Hydrogen as a Co-electron Donor for Chain Elongation With Complex Communities

Abstract

Electron donor scarcity is seen as one of the major issues limiting economic production of medium-chain carboxylates from waste streams. Previous studies suggest that co-fermentation of hydrogen in microbial communities that realize chain elongation relieves this limitation. To better understand how hydrogen co-feeding can support chain elongation, we enriched three different microbial communities from anaerobic reactors (A, B, and C with ascending levels of diversity) for their ability to produce medium-chain carboxylates from conventional electron donors (lactate or ethanol) or from hydrogen. In the presence of abundant acetate and CO₂, the effects of different abiotic parameters (pH values in acidic to neutral range, initial acetate concentration, and presence of chemical methanogenesis inhibitors) were tested along with the enrichment. The presence of hydrogen facilitated production of butyrate by all communities and improved production of i-butyrate and caproate by the two most diverse communities (B and C), accompanied by consumption of acetate, hydrogen, and lactate/ethanol (when available). Under optimal conditions, hydrogen increased the selectivity of conventional electron donors to caproate from 0.23 ± 0.01 mol e^-/mol e^- to 0.67 ± 0.15 mol e^-/mol e^- with a peak caproate concentration of 4.0 g L^-1. As a trade-off, the best-performing communities also showed hydrogenotrophic methanogenesis activity by Methanobacterium even at high concentrations of undissociated acetic acid of 2.9 g L^-1 and at low pH of 4.8. According to 16S rRNA amplicon sequencing, the suspected caproate producers were assigned to the family Anaerovoracaceae (Peptostreptococcales) and the genera Megasphaera (99.8% similarity to M. elsdenii), Caproiciproducens, and Clostridium sensu stricto 12 (97-100% similarity to C. luticellarii). Non-methanogenic hydrogen consumption correlated to the abundance of Clostridium sensu stricto 12 taxa (p < 0.01). If a robust methanogenesis inhibition strategy can be found, hydrogen co-feeding along with conventional electron donors can greatly improve selectivity to caproate in complex communities. The lessons learned can help design continuous hydrogen-aided chain elongation bioprocesses.

Keywords: acidogenesis; caproate; carboxylate platform; isobutyrate; methanogenesis; microbial consortia; microbiome; syngas fermentation.

FIGURE 1

Enrichment scheme and overview of experiments. Reactor A was fed with lactate and xylan, reactor B with corn silage, and reactor C with corn silage and cow manure. The syringe icon symbolizes addition of conventional electron donors (lactate and ethanol). One bottle with H₂/CO₂ is depicted for each condition and experiment. Duplicates, H₂-free, and abiotic controls are not shown. Full arrows mean transfer from selected bottles with 10% inoculation. Dashed arrows represent new experiments without dilution of the inoculum.

FIGURE 2

Effect of pH on the production rates (positive values) and consumption rates (negative values) of butyrate, caproate, CH₄, and H₂. Community A (A) and Community C (B) are shown. Results are not shown for community B, because it showed no activity after acidification to pH values of 4.8 and 5.5. Error bars represent standard errors.

FIGURE 3

Effect of acetate concentration on production and consumption rates of butyrate, caproate, CH₄, and H₂. Community A (A), Community B (B), and Community C (C) are shown.

FIGURE 4

Effect of methanogenesis inhibition on production and consumption rates of butyrate, caproate, CH₄, and H₂. Community B (A) and Community C (B) are shown.

FIGURE 5

Effect of methanogenesis inhibition in the presence of lactate and ethanol on production and consumption rates of butyrate, caproate, CH₄, and H₂. Community B (A) and Community C (B) are shown. One of the duplicates of community C with H₂ and inhibitor did not grow and no standard error bar is shown for this condition.

FIGURE 6

Selectivity of conventional electron donors (lactate, ethanol, and yeast extract) to caproate and maximum caproate concentration achieved by Community A (A), Community B (B), and Community C (C).

FIGURE 7

Diversity of Community A, Community B, and Community C throughout the enrichment in terms of richness (observed ASVs), Shannon index, and Simpson index.

FIGURE 8

Community profiles resolved to the ASV level (ASV numbers in parentheses) at each condition tested with H₂. The 30 most abundant ASVs in the dataset are shown. Duplicates are shown. Slots left blank represent samples that could not be sequenced.