The diesel-degrading capabilities of Acinetobacter calcoaceticus isolates LT1, LT1A and V2 were established in previous studies. LT1 and LT1A were isolated from diesel-contaminated soil and V2 was from soil contaminated with used engine oil. Isolates were grown in Bushnell-Haas medium supplemented with 1% sterile diesel. Determination of diesel-degradation patterns by gravimetric analysis and harvesting of cells for RNA extraction were performed at regular time intervals over a 60 day period. The involvement of genes alkM, alkR, rubA, rubB, estB, lipA, lipB, and xcpR in hydrocarbon degradation has been reported in previous studies. LT1, LT1A, and V2 were compared in terms of gene expression levels by real-time quantitative PCR. Expression levels were assessed by relative quantification and normalized against the 16S rRNA reference gene using the Relative Expression Software Tool - XL (REST-XL). Amplification of all genes, except rubB, was achieved with a high degree of efficiency. The expression of rubA, alkM, alkR, xcpR, and lipB based on pair-wise randomization, was all down-regulated in LT1A in relation to LT1. Highest expression levels of the aforementioned genes were documented during the initial stages of incubation for LT1 while LT1A showed highest expression levels midway through the study period. LT1, LT1A, and V2 achieved 58.6%, 51.7%, and 48.3% diesel degradation after 5 days of incubation, respectively. The higher percentage of diesel degradation achieved by LT1 can be attributed to higher levels of overall gene expression in the initial stages of degradation. Amplification of alkane hydroxylase alkM of V2 revealed a possible second hydroxylase gene that was expressed after 20 days of incubation. Amplification of alkR and xcpR in V2 isolates also resulted in multiple product formation. Very low lipB and lipA expression was detected in LT1 and LT1A and the absence of lipA expression in V2 suggests that lipases were not involved in diesel degradation. In contrast, estB was predominantly expressed in V2, and suspected to be involved in the release of a bioemulsifier that was only observed in V2 samples. Although all three isolates were comparably efficient in degrading diesel, the results of this study suggest that different mechanisms may be employed in the degradation process. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2009.

Bacteria of the genus Acinetobacter are known to be involved in the degradation, leaching and removal of various hazardous compounds from the environment. Several studies of Acinetobacter spp. have reported on the genes involved in alkane degradation; but less is known about the proteins that are expressed at certain points within the degradation period. Acinetobacter sp. LT1A and Acinetobacter sp. V2 were isolated from diesel- and used engine oil-contaminated soils respectively. In a previous investigation (Toolsi, 2008), these isolates have been shown to demonstrate different gene expression patterns during diesel degradation using real time PCR. The real time PCR data showed that isolate V2 made use of multiple alkane hydroxylases whereas LT1A made use of only one, and the expression of the alkane hydroxylase regulator alkR and secretory protein xcpR also revealed multiple product formations in isolate V2 as compared to LT1A. Thus the objectives for the current investigation were to monitor the hydrocarbon degradation ability of Acinetobacter sp. isolates V2 and LT1A using medium chain (C14) and long chain (C28) hydrocarbon substrates and to compare the hydrocarbon degradation abilities and protein expression patterns of both isolates. To achieve this, the isolates were grown for 20 days in Bushnell Haas liquid medium supplemented with tetradecane (C14) or octocosane (C28) as a sole carbon source. Gravimetric analysis was used to monitor degradation and whole cell protein was extracted from the culture medium throughout the 20-day study period. The protein expression patterns were visualized using 1D and 2D PAGE. The 2D PAGE images were analyzed using the PDQuest Advanced 2D image analysis software (BIORAD). By day 20, approximately 90% of C14 was degraded by both isolates, whereas only 36% of C28 had been broken down. In both the C14 and C28 degradation assays, the isolates achieved significant amounts of hydrocarbon degradation as compared to the abiotic controls. One-dimensional and 2D SDS-PAGE gels indicated that there are observable differences in protein expression patterns between the isolates during C14 and C28 degradation. Both isolates achieved similar rates of hydrocarbon usage, but appear to do so using different, unidentified, protein systems. Analysis of the 2D-SDS PAGE gel images revealed that more proteins were required for the utilization of the long chain alkane (C28) as compared to the medium chain alkane (C14) for both isolates. Potential spots of interest were identified from the 2D SDS-PAGE images and sequenced. The identities of these proteins were found to be: a conserved hypothetical protein, TonB-dependent receptor protein, Peptidyl-prolyl-cis-trans isomerase and a Protein containing DUF1559. No alkane hydroxylase components were detected in this study. This investigation demonstrated the need for more studies at the proteomic level. Future investigations should focus on the insoluble subproteome of the isolates and make use of larger sample sizes (replicates) to reduce variation in spot detection and quantification. Genomic sequencing of the isolates will also shed light on the genetics and biochemistry of alkane metabolism in these Acinetobacter sp. isolates. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2012.

The possibility of using waste vegetable oil from deep-frying processes as a fuel for long term use in diesel engines was investigated. Research was aimed at using existing technology in terms of engine design in order to utilize a maximum amount of waste vegetable oil as the energy source with a minimum of processing. A small swirl-chamber diesel engine was selected and used to run the 200-hour test recommended by the EMA for testing vegetable oil-based fuels. A blend of 20/80 (waste oil/diesel fuel) was tested as well as a 50/50 blend. BSFC data for both blends did not indicate any significant deterioration in engine performance during the 200 hour tests for ail the fuels tested. However, the 50/50 blend BSFC data had more spread than the data from the 20/80 or the diesel baseline test. This was attributed to variable amounts of deposits on the injector nozzle throughout this test Carbon deposits on all other parts of the combustion chamber were comparable for all the fuels tested. Wear of the engine parts was also comparable except for the piston rings. Piston ring wear was greater with diesel fuel and smaller when burning the 50/50 blend. This was attributed to a film of unburned fuel on the cylinder wall that improved lubrication. Lower -lubricating oil consumption was also attributed to this film. The alternate fuel blends completed the 200 hour EMA screening test and could be considered as possible candidates for long-term use in I.D.I, engines. / Applied Science, Faculty of / Graduate