Abstract:Immobilized microbial technology has advantages such as strong toxicity resistance, higher stability, and superior degradation performance. This study developed an innovative embedding-crosslinking co-immobilization strategy using sodium alginate (SA), chitosan (CS), and biochar (BC) to construct SA/CS@BC composite microspheres for encapsulating the deep-sea phenol-degrading bacterial consortium SP-1. Key parameters were optimized through single-factor experiments: SA concentration (1.0%, 2.0%, 3.0%, 4.0%, 5.0%), CS concentration (0.25%, 0.5%, 0.75%, 1.0%, 1.25%), CaCl2 crosslinker concentration (1.0%, 2.0%, 3.0%, 4.0%, 5.0%), and BC dosage (0.25%, 0.5%, 0.75%, 1.0%, 1.25%). Following degradation screening at 800 mg/L phenol, the performance of free bacteria, SA/CS microspheres, and SA/CS@BC microspheres was comparatively analyzed at phenol concentrations of 200, 400, 600, 800, 1 000, and 1 200 mg/L. The mechanisms were investigated via SEM for microstructural morphology, FTIR for functional group analysis, the BET method for specific surface area measurement, LC-MS for metabolic intermediate identification, TOC analysis for mineralization quantification, and 5-cycle reuse tests for operational stability assessment. The optimized SA/CS@BC microspheres at 3.0% SA, 0.75% CS, 4.0% CaCl2, and 1.0% BC achieved 94.6% degradation at 1 200 mg/L phenol, which was 3.3-fold higher than that of free bacteria and 76.2% higher than that of SA/CS microspheres. At 1 000 mg/L, SA/CS@BC maintained 95.2% efficiency, significantly exceeding that of SA/CS microspheres and free bacteria. BC incorporation increased the specific surface area from 4.936 m2/g to 32.829 m2/g; SEM confirmed the dense colonization of SP-1 within the BC porous architecture, whereas SA/CS microspheres exhibited a compact structure with sparse microbial loading. FTIR spectra revealed intensified absorption peaks at 3 335 cm−1 (—OH), 1 590 cm−1 (—COOH), and 1 004 cm−1 (C—O—C) for SA/CS@BC, with blue shifts compared to SA/CS (3 420 cm−1, 1 622 cm−1, 1 024 cm−1), indicating enhanced hydrogen-bonding networks. Elevated functional group abundance and strengthened hydrogen bonds jointly promoted phenol adsorption. LC-MS detected key intermediates including catechol, muconic acid, and succinic acid. Phenol was first hydroxylated to catechol, followed by ortho-cleavage of catechol to form muconic acid, which was further oxidized to succinic acid entering the tricarboxylic acid cycle, and ultimately mineralized to CO2 and H2O. TOC analysis demonstrated 93.9% removal after 9 days treatment at 1 200 mg/L phenol, with the IC/TC ratio increasing from 5.8% to 68.5%, verifying complete mineralization to CO2 and H2O. SA/CS@BC microspheres retained 90.5% degradation efficiency after 5 reuse cycles at 1 200 mg/L phenol. This work establishes that SA/CS@BC microspheres enhance phenol degradation: the SA/CS hydrogel shields microbes from acute phenol toxicity; BC rapidly concentrates phenol, while SA/CS gel controls the gradual release to maintain sub-inhibitory concentrations. BC′s pores increase microbial loading density, and surface oxygen groups facilitate microbe-pollutant interactions. This technology provides an efficient, stable, and engineerable solution for bioaugmentation of high-concentration phenol-laden wastewater. This research presents an innovative immobilization technique for the biological treatment of high phenol wastewater, demonstrating high degradation efficiency and stable operational reliability. Close-