In vitro degradation of hexacosane (C26H54), a HMW n-alkane, was studied in MSM by two bacterial strains i.e., Pseudomonas sp. BP10 and Stenotrophomonas nitritireducens E9, isolated from petroleum sludge, in isolation and combination. The results revealed that both the strains were able to metabolize hexacosane by 82% in isolation and 98% in their consortium after 7 days. An enhancement of 16% in hexacosane degradation by the consortium indicated an additive action of bacterial strains. However, in control, a degradation of 21% was attributed to abiotic factors. During incubation with hexacosane, both the bacteria continued to multiply in isolation and consortium, which reflected that hexacosane was utilized by bacteria as a carbon and energy source. Activities of alkane hydroxylase and alcohol dehydrogenase were differentially expressed in isolation and combination, indicating their involvement in hexacosane degradation. Enhanced cell surface hydrophobicity and emulsification index and reduced surface tension also supported the degradation process. (C) 2014 Elsevier Ltd. All rights reserved.