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.

Developing a curriculum for the real world : a whole of institution approach

Keynote address at University of Surrey, Real World Education Symposium, 29 September 2010. Full-text in audio form only. Link to audio provided in Official URL field.

Analysis of productive performance of crop and animal production systems : an integrated analytical framework

This article presents a two-stage analytical framework that integrates ecological crop (animal) growth and economic frontier production models to analyse the productive efficiency of crop (animal) production systems. The ecological crop (animal) growth model estimates "potential" output levels given the genetic characteristics of crops (animals) and the physical conditions of locations where the crops (animals) are grown (reared). The economic frontier production model estimates "best practice" production levels, taking into account economic, institutional and social factors that cause farm and spatial heterogeneity. In the first stage, both ecological crop growth and economic frontier production models are estimated to calculate three measures of productive efficiency: (1) technical efficiency, as the ratio of actual to "best practice" output levels; (2) agronomic efficiency, as the ratio of actual to "potential" output levels; and (3) agro-economic efficiency, as the ratio of "best practice" to "potential" output levels. Also in the first stage, the economic frontier production model identifies factors that determine technical efficiency. In the second stage, agro-economic efficiency is analysed econometrically in relation to economic, institutional and social factors that cause farm and spatial heterogeneity. The proposed framework has several important advantages in comparison with existing proposals. Firstly, it allows the systematic incorporation of all physical, economic, institutional and social factors that cause farm and spatial heterogeneity in analysing the productive performance of crop and animal production systems. Secondly, the location-specific physical factors are not modelled symmetrically as other economic inputs of production. Thirdly, climate change and technological advancements in crop and animal sciences can be modelled in a "forward-looking" manner. Fourthly, knowledge in agronomy and data from experimental studies can be utilised for socio-economic policy analysis. The proposed framework can be easily applied in empirical studies due to the current availability of ecological crop (animal) growth models, farm or secondary data, and econometric software packages. The article highlights several directions of empirical studies that researchers may pursue in the future.

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.

Cellulosic ethanol from sugarcane bagasse in Australia : exploring industry feasibility through systems analysis, techno-economic assessment and pilot plant development

Overcoming many of the constraints to early stage investment in biofuels production from sugarcane bagasse in Australia requires an understanding of the complex technical, economic and systemic challenges associated with the transition of established sugar industry structures from single product agri-businesses to new diversified multi-product biorefineries. While positive investment decisions in new infrastructure requires technically feasible solutions and the attainment of project economic investment thresholds, many other systemic factors will influence the investment decision. These factors include the interrelationships between feedstock availability and energy use, competing product alternatives, technology acceptance and perceptions of project uncertainty and risk. This thesis explores the feasibility of a new cellulosic ethanol industry in Australia based on the large sugarcane fibre (bagasse) resource available. The research explores industry feasibility from multiple angles including the challenges of integrating ethanol production into an established sugarcane processing system, scoping the economic drivers and key variables relating to bioethanol projects and considering the impact of emerging technologies in improving industry feasibility. The opportunities available from pilot scale technology demonstration are also addressed. Systems analysis techniques are used to explore the interrelationships between the existing sugarcane industry and the developing cellulosic biofuels industry. This analysis has resulted in the development of a conceptual framework for a bagassebased cellulosic ethanol industry in Australia and uses this framework to assess the uncertainty in key project factors and investment risk. The analysis showed that the fundamental issue affecting investment in a cellulosic ethanol industry from sugarcane in Australia is the uncertainty in the future price of ethanol and government support that reduces the risks associated with early stage investment is likely to be necessary to promote commercialisation of this novel technology. Comprehensive techno-economic models have been developed and used to assess the potential quantum of ethanol production from sugarcane in Australia, to assess the feasibility of a soda-based biorefinery at the Racecourse Sugar Mill in Mackay, Queensland and to assess the feasibility of reducing the cost of production of fermentable sugars from the in-planta expression of cellulases in sugarcane in Australia. These assessments show that ethanol from sugarcane in Australia has the potential to make a significant contribution to reducing Australia’s transportation fuel requirements from fossil fuels and that economically viable projects exist depending upon assumptions relating to product price, ethanol taxation arrangements and greenhouse gas emission reduction incentives. The conceptual design and development of a novel pilot scale cellulosic ethanol research and development facility is also reported in this thesis. The establishment of this facility enables the technical and economic feasibility of new technologies to be assessed in a multi-partner, collaborative environment. As a key outcome of this work, this study has delivered a facility that will enable novel cellulosic ethanol technologies to be assessed in a low investment risk environment, reducing the potential risks associated with early stage investment in commercial projects and hence promoting more rapid technology uptake. While the study has focussed on an exploration of the feasibility of a commercial cellulosic ethanol industry from sugarcane in Australia, many of the same key issues will be of relevance to other sugarcane industries throughout the world seeking diversification of revenue through the implementation of novel cellulosic ethanol technologies.

