Despite being the largest ecosystem on earth, the deep biosphere is considered to be energy limited. Chemoautotrophy is an important source of organic carbon in the deep biosphere, and significantly contributes to the deep carbon cycle. We investigated the carbon fixation strategies in the model organism Themovibrio ammonificans in relationship to the presence of different terminal electron acceptor. T. ammonicans uses the reverse Tricarboxylic Acid cycle (rTCA) as carbon fixation pathways, however our comparative genomic analysis reveals the presence of an incomplete Wood-Ljungdahl (WL) pathway. Carbon isotopic fractionation value support the rTCA cycle as carbon fixation pathway, however a difference in carbon fractionation is present when growing T. ammonificans with sulfur instead of nitrate as terminal electron acceptor. Transcriptomic analysis showed that the putative carbon monoxide dehydrogenase (type V) is expressed under both conditions. We also identified a putative oxidoreductase involved in the respiration of elemental sulfur, and propose a new pathway of sulfur reduction. The presence of incomplete yet functional alternative pathways of carbon fixation in subsurface organisms may be more widespread that previously thought, and may provide an evolutionary advantage in surviving under energy limiting conditions.