Experimental study of Australian sugarcane bagasse pulp permeability

In this experimental study the permeability of Australian bagasse chemical pulps obtained from different bagasse fractions were measured in a simple permeability cell and the results compared to one another as well as to eucalypt, Argentinean bagasse and pine pulps. The pulps were characterised in terms of the permeability parameters, the specific surface area, Sv, and the swelling factor, α. It was found that the bagasse fraction used affects these parameters. Fractionation of whole bagasse prior to pulping produced pulps that have permeability properties that compare favourably with eucalypt pulp. The values of Sv and α for bagasse pulp also depend on whether a constant or a variable Kozeny factor is used.

Phylogenetic placement of the sugarcane orange rust pathogen Puccinia kuehnii in a historical and regional context

Sugarcane orange rust, caused by Puccinia kuehnii, was once considered a minor disease in the Australian sugar industry. However, in 2000 a new race of the pathogen devastated the high-performing sugarcane cultivar Q124, and caused the industry Aus$150–210 million in yield losses. At the time of the epidemic, very little was known about the genetic and pathogenic diversity of the fungus in Australia and neighbouring sugar industries. DNA sequence data from three rDNA regions were used to determine the genetic relationships between isolates within two P. kuehnii collections. The first collection comprised only recent Australian field isolates and limited sequence variation was detected within this population. In the second study, Australian isolates were compared with isolates from Papua New Guinea, Indonesia, China and historical herbarium collections. Greater sequence variation was detected in this collection and phylogenetic analyses grouped the isolates into three clades. All isolates from commercial cane fields clustered together including the recent Australianfield isolates and the Australian historical isolate from 1898.The other two clades included rust isolates from wild and garden canes in Indonesia and PNG. These rusts appeared morphologically similar to P. kuehnii and could potentially pose a quarantine threat to the Australian sugar industry. The results have revealed greater diversity in sugarcane rusts than previously thought.

An ecological assessment of secondary poisoning risk in the Australian sugarcane industry

Rodenticide use in agriculture can lead to the secondary poisoning of avian predators. Currently the Australian sugarcane industry has two rodenticides, Racumin® and Rattoff®, available for in-crop use but, like many agricultural industries, it lacks an ecologically-based method of determining the potential secondary poisoning risk the use of these rodenticides poses to avian predators. The material presented in this thesis addresses this by: a. determining where predator/prey interactions take place in sugar producing districts; b. quantifying the amount of rodenticide available to avian predators and the probability of encounter; and c. developing a stochastic model that allows secondary poisoning risk under various rodenticide application scenarios to be investigated. Results demonstrate that predator/prey interactions are highly constrained by environmental structure. Rodents used crops that provided high levels of canopy cover and therefore predator protection and poorly utilised open canopy areas. In contrast, raptors over-utilised areas with low canopy cover and low rodent densities, but which provided high accessibility to prey. Given this pattern of habitat use, and that industry baiting protocols preclude rodenticide application in open canopy crops, these results indicate that secondary poisoning can only occur if poisoned rodents leave closed canopy crops and become available for predation in open canopy areas. Results further demonstrate that after in-crop rodenticide application, only a small proportion of rodents available in open areas are poisoned and that these rodents carry low levels of toxicant. Coupled with the low level of rodenticide use in the sugar industry, the high toxic threshold raptors have to these toxicants and the low probability of encountering poisoned rodents, results indicate that the risk of secondary poisoning events occurring is minimal. A stochastic model was developed to investigate the effect of manipulating factors that might influence secondary poisoning hazard in a sugarcane agro-ecosystem. These simulations further suggest that in all but extreme scenarios, the risk of secondary poisoning is also minimal. Collectively, these studies demonstrate that secondary poisoning of avian predators associated with the use of the currently available rodenticides in Australian sugar producing districts is minimal. Further, the ecologically-based method of assessing secondary poisoning risk developed in this thesis has broader applications in other agricultural systems where rodenticide use may pose risks to avian predators.

Some aspects of calcium phosphate chemistry in sugarcane clarification

This paper reviews some aspects of calcium phosphate chemistry since phosphate in juice is an important parameter in all sugar juice clarification systems. It uses basic concepts to try and explain the observed differences in clarification performance obtained with various liming techniques. The paper also examines the current colorimetric method used for the determination of phosphate in sugar juice. In this method, a phosphomolybdate blue complex formed due to the addition of a dye is measured at 660 nm. Unfortunately, at this wavelength there is interference of the colour arising from within the juice and results in the underestimation of the amount of soluble inorganic phosphate content of juice. It is suggested that phosphate analysis be conducted at the higher wavelength of 875 nm where the interference of the juice colour is minimised.

