Metabolism and Occurrence of Methanogenic and Sulfate-Reducing Syntrophic Acetate Oxidizing Communities in Haloalkaline Environments

Abstract

Anaerobic syntrophic acetate oxidation (SAO) is a thermodynamically unfavorable process involving a syntrophic acetate oxidizing bacterium (SAOB) that forms interspecies electron carriers (IECs). These IECs are consumed by syntrophic partners, typically hydrogenotrophic methanogenic archaea or sulfate reducing bacteria. In this work, the metabolism and occurrence of SAOB at extremely haloalkaline conditions were investigated, using highly enriched methanogenic (M-SAO) and sulfate-reducing (S-SAO) cultures from south-western Siberian hypersaline soda lakes. Activity tests with the M-SAO and S-SAO cultures and thermodynamic calculations indicated that H₂ and formate are important IECs in both SAO cultures. Metagenomic analysis of the M-SAO cultures showed that the dominant SAOB was 'Candidatus Syntrophonatronum acetioxidans,' and a near-complete draft genome of this SAOB was reconstructed. 'Ca. S. acetioxidans' has all genes necessary for operating the Wood-Ljungdahl pathway, which is likely employed for acetate oxidation. It also encodes several genes essential to thrive at haloalkaline conditions; including a Na⁺-dependent ATP synthase and marker genes for 'salt-out' strategies for osmotic homeostasis at high soda conditions. Membrane lipid analysis of the M-SAO culture showed the presence of unusual bacterial diether membrane lipids which are presumably beneficial at extreme haloalkaline conditions. To determine the importance of SAO in haloalkaline environments, previously obtained 16S rRNA gene sequencing data and metagenomic data of five different hypersaline soda lake sediment samples were investigated, including the soda lakes where the enrichment cultures originated from. The draft genome of 'Ca. S. acetioxidans' showed highest identity with two metagenome-assembled genomes (MAGs) of putative SAOBs that belonged to the highly abundant and diverse Syntrophomonadaceae family present in the soda lake sediments. The 16S rRNA gene amplicon datasets of the soda lake sediments showed a high similarity of reads to 'Ca. S. acetioxidans' with abundance as high as 1.3% of all reads, whereas aceticlastic methanogens and acetate oxidizing sulfate-reducers were not abundant (≤0.1%) or could not be detected. These combined results indicate that SAO is the primary anaerobic acetate oxidizing pathway at extreme haloalkaline conditions performed by haloalkaliphilic syntrophic consortia.

Keywords: SAOB; genome-centric metagenomics; haloalkaliphiles; soda lakes; syntrophic acetate oxidation; syntrophic acetate oxidizing bacteria; syntrophy.

FIGURE 1

Activity test results with pre-grown syntrophic acetate oxidizing cultures with a methanogenic partner (M-SAO) showing acetate (blue line), H₂ (black line), formate (gray line) and methane (red line) evolution in cultures supplemented with (A) acetate, (B) acetate and formate – 7 mM, (C) acetate and BES, (D) acetate and formate – 80 mM, (E) acetate and 100% H₂. Standard deviations represent biological triplicate incubations.

FIGURE 2

Activity test results with pre-grown syntrophic acetate oxidizing cultures with a sulfate-reducing partner (S-SAO) showing acetate (blue line), H₂ (black line), formate (gray line) and sulfide (red line) evolution in cultures supplemented with (A) acetate, (B) acetate and molybdate, (C) acetate and formate – 80 mM, (D) acetate and 100% H₂ showing acetate, H₂ and sulfide and (E) acetate and 100% H₂ showing only acetate and formate evolution. Standard deviations represent biological triplicate incubations.

FIGURE 3

Summary of the Wood–Ljungdahl pathway and acetate activation. Red lines and red names indicate absence of these conversions or genes in the partial genome of ‘Ca. Syntrophonatronum acetioxidans.’

FIGURE 4

Scheme showing transmembrane transporters, energy conserving enzymes and electron transport mechanisms found in the genome of ‘Ca. Syntrophonatronum acetioxidans.’

FIGURE 5

Maximum likelihood tree based on the phylogeny of 16 ribosomal proteins found in selected reference genomes from the family Syntrophomonadaceae and the draft genome of “Ca. S. acetioxidans” (MSAO_Bac 1). The tree was rooted to the proteins found in Natranaerobius thermophilus. The estimated relative abundance of each organism in five different hypersaline soda lake sediment samples is expressed as reads per Kb of genome sequence per Gb of mapped reads (RPGK). Black dots at the nodes show the ≥90% confidence of 100x bootstraps. The reference genomes indicated as bin X Y are MAGs obtained from the same metagenomes (CSSed10, CSSed11, T3Sed10, T1Sed10, and B1Sed10) for which the recruitment experiment was performed [33]. MAG IDs with an asterisk show the presence of a partial 16S rRNA gene with 94% identity to ‘Ca. Syntropholuna alkaliphila’. Presence and absence of genes and pathways are indicated by the symbols on the right hand side. Full = present, empty = partially present. Gene abbreviations for acetate activation: ack/pta = acetate kinase/phosphate acetyltransferase, ACSS/hppA = acetyl-coA synthetase/H⁺/Na⁺ translocating pyrophosphatases, paaK = phenylacetate-CoA ligase. WL-carbonyl and WL-methyl are the carbonyl and methyl branch of the Wood–Ljungdahl pathway, respectively. PFOR = pyruvate ferredoxin oxidoreductase. Gene abbreviations encoding for formate dehydrogenases: fdoGH, fdhAB. Gene abbreviations encoding for [NiFe] hydrogenase: B-cyt = b-type cytochrome subunit, hyaBA.

FIGURE 6

Relative abundance (%) of OTUs of Firmicutes, Deltaproteobacteria, and Euryarchaeota in five different soda lake sediment samples with different salinity. Only reads with higher relative abundance than 0.1% in at least one of the sediment samples are shown.