Investigation of lignin biosynthesis in sugarcane for improved lignocellulosic ethanol production

As oil use increases at a rate unsustainable for the environment and unmatchable by current levels of oil production, a major shift towards renewable energy is necessary. By expanding the current knowledge of lignin biosynthesis and its manipulation in sugarcane, this PhD contributes to the production of economically viable second generation bioethanol, a fuel produced from plant biomass. The findings of this thesis contribute to the limited knowledge of lignin biosynthesis and deposition in sugarcane, and the application of biotechnology to produce sugarcane, and the resulting bagasse, with a modified cell wall. Reducing or modifying the lignin content in the cell wall of bagasse can reduce production costs and increase yields of bioethanol. This makes bioethanol more economically competitive with oil as an alternative energy source. A move to using bioethanol over fossil based transport fuels will have global economic and environmental benefits.

Characterisation of lignins isolated from sugarcane bagasse pretreated with acidified ethylene glycol and ionic liquids

Sugarcane bagasse pretreatment processes using acidified aqueous ethylene glycol (EG) and ionic liquids (ILs) have been reported recently. In this study, recovery of lignins from these processes was conducted, as well as determination of their physico-chemical properties. The amount of lignins recovered from 1-butyl-3-methylimidazolium chloride ([bmim]Cl) with HCl as a catalyst and [bmim][CH3SO3] was ∼42%, and ∼35%–36% by EG with HCl or H2SO4 as a catalyst, respectively. The isolated lignins were characterised using wet chemistry, spectroscopy and thermogravimetry analysis (TGA), and the results compared to soda lignin from NaOH pretreatment of bagasse. The IL and EG lignins contained no or trace amounts of carbohydrates, slightly lower hydrogen content but slightly higher oxygen contents than soda lignin. The IL and EG lignins contained more C-3 and C-5 reactive sites for Mannich reaction and had more p-hydroxypheny propane unit structures than soda lignin. Two-dimensional heteronuclear single quantum coherence (2D HSQC) nuclear magnetic resonance (NMR) identified the major substructural units in the lignins, and allowed differences among them to be studied. As EG lignins were extracted in very reactive environment, intermediate enol ethers were formed and led to cleavage reactions which were not apparent in the other lignins. 31P NMR and infra-red spectroscopy results showed that IL and EG lignins had lower total hydroxyl content than soda lignin, probably indicating that a higher degree of self-polymerisation occurred during bagasse pretreatment, despite the use of lower temperature and shorter reaction time. On the basis of the salient features of these lignins, potential applications were proposed.

Issues associated with using trash as a cogeneration fuel

Considerable work has been undertaken to determine an economical process to provide sugarcane trash as a fuel for cogeneration. This paper reviews efforts to provide that trash fuel by harvesting, transporting and processing the trash with the cane. Harvesting trash with the cane has the advantage that cane that would otherwise be lost by extracting it with the trash is captured and sugar can be produced from that cane. Transporting trash with the cane significantly reduces the bulk density of the cane, requiring substantial changes and costs to cane transport. Shredding the trash at the harvester and compacting the cane in the bin prior to transport are possible methods to increase the bulk density but both have considerable cost. Processing trash through the sugar factory with the cane significantly reduces sugar recovery and sugar quality. Although considerable knowledge has been gained of these effects and further analysis has provided insights into their causes, much more work is required before whole crop harvesting and transport is an economically viable means of trash recovery.

Hybrid metaheuristic techniques for optimising sugarcane rail operations

Mixed integer programming and parallel-machine job shop scheduling are used to solve the sugarcane rail transport scheduling problem. Constructive heuristics and metaheuristics were developed to produce a more efficient scheduling system and so reduce operating costs. The solutions were tested on small and large size problems. High-quality solutions and improved CPU time are the result of developing new hybrid techniques which consist of different ways of integrating simulated annealing and Tabu search techniques.

