Strain 761M, based on its 16S rRNA gene sequence, was later found to group with the Gammaproteobacteria (Bowman et al., 1995). To the best of our knowledge, the phylogenetic grouping of strain R6 was never determined (although enzymatic analyses suggested its affiliation to Alphaproteobacteria). None of these strains appear to be still extant, making it impossible to repeat these experiments. Two methanotrophs isolated from freshwater lake sediments were also described as being facultative, i.e., able to utilize not
only methane, but also casamino acids, nutrient HM781-36B cost agar, and a variety of organic acids and sugars for carbon and energy (Lynch et al., 1980). However, one of these isolates, Methylobacterium ethanolicum, was
later found by members of the same laboratory to actually consist of a stable syntrophic consortium of two methylotrophs, i.e., a Methylocystis strain capable of utilizing methane, and a Xanthobacter Navitoclax strain capable of utilizing a variety of multicarbon compounds for growth (Lidstrom-O’Connor et al., 1983). Collectively, the inability of putative facultative methanotrophs to grow on methane after growth on multicarbon substrates, the lack of extant strains, and evidence of stable mixed cultures initially originally described as pure methanotrophic strains all cast serious doubts on the possibility of facultative methanotrophy. As a result, research in this area was severely limited for the next 20 years. Efforts to identify novel methanotrophs
significantly regained momentum in the 1990s with the discovery of acidophilic methanotrophs from Sphagnum peat bogs (Dedysh et Sclareol al., 1998a, b). The first characterized acidophilic methanotroph was found to represent a new genus and species within Alphaproteobacteria, Methylocella palustris (Dedysh et al., 2000), and subsequently two further strains of the same genus were isolated, Methylocella silvestris and Methylocella tundrae (Dunfield et al., 2003; Dedysh et al., 2004). All three strains were considered novel methanotrophs as their optimal pH for growth was <6.0. Even more remarkably, all three isolates could only express the sMMO, and not the pMMO. This finding was quite unexpected as it showed that these were the first methanotrophs that did not express pMMO. Initial screens of each isolate showed that they could not grow on sugars or multicarbon substrates, but could grow on methane and methanol, as well as on methylamine to a variable degree, thus they were considered obligate methanotrophs. These methanotrophs, however, were later shown to be facultative as they could utilize not only C1 compounds for growth, but also acetate, pyruvate, succinate, malate, and ethanol (Dedysh et al., 2005 and Table 1).