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Title: Towards engineering Rhodococcus for biodiesel production from lignin
Author: Somani, Hannah
ISNI:       0000 0004 7659 1441
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2019
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There is a pressing need for sustainable liquid transportation fuels to address climate change concerns and meet increasing global energy demand. Biofuels offer a low carbon alternative to fossil fuels, and microbial biodiesel has the potential to play a key role in their future. This thesis first assesses the economic and environmental considerations associated with producing biodiesel from lignin on a large scale. The analysis concludes that it could be commercially viable by sourcing underutilised waste lignin as a feedstock and using existing pulp mill or biorefinery infrastructure in the process, bringing costs in line with current cellulosic bioethanol production at around $140 per barrel of oil equivalent. Based on this conclusion the oleaginous bacteria Rhodococcus opacus PD630 was then selected to explore the potential use of this genus of microorganisms to carry out this process. We demonstrate its capacity to metabolise, and accumulate intracellular lipids, when provided with lignin breakdown model compounds as a sole carbon source under nitrogen limiting conditions. Cells reached 22% lipid content (dry weight) after 36 hours when grown on 4-hydroxybenzoic acid, 14% after 48 hours on vanillic acid and were able to grow on, but not synthesise lipids from, vanillin. Although this demonstrated that the strain could use lignin breakdown products for lipid biosynthesis it was unable use untreated Kraft lignin for this purpose. To remedy this we sought to express the DypB lignin peroxidase from the ligninolytic strain, R. jostii RHA1, in R. opacus PD630 to catalyse depolymerisation. Attempts at plasmid construction, cloning and protein expression were challenging, and creating a single organism able to perform the whole bioconversion pathway proved elusive. A mixed enzymatic and microbial approach using T. versicolor laccase was more successful. The laccase catalysed lignin depolymerisation, in the presence of syringaldehyde as a redox mediator, allowing R. opacus PD630 to metabolise the breakdown products and accumulate lipids. A 96-hour depolymerisation reaction followed by fermentation yielded 20% cell lipid content after 60 hours. Combining these steps into a simultaneous process slowed culture growth, but cells reached a higher lipid content of 23% after 72 hours. These results demonstrate that a mixed approach of enzymatic lignin depolymerisation and microbial fermentation is a potential route for converting waste lignin, a renewable feedstock, into biodiesel, a sustainable alternative to fossil fuels.
Supervisor: Cass, Tony ; Hassard, John Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral