Methane (CH4) is an important greenhouse gas, and the majority (ca. 60%) of its emission originates from anthropogenic sources. Methane oxidizing bacteria (MOB) are characterized by their unique ability to use CH4 as a sole carbon and energy source. Recently, accumulating evidence demonstrated that methane oxidation is stimulated when MOB are interacting with non-methanotrophic microbes. These interactions can be very specific, although it is not yet entirely elucidated what the determining factors to a successful partnership are. Hence, it seems that a methanotrophic interactome is required for effective biological aerobic methane oxidation, rather than individual obligate methanotrophic bacteria. In this dissertation, we focused on bacterial interactions between MOB and non-MOB and try to assess what are the determinants for non-MOB partner selection in the methanotrophic interactome. We focus specifically on representatives of alpha- and gammaproteobacterial MOB, which are known to exhibit differential functional traits which can in turn be conceptualized as distinct life strategies. The impact of these differences in MOB life strategies on interactions with non-MOB has not been studied. An increased insight in the co-existence and the extent of co-dependence of MOB and non-MOB partners is required to fully understand the essential ecosystem service of biological methane oxidation. In PART 1 (TOP-DOWN APPROACH), we dissected a methanotrophic enrichment culture (optimal methanotrophic interactome) by means of time-resolved stable isotope probing combined with 454-pyrosequencing of the 16S rRNA genes to assess which interactome partners are most intricately involved in the assimilation of 13CH4-derived carbon. In PART 2 (BOTTOM-UP APPROACH), we artificially assembled an optimal methanotrophic interactome by piecing together multiple non-MOB strains with MOB (co-cultivation). Firstly, we supervised the selection of MOB for non-MOB partners by establishing compatibility and matching selected MOB with non-MOB partners. Microbial adaptation and abundance of all partners was investigated using q-PCR. Secondly, we allowed an unsupervised selection for non-MOB partners by the MOB and these interactions were monitored by DGGE. In PART 3 (CRYOPRESERVETION), we develop a cryopreservation protocol based on DMSO (and DMSO+TT) as a cryopretactant, which can used to adequately preserve optimal interactomes (preservation of key functional characteristics as well as composition) to allow possible biotechnological applications. In PART 1, we found that 13CH4-derived carbon is differentially distributed among interactome partners through time. We suggested that the most intricately associated non-MOB partners would be the first to assimilate 13CH4-C and could hence be considered “primary consumers” of whereas organisms labeled later in the time-course SIP experiment are more loosely associated with the MOB and may be “secondary consumers” of organic carbon derived from both the MOB and the primary consumers. In PART 2a, we could not observe adaptation of a moderately compatible partner to MOB (or vice versa) during repeated co-cultivation with a gammaproteobacterial MOB. Conversely, clear (though limited) adaptation was observed with an alphaproteobacterial MOB. Regardless of its initial compatibility with the MOB, a third partner nearly always completely obliterated the non-MOB partner in the existing MOB:non-MOB interactome. In PART 2b, we showed a clear partner selection where some of the persisting partners were “promiscuous” and could persist regardless of MOB type , whereas others were more specifically associated with either MOB type. Additionally, we observed that alpha- and gammaproteobacterial MOB differentially route CH4-derived carbon to the interactome. Finally, while repeated co-cultivation did not significantly impact the magnitude of overall methane oxidation rates (MOR), it did appear to stabilize the biological variability in MOR as compared to acenically grown MOB. In PART 3, the optimized cryopreservation protocol for storage of mixed microbial cultures adequately preserved both community structure and functionality of a methanotrophic interactome (among others) for 3 months at -80°C. These findings may be of significance for methanotrophic interactome Microbial Resource Management, if biotechnological application of non-MOB partners is the ultimate goal. Overall, unsupervised synthetic interactome assembly approaches should be preferred, as they specifically restrict only non-MOB partners that can persist with the MOB from other non-MOB partners. If specific biotechnological applications can be found for such an optimal combination of interactome partners, it is of great interest to preserve a reproducible sample of it for extended periods of time. Exploitation and engineering of these methanotrophic interactomes could lead to improved and sustainable mitigation and recovery of CH4 in the form of metabolic energy or CH4-derived carbon.