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Additional file 2: Table S1. Chemical composition analysis of untreated South African sugarcane bagasse preparations used in this study. Table S2. Pretreatment outputs.

Additional file 1: Figure S1. HSQC NMR spectra (side chain region) of lignins isolated from depithed sugarcane bagasse (DB) using [TEA][HSO4] containing 20 wt% water and a solids loading of 20 wt% at 120 °C for 4 and 8 h. Representative substructures are shown. Figure S2. Particle size distribution of untreated pith (PB), long fiber (LFB) and short fiber bagasse (SFB) obtained after sieving within the pretreatment size fraction (0.18–0.85 mm). Inset values show calculated geometric mean particle length D50. Figure S3. Area-normalized size exclusion chromatograph of ionoSolv lignins isolated from industrially depithed (DB) and long fiber bagasse (LFB) pretreated in [TEA][HSO4] containing 20 wt% water at 120 °C for 4 h with 10 wt% solids loading. Mixed-D column, NMP eluent, 300 nm. Figure S4. Scanning electron micrographs of longitudinal view of, a pith bagasse before pretreatment, b pith bagasse after pretreatment, c long fiber bagasse before pretreatment and, d long fiber bagasse after pretreatment. Treatments were conducted in [TEA][HSO4] containing 20 wt% water and solids loading of 10 wt% at 120 °C for 4 h

In order to transition energy use away from fossil fuels, the transformation of renewable lignocellulosic biomass needs to be improved and its supply of sustainable bioenergy into a wider bioeconomy increased. Achieving this will require: (1) renewable, cheap and high-quality lignocellulosic feedstocks, (2) a robust process to transform this feedstock into bioenergy and finally (3) a system to recover and use this bioenergy as well as value-added products. In this PhD, multiple examples were used to explore unconventional feedstocks, novel transformation processes and opportunities for value-added products. Feedstocks ranging from invasive species threatening the UK environment, Rhododendron and Japanese Knotweed (Chapter 3), metal contaminated waste wood (Chapter 4), metal enriched biomass grown on marginal land (Chapter 5), and wastewater irrigated willow, a leading dedicated bioenergy crop (Chapter 6). While conventional bioenergy systems often burn wood pellets for energy co-generation, the innovative transformation process of ionic liquid-based technologies are explored as flexible enough to fractionate unconventional biomass feedstocks and deliver high yields of sustainable bioenergy and bioproducts. This was allowed by the unique and tuneable properties of protic ionic liquids. Here dimethylbutyl-hydrogen sulphate - [DMBA][HSO4], a cheap hydrogen sulphate [HSO4]- based ionic liquid, and 1-methylimidazolium chloride - [C1Him][Cl], were used in the ionic-liquid based ionoSolv process. Key efficiency parameters such as temperature, reaction time, biomass to solvent loading and solvent recycling, were explored. The process was also challenged with the presence of diverse metal contamination to determine the potential to extract the metals and produce a fermentable pulp and lignin in parallel. Bioenergy recovery from the ionoSolv process was explored as well as the potential to recover multiple value-added products. In addition to determination of heating values of isolated lignin as well as hydrolysis and fermentation yields of cellulose rich pulps into bioethanol, interactions of contaminating metals and their impact on yeast fermentation yields were investigated. This investigation highlights the benefits of [C1Him][Cl] ionic liquid pretreatment for the production of clean bioenergy and bioproducts from highly contaminated feedstocks. As an important property of ionic liquids is that they can act as media for electrochemical reactions, electrodeposition of metals from ionic liquid liquor, metal extraction efficiencies and any detrimental interactions with ionic liquid recycling were assessed. To further diversify system outputs beyond bioenergy alone, production of bio-oils, char and gases from pyrolysis of post-hydrolysis residue was determined, as well as the possibility for recovery of phytochemicals as potential complementary value-added products. This research highlights that unconventional feedstocks have the potential to support a developing bioeconomy and that the reallocation of waste and reclamation of contaminated soils and waters could act as a financial, social and environmental levers to improve the sustainability of bioenergy production. The studies also showcase how a versatile engineered ionic-liquid pretreatment has the potential to transform environmental burdens into resources that are compatible with the diversification of multiple product streams. Taken together, these findings can serve as a proof-of-concept for integrated scale-up of sustainable ionic-liquid based biorefinery. Open Access

