Exogenous cyclic di-GMP promotes multilevel functional restoration of algal-bacterial symbiotic system under prolonged ciprofloxacin stress.

Elevated concentrations of antibiotics exert significant stress on algal-bacterial interactions, thereby threatening the functional stability of biological wastewater treatment systems. However, robust and scalable strategies for restoring the functionality of such impaired systems remain inadequately developed. In this study, an algal-bacterial symbiotic system (ABSS) was subjected to continuous ciprofloxacin (CIP) stress at 50 mg/L, followed by exogenous supplementation of cyclic di-GMP (c-di-GMP) at 100 μg/L. Exposure to a high concentration of CIP significantly decreased intracellular c-di-GMP and autoinducer-2 (AI-2) levels, concomitantly reducing extracellular polymeric substances (EPS) production, indole-3-acetic acid (IAA) synthesis, microalgal photosynthetic efficiency, inorganic carbon assimilation, and overall pollutant removal performance. Following exogenous c-di-GMP supplementation, intracellular c-di-GMP and AI-2 levels in algal-bacterial symbionts increased by 156.8% and 137.4%, respectively, concomitant with recovery of EPS, IAA, chlorophyll a (Chl a) content, maximum quantum yield of PSII (Fv/Fm), carbonic anhydrase activity, and inorganic carbon assimilation. Piecewise regression analysis revealed significantly positive slope differences for 16 of 17 functional indicators relative to the non-c-di-GMP-treated control, demonstrating coordinated functional restoration across microbial signaling, extracellular matrix regulation, and system-level performance. Metatranscriptomic analysis further revealed that the ABSS exhibited a transcriptionally distinct community state. The transcriptional response was functionally selective, with positive responses in biofilm formation, EPS biosynthesis, photosynthesis, and carbon fixation rather than global enhancement of quorum sensing (QS). These findings indicate that exogenous c-di-GMP supplementation conferred coordinated restoration of interfacial communication and metabolic functionality in the ABSS, thereby offering a promising intervention strategy to enhance system resilience under high-concentration antibiotic stress.