The rapid development of the bioethanol industry globally demonstrates the importance of bioethanol as an alternate energy source to the depleting fossil fuels. To decrease costs and avoid undue pressure on the global food supply, the renewable lignocelluloses appear to be a better substrate for bioethanol production compared to others being investigated. This study investigated the conversion of lignocellulosic material, sugarcane bagasse, to ethanol by the use of Zymomonas mobilis and Pichia stipitis. The investigation of fermentation characteristics of the two strains revealed that their performance on the ethanol production was closely related to the viable cell concentration in the medium. The increase of inoculum size to five fold resulted in an increase in the system co-efficiency to 2.2 fold and 5.2 fold respectively for Z. mobilis and P. stipitis. A theoretical value de (the cell instantaneous ethanol production rate) was introduced to describe the ethanol productivity based on biomass. System co-efficiency proved to be only affected by the viable cell concentration (xC) and de, regardless of ethanol re-assimilation. Immobilized culture of Z. mobilis and P. stipitis showed distinct differences in their characteristics. The bacterium acclimatized to the interior of gel beads; the biomass concentration within the beads increased greater than 10 fold during the reuse of the beads, resulting in an improved fermentation performance. The immobilized P. stipitis gave a similar system co-efficiency level of approximately 0.5 g/l/h under different culture conditions; cell growth in the medium was considerably more vigorous compared to that within the beads. P. stipitis sole-culture on the glucose/xylose medium with a high inoculum size showed a comparable fermentation efficiency with the best result of the co-culture processes. Fermentation of 50.0 g/l of sugar mixture (30.0 g/l glucose and 20.0 g/l xylose) was completed in 20 h with an ethanol yield of 0.44 g/g. No catabolite repression due to glucose was observed for the xylose assimilation. Co-culture of immobilized Z. mobilis and free cells of P. stipitis proved to be the best fermentation scheme on the glucose/xylose sugar mixture co-fermentation. The removal of Z. mobilis after the utilization of glucose improved the stability of the performance. The best result showed that 50.0 g/l sugars were fully converted to ethanol within 19 h, giving an ethanol yield of 0.49 g/g, which is 96% of the theoretical rate. When co-cultured, viable cells of Z. mobilis inhibited the cell activity of P. stipitis, and were capable of growing to high concentration levels without an appropriate carbon source. Acid and enzymatic hydrolysates of sugarcane bagasse showed similar fermentability, but the hydrolysate without overliming significantly inhibited both cell growth and ethanol production of P. stipitis. The co-culture process on the hydrolysate medium successfully utilized 53.56 g/l sugars (32.14 g/l glucose and 21.42 g/l xylose) in 26 h with a yield of 0.43 g/g; this value further increased to 0.49 g/g when ethanol peaked at 40 h. A high cell density proved to be an effective method to improve the system co-efficiency for ethanol production. For the fermentation processes on the sugar medium, results achieved in this study, 10.54 g/l/h for Z. mobilis free cell culture on glucose, 0.755 g/l/h for P. stipitis free cell culture on xylose, 1.092 g/l/h for P. stipites free cell culture on the glucose/xylose mixture and 1.277 g/l/h for glucose/xylose co-fermentation using co-culture, are higher than the best values reported in the literature in batch culture. In the fermentation of the hydrolysate, the system co-efficiency of 0.879 g/l/h achieved with co-culture is comparable to the best values reported for the fermentation of lignocellulosic hydrolysates. / Master of Science (Hons)

Copy of author's previously published article inserted. Bibliography: leaves 180-195. Investigation of the contribution of polyphenol oxidase (PPO) and peroxidase (POD) to enzymic browning in sugarcane juice.

Thesis (Ph. D.)--Brigham Young University. Dept. of History, 2005. / Includes bibliographical references (p. 289-299).