This work proposed and investigated key strategies that contribute to the advancements of low-cost protic ILs (PILs) for use in the future sustainable chemical industry, particularly in the areas of waste valorization and decontamination. In large, this PhD research contributed to the ongoing development of a lignocellulose fractionation process using PILs. First, the use of contaminated waste wood was investigated as a low-cost alternative feedstock to expensive virgin biomass. Fractionation of post-consumer waste wood collected from construction activities was shown to be highly effective using 1-methylimidazolium chloride [H1Cim]Cl, producing a highly digestible metal-free cellulose pulp, with >70% glucose yield during enzymatic hyrolysis. Evaluation of key process parameters such as solid loading, waste wood composition variation, metal chelation with lignin and IL-clean up were also investigated. The study was expanded to include the valorization of hazardous creosote waste wood using the low-cost PIL N,N,N-dimethylbutylammonium hydrogen sulfate [DMBA][HSO4]. The fractionation produced a highly digestible, PAH-free cellulose pulp stream with 70% glucose release, and a PAH-lignin stream. Second, to develop a better understanding of the process boundary conditions, water use as co-solvent and anti-solvent was investigated using a variety of promising lignocellulosic biomass. It was shown that the impact of water as a co-solvent on the fractionation ability of [DMBA][HSO4] is feedstock-dependent. A reduced water input for lignin precipitation was found not to compromise the cellulose digestibility, while significantly reducing the process energy. In addition, the impact of ionoSolv pretreatment severity on fractionation performance was evaluated using a modified pretreatment severity factor, incorporating the Hammett acidity of the aqueous IL solution. The modified severity factor can better predict the fractionation outcome compared to the classical pretreatment severity factor, particularly regarding delignification and hemicellulose removal. Attention was then turned to utilization of the cellulose pulp derived from the ionoSolv process to produce functionalized nanocellulose crystals (CNCs). Alkaline-H2O2 oxidation was used as a simple and more environmentally friendly method for facile extraction of carboxylated CNCs. The impact of pretreatment severity and cellulose composition on the properties of extracted CNCs was evaluated. The produced CNCs had the ability to form self-standing nanofilms and exhibited similar thermal and colloidal stability to CNCs produced by TEMPO-mediated oxidation. Lastly, a novel approach for textile waste decontamination and synthetic dye reuse using PILs was developed. The PIL [DMBA][HSO4] was used to selectively extract dyes from polyester-based synthetic textiles, leaving the dye-free polyester fiber behind for upcycling. Subsequent dyeing using the dye-rich [DMBA][HSO4] solutions was shown to be possible, achieving a similar color strength to commercially dyed products. The process provides key and novel advantages that can provide a new circular dimension to the textile recycling sector by eliminating virgin dye use, applying a closed-loop solvent-based dyeing process, and creating dye-free polyester fibers. Open Access

