Acetate Shock Loads Enhance CO Uptake Rates of Anaerobic Microbiomes

Abstract

Pyrolysis of lignocellulosic biomass commonly produces syngas, a mixture of gases such as CO, CO₂ and H₂, as well as an aqueous solution generally rich in organic acids such as acetate. In this study, we evaluated the impact of increasing acetate shock loads during syngas co-fermentation with anaerobic microbiomes at different pH levels (6.7 and 5.5) and temperatures (37°C and 55°C) by assessing substrates consumption, metabolites production and microbial community composition. The anaerobic microbiomes revealed to be remarkably resilient and were capable of converting syngas even at high acetate concentrations of up to 64 g/L and pH 5.5. Modifying process parameters and acetate loads resulted in a shift of the product spectrum and microbiota composition. Specifically, a pH of 6.7 promoted methanogens such as Methanosarcina, whereas lowering the pH to 5.5 with lower acetate loads promoted the enrichment of syntrophic acetate oxidisers such as Syntrophaceticus, alongside hydrogenotrophic methanogens. Increasing acetate loads intensified the toxicity of undissociated acetic acid, thereby inhibiting methanogenic activity. Under non-methanogenic conditions, high acetate concentrations suppressed acetogenesis in favour of hydrogenogenesis and the production of various carboxylates, including valerate, with product profiles and production rates being contingent upon temperature. A possible candidate for valerate production was identified in Oscillibacter. Across all tested conditions, acetate supplementation provided additional carbon and energy to the mixed cultures and consistently increased carboxydotrophic conversion rates up to about 20-fold observed at pH 5.5, 55°C and 48 g/L acetate compared to control experiments. Species of Methanobacterium, Methanosarcina and Methanothermobacter may have been involved in CO biomethanation. Under non-methanogenic conditions, the bacterial species responsible for CO conversion remain unclear. These results offer promise for integrating process streams, such as syngas and wastewater, as substrates for mixed culture fermentation allowing for enhanced resource circularity, mitigation of environmental impacts and decreased dependence on fossil fuels.

Keywords: acetic acid; acetogenesis; anaerobic digestion; hydrogenogenesis; methanogenesis; open mixed cultures; syngas; syntrophic acetate oxidation.

FIGURE 1

Electron mol balancing between substrates (CO; H₂ and acetate if consumed) and products (CH₄, formate, ethanol, propionate, butyrate and valerate; H₂ and acetate if produced) at different process conditions and increasing acetate concentrations. Product recoveries higher that 100% may result from extra electron sources, such as solids in the inoculum. Error bars represent standard deviation among replicates (n = 3).

FIGURE 2

Consumption and formation rates of gaseous components (CO, H₂ and CH₄) and of some carboxylates (formate, acetate, propionate, butyrate and valerate) and ethanol at different process conditions and increasing acetate concentrations. Negative values indicate consumption. Error bars represent standard deviation among replicates (n = 3).

FIGURE 3

Average relative abundance of the enriched methanogenic genera (based on mcrA gene amplicon sequencing variants). Only the top five most abundant genera are shown. The rest are grouped in ‘Others’. Community analysis was performed only for cultures with methanogenic activity for the last samples collected at day 16. The microbial community composition for each replicate is available in the Figure S4.

FIGURE 4

Average relative abundance of triplicate samples of the enriched bacterial genera (based on 16S rRNA amplicon sequencing variants). Only the top 15 most abundant genera are shown. The rest are grouped in ‘Others’. Community analysis was performed only for the last samples collected at day 16. The microbial community composition for each replicate is available in the Figure S5.

FIGURE 5

Cumulative e‐M for CO, H₂, CH₄ and acetate normalised to control experiments in the absence of supplemented acetate. Negative values indicate consumption while positive values production. Changes in sign (positive to negative, for instance) for a compound mark the switch of the metabolism compared to the control experiments.