Stable carbon isotopes can provide insight into carbon cycling pathways in natural environments. We examined carbon isotope fractionations associated with a hyperthermophilic fermentative bacterium, Thermotoga maritima, and a thermophilic chemolithoautotrophic bacterium Persephonella marina. In T. maritima, phospholipid fatty acids (PLFA) are slightly enriched in 13C relative to biomass (ε =0.1-0.8‰). However, PLFA and biomass are depleted in 13C relative to the substrate glucose by ∼8‰. In P. marina, PLFA are 1.8-14.5‰ enriched in 13C relative to biomass, which suggests that the reversed tricarboxylic acid (TCA) cycle or the 3-hydroxypropionate pathway may be used for CO2 fixation. This is supported by small fractionation between biomass and CO2 (ε = -3.8‰ to -5.0‰), which is similar to fractionations reported for other organisms using Similar CO2 fixation pathways. Identification of the exact pathway will require biochemical assay for specific enzymes associated with the reversed TCA cycle or the 3-hydroxypropionate pathway.