Valorization of acid-hydrolyzed tea stem waste for sustainable biodiesel production using Chlorella vulgaris: a biorefinery approach

The prohibitive cost of synthetic cultivation media remains a fundamental bottleneck in the commercial deployment of microalgal biodiesel. This study investigates the valorization of recalcitrant tea stem waste, an abundant agro-industrial by-product, as a low-cost, nutrient-rich medium for Chlorella vulgaris within an integrated biorefinery framework. Following thermochemical acid hydrolysis, a two-stage optimization of hydrolysate concentration and incident irradiance was conducted to maximize biomass production. Undiluted (100%) hydrolysate under elevated irradiance (240 µmol photons m -2 s -1 ) compensated for optical attenuation in the dark medium and yielded a maximum biomass concentration of 1.65 ± 0.07 g L -1 , representing an approximately 5-fold increase over the synthetic Bold’s Basal Medium (BBM) control. Concurrently, substantial nutrient recovery was achieved, with 83.23% nitrate and 95.60% phosphate assimilation by Day 10. The resulting nutrient limitation acted as a secondary abiotic stressor, triggering enhanced intracellular lipid accumulation and yielding a peak volumetric lipid concentration of 0.094 ± 0.005 g L -1 , approximately 4.5-fold higher than the autotrophic control. Fatty acid methyl ester (FAME) profiling revealed a saturated-dominant composition (85.57% SFA), corresponding to favorable predicted biodiesel properties, including low iodine value and high cetane number, consistent with international fuel standards. Overall, this study establishes tea stem hydrolysate as an efficient integrated cultivation matrix for simultaneous mixotrophic growth and lipid biosynthesis, advancing a scalable circular waste-to-energy pathway for agro-industrial systems.