Managing the impacts of sugarcane farming on the Great Barrier Reef: An evaluation of the implementation of the Polluter Pays Principle

This report provides an evaluation of the implementation of the Polluter Pays Principle (PPP) – a principle of international environmental law – in the context of pollution from sugarcane farming affecting Australia’s Great Barrier Reef (GBR). The research was part of an experiment to test methods for evaluating the effectiveness of environmental laws. Overall, we found that whilst the PPP is reflected to a limited extent in Australian law (more so in Queensland law, than at the national level), the behaviour one might expect in terms of implementing the principle was largely inadequate. Evidence of a longer term, explicit commitment to the PPP was particularly weak.

Abacá mosaic virus: a distinct strain of Sugarcane mosaic virus

Abacá mosaic virus (AbaMV) is related to members of the sugarcane mosaic virus subgroup of the genus Potyvirus. The ~2 kb 3′ terminal region of the viral genome was sequenced and, in all areas analysed, found to be most similar to Sugarcane mosaic virus (SCMV) and distinct from Johnsongrass mosaic virus (JGMV), Maize dwarf mosaic virus (MDMV) and Sorghum mosaic virus (SrMV). Cladograms of the 3′ terminal region of the NIb protein, the coat protein core and the 3′ untranslated region showed that AbaMV clustered with SCMV, which was a distinct clade and separate from JGMV, MDMV and SrMV. The N-terminal region of the AbaMV coat protein had a unique amino acid repeat motif different from those previously published for other strains of SCMV. The first experimental transmission of AbaMV from abacá (Musa textilis) to banana (Musa sp.), using the aphid vectors Rhopalosiphum maidis and Aphis gossypii, is reported. Polyclonal antisera for the detection of AbaMV in western blot assays and ELISA were prepared from recombinant coat protein expressed in E. coli. A reverse transcriptase PCR diagnostic assay, with microtitre plate colourimetric detection, was developed to discriminate between AbaMV and Banana bract mosaic virus, another Musa-infecting potyvirus. Sequence data, host reactions and serological relationships indicate that AbaMV should be considered a distinct strain of SCMV, and the strain designation SCMV-Ab is suggested.

The environmental fate of diuron under a conventional production regime in a sugarcane farm during the plant cane phase

BACKGROUND: Field studies of diuron and its metabolites 3-(3,4-dichlorophenyl)-1-methylurea (DCPMU), 3,4-dichlorophenylurea (DCPU) and 3,4-dichloroaniline (DCA) were conducted in a farm soil and in stream sediments in coastal Queensland, Australia. RESULTS: During a 38 week period after a 1.6 kg ha^-1 diuron application, 70-100% of detected compounds were within 0-15 cm of the farm soil, and 3-10% reached the 30-45 cm depth. First-order t1/2 degradation averaged 49 ± 0.9 days for the 0-15, 0-30 and 0-45 cm soil depths. Farm runoff was collected in the first 13-50 min of episodes lasting 55-90 min. Average concentrations of diuron, DCPU and DCPMU in runoff were 93, 30 and 83-825 µg L^-1 respectively. Their total loading in all runoff was >0.6% of applied diuron. Diuron and DCPMU concentrations in stream sediments were between 3-22 and 4-31 µg kg^-1 soil respectively. The DCPMU/diuron sediment ratio was >1. CONCLUSION: Retention of diuron and its metabolites in farm topsoil indicated their negligible potential for groundwater contamination. Minimal amounts of diuron and DCMPU escaped in farm runoff. This may entail a significant loading into the wider environment at annual amounts of application. The concentrations and ratio of diuron and DCPMU in stream sediments indicated that they had prolonged residence times and potential for accumulation in sediments. The higher ecotoxicity of DCPMU compared with diuron and the combined presence of both compounds in stream sediments suggest that together they would have a greater impact on sensitive aquatic species than as currently apportioned by assessments that are based upon diuron alone.

