| dc.description.abstract | Lipid-based biofuel is a clean and renewable energy source that has been recognized as a promising replacement for petroleum-based fuels. Lipid-based biofuel can be made from intracellular biolipids from lignocellulosic biomass using lipid-producing microorganisms. However, large scale production of lipid-based biofuel remains expensive due to several technical challenges such as inefficient pretreatment of lignocellulose, sterile cultivation of lipid-producing microorganisms, and costly biolipid extraction from the biolipid-filled microorganisms. This research explored several approaches to overcome these challenges in order to enable economical and sustainable production and release of biolipids for large scale lipid-based biofuel.
The first approach was to improve sugar release during enzymatic hydrolysis of pretreated lignocellulose while reducing the cost of enzymes for hydrolysis. Specifically, this study explores a one-step saccharification of pretreated lignocellulose using immobilized biocatalysts containing five different saccharifying enzymes (SEs) with a similar optimum operating condition. The five SEs were successfully produced from engineered Escherichia coli in quantity, worked synergistically to saccharify pretreated biomass to release more reduced sugars than those observed using two commercial enzymes. Additionally, when cross-linked in the absence or the presence of magnetic nanoparticles, the five SEs can be reused at least three times.
To overcome challenges related to sterile cultivation and biolipid extraction, this study successfully demonstrated non-sterile cultivation of an engineered Rhodococcus opacus PD631 and released its biolipids triacylglycerols (TAGs) using a solvent-free, phage-based cell lysis approach. The newly developed salt-tolerant R. opacus PD631 carrying an inducible plasmid with a phage lytic gene cassette was able to grow in non-sterile saline growth medium while producing TAGs. The TAG-filled strain was able to release its TAGs into the culture medium on demand. This newly developed strain is a promising candidate to overcome the two major challenges of current biolipid-based biofuel production.
Lastly, this study explored the possibility of editing prophage Mushu4 DNA in the genome of Rhodococcus opacus strains to produce and release lignin peroxidase in quantity for effective depolymerization of lignin, as an efficient biological pretreatment of lignocellulose. Two homologous recombination approaches, i.e., using an inserted phage recombinase or own recombinases of prophage, were used but unsuccessful. Based on the results, new approaches were suggested for future study.
Overall, results of this study suggest that non-sterile biolipids can be produced and released from the engineered salt-tolerant lipid-producing bacteria using pretreated lignocellulose, prepared by an efficient one-step saccharification. Also, the trial of prophage genome editing provided knowledge to serve as the first step for the development of a Rhodococcus-based factory for production and release of needed enzymes for effective depolymerization of lignin. Results of the approaches explored in this study not addressed two major challenges and offered a new strategy to reduce the overall production cost of biolipid-based biofuel from lignocellulose. | |
