利用废弃生物质通过链延长（chain elongation, CE）生产的正己酸可以供应各种化石衍生产品，从而推动碳中和的实现。富氮废弃生物质降解释放的氨可以充当厌氧生物过程（包括CE过程）中的营养物质或抑制剂，二者的区别主要取决于氨的浓度。目前，使用乙醇作为电子供体进行开放培养正己酸生产的最佳氨浓度、毒性阈值以及潜在机制尚不清楚。本研究表明，生产正己酸的最佳氨浓度为2 g∙L^-1，而超过此阈值的氨浓度会显著抑制CE性能。对该机制的探索揭示了两种形式的氨（即铵离子和游离氨）参与了这种抑制行为。高氨浓度（5 g∙L^-1）诱导乙醇过度氧化并抑制逆β氧化（reverse β-oxidation, RBO）途径，直接导致负责乙酸形成的酶（磷酸转乙酰酶和乙酸激酶）的活性增强，丁酰辅酶A∶乙酰辅酶A转移酶、己酰辅酶A∶丁酰辅酶A转移酶和己酰辅酶A∶乙酰辅酶A转移酶活性降低，这些酶参与正丁酸和正己酸合成。此外，微生物群落的优势菌属由Paraclostridium（氨浓度为0.1 g∙L^-1）转变为Fermentimonas, Clostridium sensu stricto 12和Clostridium sensu stricto 15（氨浓度为2 g∙L^-1）。然而，在过量氨（氨浓度为5 g∙L^-1）条件下，这些具有CE功能的细菌大多不存在。宏基因组分析揭示了在氨浓度为2 g∙L^-1的条件下RBO、脂肪酸合成、K⁺外流、腺苷三磷酸酶（ATPase）代谢和金属阳离子输出等的功能基因上调，共同促进了正己酸产量的增加。相反，过量的氨会抑制上述功能（不包括金属阳离子输出）和K⁺流入，从而削弱氨解毒和正己酸生物合成。本研究对影响正己酸生产的氨驱动机制进行了全面的阐明，预计将激励研究人员设计出有效的策略来减轻氨诱导的抑制作用。

n-Caproate, which is produced via chain elongation (CE) using waste biomass, can supply various fossil-derived products, thus advancing the realization of carbon neutrality. Ammonia released from the degradation of nitrogen-rich waste biomass can act as a nutrient or an inhibitor in anaerobic bioprocesses, including CE, with the distinction being primarily dependent on its concentration. Currently, the optimal concentration of ammonia and the threshold of toxicity for open-culture n-caproate production using ethanol as an electron donor, along with the underlying mechanisms, remain unclear. This study revealed that the optimal concentration of ammonia for n-caproate production was 2.0 g∙L⁻¹, whereas concentrations exceeding this threshold markedly suppressed the CE performance. Exploration of the mechanism revealed the involvement of two forms of ammonia (i.e., ammonium ions and free ammonia) in this inhibitory behavior. High ammonia levels (5.0 g∙L⁻¹) induced excessive ethanol oxidation and suppressed the reverse β-oxidation (RBO) process, directly leading to the enhanced activities of enzymes (phosphotransacetylase and acetate kinase) responsible for acetate formation and diminished activities of butyryl-coenzyme A (CoA):acetyl-CoA transferase, caproyl-CoA:butyryl-CoA transferase, and caproyl-CoA:acetyl-CoA transferase that are involved in the syntheses of n-butyrate and n-caproate. Furthermore, the composition of the microbial community shifted from Paraclostridium dominance (at 0.1 g∙L⁻¹ ammonia) to a co-dominance of Fermentimonas, Clostridium sensu stricto 12, and Clostridium sensu stricto 15 at 2.0 g∙L⁻¹ ammonia. However, these CE-functional bacteria were mostly absent in the presence of excessive ammonia (5.0 g∙L⁻¹ ammonia). Metagenomic analysis revealed the upregulation of functions such as RBO, fatty acid synthesis, K⁺ efflux, adenosine triphosphatase (ATPase) metabolism, and metal cation export in the presence of 2.0 g∙L⁻¹ ammonia, collectively contributing to enhanced n-caproate production. Conversely, the aforementioned functions (excluding metal cation export) and K⁺ influx were suppressed by excessive ammonia, undermining both ammonia detoxification and n-caproate biosynthesis. The comprehensive elucidation of ammonia-driven mechanisms influencing n-caproate production, as provided in this study, is expected to inspire researchers to devise effective strategies to alleviate ammonia-induced inhibition.

・2 g/L ammonia was the optimal dose and inhibitory threshold for MCFAs production.

・FNA and NH₄⁺ were confirmed to be involved in the inhibitory effect.

・5 g/L ammonia could induce the process of excessive ethanol oxidation.

・Ammonia shifted the dominant CE functional bacteria.

・Functions of RBO, FAS, K+ transport and ATPase metabolism were inhibited by high ammonia.