Population based association studies in crops that were established by domestication and early breeding can be a valuable basis for the identification of QTLs. A case control design in a population is an ideal way to identify maximum candidate sites contributing to a complex polygenic trait such as drought. In the current study, marker loci associated with drought related QTLs were identified in sugarcane (Saccharum spp), one of the most complex crop genomes, with its polyploid nature (>8), chromosome number (>100) and interspecific origin. The objectives of this investigation were: 1) development of genic markers, which can be used for marker-assisted selection of drought tolerant genotypes of sugarcane. 2) genotypic characterization of sugarcane population at drought related loci using EST-SSR markers. Using 55 microsatellite markers, 56 polymorphisms were scored among 80 modern sugarcane genotypes. Homogeneity of the population was confirmed by determining the distribution of allele frequencies obtained by random genomic microsatellite markers. This analysis was conducted in the STRUCTURE program and the population was divided in 3 subgroups based on the allelic distribution. Phenotypic data to evaluate drought tolerance among the genotypes was collected by measuring chlorophyll content, chlorophyll fluorescence, leaf temperature and leaf relative water content. A generalized linear model in SPSS was used to find association between marker loci and phenotypic data. Markers with significant association (P 0.001 level) with the trait were subjected to linear regression to screen the spurious associations. Based on the results, 21 EST-SSR markers and 11 TRAP markers related to drought-defining physiological parameters were considered as genuine associations in this study. Fifty-six polymorphisms produced by 13 EST-SSR primers were used to produce genetic similarity matrix for 80 genotypes. Dendrogram prepared from this genetic similarity matrix will be useful in selecting parents carrying diversity at drought specific loci.

Sweet sorghum is a highly versatile C4 grass noted for its improved drought tolerance and water use efficiency relative to sugarcane. Sweet sorghum is well suited for ethanol production due to a rapid growth rate, high biomass production, and a wide range of adaptation. Unlike the 12-18 month growth cycle of sugarcane, sweet sorghum produces a harvestable crop in three to five months. Sweet sorghum and sugarcane crops are complementary and in combination can extend the sugar mill seasons in many regions of the world to an estimated 8 months. Seasonal growth and weather patterns both optimize and restrict production of each crop to specific times of the year, however these are different for the two crops. In addition to temporally spacing the date of harvest between crops, the genetic variability of maturity within the crops may also be used to extend the mill seasons; specific hybrids can be used and selected to maximize yield throughout the harvest season. Under favorable growing environments, sweet sorghum hybrids of all maturity groups produced sugar yields ranging from 2.8 to 4.9 MT/ha. Early/medium, late, and very late maturity hybrids planted during April, May, and June planting dates are necessary to maximize the mill season. In this study, early/medium maturity hybrids planted during April and May matured for harvest between late July and mid-August. June planting dates were unfavorable for early/medium maturity hybrids. In addition, late and very late maturity hybrids planted during April matured for harvest in late August; the additional growing season thus resulted in higher sugar yields. Timely planting of late and very late maturity hybrids in April, May, and June produce the maximum yields for harvests after mid August. Intermittent use of late and very late maturity hybrids can therefore extend sugar milling seasons into mid November if so desired.

Aflatoxin is a carcinogenic mycotoxin. Aflatoxin contamination of susceptible crops is the product of communities of Aspergillus section Flavi and average aflatoxin-producing potential of these communities influence aflatoxin contamination risk. In 2004 and 2005, Sugarcane producing counties in the Rio Grande Valley of Texas (RGV) had unique aflatoxin-producing communities containing Aspergillus parasiticus. Sugarcane fields or those rotated for less than two years had Aspergillus section Flavi communities dominated by A. parasiticus. A. parasiticus was rarely detected in long-term rotation fields and not detected in counties without sugarcane crops. Aflatoxin-producing fungi infecting RGV sugarcane stems ranged from 52 - 95% A. parasiticus in hand-collected samples and billets for commercial planting, respectively. Identical A. parasiticus fungi found in Japan caused aflatoxin contamination of raw sugar there. Population genetics and phylogenetics were used to characterize a global sampling of 112 A. parasiticus and identify geographic distributions and crop associations within the species. One population shows clear association with sugarcane and is distributed to Asia, Africa and North America, implicating human involvement in its distribution. A. parasiticus populations from maize and peanut have broad geographic distribution but crop specific lineages and/or populations were not detected. One A. parasiticus population isolated from maize has a distribution limited to Mexico. A phylogeny generated from a partial nitrate reductase gene resolves a lineage that correlates with the sugarcane population and suggests crop association and geographic distribution may drive divergence within A. parasiticus. Crop associations shape fungal communities and must be considered for aflatoxin management. Native food enthusiasts in Arizona conduct public millings of wild- and landscape-collected mesquite pods (Prosopis spp.) to produce mesquite flour, which is often consumed in the same localities where it is produced without conventional food safety inspection. Aflatoxin was found in imported, domestic, and non-commercial mesquite flour batches, with 10% above the FDA action level for human food (>20 ppb), and 95% could not be exported to Europe (>2 ppb). Aflatoxin content in Tucson was largely explained (63%) by harvest date with those harvested later yielding more aflatoxin. Lateral flow aflatoxin assay of mesquite flour proved viable for lab and public testing.