Enhancing agrobacterium-mediated transformation efficiency of sugarcane : progress towards an efficient, genotype-independent method

Several key issues need to be resolved before an efficient and reproducible Agrobacterium-mediated sugarcane transformation method can be developed for a wider range of sugarcane cultivars. These include loss of morphogenetic potential in sugarcane cells after Agrobacterium-mediated transformation, effect of exposure to abiotic stresses during in vitro selection, and most importantly the hypersensitive cell death response of sugarcane (and other nonhost plants) to Agrobacterium tumefaciens. Eight sugarcane cultivars (Q117, Q151, Q177, Q200, Q208, KQ228, QS94-2329, and QS94-2174) were evaluated for loss of morphogenetic potential in response to the age of the culture, exposure to Agrobacterium strains, and exposure to abiotic stresses during selection. Corresponding changes in the polyamine profiles of these cultures were also assessed. Strategies were then designed to minimize the negative effects of these factors on the cell survival and callus proliferation following Agrobacterium-mediated transformation. Some of these strategies, including the use of cell death protector genes and regulation of intracellular polyamine levels, will be discussed.

Demonstration of cellulosic ethanol production from sugarcane bagasse in Australia : the Mackay Renewable Biocommodities Pilot Plant

The ready availability of sugarcane bagasse at an existing industrial facility and the potential availability of extra fibre through trash collection make sugarcane fibre the best candidate for early stage commercialisation of cellulosic ethanol technologies. The commercialisation of cellulosic ethanol technologies in the sugar industry requires both development of novel technologies and the assessment of these technologies at a pre-commercial scale. In 2007, the Queensland University of Technology (QUT) received funding from the Australian and Queensland Governments to construct a pilot research and development facility for the production of bioethanol and other renewable biocommodities from biomass including sugarcane bagasse. This facility has been built on the site of the Racecourse Sugar Mill in Mackay, Queensland and is known as the Mackay Renewable Biocommodities Pilot Plant (MRBPP). This research facility is capable of processing cellulosic biomass by a variety of pretreatment technologies and includes equipment for enzymatic saccharification, fermentation and distillation to produce ethanol. Lignin and fermentation co-products can also be produced in the pilot facility.

Development of a high-level gene expression system for the production of bioplastics in sugarcane

