Methane is an essential component of the global carbon cycle and one of the most powerful greenhouse gases, yet it is also a promising alternative source of carbon for the biological production of value-added chemicals. Aerobic methane-consuming bacteria (methanotrophs) represent a potential biological platform for methane-based biocatalysis. Here we use a multi-pronged systems-level approach to reassess the metabolic functions for methane utilization in a promising bacterial biocatalyst. We demonstrate that methane assimilation is coupled with a highly efficient pyrophosphate-mediated glycolytic pathway, which under oxygen limitation participates in a novel form of fermentation-based methanotrophy. This surprising discovery suggests a novel mode of methane utilization in oxygen-limited environments, and opens new opportunities for a modular approach towards producing a variety of excreted chemical products using methane as a feedstock.

In the past few years, the field of methylotrophy has undergone a significant transformation in terms of discovery of novel types of methylotrophs, novel modes of methylotrophy, and novel metabolic pathways. This time has also been marked by the resolution of long-standing questions regarding methylotrophy and the challenge of long-standing dogmas. This chapter is not intended to provide a comprehensive review of metabolism of methylotrophic bacteria. Instead we focus on significant recent discoveries that are both refining and transforming the current understanding of methylotrophy as a metabolic phenomenon. We also review new directions in methylotroph ecology that improve our understanding of the role of methylotrophy in global biogeochemical processes, along with an outlook for the future challenges in the field.

Active members of the bacterial community in the sediment of Lake Washington, with special emphasis on C ₁ utilizers, were identified by employing two complementary culture-independent approaches: reverse transcription of environmental mRNA and 16S rRNA combined with PCR (RT-PCR) and stable-isotope probing (SIP) of DNA with the ¹³ C-labeled C ₁ substrates methanol, methylamine, formaldehyde, and formate. Analysis of RT-PCR-amplified fragments of 16S rRNA-encoding genes revealed that gammaproteobacterial methanotrophs belonging to Methylobacter and Methylomonas dominate the active methylotroph population, while only one other known methylotrophic lineage, Methylophilaceae , was detected via this approach. Analysis of RT-PCR-amplified functional genes, pmoA and fae , allowed detection of alphaproteobacterial ( Methylosinus ) and gammaproteobacterial ( Methylobacter , Methylomonas , and Methylomicrobium ) methanotrophs, methylotrophs of the genus Methylobacterium , and yet-unidentified proteobacteria. SIP experiments allowed detection of a broad variety of groups actively metabolizing C ₁ compounds. Comparisons between 16S rRNA gene pools amplified from [ ¹³ C]DNA and from [ ¹² C]DNA revealed that the proportion of Methylophilus -related sequences increased in the presence of [ ¹³ C]methanol, [ ¹³ C]methylamine, and [ ¹³ C]formaldehyde; Novosphingobium -related sequences were enriched in the presence of [ ¹³ C]methanol; Gemmatimonadaceae -related sequences were enriched in the presence of [ ¹³ C]formaldehyde and [ ¹³ C]formate; and Xanthomonadaceae -related sequences were enriched in the presence of [ ¹³ C]formate. Analysis of fae genes amplified from [ ¹³ C]DNAs isolated from different microcosms revealed specific shifts in populations in response to a specific C ₁ compound: Methylosinus sequences dominated the [ ¹³ C]methanol microcosm pool, and beta- and gammaproteobacterial sequences dominated the [ ¹³ C]methylamine microcosm pool. The [ ¹³ C]formaldehyde microcosm was dominated by betaproteobacterial sequences and by sequences of a nonaffiliated group, while the [ ¹³ C]formate microcosm was dominated by alpha- and betaproteobacterial sequences. Overall, these data point toward the presence of a diverse population of active methylotrophs in Lake Washington sediments and toward the existence of yet-uncultivated organisms.