The soil being delivered with sugar cane consignments. from the cane fields to the factories, has been a recent cause for concern in the South African sugar industry. The soil impurities increase the wear of processing machinery reduce extraction efficiency and represents an unnecessary transport of material. The cost due to soil was estimated at R63 million (about US $8 million) over the 1996/97-season. The need to reduce costs, due to the unwanted soil component, has been given a high priority. Ashing is currently used by the sugar industry to estimate the amount of soil in cane. Although simple to implement, the method is destructive, requires long processing times and limited to small sample sizes. In fecent times, non-destructive techniques have become more prominent in industry. Hence, the decision to apply such techniques to the soil in cane-problem. This dissertation describes an experimental investigation into Dual-Energy Transmission (DuET) and X-ray lmaging for quantifying the amount of soil in cane. DuET can determine the relative concentrations of the components of a binary mixture by measuring the transmission of low- and high-energy gamma photons through the mixture. The principle of DuET was successfully demonstrated with aqueous solutions of ferric chloride. Experimentally-determined mass attenuation coefficients of water and ferric chloride were compared to theoretical values. DuET was then applied to dried, shredded sugar cane spiked with various amounts of soil. Results showed large variations in the predicted soil concentrations. These variations were attributed to radiation scatter and incomplete volume sampling by the radioactive source. However, new experimental arrangements are expected to improve the technique: initial test results are given of a sample holder that continuously rotates a sample up and down through the source-detector axis. An alternative approach to processmg DuET-spectra, using the discrete wavelet transform coupled with an artificial neural network, is also introduced. X-ray Imaging was the second technique investigated. A literature survey revealed that this technique had not previously been applied to the soil in cane-problem. The present work constituted an initial investigation to determine the feasibility of applying X-ray imaging to measure the amount of soil in cane. The soil/cane-samples, that were used for DuET, were imaged us ing a commercial mammography unit, and the resulting radiographs were analysed using image processing techniques. Although the results are promising, a more comprehensive investigation is foreseen. / Thesis (M.Sc.)-University of Natal, Durban, 2001.

The C4 plant, sugarcane (Saccharum spp. hybrids), accumulates sucrose to high concentrations and, as a result, has been the focus of extensive research into the biochemistry and physiology of sucrose accumulation. Despite this, the relationship between source leaf photosynthetic rates and sucrose accumulation in the culm has not been well documented. The observations that photosynthetic activity declines during culm maturation in commercial cultivars and that high-sucrose accumulating ancestral genoptypes photosynthesize at rates two-thirds of those of low-sucrose ancestral Saccharum species indicate that source-sink communication may play a pivotal role in determining sucrose yield. The relationship between source and sink tissues in sugarcane was investigated using a supply-demand paradigm, an approach novel in the study of the crop. The demand for photosynthate from the primary culm growth sink was shown to be closely linked to photosynthetic rates, sucrose export and the eventual physiological decline of source leaves. Results from initial field experiments revealed that leaf assimilation rates were negatively correlated with leaf hexose concentrations, but not those of sucrose. Further manipulation of leaf sugar status, through sugar-feeding and cold-girdling techniques, demonstrated the regulatory role of leaf sugar concentrations on photosynthetic activity, thus revealing sucrose, and particularly hexose, as key signal molecules in the modulation of the amount of photosynthate available for export to the sink. Gene expression profiling, by means of array technologies, indicated that changes in leaf sugar status and photosynthetic rates result in concurrent modifications in the expression of several genes involved in fundamental metabolic pathways, including photosynthesis, carbohydrate metabolism, stress response and sugar-signaling. Notable amongst these, was the identification of a potential trehalose 6-phosphate (T6P) sugar-signaling mechanism, thus implicating the trehalose pathway as a central regulatory system in the communication of sink carbon requirements to the source leaf. This study demonstrated that maturation of the culm results in a decreased demand for sucrose, which invokes a sugar-mediated feedback signal to decrease leaf photosynthetic supply processes. However, sugarcane leaves appear to retain the capacity to increase the supply of assimilate to culm tissues under conditions of increased assimilate demand. Uncoupling of the signaling pathways that mediate negative feedback between source and sink tissues may result in improved leaf assimilation rates and, consequently, lead to increased sugarcane sucrose yields. / Thesis (Ph.D.) - University of KwaZulu-Natal, Westville, 2007.

