Terrestrial ecosystems represent a major sink of CO2 through fixation by plants but they have been shown to mitigate the rise of atmospheric CO2 also via microbial CO2 fixation (Ge et al., 2016; Yuan et al., 2012). Microbial CO2 fixation has been mostly ascribed to autotrophic microorganisms (Ge et al., 2016), but fundamentally all microorganisms may use inorganic C (IC; i.e. CO2 or bicarbonate) in their metabolism. All these fixations require energy generated by phototrophic, autotrophic or heterotrophic energy sources. IC is the main or even the only C source for chemoautotrophs and photoautotrophs, while heterotrophs and mixotrophs rely on organic C (OC) but also incorporate IC via a variety of carboxylation reactions that are part of their central or peripheral metabolic pathways (for review see Erb, 2011; Wood and Stjernholm, 1962). The importance of carboxylases in heterotrophic metabolism increases whenever microorganisms experience C limitation through a disproportion between C demand for energy generation and growth and its availability, caused by deficiency or complexity of OC sources, or fast growth (Alonso-Saez et al., 2010; Feisthauer et al., 2008; Merlin et al., 2003). Even though the occurrence of dark and largely heterotrophic CO2 fixation in soils is generally accepted, very few studies have assessed its relevance for soil microorganisms (Miltner et al., 2004, 2005a,b; Šantrůčková et al., 2005). Estimates of the importance of soil CO2 fixation for the C balance in certain ecosystems or within an entire soil profile are rare (Ge et al., 2016; Yuan et al., 2012) and analyses of diversity and abundance of carboxylases are missing entirely. Soil OC becomes progressively enriched in 13C with increasing soil depth (Bird et al., 2002; Gentsch et al., 2015; Nadelhoffer and Fry, 1988; Torn et al., 2002). There are several explanations but no one can fully explain the measured isotopic shift. The enrichment of soil OC with depth can be connected with decrease of δ ۱۳C of atmospheric CO2 by 1.3‰ due to Suess effect (McCarroll and Loader, 2004), with preferential decomposition of different organic compounds and microbial fractionation during litter decomposition or mixing of new C input with old soil OC (Buchmann et al., 1997; Ehleringer et al., 2000; Šantrůčková et al., 2000). Another hypothesis that has been discussed but never supported experimentally states that soil microbes should be isotopically heavier as a result of carboxylation reactions (Ehleringer et al., 2000). Whenever carboxylation reactions are involved, CO2 molecules used in the reactions likely originate from the soil atmosphere, which is isotopically heavier than the organic materials being decomposed (Cerling et al., 1991). The 13CO2 enrichment of bulk soil atmosphere is highest in the uppermost soil horizons, where CO2 originates mostly from the atmospheric air. In deeper horizons of the soil profile, CO2 originates from organic matter decomposition and carries the isotopic signal of decomposed material. But still CO2 remaining in the soil that surrounds microbes is 4.4‰ heavier than organic matter at the location due to slower diffusion of heavier 13CO2 than lighter 12CO2 (Cerling et al., 1991). CO2 hydrogenation causes further enrichment of 13C in HCO3 − by 8–۱۲‰, depending on temperature (Mook et al., 1974).