The philosophy and practice of water sensitive urban design : is it consistent with a whole system approach

This paper provides a critique of the Water Sensitive Urban Design (WSUD) paradigm by discussing its congruence with an established sustainable design principle called 'whole system design'. It was found that WSUD is congruent with the whole system design approach as a philosophy, but not in practice. Future improvement of WSUD practice may depend on the adoption of a front-loaded, teamwork-based design and planning process that is embedded in the principle of whole system design.

Effects of whole-body cryotherapy (-110 degrees C) on proprioception and indices of muscle damage

The purpose of this study was to investigate the effects of whole-body cryotherapy (WBC) on proprioceptive function, muscle force recovery following eccentric muscle contractions and tympanic temperature (TTY). Thirty-six subjects were randomly assigned to a group receiving two 3-min treatments of −110 ± 3 °C or 15 ± 3 °C. Knee joint position sense (JPS), maximal voluntary isometric contraction (MVIC) of the knee extensors, force proprioception and TTY were recorded before, immediately after the exposure and again 15 min later. A convenience sample of 18 subjects also underwent an eccentric exercise protocol on their contralateral left leg 24 h before exposure. MVIC (left knee), peak power output (PPO) during a repeated sprint on a cycle ergometer and muscles soreness were measured pre-, 24, 48 and 72 h post-treatment. WBC reduced TTY, by 0.3 °C, when compared with the control group (P<0.001). However, JPS, MVIC or force proprioception was not affected. Similarly, WBC did not effect MVIC, PPO or muscle soreness following eccentric exercise. WBC, administered 24 h after eccentric exercise, is ineffective in alleviating muscle soreness or enhancing muscle force recovery. The results of this study also indicate no increased risk of proprioceptive-related injury following WBC.

Public participation in infrastructure and construction projects in China: From an EIA-based to a whole-cycle process

Many governments world-wide are increasingly encouraging the involvement of interested individuals, groups and organisations in their publicinfrastructure and construction (PIC) projects as a means of improving the openness, transparency and accountability of the decision-making process and help improve the projects’ long-term viability and benefits to the community. In China, however, the current participatory mechanism at the project level exists only as part of the environmental impact assessment (EIA) process. With an increasing demand for PIC projects and social equality in China, this suggests a need to bring the participatory process into line with international practice. The aim of this paper, therefore, is to identify the weaknesses of EIA-basedpublicparticipation in China and the means by which it may be improved for the whole lifecycle of PIC schemes. To do this, the results of a series of interviews with a diverse group of experts is reported which analyse the nature and extent of existing problems of publicparticipation in EIA and suggestions for improvement. These indicate that the current level of participation in PIC projects is quite limited, particularly in the crucial earlier stages, primarily due to traditional culture and values, uneven progress in the adoption of participatory mechanisms, the risk of not meeting targets and lack of confidence in public competence. Finally, aprocess flowchart is proposed to guide construction practitioners and the community in general.

