CO2 agitation combined with magnetized biochar to alleviate "ammonia inhibited steady-state": Exploring the mechanism by combining metagenomics with macroscopic indicators.

The "ammonia inhibited steady-state" phenomenon is frequently observed in the anaerobic digestion (AD) process of nitrogen-rich substrates. Reconfiguring microbial ecosystems has proven to be an effective strategy for mitigating ammonia inhibition. In the current study, biochars were screened and targeted for modification. CO2 agitation combined with magnetized biochar was used to aid the semi-continuous AD systems with "ammonia inhibited steady-state." The results indicated that coconut shell biochar had the best stimulating effect on AD performance. The content of oxygen-containing functional groups (OCFGs), which had a positive correlation with the electron donating capacity (EDC), was targeted to be regulated. This strategy significantly increased the CH4 yield by 31.7 % (from 344 to 278 mL/g VS) (p < 0.05). Isotope tracing and KEGG gene annotation indicated that this strategy stimulated the efficiency of the hydrogenotrophic pathway. Simultaneously, it accelerated the attachment of microorganisms, which made the DIET pathway between bacteria and archaea efficient. Under CO2 agitation, the attachment of functional microorganisms to the biochar accelerated. Biochar weakened the synthesis of bioelectronic carriers (Cyt-c and chemosensory pili), while the electroactivity of the AD system was enhanced. This means that biochar replaced bioelectronic carriers and improved the DIET efficiency. In addition, the strategy had a positive effect on the colonization of simultaneous nitrification-denitrifying bacteria (Georgenia), which led to a decrease in ammonia nitrogen concentrations. This study revealed the mechanism by which this strategy alleviates ammonia inhibition and provided a promising strategy for the efficient AD of nitrogen-rich substrates.