Modelling the flow of cane constituents through the milling process of a raw sugar factory

This thesis reports on an investigation to develop an advanced and comprehensive milling process model of the raw sugar factory. Although the new model can be applied to both, the four-roller and six-roller milling units, it is primarily developed for the six-roller mills which are widely used in the Australian sugar industry. The approach taken was to gain an understanding of the previous milling process simulation model "MILSIM" developed at the University of Queensland nearly four decades ago. Although the MILSIM model was widely adopted in the Australian sugar industry for simulating the milling process it did have some incorrect assumptions. The study aimed to eliminate all the incorrect assumptions of the previous model and develop an advanced model that represents the milling process correctly and tracks the flow of other cane components in the milling process which have not been considered in the previous models. The development of the milling process model was done is three stages. Firstly, an enhanced milling unit extraction model (MILEX) was developed to access the mill performance parameters and predict the extraction performance of the milling process. New definitions for the milling performance parameters were developed and a complete milling train along with the juice screen was modelled. The MILEX model was validated with factory data and the variation in the mill performance parameters was observed and studied. Some case studies were undertaken to study the effect of fibre in juice streams, juice in cush return and imbibition% fibre on extraction performance of the milling process. It was concluded from the study that the empirical relations developed for the mill performance parameters in the MILSIM model were not applicable to the new model. New empirical relations have to be developed before the model is applied with confidence. Secondly, a soluble and insoluble solids model was developed using modelling theory and experimental data to track the flow of sucrose (pol), reducing sugars (glucose and fructose), soluble ash, true fibre and mud solids entering the milling train through the cane supply and their distribution in juice and bagasse streams.. The soluble impurities and mud solids in cane affect the performance of the milling train and further processing of juice and bagasse. New mill performance parameters were developed in the model to track the flow of cane components. The developed model is the first of its kind and provides some additional insight regarding the flow of soluble and insoluble cane components and the factors affecting their distribution in juice and bagasse. The model proved to be a good extension to the MILEX model to study the overall performance of the milling train. Thirdly, the developed models were incorporated in a proprietary software package "SysCAD’ for advanced operational efficiency and for availability in the ‘whole of factory’ model. The MILEX model was developed in SysCAD software to represent a single milling unit. Eventually the entire milling train and the juice screen were developed in SysCAD using series of different controllers and features of the software. The models developed in SysCAD can be run from macro enabled excel file and reports can be generated in excel sheets. The flexibility of the software, ease of use and other advantages are described broadly in the relevant chapter. The MILEX model is developed in static mode and dynamic mode. The application of the dynamic mode of the model is still under progress.

Adsorption of heavy metals by road deposited solids

The research study discussed in the paper investigated the adsorption/desorption behaviour of heavy metals commonly deposited on urban road surfaces, namely, Zn, Cu, Cr and Pb for different particle size ranges of solids. The study outcomes, based on field studies and batch experiments confirmed that road deposited solids particles contain a significantly high amount of vacant charge sites with the potential to adsorb additional heavy metals. Kinetic studies and adsorption experiments indicated that Cr is the most preferred metal element to associate with solids due to the relatively high electro negativity and high charge density of trivalent cation (Cr3+). However, the relatively low availability of Cr in the urban road environment could influence this behaviour. Comparing total adsorbed metals present in solids particles, it was found that Zn has the highest capacity for adsorption to solids. Desorption experiments confirmed that a low concentration of Cu, Cr and Pb in solids was present in water-soluble and exchangeable form, whilst a significant fraction of adsorbed Zn has a high likelihood of being released back into solution. Among heavy metals, Zn is considered to be the most commonly available metal among road surface pollutants.

Poly(2-oxazoline) hydrogels for controlled fibroblast attachment

Currently there is a lack of choice when selecting synthetic materials with the cell-instructive properties demanded by modern biomaterials. The purpose of this study was to investigate the attachment of cells onto hydrogels prepared from poly(2-oxazoline)s selectively-functionalized with cell adhesion motifs. A water-soluble macromer based on the microwave-assisted cationic ring-opening polymerization of 2-methyl-2-oxazoline and 2-(dec-9-enyl)-2-oxazoline was functionalized with the peptide CRGDSG or controls using thiol-ene photochemistry followed by facile crosslinking in the presence of a dithiol crosslinker. The growth of human fibroblasts on the hydrogel surfaces was dictated by the structure and amount of incorporated peptide. Controls without any peptide showed resistance to cellular attachment. The benignity of the crosslinking conditions was demonstrated by the incorporation of fibroblasts within the hydrogels to produce three-dimensional cell-polymer constructs.

