Abstract
Commercial production of biodiesel has a few drawbacks, as most biodiesel production utilizes feedstocks as carbon sources. Ethical concerns arise as taking advantage of food sources for biodiesel decreases availability of food for those who truly need it, as 13.8 million households struggled with food insecurity in 2020 alone. Chitin, a polysaccharide from crustacean shell waste, is a much more economical and ethical choice for a carbon source. Chitin is broken down into monomers of N-acetyl glucosamine (GlcNAc) by chitinases ChiA, ChiB, and ChiC. The monomers produced by these enzymes can be used to produce triacylglycerols (TAG) in Rhodococcus opacus. TAG can then be harvested and trans-esterified into biodiesel. Previous research has resulted in a non-chemical procedure to separate chitin from shell waste and removal of protein from the sample. The purified colloidal chitin can be used as a feedstock for bacterial growth. This ecofriendly extraction method has given us the ability to utilize crustacean waste from human consumption to isolate chitin and use it as a steady carbon source for TAG synthesis in Rhodococcus opacus (R. opacus) strain PD630. The high lipid storage ability and rapid turnover rate of R. opacus make it an excellent candidate for biodiesel production. The wildtype R. opacus is unable to produce the chitinases necessary to breakdown chitin into its monomer counterparts for TAG utilization and biodiesel production. Therefore, this project has developed a plasmid containing the primers necessary for insertion of ChiA into R. opacus. The plasmid has the potential to undergo Gibson Assembly with the pmyc plasmid which will enable secretion of ChiA upon insertion into R. opacus. This will ultimately break down chitin into its constitutive monomers, enabling TAG synthesis in R. opacus.