BTEX biodegradation by biofilms from constructed wetlands treating petroleum refinery wastewater: insights from compound-specific isotope analysis

Constructed wetlands are increasingly employed to treat petroleum refinery wastewater containing benzene, toluene, ethylbenzene, and xylenes (BTEX). However, distinguishing biodegradation from physical removal processes remains challenging, and the effect of constructed wetland configuration and associated microbial communities on BTEX transformation is still poorly understood. This study examined anoxic BTEX degradation in microcosms inoculated with biofilms from planted and unplanted horizontal- and vertical-flow constructed wetlands. Dual-element compound-specific isotope analysis (CSIA) was combined with microbial community profiling to evaluate biodegradation and differentiate it from volatilization and sorption. Biodegradation accounted for approximately 50 % of total BTEX removal, with ethylbenzene and xylene exhibiting greater transformation than benzene and toluene. Dual δ¹³C-δ²H fractionation confirmed biodegradation, with carbon isotope enrichment factors (ε_C) ranging from -1.0 ± 0.2 ‰ to -3.6 ± 0.2 ‰ and hydrogen isotope enrichment factors (ε_H) from -9.6 ± 2.7 ‰ to -39.8 ± 1.3 ‰. Λ values (4.9 ± 2.2 to 24.3 ± 2.6) indicated predominantly anoxic degradation pathways. Microcosms with biofilms from planted wetlands displayed higher microbial activity and biodegradation, likely stimulated by root exudates. Proteobacteria, Actinobacteriota, and Desulfobacterota were identified as key phyla associated with BTEX transformation under anoxic conditions. Although biodegradation was the dominant BTEX removal pathway, predicted estimates showed that volatilization and sorption may also contribute significantly in constructed wetlands. The integrated CSIA-microbial approach provides a framework to elucidate BTEX degradation mechanisms and supports the optimized design of constructed wetlands as sustainable bioremediation systems for hydrocarbon-contaminated effluents.