The effects of C/N (10–25) on the relationship of substrates, metabolites, and microorganisms in “inhibited steady-state” of anaerobic digestion

The carbon/nitrogen ratio (C/N) is a key parameter that affects the performance of anaerobic digestion (AD). Recent AD research has focused on optimizing the C/N of feedstock. The so-called inhibited steady-state refers to a special state of ammonia inhibition of AD that often occurs at low-C/N (below 25) when degradable nitrogen-rich substrates, such as livestock manure, are used as feedstock. However, the mechanism behind the inhibited steady-state is still unknown. In the current study, co-digestion and recirculation were used to create a C/N gradient in the influent to explore the relationship between substrates, metabolites, and microorganisms in the inhibited steady-state. Data were collected at the macro, microbial, and genetic levels. Three CSTRs were successfully made run into the inhibited steady-state using influent C/Ns of 10-12. Digestion performance levels of R10-R12 were low and stable, transitioning from an aceticlastic methane-producing pathway to a hydrogenotrophic pathway as the C/N gradually decreased. As the abundance of the hydrogenophilic methanogens increased, the abundance of syntrophic acetate-oxidizing bacteria (SAOB) also increased. The succession between populations of Methanosaeta and Methanosarcina may be used as a microbiological indicator of ammonia inhibition. Under high-C/Ns, cooperation among bacteria was high, while under low-C/Ns, competition among bacteria was high. These results clarify the processes underlying the inhibited steady-state, which is a condition often faced in actual large-scale biogas facilities that use degradable nitrogen-rich substrates. Moreover, practical guidelines for evaluating ammonia inhibition are provided, and strategies to alleviate ammonia suppression are developed.