Biosurfactant slows down n-hexadecane biodegradation: ¹³C-labeled rhamnolipid tracing

Rhamnolipids are globally applied in bioremediation, but their specific metabolic fate and regulatory mechanisms remain unclear. This study addresses this gap by using ¹³C stable isotope technology to trace rhamnolipid partitioning and evaluate its impact on n-hexadecane mineralization. A method to produce ¹³C-labeled rhamnolipid (δ¹³C= 2490 ‰) using Pseudomonas aeruginosa ATCC 27853 was established to determine the bioavailability and metabolic pathways of rhamnolipid in environmental matrices. P. aeruginosa ATCC 27853 was acclimated to optimize rhamnolipid production and enhance n-hexadecane degradation. Rhamnolipid addition slowed n-hexadecane degradation by 18 % over 12 days. This is because rhamnolipids are more degradable and encapsulate bacterial surfaces (indicated by SEM images), reducing contact with n-hexadecane. Concurrently, a 0.3-unit pH decrease and the stimulated secretion of long-chain fatty acids (e.g., oleic acid) altered bacterial membrane permeability, further inhibiting n-hexadecane uptake. Despite this, complete mineralization of n-hexadecane to CO₂ increased by 20 % after rhamnolipid addition. ¹³C tracing revealed that 84 % of the added rhamnolipid was mineralized to CO₂, while only 5.5 % was assimilated into biomass. Structural equation model demonstrated that the increased oxidase activity after rhamnolipid addition contributes to this priming effect. These findings provide a methodology to accurately track the fate of carbon-based additives, revealing the priming effect as a critical factor in wastewater bioremediation. This underscores the necessity of evaluating total carbon fate to optimize remediation efficiency and minimize hazards in bioremediation strategies.