The relationship between extractable invertase activities and sucrose accumulation in the sugarcane (Saccharum spp. hybrids) culm and in vivo invertase mediated sucrose hydrolysis was investigated to determine the significance of invertases in sucrose utilisation and turnover. In vitro activities were determined by assaying the soluble acid invertase (SAI), cell wall bound acid invertase (CWA) and neutral invertase (NI) from internodes three to ten in mature sugarcane plants of cultivar NCo376. Extractable activities were verified by immunoblotting. In vivo invertase mediated sucrose hydrolysis was investigated in tissue discs prepared from mature culm tissue of the same cultivar. Sugarcane NI had a higher specific activity than SAI (apoplastic and vacuolar) in the sucrose accumulating region of the sugarcane culm. CWA was also present in significant quantities in both immature and mature tissue. Sugarcane NI was partially purified from mature sugarcane culm tissue to remove any potential competing activity. The enzyme is non-glycosylated and exhibits catalytic activity as a monomer, dimer and tetramer. Most of the activity elutes as a monomer of native Mr ca 60 kDa. The enzyme displays typical hyperbolic saturation kinetics for sucrose hydrolysis. It has a Km of 9.8 mM for sucrose and a pH optimum of 7.2. An Arrhenius plot shows the energy of activation of the enzyme for sucrose to be 62.5 kJ.mol-1 below 30°C and -11.6 kJ.mol-1 above 30°C. Sugarcane NI is inhibited by its products, with fructose being a more effective inhibitor than glucose. Sugarcane NI is significantly inhibited by HgCI2, AgNO-3, ZnCI2, CuSO4 and CoCI2 but not by CaCI2, MgCI2 or MnCI2. Sugarcane NI showed no significant hydrolysis of cellobiose or trehalose. When radiolabelled fructose was fed to sugarcane internodal tissue, label appeared in glucose which demonstrates that invertase mediated hydrolysis of sucrose occurs. A combination of continuous feeding and pulse chase experiments was used to investigate the in vivo contribution of the invertases and the compartmentation of sugars. Sucrose is synthesised at a rate greater than the rate of breakdown at all stages of maturity in sugarcane culm tissue. The turnover time of the total cytosolic label pool is longer for internode three than internode six. A higher vacuolar:cytosolic sugar molar ratio than previously assumed is indicated. Developmentally, the greatest change in carbon allocation occurs from internodes three to six. The main competing pools are the insoluble and neutral fractions. As the tissue matures, less carbon is allocated to the insoluble and more to the neutral fraction. The neutral fraction consists mainly of sucrose, glucose and fructose. The compartmented nature of sugarcane storage parenchyma carbohydrate metabolism results in a system that is complex and difficult to investigate. A computer based metabolic flux model was developed to aid in the interpretation of timecourse labelling studies. A significant obstacle was the global optimization of the model, while maintaining physiologically meaningful flux parameters. Once the vacuolar:cytosolic molar ratio was increased, the model was able to describe the internode three and six labelling profiles. The model results were in agreement with experimental observation. An increase in the rate of sucrose accumulation was observed with tissue maturation. Only the internode three glucokinase activity was greater than the experimentally determined limit. The rate was however physiologically feasible and may reflect the underestimation of the in vivo rate. SAI and NI contributed to sucrose hydrolysis in internode three but not in internode six. The rates in internode six were set to fixed low values to enable the model to fit the experimental data. This does not however preclude low levels of in vivo SAI and NI activity, which would prove significant over a longer time period. The flow of label through the individual pools, which comprise the experimentally measured composite pools could be observed. This provides insight into the sucrose moiety label ratio, SPS:SuSy sucrose synthesis ratio, and the rate of 14CO2 release. The model provides a framework for the investigation and interpretation of timecourse labelling studies of sugarcane storage parenchyma. / Thesis (Ph.D.)-University of Natal, Durban, 2000.