Phenolics in sugar cane juice : potential degradation by hydrogen peroxide and Fenton's reagent

The presence of colour in raw sugar plays a key role in the marketing strategy of the Australian raw sugar industry. Some sugars are relatively difficult to decolourise during refining and develop colour during storage. A new approach that might result in efficient and cost-effective colour removal during the sugar manufacturing process is the use of an advanced oxidation process (AOP), known as Fenton oxidation, that is, catalytic production of hydroxyl radicals from the decomposition of hydrogen peroxide using ferrous iron. As a first step towards developing this technology, this study determined the composition of colour precursors present in the juice of cane harvested by three different methods. The methods were harvesting cane after burning, harvesting the whole crop with half of the trash extracted and harvesting the whole crop with no trash extracted. The study also investigated the degradation at pH 3, 4 and 5 of a phenolic compound, caffeic acid (3,4–dihydroxycinnamic acid), which is present in sugar cane juice, using both hydrogen peroxide and Fenton’s reagent. The results show that juice expressed from whole crop cane has significantly higher colour than juices expressed from burnt cane. However, the concentrations of phenolic acids were lower in the juices expressed from whole crop cane. The main phenolic acids present in these juices were p-coumaric, vanillic, 2,3–dihydroxybenzoic, gallic and 3,4–dihydroxybenzoic acids. The degradation of caffeic acid significantly improved using Fenton’s reagent in comparison to hydrogen peroxide alone. The Fenton oxidation was optimum at pH 5 when up to ~86 % of caffeic acid degraded within 5 min.

Phenolics in sugar cane juice : potential degradation by hydrogen peroxide and Fenton’s reagent

The presence of colour in raw sugar plays a key role in the marketing strategy of the Australian raw sugar industry. Some sugars are relatively difficult to decolourise during refining and develop colour during storage. A new approach that might result in efficient and cost-effective colour removal during the sugar manufacturing process is the use of an advanced oxidation process (AOP), known as Fenton oxidation, that is, catalytic production of hydroxyl radicals from the decomposition of hydrogen peroxide using ferrous iron. As a first step towards developing this technology, this study determined the composition of colour precursors present in the juice of cane harvested by three different methods. The methods were harvesting cane after burning, harvesting the whole crop with half of the trash extracted and harvesting the whole crop with no trash extracted. The study also investigated the degradation at pH 3, 4 and 5 of a phenolic compound, caffeic acid (3,4–dihydroxycinnamic acid), which is present in sugar cane juice, using both hydrogen peroxide and Fenton’s reagent. The results show that juice expressed from whole crop cane has significantly higher colour than juices expressed from burnt cane. However, the concentrations of phenolic acids were lower in the juices expressed from whole crop cane. The main phenolic acids present in these juices were p-coumaric, vanillic, 2,3–dihydroxybenzoic, gallic and 3,4–dihydroxybenzoic acids. The degradation of caffeic acid significantly improved using Fenton’s reagent in comparison to hydrogen peroxide alone. The Fenton oxidation was optimum at pH 5 when up to ~86% of caffeic acid degraded within 5 min.

Dating of divergences within the Rattus genus phylogeny using whole mitochondrial genomes

The timing and order of divergences within the genus Rattus have, to date, been quite speculative. In order to address these important issues we sequenced six new whole mitochondrial genomes from wild-caught specimens from four species, Rattus exulans, Rattus praetor, Rattus rattus and Rattus tanezumi. The only rat whole mitochondrial genomes available previously were all from Rattus norvegicus specimens. Our phylogenetic and dating analyses place the deepest divergence within Rattus at ∼3.5 million years ago (Mya). This divergence separates the New Guinean endemic R. praetor lineage from the Asian lineages. Within the Asian/Island Southeast Asian clade R. norvegicus diverged earliest at ∼2.9 Mya. R. exulans and the ancestor of the sister species R. rattus and R. tanezumi subsequently diverged at ∼2.2 Mya, with R. rattus and R. tanezumi separating as recently as ∼0.4 Mya. Our results give both a better resolved species divergence order and diversification dates within Rattus than previous studies.

