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.

碳/氮比（C / N）是影响厌氧消化（AD）性能的关键参数。最近的AD研究集中在优化原料的C / N。所谓的“抑制稳态”是指当将可降解的富氮底物（例如牲畜粪便）用作原料时，通常在低C / N（25以下）时发生的一种特殊的氨抑制AD的状态。但是，“抑制稳态”背后的机制仍然未知。在当前研究中，共消化和再循环用于在进水中创建C / N梯度，以探索“抑制稳态”中底物，代谢物和微生物之间的关系。在宏观，微生物和遗传水平上收集数据。通过使用10-12的进水C / N，成功地使三个CSTR进入“抑制稳态”。R10–R12的消化性能水平较低且稳定，随着C / N逐渐降低，其从产甲烷的途径转变为氢营养途径。随着亲水性产甲烷菌的丰度增加，同养型乙酸氧化细菌（SAOB）的丰度也增加。人口之间的继承Methanosaeta和Methanosarcina可用作氨抑制的微生物指标。在高C / Ns下，细菌之间的协作较高，而在低C / Ns下，细菌之间的竞争较高。这些结果阐明了“抑制稳态”的过程，而“抑制稳态”是使用可降解富氮底物的实际大型沼气设施经常面临的条件。此外，提供了评估氨气抑制的实用指南，并开发了缓解氨气抑制的策略。