deployment at O&G installations.