A near Optimal Cane Rail Scheduler under Limited and Unlimited Capacity Constraints

Australia is the world’s third largest exporter of raw sugar after Brazil and Thailand, with around $2.0 billion in export earnings. Transport systems play a vital role in the raw sugar production process by transporting the sugarcane crop between farms and mills. In 2013, 87 per cent of sugarcane was transported to mills by cane railway. The total cost of sugarcane transport operations is very high. Over 35% of the total cost of sugarcane production in Australia is incurred in cane transport. A cane railway network mainly involves single track sections and multiple track sections used as passing loops or sidings. The cane railway system performs two main tasks: delivering empty bins from the mill to the sidings for filling by harvesters; and collecting the full bins of cane from the sidings and transporting them to the mill. A typical locomotive run involves an empty train (locomotive and empty bins) departing from the mill, traversing some track sections and delivering bins at specified sidings. The locomotive then, returns to the mill, traversing the same track sections in reverse order, collecting full bins along the way. In practice, a single track section can be occupied by only one train at a time, while more than one train can use a passing loop (parallel sections) at a time. The sugarcane transport system is a complex system that includes a large number of variables and elements. These elements work together to achieve the main system objectives of satisfying both mill and harvester requirements and improving the efficiency of the system in terms of low overall costs. These costs include delay, congestion, operating and maintenance costs. An effective cane rail scheduler will assist the traffic officers at the mill to keep a continuous supply of empty bins to harvesters and full bins to the mill with a minimum cost. This paper addresses the cane rail scheduling problem under rail siding capacity constraints where limited and unlimited siding capacities were investigated with different numbers of trains and different train speeds. The total operating time as a function of the number of trains, train shifts and a limited number of cane bins have been calculated for the different siding capacity constraints. A mathematical programming approach has been used to develop a new scheduler for the cane rail transport system under limited and unlimited constraints. The new scheduler aims to reduce the total costs associated with the cane rail transport system that are a function of the number of bins and total operating costs. The proposed metaheuristic techniques have been used to find near optimal solutions of the cane rail scheduling problem and provide different possible solutions to avoid being stuck in local optima. A numerical investigation and sensitivity analysis study is presented to demonstrate that high quality solutions for large scale cane rail scheduling problems are obtainable in a reasonable time. Keywords: Cane railway, mathematical programming, capacity, metaheuristics

Growth responses of sugarcane to mycorrhizal spore density and phosphorus rate

Arbuscular mycorrhizal (AM) fungi, commonly found in long-term cane-growing fields in northern Queensland, are linked with both negative and positive growth responses by sugarcane (Saccharum spp.), depending on P supply. A glasshouse trial was established to examine whether AM density might also have an important influence on these growth responses. Mycorrhizal spores (Glomus clarum), isolated from a long-term cane block in northern Queensland, were introduced into a pasteurised low-P cane soil at 5 densities (0, 0.06, 0.25, 1, 4 spores/g soil) and with 4 P treatments (0, 8.2, 25, and 47 mg/kg). At 83 days after planting, sugarcane tops responded positively to P fertilizer, although responses attributable to spore density were rarely observed. In one case, addition of 4 spores/g led to a 53% yield response over those without AM at 8 mgP/kg, or a relative benefit of 17 mg P/kg. Root colonisation was reduced for plants with nil or 74 mg P/kg. For those without AM, P concentration in the topmost visible dewlap (TVD) leaf increased significantly with fertiliser P (0.07 v. 0.15%). However, P concentration increased further with the presence of AM spores. Irrespective of AM, the critical P concentration in the TVD leaf was 0.18%. This study confirms earlier reports that sugarcane is poorly responsive to AM. Spore density, up to 4 spores/g soil, appears unable to influence this responsiveness, either positively or negatively. Attempts to gain P benefits by increasing AM density through rotation seem unlikely to lead to yield increases by sugarcane. Conversely, sugarcane grown in fields with high spore densities and high plant-available P, such as long-term cane-growing soils, is unlikely to suffer a yield reduction from mycorrhizal fungi.

Row spacing and planting density effects on the growth and yield of sugarcane. 1. Responses in fumigated and non-fumigated soil.