In the context of climate change, it is essential to use renewable materials and to reduce the environmental footprint of industrial processes. This work focuses on the feasibility of implementing a low-cost Ionic Liquid (IL) in a large-scale biorefinery for bioethanol production (the ionoSolv process). The selected feedstock was Eucalyptus red grandis, a fast-growing hardwood. The lignocellulosic biomass was fractionated at laboratory scale, using aqueous N,N,N-trimethylammonium hydrogen sulfate (20 wt% water), at different temperatures and reaction times, to maximize glucose recovery (86%). Experiments under CO2 atmospheres (sub and supercritical) revealed that the ionoSolv process is pressure insensitive. A detailed Techno-Economic Analysis (TEA) for a biorefinery using the ionoSolv pretreatment was performed and compared to one using the acid-catalysed steam explosion pretreatment. With the ionoSolv pretreatment, the composition of the cellulose-rich pulps can be tailored and high-purity lignins can be recovered. The economic performance of both pretreatments are similar. From a sustainability perspective there are trade-offs: the ionoSolv process consumes 25% more energy (with potential for optimization) but consumes less chemicals and produces less waste. These results indicate that this process can be a competitive alternative. During the development of this process, and other IL-based processes, the interaction of ILs (neat and aqueous) with metals was investigated to establish suitable materials of construction. It was observed that the corrosion behaviour of metals exposed to ILs is system dependent. Surprisingly, water can act either as a corrosion inhibitor or promoter. A semi-quantitative classification method for the different corrosion behaviours observed was developed. Some metals exposed to aqueous ILs formed particles, resulting in the inadvertent development of a novel process for metal-based materials at large-scale: Oxidative Ionothermal Synthesis (OIS). A high-level TEA suggests that OIS offers economic and environmentally advantageous production of bulk and advanced metal-based materials, such as zinc oxide. Open Access

The temperature sensitivity of proteins hinders the bioavailability and safety of therapeutic antibodies and vaccines. These biomolecules require storage at 2 – 8 °C from manufacturing to administration to prevent thermal degradation, known as the ‘cold chain’. However, the cold chain cannot be implemented in resource-limited areas with inadequate infrastructure. Stabilising proteins at room temperature would break the ‘cold chain’ and allow life-saving treatments to be distributed to resource-limited areas, where infectious diseases can account for over half the mortality rate. In this thesis, surface engineering and ionic liquids were used to improve the storage-lifetime of proteins (Chapter 3 – 5). Surface engineering can improve thermal stability whilst increasing solubility in ionic liquids; however, ionic liquid-protein interactions are misapprehended and often contradictory. Therefore, a holistic multi-technique framework — a combination of scattering and spectroscopic techniques — was developed on the green fluorescent protein and avidin as an archetypal probe to decipher the complex interactions (Chapter 3). The framework revealed the effect of ionic liquid anion nuances on protein structure and stability. Surface engineering has been applied for the first time to ligand‐binding proteins (Chapter 4) and antibodies (Chapter 5) to create a thermally stable biofluid. Surface-engineering of avidin revealed a nanoconjugate with retention of activity and a half denaturation temperature of 139.0 °C and observed no denaturation after equivalent storage at 25 °C for 160 days. For the surface-engineering of antibodies, a maximum half denaturation temperature of 198.2 °C, while retaining binding activity, revealing that the high stability was transferable. However, a potential trade-off between thermal stability and bioavailability was observed. Regardless, the unprecedented transferable thermal stability presented herein provides a blueprint for stabilising vaccine candidates, avoiding the use of ultra-low temperature freezers. The stabilisation of these vaccines will be necessary for their dissemination to the unimmunised population to prevent or end a pandemic. Open Access

Petrochemical industry has experienced huge development in the last century, dominating different sectors such as plastics, detergents, pharmaceuticals and fertilizers. While technologies for processing of fossil feedstocks are now well established with marginal room for improvements, biorenewable chemicals derived from biomass struggle to be commercialised at large scale due to strong barriers experienced in R&D. Much research is in place to develop catalysts which can achieve high turnover numbers and high yield which can compete with the traditional processes, but the usage of lignocellulosic biomass requires innovative approaches which combine catalyst and solvent design with optimal separation minimizing emissions and waste streams. In this work, we will investigate ionic liquids as valuable solvents for the synthesis of high value biomonomers such as diformylfuran (DFF) and 2,5-furandicarboxylic acid (FDCA) starting from sugars. So far, the synthesis of these chemicals has seen huge challenges due to the difficulties in isolating the intermediate 5-HMF derived from sugar dehydration, which is the key molecule to produce these monomers. The usage of ionic liquids as solvents can lead to multiple advantages which can allow the production of these molecules at large scale, simplifying process separation and favouring high yield of reaction. Other bioderived products such as humins and surfactants will be studied as potential applications in the wastewater treatment for removal of heavy metals and detergents industry. A new category of biosurfactants derived from furfural exhibits remarkable proprieties which can have a potential higher cleaning ability compared the current oil derivatives ones. This thesis copes different aspects for an efficient sugar biorefinery, focused to substitute oil derivative consumer products, such as plastic and detergents, with the aim to achieve the reduction in CO2 emissions. Open Access

