Isolation and characterization of novel acetogenic strains of the genera Terrisporobacter and Acetoanaerobium

Abstract

Due to their metabolic versatility in substrate utilization, acetogenic bacteria represent industrially significant production platforms for biotechnological applications such as syngas fermentation, microbial electrosynthesis or transformation of one-carbon substrates. However, acetogenic strains from the genera Terrisporobacter and Acetoanaerobium remained poorly investigated for biotechnological applications. We report the isolation and characterization of four acetogenic Terrisporobacter strains and one Acetoanaerobium strain. All Terrisporobacter isolates showed a characteristic growth pattern under a H₂ + CO₂ atmosphere. An initial heterotrophic growth phase was followed by a stationary growth phase, where continuous acetate production was indicative of H₂-dependent acetogenesis. One of the novel Terrisporobacter isolates obtained from compost (strain COM^T) additionally produced ethanol besides acetate in the stationary growth phase in H₂-supplemented cultures. Genomic and physiological characterizations showed that strain COM^T represented a novel Terrisporobacter species and the name Terrisporobacter vanillatitrophus is proposed (=DSM 116160^T = CCOS 2104^T). Phylogenomic analysis of the novel isolates and reference strains implied the reclassification of the T. petrolearius/T. hibernicus phylogenomic cluster to the species T. petrolearius and of the A. noterae/A. sticklandii phylogenomic cluster to the species A. sticklandii. Furthermore, we provide first insights into active prophages of acetogens from the genera Terrisporobacter and Acetoanaerobium.

Keywords: Acetoanaerobium; Terrisporobacter; Wood-Ljungdahl pathway; acetogen; ethanol; prophage.

Figure 1

Phylogenomic analysis of novel Terrisporobacter and Acetoanaerobium isolates. The heatmaps visualize ANIm and dDDH values for the genomes of Terrisporobacter isolates HSE, ELB, DSL and COM (A) and the Acetoanaerobium isolate DSL2 genome (B). The top part of the heatmaps visualize the ANIm percentage identity values from 80–100% and the lower part of the heatmaps visualize dDDH values (d₄) from 20–100%. The legend for the color code is shown on top (ANIm) and to the left (dDDH). Novel isolates from this study are highlighted in green.

Figure 2

Phylograms of novel Terrisporobacter and Acetoanaerobium isolates. Whole-genome sequence-based phylogenetic tree of novel Terrisporobacter isolates (A). Branch lengths are scaled in terms of GBDP distance formula d₅. Red numbers above branches are GBDP pseudo-bootstrap support values >60% from 100 replications, with an average branch support of 73.9%. Black numbers below branches are confidence scores. The tree was rooted at the midpoint. Whole-genome sequence-based phylogenetic tree of the novel Acetoanaerobium isolate (B). Branch lengths are scaled in terms of GBDP distance formula d₅. Red numbers above branches are GBDP pseudo-bootstrap support values >60% from 100 replications, with an average branch support of 61.0%. Black numbers below branches are confidence scores. The tree was rooted at the midpoint.

Figure 3

Pan/core genome analysis of novel isolates. Detected unique OGs in the novel and reference Terrisporobacter (A) and Acetoanaerobium (B) genomes colored by species. Novel isolates from this study are highlighted in green and the species color code is shown on the top.

Figure 4

Growth in H₂-supplemented cultures and control cultures of the novel Terrisporobacter isolate COM. Measured OD₆₀₀ growth values for COM cultures grown with an atmosphere of H₂ + CO₂ (solid red line) and control cultures with an atmosphere of N₂ + CO₂ (solid blue line) (A). GC-measurements detecting the amount of produced acetate (B) and ethanol (C) in H₂ + CO₂ (dotted red line) and N₂ + CO₂ (dotted blue line) cultures.

Figure 5

Phylogram of novel Terrisporobacter and Acetoanaerobium phage genomes and related phages. Branch lengths are scaled in terms of GBDP distance formula d₀ and the black numbers below branches are confidence scores yielding an average branch support of 71%. The tree was rooted at the midpoint.