Perchlorate Anaerobiosis in Haloferax volcanii: A Novel Metabolic Capability for A Model Haloarchaeon

Haloferax volcanii ( H. volcanii) is a facultatively anaerobic model halophilic archaeon capable of anaerobic growth using nitrate, chlorate, fumarate, trimethylamine N-oxide (TMAO), and dimethyl sulfoxide (DMSO) as alternative electron acceptors. H. volcanii has been previously documented to tolerate high concentrations of perchlorate during aerobic respiration, but has not been previously documented to grow anaerobically using perchlorate as an alternative electron acceptor. Here, we document the novel metabolic capability of H. volcanii to grow anaerobically using perchlorate and show the initial preferred conditions with respect to NaCl concentration, pH, carbon sources, and perchlorate concentration. Additionally, we investigate changes in carotenoid composition during anaerobic growth on perchlorate with relevance for the search for signs of extinct and extant life on Mars. Our results show that NaCl concentrations of > 175 g/l are required to induce anaerobic growth on perchlorate. We show a preference for a pH of 7.0 and a combination of yeast extract and casamino acids as preferred carbon sources. Furthermore, we document anaerobic growth and perchlorate reduction in the presence of perchlorate concentrations (200 mM) that exceed the currently accepted limit for any organism (100 mM). Raman spectra of cultures grown anaerobically on perchlorate show significant decreases in the intensity of the carotenoid peaks corresponding to bacterioruberin at ~ 1505 cm ^-1 , ~ 1150 cm ^-1 , and ~ 1000 cm ^-1 , highlighting how extreme Martian conditions may cause biosignature degradation. Notably, we demonstrate the previously unreported ability of the model halophilic archaeon Haloferax volcanii to grow anaerobically using perchlorate and extend the known limits of biological perchlorate tolerance under anoxic conditions. The discovery that H. volcanii is capable of perchlorate reduction has potential implications for the development of biological strategies for perchlorate remediation and for the interpretation of potential biosignatures in perchlorate-rich environments, including those hypothesized to exist on Mars.