In Plant Activation: An inducible, hyperexpression platform for recombinant protein production in plants

In this study, we describe a novel protein production platform that provides both activation and amplification of transgene expression in planta. The In Plant Activation (INPACT) system is based on the replication machinery of tobacco yellow dwarf mastrevirus (TYDV) and is essentially transient gene expression from a stably transformed plant, thus combining the advantages of both means of expression. The INPACT cassette is uniquely arranged such that the gene of interest is split and only reconstituted in the presence of the TYDV-encoded Rep/RepA proteins. Rep/RepA expression is placed under the control of the AlcA:AlcR gene switch, which is responsive to trace levels of ethanol. Transgenic tobacco (Nicotiana tabacum cv Samsun) plants containing an INPACT cassette encoding the b-glucuronidase (GUS) reporter had negligible background expression but accumulated very high GUS levels (up to 10% total soluble protein) throughout the plant, within 3 d of a 1% ethanol application. The GUS reporter was replaced with a gene encoding a lethal ribonuclease, barnase, demonstrating that the INPACT system provides exquisite control of transgene expression and can be adapted to potentially toxic or inhibitory compounds. The INPACT gene expression platform is scalable, not host-limited, and has been used to express both a therapeutic and an industrial protein.

Application of kaolin amorphous derivative as a nitrogen carrier

Sandy soils have low nutrient holding capacity and high water conductivity. Consequently, nutrients applied as highly soluble chemical fertilisers are prone to leaching, particularly in heavily irrigated environments such as horticultural soils and golf courses. Amorphous derivatives of kaolin with high cation exchange capacity may be loaded with desired nutrients and applied as controlledrelease fertilisers. Kaolin is an abundant mineral, which can be converted to a meso-porous amorphous derivative (KAD) using facile chemical processes. KAD is currently being used to sequester ammonium from digester effluent in sewage treatment plants in a commercial environment. This material is also known in Australia by the trade name MesoLite. The ammonium-saturated form of KAD may be applied to soils as a nitrogen fertiliser. Up to 7% N can be loaded onto KAD by contacting it with high-ammonia concentration wastewater from sewerage treatment plants. This poster paper demonstrates plant uptake of nitrogen from KAD and compares its efficiency as a fertiliser with NH4SO4. Rye grass was grown in 1kg pots in a glass-house. Nitrogen was applied at a range of rates using NH4SO4 and two KAD materials carrying 7% and 3% nitrogen, respectively. All other nutrients were applied in adequate amounts. All treatments were replicated three times. Plants were harvested after four weeks. Dry mass and N concentrations were determined by standard methods. At all N application rates, ammonium-loaded KAD produced significantly higher plant mass than for NH4SO4. The lower fertiliser effectiveness of NH4SO4 is attributed to possible loss of some N through volatilisation. Of the two KAD types, the material with lower CEC value supported slightly higher plant yields. The KAD materials did not show any adverse effect on availability of trace elements, as evidenced by lack of deficiency symptoms and plant analyses. Clearly, nitrogen loaded on to KAD in the form of ammonium is likely to be protected from leaching, but is still available to plants. These data suggest that KAD-based fertilisers may be suitable substitutes for water soluble N, K and other cation fertilisers for leaching soils.

