Comparing the mechanisms of syntrophic volatile fatty acids oxidation and methanogenesis recovery from ammonia stress in regular and biochar-assisted anaerobic digestion: Different roads lead to the same goal

Abstract

Biochar has been recognized as a promising additive to mitigate ammonia inhibition during syntrophic methanogenesis, while the key function of biochar in this process is still in debates. This study clarified the distinct mechanisms of syntrophic volatile fatty acids -oxidizing and methanogenesis recovery from ammonia inhibition in regular and biochar-assisted anaerobic digestion. Under 5 g/L ammonia stress, adding biochar shortened the methanogenic lag time by 10.9% and dramatically accelerated the maximum methane production rate from 60.3 to 94.7 mLCH₄/gVS_sludge/d. A photometric analysis with a nano-WO₃ probe revealed that biochar enhanced the extracellular electron transfer (EET) capacity of suspended microbes (Pearson's r = -0.98), confirming that biochar facilitated methanogenesis by boosting EET between syntrophic butyrate oxidizer and methanogens. Same linear relationship between EET capacity and methanogenic rate was not observed in the control group. Microbial community integrating functional genes prediction analysis uncovered that biochar re-shaped syntrophic partners by enriching Constridium_sensu_stricto/Syntrophomonas and Methanosarcina. The functional genes encoding Co-enzyme F₄₂₀ hydrogenase and formylmethanofuran dehydrogenase were upregulated by 1.4-2.3 times, consequently enhanced the CO₂-reduction methanogenesis pathway. Meanwhile, the abundances of gene encoding methylene-tetrahydrofolate transformation, a series of intermediate processes involved in acetate oxidation, in the biochar-assisted group were 28.2-63.7% higher than these in control group. Comparatively, Methanosaeta played a pivotal role driving aceticlastic methanogenesis in the control group because the abundance of gene encoding acetyl-CoA decarbonylase/synthase complex increased by 1.9 times, suggesting an aceticlastic combining H₂-based syntrophic methanogenesis pathway was established in control group to resist ammonia stress. A 2^nd period experiment elucidated that although depending on distinct mechanisms, the volatile fatty acid oxidizers and methanogens in both groups developed sustained and stable strategies to resist ammonia stress. These findings provided new insights to understand the distinct methanogenic recovery strategy to resist toxic stress under varied environmental conditions.

Keywords: Ammonia stress; Anaerobic digestion; Biochar; Extracellular electron transfer; Methanogen evolution; Methanogenic pathway.