It is estimated that around 2 ×109 hectares of land (15% of global land area) has been degraded by human activity, and rates of soil degradation are increasing. Soil is a precious resource, it sustains almost all terrestrial life and provides 90% of the food that feeds humanity. New methods are needed to improve degraded soils and recover desertified soils, particularly in higher risk areas such as deforested areas in the tropics. Waste products of industrial bio-refining may provide a convenient and abundant material for use as a soil amendment, with the added benefit of increasing the carbon sequestration potential of bio-based products. Unfortunately, knowledge of how soils can be restored in a targeted manner are missing. This thesis aims to determine the key inter-particulate interactions which promote the formation of stable aggregates, with a focus on bio-refinery products as soil amendments. New methodologies are established in order to explore particulate interactions in the context of soils, and tested on artificial soils and aggregates with defined compositions. Abiotic artificial soils are studied, to identify the physical and chemical aggregate forming processes separately of biological processes. Particulate - clay interactions are found to be complex, and the mechanical properties of the aggregate are found not only to be dependent on surface chemistry but also on the fabric morphology, pore space and particle shape. Organosolv lignin, a biorefinery waste product, was shown to bind to the silica face of kaolinite, driven by hydrophobic interactions in suspension, and were found to strengthen kaolinite aggregates up to a percolation threshold. Video image analysis is used to determine slaking kinetics of aggregates submerged in a flow cell. The role of these interactions for soil development is also investigated, in order to determine if aggregates form by accumulating stable organo-mineral interactions over repeated wet/dry cycles. The nature of lignin-kaolinite interactions are investigated using solid-state magic angle spinning NMR and T1 relaxation NMR. Finally, a soil microcosm experiment was carried out, in order to determine the microbial response to the addition of lignin, which present a challenging substrate for ecosystems of degraded soils. These experiments illustrate the importance of particulate interactions for forming stable soil aggregates and soil structure, and the results provide insights into how particulates may be engineered to improve soils with a targeted approach. Open Access

Biofuel technology has been introduced to reduce the reliance on fossil fuels. It is also superior for addressing environmental issues, such as greenhouse gas (GHG) emissions. Biofuel production from lignocellulosic biomass has been attracting attention due to the high abundance of the feedstock and the incredible GHG emission reduction associated with the production process. Ionic liquid pretreament, the ionoSolv process, and organosolv pretreatment are well-known for their selective fractionation performance. The ionoSolv process is able to generate a highly digestible cellulose fraction and the organosolv process is famous for producing high quality lignin as the side product, where the lignin generated is suitable for value-added applications. Here, a hybrid pretreament process has been developed based on these two processes, where two protic ionic liquids and three organic solvents were the selected solvents. The new process has been tested on three classes of feedstocks, miscanthus, pine and agricultural residues. The pretreatment effectiveness was determined by enzymatic saccharification and compositional analysis. The isolated lignin fraction was subjected to HSQC and GPC analysis. For miscanthus, ethanol/butanol-IL process was able to produce a highly digestible pulp with a glucose yield of up to 85%, 10% higher than the standard ionoSolv pulp, due to more profound lignin removal for the hybrid process. The process maintained its functionality with a range of IL acidities, 1.00 to 1.02 (acid/base) and up to 50% wt biomass loading. Similar glucose yield increases were observed for pine, rice husk and bagasse. For the process of two straws, additional hemicellulose releases were detected in enzymatic hydrolysis while the level of glucose yields remained the same as for the ionoSolv ones. HSQC NMR of the lignin indicated that α-alkoxylation took place during ethanol/butanol-IL fractionation, inhibiting lignin condensation. Three major monolignols were synthesised and radical polymerisation induced by horseradish peroxidases was conducted for the monolignols synthesised. The lignin-like polymer was analysed by GPC. Open Access

