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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - EcOILogy (Microbial life in oil)

Teaser

Summary

Microbial biodegradation is a key factor influencing the quality of oil and, according to current concepts, takes place mostly at the continuous oil-water transition zone in oil reservoirs.
I recently discovered microorganisms in minuscule water droplets (1-3 Âµl) entrapped in oil from a natural oil seep. In EcOILogy, I propose that biodegradation of oil resources takes place in such minuscule water droplets dispersed in the oil phase which is a shift of paradigm and a new conceptional view for environmental science, -life in oil-. EcOILogy aims to explore this new world investigating the generic principles of life in oil. I will study if such droplets are a common phenomenon in degraded oil resources and how significant the respective degradation activities are. To this end, I will develop reverse stable isotope labelling as a novel method for quantifying minute microbial activities (WP 1). The droplets provide a unique test system of micro-ecosystem, all experiencing identical boundary conditions in the oil with no dispersion of microorganisms between the isolated droplets. I will study how microbial communities for oil degradation assemble in the droplets allowing for unprecedented testing of ecological theory including a new bimodal hypothesis of community assembly. To tackle the big challenge of metabolic traits in systems ecology, I will make use of metagenomics, single cell sequencing, and high resolution metabolomics to assess the functions in single water droplets (WP 2). Finally, I will study how microorganisms adapt to this extreme environment under saturated hydrocarbon concentrations by isolation and comparative genome analysis of strains and study the role of different organisms in the droplets by Raman-CLSM (WP 3).
Thus, EcOilogy opens new horizons for microbial degradation of our most important energy resources with far-reaching implications for fundamental, interdisciplinary understanding of ecological processes, bioremediation, and oil exploration.

Work performed

The ERCadv grant EcOILogy aims at elucidating how microorganisms can make a living in oil and how these microbial ecosytems are assembled. Our first discovery of life in oil in a natural oil seep in Trinidad, revealed small water droplets dispersed in the oil that inhabited complex microbial communities. This raised the novel concept that microbial degradation of oil in reservoirs is not only taking place at the oil-water transition zone at the bottom of the oil leg but also in small water pockets that are dispersed in the oil-bearing rock.
Analysing several droplets from oil sampled at the Pitch Lake in Trinidad-Tobago, we found that they contained a surprisingly high microbial density of approximately 109 cells/ml, which is equivalent to a dense Escherichia coli culture of OD 1. Life-dead staining revealed that the cells are alive, which is supported by the ATP content per cell, which is equivalent to average values reported in the literature for microbial cultures.
Biofilm formation is a frequent protection mechanism against environmental stress. We therefore studied the spatial structures of the microbial communities in the water droplets enclosed in oil and managed to visualize the cells with confocal laser scanning microscopy (CLSM) directly in the water droplets. Thin biofilms were found directly on the oil-water interface with approximately 100 times more cells at the droplet wall compared to suspended cells in the droplet volume. This makes the water droplets enclosed in oil a densely populated environment that is actively degrading the oil from within.
Nevertheless, the question remains how active the microbial populations in the droplets are and to which extent they are contributing to the overall degradation in an oil reservoir as compared to the microbial degradation at the oil-water transition zone. To measure the overall degradation activity of microorganisms in oil, we first developed a new method, the reverse stable isotope labelling (RIL). We bought a Delta Ray near infra-red laser absorption spectrometer with URI connect from Thermo Fisher, Bremen, Germany for measuring stable isotope ratios of CO2. We established the instrument in the lab and developed the technology for measuring evolution of CO2 from biodegradation. To this end, 13C-bicarbonate was added to the buffer system at distinct atom percent of 13C/12C. Biodegradation of oil leads to the evolution of 12C-CO2 and 13C-CO2 in a ratio of ca. 99/1 % which changes the stable isotope ratio of the buffer system (set to 10 atom %).
We first established the method with anaerobic cultures degrading aromatic hydrocarbons and showed that RIL is well suited to measure the degradation of hydrocarbons with non-defined cultures (Dong et al., 2017). We could even show that the method is suitable for analysing microbial degradation activities on solid specimen. However, it became obvious that we have to refine the method for assessing very small degradation activities as expected for oil degradation. Thus, we spend time on the optimization of the sensitivity and accuracy of RIL by: defining the optimal concentration of the carbonate buffer, the 13C/12C ratio of the bicarbonate buffer, ways to increase the sample volume that can be analysed, improving the precision of the instrumental analysis by changing flushing modes, and other parameters. The RIL method is now developed to a technical state where it can be applied to standard applications where extremely low degradation activities are to be measured.
First measurements with oil from the Pitch Lake in Trinidad took almost one year and showed that RIL can be applied to measure oil degradation with autochthonous microbial communities.
During EcOILogy project, we realized that it is not enough to focus on the microorganisms degrading hydrocarbons but that the majority of the species is not directly dealing with the anaerobic degradation of hydrocarbons. Thus, we performed an extensive study in order to elucidate

Final results

As a groundbreaking discovery we were able to develop a new method for surface enhance raman spectroscopy (SERS) which allows an easy and reproducible generation of a SERS for single microbes. This has not been achieved so far and allows for a new dimension in Raman-microscopy. This opens wide opportunities to link the function and phylogeny of microbial communities but also beyond microbiological applications.
The subsurface microbial loop, a new ecological concept.
During the work in EcOILogy, we realized that it is not enough to focus on the microorganisms degrading hydrocarbons but that the majority of the species is not directly dealing with the anaerobic degradation of hydrocarbons. Thus, we performed an extensive study in order to elucidate the function of such non-hydrocarbon degraders. During this study, we discovered a new ecological principle which we call the subsurface microbial loop and which is somewhat similar to the microbial loop in surface waters (Dong et al., 2018).
In contrast to surface waters, there is only a very restricted food chain in the subsurface, because higher organisms are mostly lacking. The question is, if and how microbial biomass is recycled. In an enrichment culture, we discovered that a subsurface microbial loop only needs two species: a primary biomass producer, which was a naphthalene-degrading sulfate reducer, and a necromass degrader, which was a spirochaete. We described the novel spirochaete as Rectinema cohabitans (Koelschbach et al., 2017) and could show that the organism ferments necromass and provides reducing equivalents and nutrients to the primary producer. About 79 % of the total biomass can be recycled via this subsurface microbial loop, despite optimal cultivation conditions. This surprising finding encouraged us to put more efforts on elucidating the subsurface microbial loop as a new ecological concept for oil microbiology and subsurface microbiology in general.
The expected outcome is a new ecological concept on the nutrient and element fluxes in the subsurface. The subsurface microbial loop is the first comprehensive theory on a subsurface microbial food web.