Abstract

Robust microalgal activity is critical for the microalgal-bacterial symbiosis system (MBSS) to enable wastewater resource recovery, but microalgal performance can be affected by high concentrations of sludge. Ca 2+ may regulate microalgal performance. Nevertheless, the mechanism of Ca 2+ -mediated regulation, particularly under high concentrations of sludge, remains unclear. This study integrated physiological and genomic analyses to investigate microalgal responses to sludge (100-800 mg/L) and Ca 2+ supplementation (10-50 mM). Results showed that high-concentration sludge (400-800 mg/L) reduced microalgal growth, pigment synthesis, and photosynthetic efficiency by 65.6%-86.6%, 20.1%-39.2%, and 1.6%-7.0%, respectively, while Ca 2+ restored these parameters by up to 39.9%, 39.7%, and 8.5%. At the genetic level, Ca 2+ activated microalgal Ca 2+ signaling pathways (43.9%-226.4% increase in CaM , CDPK , and CBL ). It upregulated antioxidant enzyme genes (76.1%-373.0% increase in SOD , CAT , and POD ) to mitigate cell damage and photosynthetic genes (e.g. 95.0%-260.9% increase in psbA and rbcL ) to restore chloroplast function. Concurrently, Ca 2+ promoted bacterial central carbon metabolism genes (e.g., 1.6%-26.2% increase in CS , IDH and OGDH ) to increase CO 2 release for microalgal carbon fixation and recruited siderophore-producing bacteria (e.g., 120.6%-154.3% increase in Sphingopyxis ) to improve iron bioavailability for microalgal photosynthesis. Therefore, a positive feedback loop was formed through the supplementation of Ca 2+ . Microalgal photosynthesis supplied organic carbon/O 2 for bacteria, while bacterial metabolism provided CO 2 and iron for microalgae. Collectively, Ca 2+ optimized microalgal activity via cross-kingdom coordination of carbon-iron metabolism, offering a mechanistic basis for optimizing MBSS applications in wastewater treatment and biological resource recovery by using Ca 2+ as an effective regulator.