# Obligate heterotrophy of hyperthermophilic archaea Pyrobaculum arsenaticum and P. aerophilum

## TL;DR

Two hyperthermophilic archaea cannot grow autotrophically and rely on organic matter for survival.

## Contribution

Demonstrates that Pyrobaculum species are obligate heterotrophs, challenging previous assumptions about their autotrophic capabilities.

## Key findings

- Pyrobaculum arsenaticum and P. aerophilum cannot grow autotrophically despite prior claims.
- Amino acid isotopologue profiling showed <30% 13C labeling, indicating heterotrophic metabolism.
- Autotrophy in Thermoproteales is linked to the presence of genes for the dicarboxylate/4-hydroxybutyrate cycle.

## Abstract

Pyrobaculum arsenaticum and P. aerophilum are two hyperthermophiles that belong to the phylum Thermoproteota (also known as Crenarchaeota), order Thermoproteales. Pyrobaculum arsenaticum is an obligate anaerobe, whereas P. aerophilum 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 P. arsenaticum 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 Thermoproteales 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.