This thesis is concerned with the valorization of the cellulose pulps produced using the ionoSolv pretreatment process. Experiments using both fresh and recycled ionic liquid, specifically triethylammonium hydrogen sulfate [TEA][HSO4], were conducted. Characterization of the isolated pulps was used to demonstrate the chemical and morphological effects of the ionoSolv pretreatment process on cellulose. Recycled ionic liquid contains solutes that impact the mechanism for lignocellulose biomass fractionation affecting the optimal pretreatment conditions. Pulp yields were consistently lower (demonstrating better pretreatment extraction, the pulps have a higher glucan content) and saccharification results were better when using recycled ionic liquid, demonstrating that the presence of these solute improved lignin extraction. In addition, it is demonstrated that protic ionic liquid pretreatment will eventually generate humins and pseudolignin, which are undesirable by-products of carbohydrate conversion which reduce saccharification yields and pulp purity. Experiments were conducted by doping the ionic liquid with 13C labelled and unlabeled sugars and their derivatives to ascertain the role of these solutes in the formation or prevention of pseudo-lignin formation. The re-deposition of these compounds onto the pulp is directly impacted by the purity of the pulp surface. The use of recycled ionic liquid in combination with additives such as HMF demonstrated a moderating effect on the formation of pseudolignin and humic compounds by increasing their solubility in the ionic liquid, preventing their re-deposition onto the surface of the pulp during pretreatment. In addition, lignin recovered from the pretreatment suggests the incorporation of carbohydrates, with lignin produced using recycled ionic liquid having higher molecular weights. The pulps produced during the pretreatment were submitted to subsequent processing to demonstrate additional potential value streams. Pulps were bleached to produce cellulose fibers and nanocellulose crystals. Fiber analysis suggests the pulps are of sufficient quality for materials production and various other industrial applications. Open Access

Reactivity of biomass relevant alcohols and esters in hydrogen sulfate based ionic liquids were successfully studied. A series of alcohols; methanol, 1-butanol, 2-butanol, isopropanol, and phenol were reacted with 1-butylimidazolium hydrogen sulfate [HC4im][HSO4] to represent cellulose hydroxyl group reactivity in the selected acidic ionic liquids. Apart from phenol, all selected alcohols were found to undergo sulfation via nucleophilic substitution by [HSO4]− anion after 24 h. Mechanistic study of the nucleophilic substitution reactions of p-substituted benzyl alcohols with [HC4im][HSO4] and [C4C1im][HSO4] were done using Hammett equation. The concave upward Hammett plots suggest there is a change in mechanism, from SN1 for electron donating substituents (OMe and Me) to SN2 for electron withdrawing substituents (H, Cl, CF3 and NO2). This chapter revealed that ILs can act as a solvent as well as a nucleophile in one reaction. Most importantly, this showed that sulfation of cellulose is probable under Ionosolv pretreatment. Hydrolysis of lignin-carbohydrate linkage model compounds, BHB and THFF were carried out in [HC4im][HSO4] with varying acid and water content. The rates of hydrolysis were increasing with increasing of mol% acid. The mechanistic insights of the reactions were studied using Zucker-Hammett, Bunnett-Olsen and Eyring equations. All three resulted plots are in agreement with A2 mechanism. Transesterification reactions of BHB and ethyl lactate and methanol in [HC4im][HSO4] were found to give low yields due to the sulfation of methanol. This again showed that the [HSO4]¯anion are reactive and nucleophilic enough to undergo nucleophilic substitution reaction with alcohol at 80 – 120 °C in aqueous or non-aqueous condition. Open Access

