Digging and identification of novel microorganisms from the soil environments with high methanol-tolerant lipase production for biodiesel preparation.

Converting renewable biomass into carbon-neutral biofuels is one of the most effective strategies to achieve zero carbon emissions and contribute to environmental protection. Microorganisms from the soil were primarily screened on the rhodamine B-plate for highly-active lipase producing strains and re-screened on a tributyrin-methanol plate using crude lipases produced from the initially screened-out strains. The lipase-producing strains with higher methanol-tolerant lipase were identified based on morphological characteristics and 16S rDNA sequencing. The crude lipases with much higher methanol-tolerance from screened top-4 strains, Stenotrophomonas maltophilia D18, Lysinibacillus fusiformis B23, Acinetobacter junii C69, and A. pittii C95 showed temperature optima of 25 °C, 35 °C, 30 °C, and 30 °C at pH 7.0, respectively, while their pH optima were 8.0, 7.0, 7.5, and 7.5 at each optimum temperature, respectively. After 24-h incubation, they retained more than 85% of their original activities in 25%, 15%, 20%, and 20% of methanol, respectively. They catalyzed the conversion of soybean oil into biodiesel by yields of 63.1%, 35.4%, 74.6%, and 78.5% after 24-h reactions, respectively. In conclusion, the as-isolated microorganisms producing high methanol-tolerant lipase are considered promising to provide robust biocatalyst for efficient biodiesel preparation and other industrial applications.