Chelator-mediated Fenton post-treatment enhances methane yield from lignocellulosic residues via microbial community modulation

Abstract

Advancing biomethane production from anaerobic digestion (AD) is essential for building a more reliable and resilient bioenergy system. However, incomplete conversion of lignocellulose-rich agricultural waste remains a key limitation, often leaving energy-dense residues in the digestate by-product. In this study, we introduce a novel application of chelator-mediated Fenton (CMF) post-treatment to recover untapped biomethane potential from these recalcitrant residues, representing a significant departure from conventional pre-treatment strategies. By systematically varying pH, iron-chelator concentration, and hydrogen peroxide dosage, we identified reaction conditions (pH 6-8, 5 mM Fe²⁺-dihydroxybenzene, 3-4 wt.% H₂O₂) that enhanced lignocellulose deconstruction and increased dissolved organic carbon (DOC) availability for methanogenesis. CMF post-treatment led to up to a tenfold increase in biomethane potential compared to untreated controls. Microbial community analysis revealed enrichment of cellulolytic species, suggesting enhanced hydrolytic activity as a driver of improved conversion. Application of the CMF post-treatment method to isolated poplar lignin further demonstrated its versatility for diverse lignocellulosic substrates. These findings position CMF post-treatment as a promising strategy to enhance AD efficiency and valorize digestate.

Scheme 1

Experimental workflow for assessing the effects of CMF treatment parameters on delignification efficiency, biomethane potential, and microbial composition

Fig. 1

Effect of CMF reaction parameters on delignification (blue), xylan removal (teal), and glucan (green) removal. In each reaction series (Series 1, 2 and 3), one parameter (pH set-point, Fe²⁺-DHB concentration, and H₂O₂ concentration) was varied, while the other parameters were held constant in the following reaction conditions: pH 6, 5 mM Fe²⁺ -DHB, and 3 wt.% H₂O₂. Standard error propagation was calculated for triplicate measurements of lignin, xylan and glucan content

Fig. 2

Effect of CMF reaction conditions on the total carbon balance. Dissolved organic carbon (dark blue), insoluble carbon (blue-grey) and lost carbon (light grey). Experiments were performed under conditions of 5 mM Fe²⁺-DHB, pH 6, and 3 wt.% H₂O₂, with one parameter varied in each series while keeping the others constant. Percentages were normalized to a total of 100% based on the initial carbon content in SD. Standard error propagation calculated for triplicate DOC measurements and for single elemental analysis measurements for insoluble carbon for duplicate reactions

Fig. 3

Anaerobic digestion organic solids analysis and biomethane potential results. A VS/TS ratio for each reaction series, representing the organic fraction of each substrate. Standard error calculated for duplicates. B Biomethane potential reported in NmL CH4/g VS for each substrate: untreated (dark grey), varied pH set-point (orange), varied Fe²⁺-DHB concentration (green) and varied H₂O₂ concentration (purple). Gas volume is automatically normalized to standard temperature and pressure conditions (NmL at STP). Standard propagated error calculated for duplicates

Fig. 4

Effect of inoculum and H₂O₂ concentration on biomethane potential and microbial composition. A Cumulative biomethane potential (NmL CH₄/g VS) of CMF-treated substrates across varying H₂O₂ concentrations using Inoculum A. B Relative abundance of microbial taxa at the end of digestion in samples treated with Inoculum A. C Cumulative biomethane potential (NmL CH₄/g VS) of CMF-treated substrates across varying H₂O₂ concentrations using Inoculum B. D Relative abundance of microbial taxa at the end of digestion in samples treated with Inoculum B. Error bars represent the standard error of duplicate measurements

Fig. 5

Final biomethane potential (NmL CH₄/g VS) of CMF-treated substrates (5 mM Fe²⁺-DHB and pH 6) under varying H₂O₂ concentrations (1–4 wt.% H₂O₂) using Inoculum A (purple) and Inoculum B (blue), both at an ISR of 1:1, pH 7. Error bars represent the standard propagated error of duplicate measurements

Fig. 6

Cumulative biomethane potential (NmL CH₄/g VS) resulting from treatment of SD-CMF (teal) vs treatment of PL-CMF (purple), compared to their respective untreated controls. CMF treatments were conducted under the following reaction conditions: 5 mM Fe²⁺-DHB, pH 6, and 3 wt.% H₂O₂. Error bars represent the standard propagated error of duplicate measurements

Fig. 7

Microbial community analysis of CMF-treated and untreated PL and SD samples. A Relative abundance of microbial taxa in untreated and CMF-treated PL and SD samples at the end of anaerobic digestion using Inoculum B. B Log-fold changes in microbial taxa from the beginning to the end of digestion for untreated and CMF-treated PL and SD samples