It has been reported that high-density planting of sugarcane can improve cane and sugar yield through promoting rapid canopy closure and increasing radiation interception earlier in crop growth. It is widely known that the control of adverse soil biota through fumigation (removes soil biological constraints and improves soil health) can improve cane and sugar yield. Whether the responses to high-density planting and improved soil health are additive or interactive has important implications for the sugarcane production system. Field experiments established at Bundaberg and Mackay, Queensland, Australia, involved all combinations of 2-row spacings (0.5 and 1.5 m), two planting densities (27 000 and 81 000 two-eyed setts/ha), and two soil fumigation treatments (fumigated and non-fumigated). The Bundaberg experiment had two cultivars (Q124, Q155), was fully irrigated, and harvested 15 months after planting. The Mackay experiment had one cultivar (Q117), was grown under rainfed conditions, and harvested 10 months after planting. High-density planting (81 000 setts/ha in 0.5-m rows) did not produce any more cane or sugar yield at harvest than low-density planting (27 000 setts/ha in 1.5-m rows) regardless of location, crop duration (15 v. 10 months), water supply (irrigated v. rainfed), or soil health (fumigated v. non-fumigated). Conversely, soil fumigation generally increased cane and sugar yields regardless of site, row spacing, and planting density. In the Bundaberg experiment there was a large fumigation x cultivar x density interaction (P<0.01). Cultivar Q155 responded positively to higher planting density in non-fumigated soil but not in fumigated soil, while Q124 showed a negative response to higher planting density in non-fumigated soil but no response in fumigated soil. In the Mackay experiment, Q117 showed a non-significant trend of increasing yield in response to increasing planting density in non-fumigated soil, similar to the Q155 response in non-fumigated soil at Bundaberg. The similarity in yield across the range of row spacings and planting densities within experiments was largely due to compensation between stalk number and stalk weight, particularly when fumigation was used to address soil health. Further, the different cultivars (Q124 and Q155 at Bundaberg and Q117 at Mackay) exhibited differing physiological responses to the fumigation, row spacing, and planting density treatments. These included the rate of tiller initiation and subsequent loss, changes in stalk weight, and propensity to lodging. These responses suggest that there may be potential for selecting cultivars suited to different planting configurations.

Row spacing and planting density effects on the growth and yield of sugarcane. 3. Responses with different cultivars.

The promotion of controlled traffic (matching wheel and row spacing) in the Australian sugar industry is necessitating a widening of row spacing beyond the standard 1.5 m. As all cultivars grown in the Australian industry have been selected under the standard row spacing there are concerns that at least some cultivars may not be suitable for wider rows. To address this issue, experiments were established in northern and southern Queensland in which cultivars, with different growth characteristics, recommended for each region, were grown under a range of different row configurations. In the northern Queensland experiment at Gordonvale, cultivars Q187((sic)), Q200((sic)), Q201((sic)), and Q218((sic)) were grown in 1.5-m single rows, 1.8-m single rows, 1.8-m dual rows (50 cm between duals), and 2.3-m dual rows (80 cm between duals). In the southern Queensland experiment at Farnsfield, cvv. Q138, Q205((sic)), Q222((sic)) and Q188((sic)) were also grown in 1.5-m single rows, 1.8-m single rows, 1.8-m dual rows (50 cm between duals), while 1.8-m-wide throat planted single row and 2.0-m dual row (80 cm between duals) configurations were also included. There was no difference in yield between the different row configurations at Farnsfield but there was a significant row configuration x cultivar interaction at Gordonvale due to good yields in 1.8-m single and dual rows with Q201((sic)) and poor yields with Q200((sic)) at the same row spacings. There was no significant difference between the two cultivars in 1.5-m single and 2.3-m dual rows. The experiments once again demonstrated the compensatory capacity that exists in sugarcane to manipulate stalk number and individual stalk weight as a means of producing similar yields across a range of row configurations and planting densities. There was evidence of different growth patterns between cultivars in response to different row configurations (viz. propensity to tiller, susceptibility to lodging, ability to compensate between stalk number and stalk weight), suggesting that there may be genetic differences in response to row configuration. It is argued that there is a need to evaluate potential cultivars under a wider range of row configurations than the standard 1.5-m single rows. Cultivars that perform well in row configurations ranging from 1.8 to 2.0 m are essential if the adverse effects of soil compaction are to be managed through the adoption of controlled traffic.