In recent years, Carbon Capture and Storage (CCS) has been proposed as a potential method to allow the continued use of fossil-fuelled power stations whilst preventing emissions of CO2 from reaching the atmosphere. Currently, CO2 capture is dominated by amine-based (e.g., monoethanolamine) technologies, which are very energy intensive and less attractive from an environmental point of view due to emissions of the used volatile solvent components. Ionic liquids have been proposed as a promising alternative to the conventional volatile solvents, because of their low volatility, wide liquid range, high thermal stabilities and tunable properties. This remarkable interest has led to a rapid growth of literature on this specific subject. The work in this thesis can be divided into two main areas: the first part is about the thermophysical properties of ionic liquids, including density, viscosity and thermal stability, for both conventional room-temperature ionic liquids and novel synthesized amine-functionalized ionic liquids. The second part of research is focus on the CO2 uptake ability of synthesized ionic liquids, including the comparison of the amine-functionalized ionic liquids to conventional ionic liquids, and more detailed study on the best performing ionic liquid, 1-ethylamine -3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([C2NH2C1im][NTf2]), including the temperature effect, pressure effect and performance after absorption/desorption cycles. In addition, donor number value is gathered for five ionic liquids to provide more information on predicting CO2 absorption performance. Open Access

Lignocellulosic biomass is considered as a promising alternative bio-based renewable resource that can be utilized for the production of energy, fuels, chemicals and materials. In order to realise its full potential, a pretreatment step which breaks up the biomass matrix and separates the lignocellulosic biopolymers is required. Research in this field has been ongoing for several decades, however, the development of cost efficient pretreatment processes and novel materials derived from lignocellulose is still a challenging and important task. The work described in this thesis focuses on two main aspects, namely biomass pretreatment using ionic liquids and synthesis of value added materials based on lignin, to aid the advancement of a biobased economy. Firstly, pretreatment of the hardwood willow was undertaken using the low-cost ionic liquid solution triethylammonium hydrogensulfate [N2220][HSO4]80%. The effect of the pretreatment severity (described by the pretreatment temperature, residence time and ionic liquid acid/base ratio) on the pulp composition and digestibility as well as the lignin properties was studied. The pretreatment conditions significantly affected the pulp properties, lignin molecular structure and molecular weight. High pulp digestibility and glucose yields were achieved for the optimised pretreatment severity. The hardwood willow is a potential feedstock for a biorefinery and characterized through its genomic diversity. The effects of ionic liquid pretreatment using the ionic liquid solution triethylammonium hydrogensulfate [N2220][HSO4]80% on 14 willow varieties was investigated to understand which lignocellulose and pulp property influences the glucose release. The lignin content of the biomass and pulp did not significantly influence the glucose release, but pulp properties such as available surface area, pore size and cellulose degree of polymerization did play a role in pulp digestibility. The use of lignin as a macromonomer for the synthesis of materials increases the cost efficiency of a potential biorefinery and the development of lignin based copolymers was investigated in the second part of this thesis. An ionic liquid mediated pathway for the synthesis of lignin-poly(furfuryl alcohol) copolymers was discovered in which poly(furfuryl alcohol) is grafted from the lignin polymer. The effect of different reaction conditions and lignin-to-fufuryl alcohol ratios on the properties of the synthesized copolymers was investigated. The material properties strongly depended on the reactant ratio. These copolymers display a high carbon content and thus are promising candidates for the production of carbon fibres or glassy carbon. Furthermore, protic ionic liquids were used as a combined solvent and catalyst system for the addition of aromatic compounds to the lignin polymer. Aromatic compounds such as 6-bromo-2-naphthol and 6,6’-dibromo-1,1'-bi-2-naphthol were successfully incorporated in the lignin polymer. This introduces a bromine functionality to the lignin structure which can be used as a reactive functionality to graft polymers from lignin via cross-coupling reactions offering a novel and simple synthesis pathway for the versatile functionalization of lignin and the production of lignin based materials. The implementation of a lignocellulosic biomass based biorefinery is still faces challenges such as high enzyme and high investment costs amongst others, thus limiting the number of the already existing biorefineries. The development of new technologies for biomass fractionation (e.g. using low-cost, recyclable ionic liquid solutions) and the utilization of lignin for the production of value added chemicals and materials are step stones to aid the realisation of a biobased economy. Open Access

