Abstract
The search for an environmentally friendly fuel source has been a major obstacle for many years as concerns surrounding climate change increase. Utilizing biofuels could provide the solution for the production of a renewable, green energy source that would limit the effects of climate change. The ever-growing levels of food waste have led to an increased amount of shellfish waste, making it a viable resource for the production of biofuels. By utilizing the polymer chitin, found in the shells of many crustaceans, food waste can be a source of biofuels without decreasing the earth’s already limited supply of feedstock, land, and water. Chitin can be isolated from shellfish using either chemical or biological methods and can be further broken down into chitosan using deacetylation. Various enzymes including Chitinases A, B, and C from the chitinase family 18 break chitin into its monomers including N-acetyl-d-glucosamine (GlcNAc) which can be utilized as a carbon source for the production of triacylglycerols (TAG). Chitinases are broken into two families, family 18 and 19, based on their amino acid sequences. Each Chitinase plays an important role in the breakdown of chitin, with Chi A degrading chitin from the reducing end, Chi B degrading chitin from the non-reducing end, and Chi C degrading more soluble chitin. The GlcNAc produced by the chitin is broken down and converted into TAGs by R. opacus which can then be easily converted into biofuels. In this work, purified Chitin previously isolated from the shells of crustaceans will be used as a feedstock for a modified Rhodococcus Opacus (R. opacus) strain. R. opacus PD630 is a gram-positive soil bacterium that can accumulate up to 78% of its dry cell weight in lipids. This allows for the bacteria to accumulate large amounts of TAGs which makes it an excellent choice for the production of biofuels. Since R. opacus does not contain the proper enzymes to breakdown chitin, this project will focus on creating a recombinant strain of R. opacus, using an electroporation protocol that was developed along with a Gibson Assembly protocol, that wil lcontain the specific Chitinase enzyme, Chitinase B (Chi B), allowing for the breakdown of chitin.