Sibpair studies implicate chromosome 18 in essential hypertension

Interest in chromosome 18 in essential hypertension comes from comparative mapping of rat blood pressure quantitative trait loci (QTL), familial orthostatic hypotensive syndrome studies, and essential hypertension pedigree linkage analyses indicating that a locus or loci on human chromosome 18 may play a role in hypertension development. To further investigate involvement of chromosome 18 in human essential hypertension, the present study utilized a linkage scan approach to genotype twelve microsatellite markers spanning human chromosome 18 in 177 Australian Caucasian hypertensive (HT) sibling pairs. Linkage analysis showed significant excess allele sharing of the D18S61 marker when analyzed with SPLINK (P=0.00012), ANALYZE (Sibpair) (P=0.0081), and also with MAPMAKER SIBS (P=0.0001). Similarly, the D18S59 marker also showed evidence for excess allele sharing when analyzed with SPLINK (P=0.016), ANALYZE (Sibpair) (P=0.0095), and with MAPMAKER SIBS (P = 0.014). The adenylate cyclase activating polypeptide 1 gene (ADCYAP1) is involved in vasodilation and has been co-localized to the D18S59 marker. Results testing a microsatellite marker in the 3′ untranslated region of ADCYAP1 in age and gender matched HT and normotensive (NT) individuals showed possible association with hypertension (P = 0.038; Monte Carlo P = 0.02), but not with obesity. The present study shows a chromosome 18 role in essential hypertension and indicates that the genomic region near the ADCYAP1 gene or perhaps the gene itself may be implicated. Further investigation is required to conclusively determine the extent to which ADCYAP1 polymorphisms are involved in essential hypertension. © 2003 Wiley-Liss, Inc.

Scanning electrochemical microscopy study of the solid--solid interconversion of TCNQ to phase I and phase II CuTCNQ

The reduction of 7,7,8,8-tetracyanoquinodimethane (TCNQ) crystals attached to a glassy carbon electrode in the presence of Cu2+(aq) to form CuTCNQ(s) has been investigated using scanning electrochemical microscopy in the substrate generation tip collection mode and shown to involve a generation of soluble TCNQ−(aq). The subsequent oxidation of CuTCNQ does not involve simple expulsion of Cu+ into solution but a soluble complex attributed to Cu2+TCNQ−(aq). Mechanistic insights relative to the electrochemical conversion of CuTCNQ phase I into phase II by repetitive cycling of potential and electrochemical formation of KTCNQ have also been established

Stepwise engineering of a Pichia pastoris D-amino acid oxidase whole cell catalyst

Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a well characterized enzyme used for cephalosporin C conversion on industrial scale. However, the demands on the enzyme with respect to activity, operational stability and costs also vary with the field of application. Processes that use the soluble enzyme suffer from fast inactivation of TvDAO while immobilized oxidase preparations raise issues related to expensive carriers and catalyst efficiency. Therefore, oxidase preparations that are more robust and active than those currently available would enable a much broader range of economically viable applications of this enzyme in fine chemical syntheses. A multi-step engineering approach was chosen here to develop a robust and highly active Pichia pastoris TvDAO whole-cell biocatalyst. As compared to the native T. variabilis host, a more than seven-fold enhancement of the intracellular level of oxidase activity was achieved in P. pastoris through expression optimization by codon redesign as well as efficient subcellular targeting of the enzyme to peroxisomes. Multi copy integration further doubled expression and the specific activity of the whole cell catalyst. From a multicopy production strain, about 1.3 x 103 U/g wet cell weight (wcw) were derived by standard induction conditions feeding pure methanol. A fed-batch cultivation protocol using a mixture of methanol and glycerol in the induction phase attenuated the apparent toxicity of the recombinant oxidase to yield final biomass concentrations in the bioreactor of >or= 200 g/L compared to only 117 g/L using the standard methanol feed. Permeabilization of P. pastoris using 10% isopropanol yielded a whole-cell enzyme preparation that showed 49% of the total available intracellular oxidase activity and was notably stabilized (by three times compared to a widely used TvDAO expressing Escherichia coli strain) under conditions of D-methionine conversion using vigorous aeration. Stepwise optimization using a multi-level engineering approach has delivered a new P. pastoris whole cell TvDAO biocatalyst showing substantially enhanced specific activity and stability under operational conditions as compared to previously reported preparations of the enzyme. The production of the oxidase through fed-batch bioreactor culture and subsequent cell permeabilization is high-yielding and efficient. Therefore this P. pastoris catalyst has been evaluated for industrial purposes.