Despite various approaches, the production of biodegradable plastics such as polyhydroxybutyrate (PHB) in transgenic plants has met with limited success due largely to low expression levels. Even in the few instances where high levels of protein expression have been reported, the transgenic plants have been stunted indicating PHB is phytotoxic (Poirier 2002). This PhD describes the application of a novel virus-based gene expression technology, termed InPAct („In Plant Activation.), for the production of PHB in tobacco and sugarcane. InPAct is based on the rolling circle replication mechanism by which circular ssDNA viruses replicate and provides a system for controlled, high-level gene expression. Based on these features, InPAct was thought to represent an ideal system to enable the controlled, high-level expression of the three phb genes (phbA, phbB and phbC) required for PHB production in sugarcane at a preferred stage of plant growth. A Tobacco yellow dwarf virus (TbYDV)-based InPAct-phbA vector, as well as linear vectors constitutively expressing phbB and phbC were constructed and different combinations were used to transform tobacco leaf discs. A total of four, eight, three and three phenotypically normal tobacco lines were generated from discs transformed with InPAct-phbA, InPAct-phbA + p1300-TaBV P-phbB/phbC- 35S T, p1300-35S P-phbA-NOS T + p1300-TaBV P-phbB/phbC-35S T and InPAct-GUS, respectively. To determine whether the InPAct cassette could be activated in the presence of the TbYDV Rep, leaf samples from the eight InPActphbA + p1300-TaBV P-phbB/phbC-35S T plants were agroinfiltrated with p1300- TbYDV-Rep/RepA. Three days later, successful activation was indicated by the detection of episomes using both PCR and Southern analysis. Leaf discs from the eight InPAct-phbA + p1300-TaBV P-phbB/phbC-35S T transgenic plant lines were agroinfiltrated with p1300-TbYDV-Rep/RepA and leaf tissue was collected ten days post-infiltration and examined for the presence of PHB granules. Confocal microscopy and TEM revealed the presence of typical PHB granules in five of the eight lines, thus demonstrating the functionality of InPActbased PHB production in tobacco. However, analysis of leaf extracts by HPLC failed to detect the presence of PHB suggesting only very low level expression levels. Subsequent molecular analysis of three lines revealed low levels of correctly processed mRNA from the catalase intron contained within the InPAct cassette and also the presence of cryptic splice sites within the intron. In an attempt to increase expression levels, new InPAct-phb cassettes were generated in which the castorbean catalase intron was replaced with a synthetic intron (syntron). Further, in an attempt to both increase and better control Rep/RepA-mediated activation of InPAct cassettes, Rep/RepA expression was placed under the control of a stably integrated alc switch. Leaf discs from a transgenic tobacco line (Alc ML) containing 35S P-AlcR-AlcA P-Rep/RepA were supertransformed with InPAct-phbAsyn or InPAct-GUSsyn using Agrobacterium and three plants (lines) were regenerated for each construct. Analysis of the RNA processing of the InPAct-phbAsyn cassette revealed highly efficient and correct splicing of the syntron, thus supporting its inclusion within the InPAct system. To determine the efficiency of the alc switch to activate InPAct, leaf material from the three Alc ML + InPAct-phbAsyn lines was either agroinfiltrated with 35S P-Rep/RepA or treated with ethanol. Unexpectedly, episomes were detected not only in the infiltrated and ethanol treated samples, but also in non-treated samples. Subsequent analysis of transgenic Alc ML + InPAct-GUS lines, confirmed that the alc switch was leaky in tissue culture. Although this was shown to be reversible once plants were removed from the tissue culture environment, it made the regeneration of Alc ML + InPAct-phbsyn plant lines extremely difficult, due to unintentional Rep expression and therefore high levels of phb expression and phytotoxic PHB production. Two Alc ML + InPAct-phbAsyn + p1300-TaBV P-phbB/phbC-35S T transgenic lines were able to be regenerated, and these were acclimatised, alcohol-treated and analysed. Although episome formation was detected as late as 21 days post activation, no PHB was detected in the leaves of any plants using either microscopy or HPLC, suggesting the presence of a corrupt InPAct-phbA cassette in both lines. The final component of this thesis involved the application of both the alc switch and the InPAct systems to sugarcane in an attempt to produce PHB. Initial experiments using transgenic Alc ML + InPAct-GUS lines indicated that the alc system was not functional in sugarcane under the conditions tested. The functionality of the InPAct system, independent of the alc gene switch, was subsequently examined by bombarding the 35S Rep/RepA cassette into leaf and immature leaf whorl cells derived from InPAct-GUS transgenic sugarcane plants. No GUS expression was observed in leaf tissue, whereas weak and irregular GUS expression was observed in immature leaf whorl tissue derived from two InPAct- GUS lines and two InPAct-GUS + 35S P-AlcR-AlcA P-GUS lines. The most plausible reason to explain the inconsistent and low levels of GUS expression in leaf whorls is a combination of low numbers of sugarcane cells in the DNA replication-conducive S-phase and the irregular and random nature of sugarcane cells bombarded with Rep/RepA. This study details the first report to develop a TbYDV-based InPAct system under control of the alc switch to produce PHB in tobacco and sugarcane. Despite the inability to detect quantifiable levels of PHB levels in either tobacco or sugarcane, the findings of this study should nevertheless assist in the further development of both the InPAct system and the alc system, particularly for sugarcane and ultimately lead to an ethanol-inducible InPAct gene expression system for the production of bioplastics and other proteins of commercial value in plants.

Congo Red adsorption by ball-milled sugarcane bagasse

The adsorption of Congo Red (CR) by ball-milled sugarcane bagasse was evaluated in an aqueous batch system. CR adsorption capacity increased significantly with small changes in bagasse surface area. CR removal decreased with increasing solution pH from 5.0 to 10.0. Maximum adsorption capacity was 38.2 mg/g bagasse at a CR concentration of 500 mg/L. The equilibrium isotherm fitted the Freundlich model and the adsorption kinetics obeyed pseudo-second order equation. CR adsorption obeyed the intra-particle diffusion model very well with bagasse surface area in the range of 0.58–0.66 m2/g, whereas it was controlled by multi-adsorption stages with bagasse surface area in the range of 1.31–1.82 m2/g. Thermodynamic analysis indicated that the adsorption process is an exothermic and spontaneous process. Fourier transform infrared analysis of bagasse containing adsorbed CR indicated interactions between the carboxyl and hydroxyl groups of bagasse and CR function groups.