Vekeman, B. (2016). Methanotrophic microbiomes from North Sea sediment. PhD Thesis. Faculty of Sciences, Ghent University: Gent. ISBN 978-94-6197-399-3. 209 pp.

235528

Vekeman, B.

2016

209 pp.

Publication

Methane is the most abundant organic greenhouse gas in our atmosphere, and has a strong infrared absorbance, being 25 to 30 times more effective than carbon dioxide on a 100 years scale. Methane therefore plays an important role in the climate warming regulation. Methanotrophs are microorganisms that can consume methane and utilize it as their sole source of carbon and energy. These organisms are the most important biological sink of methane. Their importance is especially demonstrated in marine ecosystems. While the oceans have the potential to produce enormous quantities of methane, a series of very effective microbiological oxidation processes results in the ocean being one of the smallest net global methane sources. Hence, it is of vital importance to understand who is there and what factors may positively or negatively impact the methane-oxidizing activity of these organisms. Despite the importance of the aerobic methane oxidizing bacteria (MOB) in the marine ecosystems currently only a limited amount of ex situ cultures is available. Nevertheless, they are indispensable to link physiology to genomic features and expand our knowledge about the specific habitat preferences of marine MOB. This study focused on aerobic marine MOBs and aimed at designing a large-scale enrichment and isolation strategy to retrieve a maximal MOB diversity from marine sediments. First, the effect of adhesion material and headspace composition on the methane oxidation activity in marine sediment enrichments were investigated. The addition of sterilized natural sediment as well as acid-washed silicium dioxide significantly positively influenced methane oxidation. The exact mechanism of this positive effect needs further investigation but might be the facilitation of methane, carbon dioxide and oxygen gradients in addition to adhesion. Use of adhesion material might thus facilitate the cultivation and subsequent enrichment of members of this functional guild. Next, using these insights, a large scale isolation strategy was performed from sediment collected at six different stations in the North Sea along a transect from estuary to open sea. An initial enrichment step with serial subcultivations was followed by miniaturized extinction culturing mimicking a range of nitrogen and oxygen microniches. A clear decreasing trend of cultivability and detectability was observed along the investigated transect. Furthermore widely applied pmoA primers failed to amplify biomarkers in a large number of active methanotrophic cultures, suggesting enormous underestimation of methanotrophs in situ in PCR-based molecular surveys. Unfortunately, despite the numerous attempts we were not able to obtain axenic methanotrophic cultures, most likely due to tight mutualistic interactions with heterotrophic bacteria.Shot gun sequencing of four methane-oxidizing enrichment cultures revealed the presence of a novel gammaproteobacterial MOB belonging to the deep-sea cluster 2 in two cultures and a novel alphaproteobacterial MOB belonging to the recently described methylotrophic genus Methyloceanibacter in the other two cultures. Methyloceanibacter methanicus represented the first MOB found in an exclusively methylotrophic genus, the first marine type II MOB and only the third taxon in which solely sMMO was resoponsible for methane oxidation. A targeted isolation using methanol as carbon source led to a axenic culture of the MOB, in addition to three closely related novel strict methylotrophic species, M. superfactum, M. stevinii, M. marginalis. Together with the previously described M. caenitepidi, these species exemplify an extreme niche differentiation, with a wide ecotypic variation related to growth kinetics on methanol, and preferences for nitrogen, pH, temperature and salt. Furthermore, the most striking difference of the deep-sea cluster 2 representatives demonstrated a striking difference with other gammaproteobacterial MOB in its lack of a calcium dependent methanol dehydrogenase encoded in the genome. The genome solely contained the genes for xoxF5 for the lanthanide-containing methanol dehydrogenase.Lastly, a preservation protocol was optimized for the long term storage of marine bacteria in order to successfully store the enrichments and axenic cultures obtained throughout this dissertation. Fastidious nitrite-oxidizing bacteria were used as model organisms. They demonstrated that optimal preservation conditions were strain-dependent whereby marine strains, appeared to be more sensitive to freezing than non-marine strains. Nevertheless, a general cryopreservation protocol using 10% dimethyl sulfoxide as cryoprotective agent with or without ten-fold diluted trypticase soy broth and trehalose as a preservation medium allowed successful preservation of all tested strains. Applying the same protocol on whole marine sediment samples allowed successful storage of different key players in the carbon and nitrogen cycle.In conclusion marine MOBs are notoriously difficult to cultivate and isolate. Despite numerous attempts, I was only able obtain one axenic culture, in addition to over 200 enrichment cultures, from the oxygenated zones of North Sea sediments. The availability of a successful preservation protocol allows the storage of this axenic culture, ensuring that novel diversity does not get lost, but also further guaranties archiving of the obtained enrichment cultures and environmental samples. As such these are available for future isolation when novel insights in marine MOB and isolation strategies/techniques become available.

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