Happily ever after?: how repeated subcultivation influences a methanotrophic marriage

Methane (CH4) is one of the most important greenhouse gasses, with a global warming potential of 34 times carbon dioxide over a time horizon of 100 years [1]. Methane oxidizing bacteria (MOB), characterized by their unique ability to use CH4 as both a sole carbon and energy source could be employed for both the mitigation and recovery of CH4. Recently it has been shown that methane oxidation is stimulated by (non-methanotrophic) partners in a methanotrophic interactome [2, 3]. These interacting partners can be specific depending upon the strains involved [4]. The MOB offer the partners methane-derived carbon, but it is unclear which carbon compounds are being exchanged. The partners can alleviate stress induced by self-inhibitory compounds from methane oxidation metabolism [5] or offer the MOB metabolites such as vitamins [6]. However, it is not yet elucidated what the mode of MOB and partner interaction is. To gain a better understanding of the methanotrophic partnerships we used a synthetic ecology approach to constitute “marriages” (multiple cycles of co-cultivation between MOB and their partners). We investigated how partnerships with both an alpha- (type II) and a gammaproteobacterial (type I) aerobic MOB evolved over time by means of either 16S rRNA gene-DGGE for a complex partnership with 8 partners or specific qPCR (pmoA/16S rRNA gene based) for each partner combined with Illumina MiSeq 16S rRNA gene amplicon sequencing in the case of a simple partnership with 2 partners. These partners consisted out of one fixed partner and one of 6 variable partners which were selected to be either highly, moderately or lowly compatible with the MOB. We showed that when the MOB are combined in a complex interaction marriage, a selection towards specific partners occurs, highlighting specificity of MOB and partner interactions. While no clear improvement of the methane oxidation rates (MOR) could be observed, the lag time until methane oxidation started was reduced upon addition of partners. On the other hand, in the simple partnership the “marriage” was challenged in each cycle with each of the variable partners separately. We observed, depending upon the type of the MOB, a differential impact of the variable partner on the MOR as the number of co-cultivation cycles increased. We observed that the initial partnership with the fixed partner was very easily outcompeted by a variable partner, regardless of the initial compatibility. This shows that adaptation through repeated co-cultivation did not offer an advantage to the fixed partner to make the marriage last, although with another moderately compatible partner sometimes a co-existence was possible after repeated cycles of co-cultivation with the MOB. Given the importance of biological interactions for methane oxidation in the envrionment as well as in biotechnological, our insights could be employed for microbial resource management (MRM) to steer the composition and performance of the methanotrophic microbiome in situ. REFERENCES 1. Myhre, G., et al., 2013: Anthropogenic and natural radiative forcing, Cambridge University Press. p. 659-740. 2. Ho, A., et al. ISME J, 2014. 8(9): p. 1945-1948. 3. Oshkin, I.Y., et al. ISME J, 2015. 9(5): p. 1119-1129. 4. Stock, M., et al. RES MICROBIOL, 2013. 164(10): p. 1045-1054. 5. Hanson, R.S. and T.E. Hanson. Microbiol Rev, 1996. 60(2): p. 439-+. 6. Iguchi, H., H. Yurimoto, and Y. Sakai. Appl Environ Microb, 2011. 77(24): p. 8509-8515