Obligate heterotrophy of hyperthermophilic archaea <i>Pyrobaculum arsenaticum</i> and <i>P. aerophilum</i>

<i>Pyrobaculum arsenaticum</i> and <i>P. aerophilum</i> are two hyperthermophiles that belong to the phylum Thermoproteota (also known as Crenarchaeota), order <i>Thermoproteales. Pyrobaculum arsenaticum</i> is an obligate anaerobe, whereas <i>P. aerophilum</i> is a facultatively aerobic organism. Both species have been described as capable of autotrophic growth with molecular hydrogen. Because their genomes lack genes encoding key enzymes for known autotrophic CO₂ fixation pathways, they have been discussed as organisms that may use unknown pathways. To establish reliable autotrophic cultures, we gradually reduced the supplied concentrations of yeast extract but, in our hands, autotrophy was not attainable for either of the two species. Analysis of the ¹³C-labelling of the biomass of the obtained mixotrophic cultures of <i>P. arsenaticum</i> grown on ¹³CO₂ + H₂ (20:80, v/v), using isotopologue profiling, revealed that their amino acids contained <30% of ¹³C. Amino acids were mainly labelled only in carboxyl groups, demonstrating their purely heterotrophic nature. Our data suggest that the ability to grow autotrophically in currently known <i>Thermoproteales</i> is strictly correlated with the presence of the genes for the dicarboxylate/4-hydroxybutyrate cycle. We discuss the reasons that may lead to misinterpretation of the data on the ability of prokaryotes to grow autotrophically.