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Biofiltration of n-hexane as a representative of hydrophobic volatile organic compounds (VOCs) at presence and absence of rhamnolipid biosurfactant was studied using a multilayer biofilter packed with scoria, compost, poplar tree skin and sugar beet pulp, for 131 days. The concentration of n-hexane was measured by a gas chromatograph coupled with a flame ionization detector (GC/FID). The results showed that the mean removal efficiency (RE) of n-hexane at the presence of the biosurfactant was two times higher than that at absence of the biosurfactant. According to the results, rhamnolipid can enhance the efficiency of biofiltration of VOCs from polluted air streams. Protocol name: Improved biodegradation with a multilayer biofilter, Keywords: Biodegradation, Rhamnolipid, Biofiltration, VOCs