<p>Anaerobic digestion (AD) is a commonly used technology for facilitating carbon fixation by converting complex organic matter into volatile fatty acids and CH₄; however, the issue of CO₂ emission remains unresolved in AD. The formation of amorphous carbon has been identified as a more direct method of carbon fixation in AD. This study aimed to elucidate how amorphous carbon can be formed from organic matter or CO₂ by anaerobic microorganisms. The results showed that amorphous carbon was produced in the anaerobic digestion of inorganic and mixed carbon sources, with yields of 0.38 and 3 µg/10⁵ cells, respectively. Its characteristics were analyzed using Raman microscopy. Isotope labeling revealed that CO₂ fixation into amorphous carbon primarily depends on the reversed oxidative tricarboxylic acid cycle (roTCA) and hydroxycaproate. Differential pulse voltammetry combined with gene abundance analysis indicated that flavin electron bifurcation (EB) is involved in electron transfer. The microbial isothermal calorimeter further measured the metabolic calorific value, demonstrating that anaerobic microorganisms can autotrophically fix CO₂ with energy provided by EB. Metagenomic analysis supported the large REDOX equivalents input from EB to sustain the roTCA cycle. This research contributes to understanding the mechanism of CO₂ fixation into solid carbon in anaerobic environments. Additionally, it provides new insights into the potential development of carbon-negative technologies in anaerobic biological treatment.</p>