Dual-function oleaginous biocatalysts for non-sterile cultivation and solvent-free biolipid bioextraction to reduce biolipid-based biofuel production costs.

Triacylglycerols (TAGs) are starting materials for the production of biolipid-based fuels such as biodiesel and biojet fuel. While various microorganisms can produce TAGs from renewable resources, the cultivation of TAG-producing microorganisms under sterilization conditions to avoid microbial contamination and application of solvent to extract TAGs from the TAG-filled microorganisms are costly. To overcome these challenges, this study reports the feasibility of a non-sterile cultivation of an oleaginous bacterium Rhodococcus opacus PD631SpAHB under saline conditions, followed by the use of a solvent-free, phage-lysis-protein-based bioextraction approach for TAGs release. The engineered strain PD631SpAHB was developed by introducing a recombinant plasmid carrying a phage lytic gene cassette (pAHB) into Rhodococcus opacus PD631 via transformation, followed by adaptive evolution under saline conditions. This newly developed strain is a salt-tolerant strain with the inducible plasmid pAHB to enable TAGs release into the supernatant upon induction. Cell lysis of PD631SpAHB was confirmed by the decrease of the optical density of cell suspension, by the loss of cell membrane integrity, and by the detection of TAGs in the culture medium. Up to 38% of the total TAGs accumulated in PD631SpAHB was released into supernatant after the expression of the lytic genes. PD631SpAHB strain is a promising candidate to produce TAGs from non-sterile growth medium and release of its TAGs without solvent extraction - a new approach to reduce the overall cost of biolipid-based biofuel production.