Biological CO<
sub>
2<
/sub>
fixation is an important technology that can assist in combating climate change. Here, we show an approach called anaerobic, non-photosynthetic mixotrophy can result in net CO<
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2<
/sub>
fixation when using a reduced feedstock. This approach uses microbes called acetogens that are capable of concurrent utilization of both organic and inorganic substrates. In this study, we investigated the substrate utilization of 17 different acetogens, both mesophilic and thermophilic, on a variety of different carbohydrates and gases. Compared to most model acetogen strains, several non-model mesophilic strains displayed greater substrate flexibility, including the ability to utilize disaccharides, glycerol and an oligosaccharide, and growth rates. Three of these non-model strains (<
em>
Blautia producta<
/em>
, <
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Clostridium scatologenes<
/em>
and <
em>
Thermoanaerobacter kivui<
/em>
) were chosen for further characterization, under a variety of conditions including H<
sub>
2<
/sub>
- or syngas-fed sugar fermentations and a CO<
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2<
/sub>
-fed glycerol fermentation. In all cases, CO<
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2<
/sub>
was fixed and carbon yields approached 100%. Finally, the model acetogen <
em>
C. ljungdahlii<
/em>
was engineered to utilize glucose, a non-preferred sugar, while maintaining mixotrophic behavior. This work demonstrates the flexibility and robustness of anaerobic, non-photosynthetic mixotrophy as a technology to help reduce CO<
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2<
/sub>
emissions.