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Microbial communities of the Eastern Mediterranean and their hydrocarbon degradation capability

Charalampous Georgia

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Summary

The Eastern Mediterranean Sea (EMS) is a semi-enclosed basin, usually referred to as a “miniature ocean” because of the complex oceanic activities that take place there. Increased bottom sea temperatures, salinity levels and ultraoligotrophic conditions are some of its unique characteristics that shape microbial communities. This sub-basin of the Mediterranean Sea has become a hotspot for oil and gas activities in the recent decades. Large reservoirs have already been discovered and exploited in depths reaching ~1500m while several others, including ultra-deep marine areas, have been committed and are currently under exploration (South-Southwest Crete). In the aftermath of Deepwater Horizon (DWH) blowout in 2010, deep-sea oil biodegradation studies flourished providing the scientific community with valuable information about the fate of hydrocarbons (HC) in the deep sea, microbial succession patterns and key oil-degrading taxa. One of the major lessons learned from the DWH accident was the importance of conducting site-specific research under in situ conditions to provide policy makers with realistic data for the construction of efficient bioremediation protocols. The ongoing activities, which are progressing in deeper and more challenging waters, increase the risk for a potential oil spill accident in the deep EMS with many environmental and financial consequences for the surrounding countries. This PhD thesis attempts to evaluate the microbial response and self-healing capability (natural attenuation) in the event of an accidental hydrocarbon release scenario in the deep EMS. For this purpose, seawater was retrieved from the EMS water column, down to 1000 m below sea level, in stations south of Crete (Cretan Passage) using Niskin bottles (decompressed) and a high-pressure sampling apparatus (in situ pressure). Hydrocarbon-biodegradation experiments were conducted in Erlenmeyer flasks and in high-pressure bottles or high-pressure bioreactor for incubations at 0.1MPa and 10MPa respectively, using Iranian light crude oil as carbon source. Data on microbial community analysis and hydrocarbon-degradation rates were produced via high throughput sequencing and GC-MS analysis. Prior to any hydrocarbon exposure experiments, the synthesis of the pristine microbial community along with interspecies associations were analysed across the water column in this understudied marine region and background levels of known hydrocarbon degraders were recorded. Interestingly, even though natural seepages were not present near the sampling stations, notable abundances of taxa involved in oil bioremediation were found, especially in the deep-water layers. The known hydrocarbonoclastic genus, Alcanivorax, was included in the significant nodes of the deep network.Comparison of a timeseries hydrocarbon-degradation experiment at in situ temperature conditions between surface and deep EMS-collected microbial communities indicated that the latter (deep community) responded faster to oil contamination than the surface one despite the incubation at lower in situ temperature of 14 ºC. Furthermore, incubation of the deep consortium at a higher temperature of 25 ºC (for direct comparison with the surface community) did not affect oil biodegradation levels suggesting a microbial community that is acclimatized for HC biodegradation at the lower in situ temperature. Monitoring of microbial succession patterns resulted in different key hydrocarbon-degrading taxa in each treatment. The deep consortium at 14 ºC, was dominated primarily by the generalist Vibrio and substituted later on by the slow-growing specialist Alcanivorax whereas incubation at 25 ºC led to the dominance of Pseudomonas and Pseudoalteromonas in the deep community. On the other hand, the surface consortium was enriched in Thalassospira, Halomonas, Alteromonas and Idiomarina genera.The efficacy of the deep EMS consortia in hydrocarbon bioremediation, was further tested under in situ high-pressure conditions. In particular, the impact of decompression was evaluated during deep-sea sampling and in enrichment incubations for the isolation of oil degraders. Decompression upon sampling resulted in a drastic decrease in deep microbial diversity. In addition, subjection of HC-degrading consortia for enrichment in ONR7 medium caused further decrease in biodiversity and taxa correlated with HC removal were outcompeted by Pseudoalteromonas, Halomonas, Thalassomonas and Alcanivorax which were favored under all treatments tested. Dispersant application had no significant effect in microbial community composition at any stage of the experimental process. Biodiversity loss impacted the community functionality in terms of biodegradation of the more recalcitrant oil compounds (PAHs, Heavy Alkanes). A strains of Alcanivorax venustensis (recently emended as Alloalcanivorax venustensis) that dominated the communities was isolated. Furthermore, a deep EMS hydrocarbon plume emulation experiment was conducted under in situ EMS conditions to address primary microbial responders and succession patterns in the absence and presence of dispersant. Genera belonging to Gammaproteobacteria (Oleispira, Thalassomonas, Thalassotalea, Ralstonia) were among the first responders to oil-only contamination similarly to studies in the aftermath of the DWH accident. Those were then succeeded by Alcanivorax and Methylophaga, followed by Marinobacter and Thalassospira in the late phases of exposure to crude oil. The presence of dispersant favored members of Bacteroidota along with Hyphomonas and Alcanivorax, with the latter dominating the community. In conclusion, marine areas of interest for oil and gas exploration and exploitation (off-Crete) present a microbial “seed bank” of species related to hydrocarbon removal especially in deep-water layers. This could explain why the deep EMS community responds faster to oil contamination even though at lower temperature conditions than the surface consortium. Moreover, the results of this dissertation suggest an important role for Alcanivorax in hydrocarbon bioremediation in the deep EMS along with other known obligate oil degraders such as Oleispira and Marinobacter. Overall, this PhD thesis underlines the importance of maintaining in situ pressure and temperature conditions during sampling and experimentation when conducting experiments with deep-seawater microbial communities.

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