Anaerobic hexadecane degradation by a thermophilic Hadarchaeon from Guaymas Basin

Abstract

Hadarchaeota inhabit subsurface and hydrothermally heated environments, but previous to this study, they had not been cultured. Based on metagenome-assembled genomes, most Hadarchaeota are heterotrophs that grow on sugars and amino acids, or oxidize carbon monoxide or reduce nitrite to ammonium. A few other metagenome-assembled genomes encode alkyl-coenzyme M reductases (Acrs), β-oxidation, and Wood-Ljungdahl pathways, pointing toward multicarbon alkane metabolism. To identify the organisms involved in thermophilic oil degradation, we established anaerobic sulfate-reducing hexadecane-degrading cultures from hydrothermally heated sediments of the Guaymas Basin. Cultures at 70°C were enriched in one Hadarchaeon that we propose as Candidatus Cerberiarchaeum oleivorans. Genomic and chemical analyses indicate that Ca. C. oleivorans uses an Acr to activate hexadecane to hexadecyl-coenzyme M. A β-oxidation pathway and a tetrahydromethanopterin methyl branch Wood-Ljungdahl (mWL) pathway allow the complete oxidation of hexadecane to CO2. Our results suggest a syntrophic lifestyle with sulfate reducers, as Ca. C. oleivorans lacks a sulfate respiration pathway. Comparative genomics show that Acr, mWL, and β-oxidation are restricted to one family of Hadarchaeota, which we propose as Ca. Cerberiarchaeaceae. Phylogenetic analyses further indicate that the mWL pathway is basal to all Hadarchaeota. By contrast, the carbon monoxide dehydrogenase/acetyl-coenzyme A synthase complex in Ca. Cerberiarchaeaceae was horizontally acquired from Bathyarchaeia. The Acr and β-oxidation genes of Ca. Cerberiarchaeaceae are highly similar to those of other alkane-oxidizing archaea such as Ca. Methanoliparia and Ca. Helarchaeales. Our results support the use of Acrs in the degradation of petroleum alkanes and suggest a role of Hadarchaeota in oil-rich environments.

Keywords: Archaea; alkanes; anaerobic metabolism; evolution; hydrocarbons.

Figure 1

Community composition and sulfide formation in sediment slurries incubated with hexadecane; the results correspond to cruise AT37-06; (A) community composition based on archaeal and bacterial 16S rRNA gene amplicons; (B) sulfide production in early enrichments and control incubations; control incubations did not show significant sulfide production over time; enrichments with hexadecane at 37°C and 50°C showed slow activities; enrichments at 70°C grew faster and were diluted after 90 days of incubation.

Figure 2

Community composition in Hexadecane70 cultures from AT37-06 to AT42-05; relative abundance of microbial taxa based on 16S rRNA gene fragments recruited from the metagenome; archaea dominate thermophilic alkane-degrading enrichments; Archaeoglobales were abundant in early sediment enrichments from the AT37-06 cruise (February 2018), and Hadarchaea became more dominant in later stages (September 2018, March 2020); the communities include heterotrophic Bathyarchaeia and Acetothermia, and sulfate-reducing Thermodesulfobacteriales; a second enrichment attempt from cruise AT42-05 showed similar results.

Figure 3

Methyl−/alkyl-coenzyme M reductase phylogeny, hexadecane activation by Acr, and proposed metabolism for Ca. C. oleivorans; (A) ML likelihood tree of McrA/AcrA alignment with 100 bootstraps; white circles and gray circles show bootstrap values of >70% and >90%, respectively; the clades shaded in gray include all Acr sequences (Mcr Group IV) [105]; the clade of putative long-chain alkane Acrs includes the Acr of Ca. C. oleivorans; (B) Ca. C. oleivorans activates hexadecane to hexadecyl-CoM. LC–MS analysis of hexadecane70 culture extracts shows two dominant chromatographic peaks in extracted ion chromatograms of the exact mass of hexadecyl-CoM; these peaks likely represent coenzyme M-substituted alkyls resulting from activation of the alkane in the secondary and primary position, in order of elution time [5]; (C) metabolic model for Ca. C. oleivorans; the Acr activates hexadecane to hexadecyl-CoM, which is then converted into a 16-carbon acyl-CoA (hexadecanoyl-CoA), possibly via Aor; acyl-CoA is processed to acetyl-CoA units (β-oxidation pathway); acetyl-CoA is incorporated into the downstream part of the H₄MPT mWL via the Cdh/Acs complex; the methyl group is completely oxidized to CO₂; the fate of the electrons released from this metabolism is unknown; F₄₂₀H₂ oxidation could be coupled to the production of H₂ via Frh or to the reduction of CO₂ to formate by an Fdh.

Figure 4

Pathways required for Acr-dependent alkane oxidation in Hadarchaeota; subset of a phylogenomic tree of archaea showing Hadarchaeota (including Persephonarchaea), and occurrence of pathways for alkane degradation in the class Hadarchaeia; the 95% threshold in ANI defines the 21 species of Hadarchaeota; colored squares indicate that the protein is encoded in the MAG; the Ca. Cerberiarchaeaceae family (shaded in the tree) contains MAGs encoding an Acr, a complete β-oxidation pathway, a Cdh/Acs, and a mWL pathway without methyl-H₄MPT:CoM methyltransferase (Mtr); the COGs in the β-oxidation pathway correspond to NDP-forming acyl-CoA synthetase (COG1042), AMP-forming acyl-CoA synthetase (COG0318), acyl-CoA dehydrogenase (COG1960), enoyl-CoA hydratase (COG1024), 3-hydroxyacyl-CoA dehydrogenase (COG1250), and acetyl-CoA acetyltransferase (COG0183).

Figure 5

Placement of Hadarchaea, Persephonarchaea, and Bathyarchaea in species genome tree, FwdABC phylogeny, and CdhABCDE/acetyl-CoA synthase complex (Cdh) phylogeny; maximum-likelihood phylogenetic trees with 100 bootstraps based on concatenated alignment of 38 archaeal marker genes, FwdABC, and CdhABCDE protein sequences; (A) Hadarchaea and Persephonarchaea form a clade next to Theionarchaea; (B) Hadarchaea Fwd sequences form a branch with the Persephonarchaea 2 sequences; (C) Cdh sequences from the alkane-oxidizing Hadarchaea clade cluster together and branch from Bathyarchaea sequences, probably as a consequence of an event of lateral gene transfer between subsurface alkane-oxidizing archaea.