Susceptibility of source plants to sugarcane Fiji disease virus influences the acquisition and transmission of the virus by the planthopper vector Perkinsiella saccharicida

Fiji leaf gall (FLG) caused by Sugarcane Fiji disease virus (SCFDV) is transmitted by the planthopper Perkinsiella saccharicida. FLG is managed through the identification and exploitation of plant resistance. The glasshouse-based resistance screening produced inconsistent transmission results and the factors responsible for that are not known. A series of glasshouse trials conducted over a 2-year period was compared to identify the factors responsible for the erratic transmission results. SCFDV transmission was greater when the virus was acquired by the vector from a cultivar that was susceptible to the virus than when the virus was acquired from a resistant cultivar. Virus acquisition by the vector was also greater when the vector was exposed to the susceptible cultivars than when exposed to the resistant cultivar. Results suggest that the variation in transmission levels is due to variation in susceptibility of sugarcane cultivars to SCFDV used for virus acquisition by the vector.

Is the distribution of Fiji leaf gall in Australian sugarcane explained by variation in the vector Perkinsiella saccharicida?

Fiji leaf gall (FLG) is an important virally induced disease in Australian sugarcane. It is confined to southern canegrowing areas, despite its vector, the delphacid planthopper Perkinsiella saccharicida, occurring in all canegrowing areas of Queensland and New South Wales. This disparity between distributions could be a result of successful containment of the disease through quarantine and/or geographical barriers, or because northern Queensland populations of Perkinsiella may be poorer vectors of the disease. These hypotheses were first tested by investigating variation in the ITS2 region of the rDNA fragment among eastern Australian and overseas populations of Perkinsiella. The ITS2 sequences of the Western Australian P. thompsoni and the Fijian P. vitiensis were distinguishable from those of P. saccharicida and there was no significant variation among the 26 P. saccharicida populations. Reciprocal crosses of a northern Queensland and a southern Queensland population of P. saccharicida were fertile, so they may well be conspecific. Single vector transmission experiments showed that a population of P. saccharicida from northern Queensland had a higher vector competency than either of two southern Queensland populations. The frequency of virus acquisition in the vector populations was demonstrated to be important in the vector competency of the planthopper. The proportion of infected vectors that transmitted the virus to plants was not significantly different among the populations tested. This study shows that the absence of FLG from northern Queensland is not due to a lack of vector competency of the northern population of P. saccharicida.

Revenue diversification opportunities from sugarcane

Sugarcane is a major global agricultural crop that produces significant quantities of sugar and biomass in tropical and sub-tropical regions. Over many centuries, the crop has been grown primarily for its high sugar content which traditionally contributes over 95% of the revenue derived from the crop. While the production of renewable electricity from bagasse and rum from molasses has a long history, in more recent decades significant advances have been made in the production of cogeneration products and fuel ethanol at large scale. Sugarcane biorefineries producing fuels, green chemicals, biopolymers and bio-products offer great potential for improving the profitability of sugarcane production. This paper will address the opportunities available for sugarcane biorefineries to contribute to future profitability and sustainability of the sugarcane industry.

The use of an acetoacetyl-CoA synthase in place of a -ketothiolase enhances poly-3-hydroxybutyrate production in sugarcane mesophyll cells

Engineering the production of polyhydroxyalkanoates (PHAs) into high biomass bioenergy crops has the potential to provide a sustainable supply of bioplastics and energy from a single plant feedstock. One of the major challenges in engineering C-4 plants for the production of poly[(R)-3-hydroxybutyrate] (PHB) is the significantly lower level of polymer produced in the chloroplasts of mesophyll (M) cells compared to bundle sheath (BS) cells, thereby limiting the full PHB yield-potential of the plant. In this study, we provide evidence that the access to substrate for PHB synthesis may limit polymer production in M chloroplasts. Production of PHB in M cells of sugarcane is significantly increased by replacing -ketothiolase, the first enzyme in the bacterial PHA pathway, with acetoacetyl-CoA synthase. This novel pathway enabled the production of PHB reaching an average of 6.3% of the dry weight of total leaf biomass, with levels ranging from 3.6 to 11.8% of the dry weight (DW) of individual leaves. These yields are more than twice the level reported in PHB-producing sugarcane containing the -ketothiolase and illustrate the importance of producing polymer in mesophyll plastids to maximize yield. The molecular weight of the polymer produced was greater than 2x10(6)Da. These results are a major step forward in engineering a high biomass C-4 grass for the commercial production of PHB.