# Acetoclastic versus hydrogenotrophic methanogenesis: defining how pH and alkalinity shape acetate metabolism in a haloalkaliphilic methanogenic community for biomethane production

## TL;DR

This study explores how pH and alkalinity affect methane production pathways in a haloalkaliphilic microbial community, showing that higher pH and alkalinity favor hydrogenotrophic over acetoclastic methanogenesis.

## Contribution

The study identifies the pH tipping point and the influence of alkalinity on the shift from acetoclastic to hydrogenotrophic methanogenesis in haloalkaliphilic communities.

## Key findings

- Methanocalculus abundance increases with pH, especially at higher alkalinity.
- Alkalinity has a stronger influence than pH on the metabolic shift in methanogenesis.
- Syntrophic interactions with 'Candidatus Contubernalis' support hydrogenotrophic methanogenesis.

## Abstract

In methanogenic communities, two main pathways drive methanogenesis: acetoclastic methanogenesis, which converts acetate into CH4 and CO2, and hydrogenotrophic methanogenesis, which reduces CO2 with H2 to CH4. Under high-pH conditions, a shift in dominance from acetoclastic to hydrogenotrophic methanogenesis is often observed. The goal of this work was to identify the pH tipping point for this metabolic shift and to elucidate the influence of alkalinity on this transition in a haloalkaliphilic methanogenic community enriched from anaerobic soda lake sediments. To this end, a haloalkaliphilic microbial community was cultivated across a pH range (8.20–10.00) at three different alkalinities (0.1, 0.6, 1.2 eq/L). Specific qPCR probes were developed to quantify the two dominant methanogens for each catabolism: “Ca. Methanocrinis natronophilus” (acetoclastic) and Methanocalculus alkaliphilus (hydrogenotrophic). Results showed that the relative abundance of Methanocalculus increased with the rise of pH for all alkalinities, with alkalinity exerting a stronger influence than pH. At low alkalinity (0.1 eq/L), Methanocalculus abundance doubled from 5.14 ± 1.95% to 9.15 ± 0.77% (pH 8.40–10.35). At moderate alkalinity (0.6 eq/L), it increased from 8.33 ± 1.34% to 47.92 ± 3.76% (pH 8.41–10.00), and at the highest alkalinity (1.2 eq/L), it increased from 6.78 ± 1.06% to 60.25 ± 2.00% (pH 8.26–9.68). 16S rRNA gene amplicon sequencing further identified “Candidatus Contubernalis” as a putative syntrophic acetate-oxidizing bacterium likely partnering with Methanocalculus in indirect hydrogenotrophic methanogenesis. This work highlights that haloalkaliphilic hydrogenotrophic methanogens offer a promising strategy to integrate CO2 capture in alkaline solutions with biomethanation.

The online version contains supplementary material available at 10.1007/s00253-026-13725-0.