Engineering Chlorella pyrenoidosa via atmospheric and room-temperature plasma mutagenesis and microbial microdroplet culture acclimatization for enhanced treatment of high-concentration oxytetracycline wastewater

Oxytetracycline (OTC) wastewater generated during the crystallization stage (OTC-CW) poses substantial challenges to conventional biological treatment processes. In the present study, an optimized microalgal strain (M-2) was developed through iterative atmospheric and room-temperature plasma mutagenesis and microbial microdroplet culture acclimatization, exhibiting markedly enhanced adaptability and treatment performance in OTC-CW relative to the wild-type strain (WT). Compared with WT, the tolerance of M-2 to OTC-CW was significantly enhanced, with optical density (OD680) attaining 1.20 and dry cell weight (DCW) reaching 1.13 g/L. Simultaneously, the removal rates of chemical oxygen demand, ammonia nitrogen, total nitrogen, total phosphorous and OTC were significantly enhanced, increasing by 47.06 %, 34.85 %, 41.36 %, 33.38 % and 28.85 %, respectively, relative to those of the WT. And biomass production was improved, with protein, lipid, carbohydrate and chlorophyll contents reaching 406.15, 189.47, 131.67 and 9.89 mg/g, respectively. Transcriptomic analysis identified 27 significantly differentially expressed genes in M-2, which were predominantly enriched in five KEGG pathways: ribosome biogenesis (ko03008), purine metabolism (ko00230), glyoxylate and dicarboxylate metabolism (ko00630), tryptophan metabolism (ko00380) and terpenoid backbone biosynthesis (ko00900). Significant enrichment of these pathways promoted rRNA transcription, ribosome assembly, ATP and GTP regeneration, glyoxylate-cycle activity, central carbon metabolic flux, kynurenine and terpenoid biosynthesis and reactive oxygen species scavenging capacity. Collectively, these findings demonstrate the superior adaptability and treatment efficiency of M-2, highlighting its potential for the efficient remediation of pharmaceutical OTC-CW.