Design and construction of an in-plant activation cassette for transgene expression and recombinant protein production in plants

Virus-based transgene expression systems have become particularly valuable for recombinant protein production in plants. The dual-module in-plant activation (INPACT) expression platform consists of a uniquely designed split-gene cassette incorporating the cis replication elements of Tobacco yellow dwarf geminivirus (TYDV) and an ethanol-inducible activation cassette encoding the TYDV Rep and RepA replication-associated proteins. The INPACT system is essentially tailored for recombinant protein production in stably transformed plants and provides both inducible and high-level transient transgene expression with the potential to be adapted to diverse crop species. The construction of a novel split-gene cassette, the inducible nature of the system and the ability to amplify transgene expression via rolling-circle replication differentiates this system from other DNA- and RNA-based virus vector systems used for stable or transient recombinant protein production in plants. Here we provide a detailed protocol describing the design and construction of a split-gene INPACT cassette, and we highlight factors that may influence optimal activation and amplification of gene expression in transgenic plants. By using Nicotiana tabacum, the protocol takes 6-9 months to complete, and recombinant proteins expressed using INPACT can accumulate to up to 10% of the leaf total soluble protein.

Moisture and tensile strength properties of starch-sugar cane nanofibre films

There is an increasing need for biodegradable, environmentally friendly plastics to replace the petroleum-based non-degradable plastics which litter and pollute the environment. Starch-based plastic film composites are becoming a popular alternative because of their low cost, biodegradability, the abundance of starch, and ease with which starch-based films can be chemically modified. This paper reports on the results of using sugar cane bagasse nanofibres to improve the physicochemical properties of starch-based polymers. The addition of bagasse nanofibre (2.5, 5, 10 or 20 wt%) to (modified) potato starch (‘Soluble starch’) reduced the moisture uptake by up to 17 % at 58 % relative humidity (RH). The film’s tensile strength and Young’s Modulus increased by up to 100 % and 200 % with 10 wt% and 20 wt% nanofibre respectively at 58% RH. The tensile strain reduced by up to 70 % at 20 wt% fibre loading. These results indicate that addition of sugar cane bagasse nanofibres significantly improved the properties of starch-based plastic films

Preparation and characterization of composites from starch with sugarcane bagasse nanofibres

This paper reports on the results of using unbleached sugar cane bagasse nanofibres (average diameter 26.5 nm; aspect ratio 247 assuming a dry fibre density of 1,500 kg/m3) to improve the physico-chemical properties of starch-based films. The addition of bagasse nanofibres (2.5 to 20 wt%) to modified potato starch (i.e. soluble starch) reduced the moisture uptake by up to 17 % at 58 % relative humidity. The film’s tensile strength and Young’s modulus increased by up to 100 % (3.1 to 6.2 MPa) and 300 % (66.3 to 198.3 MPa) respectively with 10 and 20 wt% fibre addition. However, the strain at yield dropped by 50 % for the film containing 10 wt% fibre. Models for composite materials were used to account for the strong interactions between the nanofibres and the starch matrix. The storage and loss moduli as well as the glass transition temperature (Tg) obtained from dynamic mechanical thermal analysis, were increased with the starch-nanofibre films indicating decreased starch chain mobility due to the interacting effect of the nanofibres. Evidence of the existence of strong interactions between the starch matrix and the nanofibres was revealed from detailed Fourier transform infra-red and scanning electron microscopic evaluation.

An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae

Background The control of plant anthocyanin accumulation is via transcriptional regulation of the genes encoding the biosynthetic enzymes. A key activator appears to be an R2R3 MYB transcription factor. In apple fruit, skin anthocyanin levels are controlled by a gene called MYBA or MYB1, while the gene determining fruit flesh and foliage anthocyanin has been termed MYB10. In order to further understand tissue-specific anthocyanin regulation we have isolated orthologous MYB genes from all the commercially important rosaceous species. Results We use gene specific primers to show that the three MYB activators of apple anthocyanin (MYB10/MYB1/MYBA) are likely alleles of each other. MYB transcription factors, with high sequence identity to the apple gene were isolated from across the rosaceous family (e.g. apples, pears, plums, cherries, peaches, raspberries, rose, strawberry). Key identifying amino acid residues were found in both the DNA-binding and C-terminal domains of these MYBs. The expression of these MYB10 genes correlates with fruit and flower anthocyanin levels. Their function was tested in tobacco and strawberry. In tobacco, these MYBs were shown to induce the anthocyanin pathway when co-expressed with bHLHs, while over-expression of strawberry and apple genes in the crop of origin elevates anthocyanins. Conclusions This family-wide study of rosaceous R2R3 MYBs provides insight into the evolution of this plant trait. It has implications for the development of new coloured fruit and flowers, as well as aiding the understanding of temporal-spatial colour change.

