Insight into c‑di-GMP Regulation in Anammox Aggregation in Response to Alternating Feed Loadings

Substrate concentrations generally fluctuate in wastewaters. However, how anammox biomass behaves to overcome the stress of alternating feed loadings remains unclear. Here, we combined long-term reactor operation, batch tests, 16S rRNA transcript sequencing, and metabolomics analysis to investigate the aggregation of anammox biomass under the regulation of c-di-GMP, a key second messenger, in response to alternating feed loadings. We demonstrated that the aggregation process was significantly faster under alternating loadings and was significantly correlated with higher levels of c-di-GMP and extracellular polymeric substances (EPS) production. The increase in c-di-GMP was positively correlated with a higher relative transcript expression level in the c-di-GMP pathway-dependent community. The targeted metabolomics results indicated that the increased production of fructose 6-phosphate and UDP-N-acetyl-d-glucosamine, the precursor substances for the synthesis of exopolysaccharides, was induced by higher levels of c-di-GMP. Consequently, the granulation process was accelerated via EPS production. Higher levels of intracellular hydrophobic amino acids were also positively correlated with increased extracellular protein levels, considering the significant increase in peptides under alternating loadings. On the basis of our findings, we believe that c-di-GMP regulation and EPS production of the anammox biomass are important mechanisms to enhance its tolerance against unfavorable feed stress. These results highlight the role of c-di-GMP in anammox biomass as it works to survive in unfavorable niches.

废水中的底物浓度通常存在波动。然而，厌氧氨氧化（anammox）微生物群落如何应对交替进料负荷带来的胁迫并存活，目前仍不明确。本研究结合长期反应器运行实验、批次试验、16S rRNA转录测序与代谢组学分析，探究了厌氧氨氧化微生物在关键第二信使环二鸟苷酸（c-di-GMP）调控下，针对交替进料负荷产生的聚集行为。研究结果表明，交替进料负荷下厌氧氨氧化微生物的聚集过程显著加快，且与环二鸟苷酸水平升高及细胞外聚合物（EPS）生成量提升显著相关。环二鸟苷酸水平的升高，与依赖其通路的菌群中更高的转录相对表达量呈正相关。靶向代谢组学分析结果显示，环二鸟苷酸水平升高可诱导胞外多糖合成前体物质——果糖6-磷酸与UDP-N-乙酰-d-葡糖胺的生成量提升。由此，通过细胞外聚合物的生成，厌氧氨氧化微生物的颗粒化过程得以加快。此外，考虑到交替进料负荷下肽类物质显著增加，胞内疏水氨基酸水平的升高也与胞外蛋白水平提升呈正相关。基于上述研究结果，我们认为厌氧氨氧化微生物的环二鸟苷酸调控与细胞外聚合物生成，是提升其应对不利进料胁迫耐受性的重要机制。本研究结果凸显了环二鸟苷酸在厌氧氨氧化微生物适应不利生境过程中的关键作用。