23The exergonic reaction of FeS with H 2 S to form FeS 2 (pyrite) and H 2 was postulated to have 24 operated as an early form of energy metabolism on primordial Earth. Since the Archean, 25 sedimentary pyrite formation played a major role in the global iron and sulfur cycles, with direct 26 impact on the redox chemistry of the atmosphere. To date, pyrite formation was considered a 27 purely geochemical reaction. Here, we present microbial enrichment cultures, which grew with 28FeS, H 2 S, and CO 2 as their sole substrates to produce FeS 2 and CH 4 . Cultures grew over 29 periods of three to eight months to cell densities of up to 2-9×10 6 cells mL −1 . Transformation of 30FeS with H 2 S to FeS 2 was followed by 57 Fe Mössbauer spectroscopy and showed a clear 31 biological temperature profile with maximum activity at 28°C and decreasing activities towards 32 4°C and 60°C. CH 4 was formed concomitantly with FeS 2 and exhibited the same temperature 33 dependence. Addition of either penicillin or 2-bromoethanesulfonate inhibited both FeS 2 and 34 CH 4 production, indicating a syntrophic coupling of pyrite formation to methanogenesis. This 35 hypothesis was supported by a 16S rRNA gene-based phylogenetic analysis, which identified 36 at least one archaeal and five bacterial species. The archaeon was closely related to the 37 hydrogenotrophic methanogen Methanospirillum stamsii while the bacteria were most closely 38 related to sulfate-reducing Deltaproteobacteria, as well as uncultured Firmicutes and 39Actinobacteria. We identified a novel type of microbial metabolism able to conserve energy 40 from FeS transformation to FeS 2 , which may serve as a model for a postulated primordial iron-41 sulfur world. 42 Microbial pyrite formation Thiel et al. 3 of 34 Significance statement 43Pyrite is the most abundant iron-sulfur mineral in sediments. Over geological times, its burial 44 controlled oxygen levels in the atmosphere and sulfate concentrations in seawater. Its 45 formation in sediments is so far considered a purely geochemical process that is at most 46 indirectly supported by microbial activity. We show that lithotrophic microorganisms can directly 47 transform FeS and H 2 S to FeS 2 and use this exergonic reaction as a novel form of energy 48 metabolism that is syntrophically coupled to methanogenesis. Our results provide insights into 49 a syntrophic relationship that could sustain part of the deep biosphere and lend support to the 50 iron-sulfur-world theory that postulated FeS transformation to FeS 2 as a key energy-delivering 51 reaction for life to emerge. 52 Microbial pyrite formation Thiel et al. 4 of 34