Page i was not in the other copies of this thesis. -Digitization Centre / Tn5 mutagenesis was used to generate four independent mutants of Rhizobium meliloti that were unable to grow on protocatechuate (Pca-). Two of the Pca- mutations were mapped to a region of the second symbiotic megaplasmid (pRmeSU47b) previously shown to be required for growth on protocatechuate. This pca locus was shown to consist of the first five structural genes of the protocatechuate branch of the B-ketoadipate pathway, in the order pcaDCHGB. This gene order is the same as determined for Agrobacterium tumefaciens. An additional reading frame with homology to LysR-type regulators was found to be upstream of, and transcribed divergently to the pcaDCGHB operon. This is likely to fulfil the same role as the regulatory gene, pcaQ, of A. tumefaciens. A cosmid plasmid which carried these pea genes failed to complement the Pca- phenotype of a strain carrying a 300 kb megaplasmid deletion encompassing this pea locus. This implies that another pca locus, perhaps pcaIJ, is present within the deleted region of the rnegaplasmid. Two Pca- Tn5 insertions which did not map to the megaplasmid locus were isolated. One of these insertions appears to be in a catalase gene. / Thesis / Master of Science (MS)

Lignin is the second most abundant biopolymer on Earth, right after cellulose, with a highly complex chemical structure that hinders its possible utilizations. Applications that utilize lignin in different manners are of great interest, due to its inexpensive nature. Present work is based on the notion of converting lignin into different biofuels that have only a few, however important, advantages over lignin as a direct energy source. The first part of current work (pyrolysis) details the analysis of lignin from a relatively new lignin isolation process called LignoBoost. It is obtained from the pulp and paper industry via CO₂ precipitation of lignin from black liquor (BL). This method is environment friendly, results lignin with minimal oxidation, eliminates the main bottleneck of the Kraft cycle (recovery boiler capacity), and yet leaves enough lignin in the process stream to recover pulping chemicals and generate energy for the pulp mill. Pyrolysis had converted this lignin into bio-oil with high aliphatic content and low oxidation level, all advantageous for application as liquid fuel. The second part of this dissertation proved the theory that lignin degradation and lipid accumulation metabolic pathways can be interconnected. Gram-positive Rhodococcus opacus species, DSM 1069 and PD630 were used to evaluate lignin to lipid bioconversion, starting with ethanol organosolv and Kraft lignin. This conversion is a first step in a multistep process towards biodiesel production, which includes transesterification, after lipids are extracted from the cells. Results clearly indicated that the lignin to lipid bioconversion pathway is viable, by cells gaining up to 4 % of their weight in lipids, while growing solely on lignin as a carbon and energy source.