For the expression of transgenes in plant cells, appropriate promoter sequences have to be introduced upstream of the gene to ensure efficient transcription. Tissue- or signal-responsive promoters are in high demand in practical plant biotechnology. The present study sought to characterise the activities of two promoters in sugarcane, namely the UBI (ubiquitin) promoter and the SUC-1 promoter (UBI linked in tandem to the cauliflower mosaic virus 35S promoter). It was hypothesised that the activity of UBI would be maintained or even increased under conditions of environmental stress, since it is well documented that ubiquitin is a stress-related protein. A further hypothesis was that SUC-1 might enhance overall gene expression since the CaMV 35S component is a constitutive promoter widely and successfully used in plant transformation. Plants of the sugarcane variety NC0310, containing the cry1A(c) (Bt) gene from Bacillus thuringiensis, were used as models in a system in which the plants were stressed by withholding water supply in a controlled manner. Since large numbers of clones of both transgenic and wild-type plants were needed for the water stress and expression experiments, three micropropagation techniques, namely, shoot tip-, callus- and node culture, were optimised and compared. The objective was to propagate genetically stable plants rapidly. Compared to shoot tip culture, node and callus culture proved slow and inefficient. Shoot tip culture was thus chosen as the most suitable for the regeneration of experimental material. Relative Water Content (RWC) determination, leaf elongation measurements and Infra Red Gas Analysis (IRGA) were compared in order to find the most appropriate method of measuring plant water status. In addition to being destructive, no observable differences were evident between the control (non-stressed) and water-stressed plants when using RWC as a measure. Results obtained from leaf elongation measurements compared favourably to the more sophisticated IRGA readings, showing that leaf elongation is as sensitive a measure of water stress. On the basis of preliminary studies with untransformed plants using the latter two techniques, water regimes for stress-induction in the final experiments were designed. Leaf elongation measurements, which are simple and non-destructive, were ultimately chosen to measure plant water status. In the final water stress experiment non-transgenic NCo310 and clonal populations of six transformants were used (three containing the UBI promoter; three the SUC-1 promoter). Exactly half of the plants of each type were stressed by withholding water supply, while the other half (controls) were watered manually twice a day. Leaf elongation measurements were made at the same time daily on the third youngest leaf of 6 plants from each population per treatment. At the same time, leaf samples were taken daily for molecular analysis. The stress regime led to marked differences in leaf elongation between control and water-stressed plants. In terms of physiological response (leaf rolling and senescing), plants containing the SUC-1 promoter appeared least affected. The reverse transcription-polymerase chain reaction (RT-PCR) and Northern hybridisation were used to assay UBI and SUC-1 activity. RT-PCR revealed that both promoters drove Bt gene expression in controls and experimentals throughout the stress period, although differences in signal intensity were not observed. The extent of expression occurring in each type of plant was revealed in Northern blots probed with two genic sequences (1) the transgene and (2) sugarcane EST ME42, homologous to heat shock protein 82 in rice. Individual transformants showed overall levels of transgene expression that were variable, possibly due to insert position in the plant genome, as well as variations in relation to the application of stress. SUC-1 seemed superior to UBI in terms of driving transgene expression under stressful environmental conditions, since UBI promoter activity appeared to decrease under stress, while SUC-1 promoter activity remained constant. In addition to the expected 2.0 kb Bt transcript, transcripts of smaller than expected size were also obtained, leading to the suggestion of premature polyadenylation signals in the coding region of the wild-type Bt234 gene. Upon inspection of the transgene sequence, a number of motifs rarely present in plant genes were observed, namely A/T rich sequences, ATTTA motifs and numerous potential polyadenylation sites. / Thesis (M.Sc.)-University of Natal, Durban, 1999.