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PETROMAKS2-Stort program petroleum

Hydrocarbon-utilizing microorganisms as oil indicators and oil spill destructors in the water and deposits of Barents Sea

Alternative title: Hydrokarbon-spisende mikroorganismer som olje-indikatorer og oljesøl-opprensere i vann og sedimenter i Barentshavet.

Awarded: NOK 2.9 mill.

Project Number:

308831

Project Period:

2020 - 2024

Partner countries:

The Barents Sea is one of the most economically attractive regions of the Arctic due to its numerous gas seepages and the large gas and oil deposits located in Russian and Norwegian sectors. Hydrocarbons can serve as carbon and energy sources for microorganisms, including those inhabiting extremely cold marine arctic environments, and their occurrence could thus be used as biological indicators of the presence of oil and gas deposits. These microorganisms participate in environmental “self-purification” of hydrocarbon contamination within their habitats. The goal of this project is to enhance the knowledge of the cold-adapted hydrocarbon (including methane)-utilizing bacterial diversity and distribution in the Barents Sea using cultivation-based approaches, as well as direct metagenomics analyses of environmental DNA aiming at assembly of complete genomic sequences and prediction of metabolic characteristics. We further aim at isolation and characterization of this type of bacteria as well as identification of active hydrocarbon-degrading populations using stable isotope probing (SIP) techniques. Data obtained would allow making correlations between the presence of hydrocarbons and hydrocarbon-utilizing microorganisms, which would in the future help to develop rapid methods for explorations in the Arctic Sea region. The ability of cold-adapted marine microbial communities to be used for biological clean-up of oil contaminations will be evaluated. The basic understanding of the diversity of cold-adapted hydrocarbon-utilizing microorganisms and their metabolism will be enhanced. The Norwegian team focuses on methane oxidizers (also called “methanotrophs”) and active populations using DNA-SIP experiments while the Russian team on degraders of higher hydrocarbons as well as direct diversity analyses based on DNA extracted from seafloor samples. From the Russian partner we received 41 seafloor samples (from 0 to 5 cm depths) from selected areas in the Eastern part of the Barents Sea. All samples were used in enrichments of psychroactive methane oxidizing bacteria at 10oC. For each sample, two enrichment cultures with methane as the sole carbon and energy source were made, with and without supplement of copper ions, this because there are two groups of methanotrophs: one (almost) copper-independent and one copper-dependent. A total of 21 enrichment cultures were positive in terms of microbial growth and consumption of methane after 2-3 months incubation, and these were used for metagenomics analyses. The results revealed the presence of methanotrophic populations in all positive enrichments, but in most of the enrichments these populations constituted a lesser part of the total bacterial community. This indicates that the added methane, which is oxidized by methanotrophic bacteria, makes a basis as primary energy source for a complex bacterial community. Using bioinformatics, we have assembled a total of 12 ”methanotophic” MAGs (metagenome-assembled-genomes) of methanotrophic bacteria representing almost complete genomes of several different taxa. We have also used key metabolic genes from these MAGs, the genes encoding enzymes participating in oxidation of methane, to map phylogenetic affiliations and diversity. All the 12 “methanotrophic” MAGs possessed genes encoding the copper-dependent key enzyme “particulate methane monooxygenase” and represent novel methanotrophic species. To isolate methane oxidizers (obtain pure cultures) from enrichments, a large number of dilutions to extinction series were performed, as well as trials to isolate methanotrophs by plating on solid media. Despite many trials isolation efforts have been unsuccessful, possibly because methanotrophs will not grow on surfaces and depend on their bacterial partners in the natural environment for growth. DNA-SIP experiments aimed at identification and analysis of active methane-oxidizing populations also failed despite many trials, for unknown reasons.

The project has provided novel genomic and taxonomic information of cold-adapted methanotrophic bacteria at the Barents Sea seafloor providing us with a better understanding of the mechanism of methane oxidation in Arctic marine regions. Marker genes from these bacteria may serve as indicators for natural gas and petroleum resources in the sub-seafloor. Enrichments of methanotrophic and hydrocarbon-degrading bacteria also have a potential for use in strategies for mitigation of petroleum spills in the Arctic region.

Natural resources, conditions and the geographic location of the Barents Sea make it the most economically attractive region of the Arctic. The Barents Sea bed is characterized by numerous gas seepages and one of the biggest gas deposits, Shtokman. The large oil deposit, Goliat, is located in the Norwegian segment. Hydrocarbons can serve as substrates for microorganisms, including those inhabiting extremely low-temperature marine environments. Some of them use hydrocarbons as sole energy and carbon source and, thus, their occurrence and numbers could be used as biological indicators of presence of oil. Hydrocarbon-degrading microorganisms, including psychrophiles, participate in self-purification of hydrocarbon contamination in natural habitats. The goal of this project is to study the psychrophilic hydrocarbon-utilizing (including methane oxidizers) bacterial diversity and their distribution in the Barents Sea by cultural and molecular approaches. Metabolism of uncultured hydrocarbon-oxidizing bacteria detected in metagenomes by the key genes of hydrocarbon oxidation will be studied using their metagenome assembled into complete genomes. Hydrocarbon degraders will also be isolated and characterized, and active hydrocarbon-degrading populations studied using DNA-stable-isotope probing. Data obtained would allow us to assess the correlation between the presence and diversity of hydrocarbons and hydrocarbon-utilizing microorganisms in water and ground samples of Barents Sea which would in the future help to develop rapid methods of oil deposits exploration in Arctic sea region. The ability of marine psychrophilic microbial communities to be used for bioremediation of oil contamination will be evaluated. The basic understanding of the diversity of psychrophilic hydrocarbon-utilizing microorganisms and their metabolism will be enhanced. The Norwegian team will focus on methanotrophs and DNA-SIP experiments while the Russian team on degraders of higher hydrocarbons.

Funding scheme:

PETROMAKS2-Stort program petroleum