Oxidation of 4-substituted TEMPO derivatives reveals modifications at the 1-and 4-positions

Potenital pathways for the deactivation of hindered amine light stabilisers (HALS) have been investigated by observing reactions of model compounds-based on 4-substituted derivatives of 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO)-with hydroxyl radicals. In these reactions, dilute aqueous suspensions of photocatalytic nanoparticulate titanium dioxide were irradiated with UV light in the presence of water-soluble TEMPO derivatives. Electron spin resonance (ESR) and electrospray ionisation mass-spectrometry (ESI-MS) data were acquired to provide complementary structural elucidation of the odd-and even-electron products of these reactions and both techniques show evidence for the formation of 4-oxo-TEMPO (TEMPONE). TEMPONE formation from the 4-substituted TEMPO compounds is proposed to be initiated by hydrogen abstraction at the 4-position by hydroxyl radical. High-level ab initio calculations reveal a thermodynamic preference for abstraction of this hydrogen but computed activation barriers indicate that, although viable, it is less favoured than hydrogen abstraction from elsewhere on the TEMPO scaffold. If a radical is formed at the 4-position however, calculations elucidate two reaction pathways leading to TEMPONE following combination with either a second hydroxyl radical or dioxygen. An alternate mechanism for conversion of TEMPOL to TEMPONE via an alkoxyl radical intermediate is also considered and found to be competitive with the other pathways. ESI-MS analysis also shows an increased abundance of analogous 4-substituted piperidines during the course of irradiation, suggesting competitive modification at the 1-position to produce a secondary amine. This modification is confirmed by characteristic fragmentation patterns of the ionised piperidines obtained by tandem mass spectrometry. The conclusions describe how reaction at the 4-position could be responsible for the gradual depletion of HALS in pigmented surface coatings and secondly, that modification at nitrogen to form the corresponding secondary amine species may play a greater role in the stabilisation mechanisms of HALS than previously considered.

Effects of inhibitors of plasminogen activator, serine proteinases, and collagenase IV on the invasion of basement membranes by metastatic cells

Using both human and murine cell lines, we show that malignant cells are able to invade through basement membrane and also secrete elevated amounts of collagenase IV, an enzyme implicated in the degradation of basement membranes. Using serine proteinase inhibitors and antibodies to plasminogen activators as well as a newly described collagenase inhibitor we demonstrate that a protease cascade leads to the activation of an enzyme(s) that cleaves collagen IV. Inhibition at each step reduces the invasion of the tumor cells through reconstituted basement membrane in vitro. Treatment with a collagenase inhibitor reduced the incidence of lung lesions in mice given i.v. injections of malignant melanoma cells.

Transmembrane/cytoplasmic domain-mediated membrane type 1-matrix metalloprotease docking to invadopodia is required for cellinvasion

The invasion of human malignant melanoma cells into the extracellular matrix (ECM) involves the accumulation of proteases at sites of ECM degradation where activation of matrix metalloproteases (MMP) occurs. Here, we show that when membrane type 1 MMP (MT-MMP) was overexpressed in RPMI7951 human melanoma cells, the cells made contact with the ECM, activated soluble and ECM-bound MMP-2, and degraded and invaded the ECM. Further experiments demonstrated the importance of localization of the MT-MMP to invadopodia. Overexpression of MT-MMP without invadopodial localization caused activation of soluble MMP-2, but did not facilitate ECM degradation or cell invasiveness. Up-regulation of endogenous MT-MMP with concanavalin A caused activation of MMP-2. However, concanavalin A treatment prevented invadopodial localization of MT-MMP and ECM degradation. Neither a truncated MT-MMP mutant lacking transmembrane (TM) and cytoplasmic domains (ΔTM(MT-MMP)), nor a chimeric MT-MMP containing the interleukin 2 receptor α chain (IL-2R) TM and cytoplasmic domains (ΔTM(MT-MMP)/TM(IL-2R)) were localized to invadopodia or exhibited ECM degradation. Furthermore, a chimera of the TM/cytoplasmic domain of MT-MMP (TM(MT-MMP)) with tissue inhibitor of MMP 1 (TIMP-1/TM(MT- MMP)) directed the TIMP-1 molecule to invadopodia. Thus, the MT-MMP TM/cytoplasmic domain mediates the spatial organization of MT-MMP into invadopodia and subsequent degradation of the ECM.