Broad-range pH/Temperature-stable Cellulase from a Novel Hydrocarbon Contaminated Mangrove soil Bacterium, Bacillus licheniformis VVA21

Abstract

Aim: This study reports the production of cellulase by Bacillus licheniformis VVA21 isolated from hydrocarbon contaminated Kegbara-Dere mangrove in Ogoniland, Nigeria. Methodology: Baseline physicochemical characteristics of the hydrocarbon contaminated soil were established. Twenty-two bacterial isolates were screened for cellulolytic activity on carboxymethyl cellulose (CMC) agar using the spread plate technique. The isolate with the highest zone of clearance was selected and assayed further. Crude cellulase was extracted and partial purification achieved by ammonium sulphate precipitation, followed by dialysis, and final purification by Sephadex G-100 chromatography. The best cellulase-producing bacterium was identified by analytical profile index (API) and 16S rRNA gene analyses. Results: The contaminated soil revealed sulphate, nitrate, phosphate, polycyclic aromatic hydrocarbon (PAH), benzene, toluene, ethylbenzene, and xylene (BTEX) and total petroleum hydrocarbon (TPH) contents of 30.23, 24.77, 17.56, 11.66, 100.22 and 11560 mg/kg, respectively. Out of the 22 bacterial isolates screened for cellulolytic activity, isolate VVA21 gave the highest zone of clearance (16 mm) with maximum cellulase activity of 871.6 U/mL after 48 h of incubation. Specific cellulase activity increased across the different purification steps (from crude fraction to Sephadex G-100 fraction), ranging between 16.8 and 68.9 U/ /mg/mL. The purified cellulase was stable over a pH range of 4 to 10 and temperature range of 30 to 100°C. The best stability for pH (102.46 U/mg) was observed at pH of 8.5 whereas the best stability for temperature (99.96 U/mg) was observed at 30°C. API and 16S rRNA gene product analyses revealed the best cellulase-producing strain as Bacillus licheniformis strain VVA21. These sequence data have been submitted to the DDBJ/EMBL/GenBank databases under accession number MF581271.1.  Conclusion: The Bacillus licheniformis VVA21, isolated from hydrocarbon contaminated mangrove soil, showed high potentials for cellulase production with high stability over broad temperature and pH ranges.