The ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) enzyme found in plants, algae, and an array of autotrophic bacteria is also encoded by a subset of methanotrophs, but its role in these microbes has largely remained elusive. In this study, we identified that CO
2
was requisite for RubisCO-encoding
Methylococcus capsulatus
Bath growth in a bioreactor with continuous influent and effluent gas flow. RNA sequencing identified active transcription of several carboxylating enzymes, including key enzymes of the Calvin and serine cycles, that could mediate CO
2
assimilation during cultivation with both CH
4
and CO
2
as carbon sources. Marker-exchange mutagenesis of
M. capsulatus
Bath genes encoding key enzymes of potential CO
2
-assimilating metabolic pathways indicated that a complete serine cycle is not required while RubisCO is essential for growth of this bacterium.
13
CO
2
tracer analysis showed that CH
4
and CO
2
enter overlapping anaplerotic pathways and implicated RubisCO as the primary enzyme mediating CO
2
assimilation in
M. capsulatus
Bath. Notably, we quantified the relative abundance of 3-phosphoglycerate and ribulose-1,5-bisphosphate
13
C isotopes, which supported that RubisCO-produced 3-phosphoglycerate is primarily converted to ribulose-1-5-bisphosphate via the oxidative pentose phosphate pathway in
M. capsulatus
Bath. Collectively, our data establish that RubisCO and CO
2
play essential roles in
M. capsulatus
Bath metabolism. This study expands the known capacity of methanotrophs to fix CO
2
via RubisCO, which may play a more pivotal role in the Earth’s biogeochemical carbon cycling and greenhouse gas regulation than previously recognized. Further,
M. capsulatus
Bath and other CO
2
-assimilating methanotrophs represent excellent candidates for use in the bioconversion of biogas waste streams that consist of both CH
4
and CO
2
.
Importance
The importance of RubisCO and CO
2
in
M. capsulatus
Bath metabolism is unclear. In this study, we demonstrated that both CO
2
and RubisCO are essential for
M. capsulatus
Bath growth.
13
CO
2
tracing experiments supported that RubisCO mediates CO
2
fixation and a non-canonical Calvin cycle is active in this organism. Our study provides insights into the expanding knowledge of methanotroph metabolism and implicates dual CH
4
/CO
2
-utilizing bacteria as more important players in the biogeochemical carbon cycle than previously appreciated. In addition,
M. capsulatus
and other methanotrophs with CO
2
assimilation capacity represent candidate organisms for the development of biotechnologies to mitigate the two most abundant greenhouse gases CH
4
and CO
2
.