A study of ionic liquids for dissolution of sugarcane bagasse

Over the last decade, Ionic Liquids (ILs) have been used for the dissolution and derivatization of isolated cellulose. This ability of ILs is now sought for their application in the selective dissolution of cellulose from lignocellulosic biomass, for the manufacture of cellulosic ethanol. However, there are significant knowledge gaps in the understanding of the chemistry of the interaction of biomass and ILs. While imidazolium ILs have been used successfully to dissolve both isolated crystalline cellulose and components of lignocellulosic biomass, phosphonium ILs have not been sufficiently explored for the use in dissolution of lignocellulosic biomass. This thesis reports on the study of the chemistry of sugarcane bagasse with phosphonium ILs. Qualitative and quantitative measurements of biomass components dissolved in the phosphonium ionic liquids (ILs), trihexyltetradecylphosphonium chloride ([P66614]Cl) and tributylmethylphosphonium methylsulphate ([P4441]MeSO4) are obtained using attenuated total reflectance-Fourier Transform Infra Red (FTIR). Absorption bands related to cellulose, hemicelluloses and lignin dissolution monitored in situ in biomass-IL mixtures indicate lignin dissolution in both ILs and some holocellulose dissolution in the hydrophilic [P4441]MeSO4. The kinetics of lignin dissolution reported here indicate that while dissolution in the hydrophobic IL [P66614]Cl appears to follow an accepted mechanism of acid catalysed β-aryl ether cleavage, dissolution in the hydrophilic IL [P4441]MeSO4 does not appear to follow this mechanism and may not be followed by condensation reactions (initiated by reactive ketones). The quantitative measurement of lignin dissolution in phosphonium ILs based on absorbance at 1510 cm-1 has demonstrated utility and greater precision than the conventional Klason lignin method. The cleavage of lignin β-aryl ether bonds in sugarcane bagasse by the ionic liquid [P66614]Cl, in the presence of catalytic amounts of mineral acid. (ca. 0.4 %). The delignification process of bagasse is studied over a range of temperatures (120 °C to 150 °C) by monitoring the production of β-ketones (indicative of cleavage of β-aryl ethers) using FTIR spectroscopy and by compositional analysis of the undissolved fractions. Maximum delignification is obtained at 150 °C, with 52 % of lignin removed from the original lignin content of bagasse. No delignification is observed in the absence of acid which suggests that the reaction is acid catalysed with the IL solubilising the lignin fragments. The rate of delignification was significantly higher at 150 °C, suggesting that crossing the glass transition temperature of lignin effects greater freedom of rotation about the propanoid carbon-carbon bonds and leads to increased cleavage of β-aryl ethers. An attempt has been made to propose a probable mechanism of delignifcation of bagasse with the phosphonuim IL. All polymeric components of bagasse, a lignocellulosic biomass, dissolve in the hydrophilic ionic liquid (IL) tributylmethylphosphonium methylsulfate ([P4441]MeSO4) with and without a catalytic amount of acid (H2SO4, ca. 0.4 %). The presence of acid significantly increases the extent of dissolution of bagasse in [P4441]MeSO4 (by ca. 2.5 times under conditions used here). The dissolved fractions can be partially recovered by the addition of an antisolvent (water) and are significantly enriched in lignin. Unlike acid catalysed dissolution in the hydrophobic IL tetradecyltrihexylphosphonium chloride there is little evidence of cleavage of β-aryl ether bonds of lignin dissolving in [P4441]MeSO4 (with and without acid), but this mechanism may play some role in the acid catalysed dissolution. The XRD of the undissolved fractions suggests that the IL may selectively dissolve the amorphous cellulose component, leaving behind crystalline material.

The chemistry of acid catalyzed delignification of sugarcane bagasse in the ionic liquid trihexyl tetradecyl phosphonium chloride

This article reports on the cleavage of lignin ß-aryl ether bonds in sugarcane bagasse by the ionic liquid (IL) trihexyl tetradecyl phosphonium chloride [P66614] Cl, in the presence of catalytic amounts of mineral acid fca. 0.4%). The deligniflcation process of bagasse was studied over a range of temperatures (120°C to 150°C) by monitoring the production of ß-ketones (indicative of cleavage of ß-aryl ethers) using FTIR spectroscopy and by compositional analysis of the undissolved fractions. Maximum deligniflcation was obtained at 150°C, with 52% of lignin removed from the original lignin content of bagasse. No deligniflcation was observed in the absence of acid, which suggests that the reaction is acid catalyzed with the IL solubilizing the lignin fragments. The rate of deligniflcation was significantly higher at 150°C, suggesting that crossing the glass transition temperature of lignin effects greater freedom of rotation about the propanoid carbon-carbon bonds and leads to increased cleavage of ß-aryl ethers. An attempt has been made to propose a probable mechanism of deligniflcation of bagasse with the phosphonuim IL. © Taylor & Francis Group, LLC.