The current dominant raw material for the energy and chemical sectors worldwide is crude oil. Within the last few years, petroleum prices have increased, geo-political stability has decreased, and supply has been threatened, driving a renewed interest across academic, government, and corporate centres to utilise biomass as an alternative raw material. The quest to make useful chemicals and fuels, which are potentially versatile intermediates between bio-based carbohydrate chemistry and petroleum-based organic chemistry, is among the most active research areas in chemistry. Many of the most promising alternatives for the sustainable supply of fuels and valuable chemicals are biomass resources. However, this process requires both economically and environmentally efficient methods before a biorefinery platform can be built on the basis of this route. In this research, the high-yield conversion of fructose, glucose and cellulose to the versatile intermediate 5-hydroxymethylfurfural (HMF) is described. The role of different transition metal salts in dithiocarbamate-based, hydrogensulfate-based and chloridebased ionic liquids in this process was investigated. Among all those investigated, the chromium(III) chloride catalyst system in hydrogensulfate-based ionic liquids was found to be the most effective method for conversion of fructose, giving an HMF yield of 96% whereas a chloride-based ionic liquid was found to be the most effective method for converting glucose to HMF, achieving a yield of 90%. Little or no side products (such as formic acid, levulinic acid, or humins) were observed, indicating the selectivity of the system. The activity of various catalysts for the conversion of fructose to 5-hydroxymethylfurfural (HMF) was investigated under different conditions including lower and higher temperatures and addition of co-solvents. The most challenging of the substrates investigated was cellulose, representing the closest substrate to untreated biomass. The best yield of HMF obtained from this substrate was 58% after one hour at 150 °C, which compares well with leading values in the literature. In these studies, the effects of temperature, substrate concentration, co-solvent addition and the nature of the ionic liquid were all investigated. Open Access

Ionic liquid (IL) pretreatment has gained significant attention for biomass deconstruction. However, residual IL left in biomass can cause enzyme deactivation, leading to problems with further processing of biomass to sugars. Cellulases are enzymes commonly used in the saccharification process and the activity of this enzyme is severely affected by ILs. This thesis focuses on understanding how ILs affect the activity of cellulases in the enzymatic saccharification process. Sigmacell cellulose was used in the enzymatic saccharification process instead of biomass plant. Seven different ILs were added in the enzymatic saccharification mixtures and the activity of a mixture of commercially available cellulases was measured using high-performance liquid chromatography (HPLC) to measure glucose release. Sulphate based ILs were more harmful for cellulase action than [EMIM][OAc], [BMIM]Cl and phosphate based ILs. [HBIM][HSO4] inactivated commercial cellulases (Celluclast®) and cellobiase (Novozyme188) in 4 h in the enzymatic saccharification process. In this thesis, it was observed that the main factor that affects the activity of cellulase is pH. To overcome this, acidophilic cellulases were suggested to improve the activity of the enzymes in ILs. Endoglucanase from Cryptococcus sp.S-2 and β-glucosidase from Thermofilum pendens were studied and tested with ILs. The result of this study has shown endoglucanase from Cryptococcus sp. as a promising enzyme in IL biomass deconstruction. There was a slight improvement in the activity of β-glucosidase from T.pendens with cellobiose in 1% [TEA][HSO4] but when both of these enzymes were combined and acting against carboxymethylcellulose (CMC) with 10% (v/v) of [TEA][HSO4], the enzymes were still active. Based from these findings, pretreatment process using low cost IL is favourable as it is cost effective and more efficient. Open Access

The environmental impact of the extensive use of fossil fuels to produce fuels and chemicals as well as the cost sensitivity of platform chemicals to oil prices remain major concerns. Sustainable biomass processing is the only viable solution for the large-scale production of bio-based products with a projected average of 30 % of the production of chemicals to derive from biomass within the EU (Joint European Biorefinery Vision for 2030). The co-production of fuels and high-value bio-derived chemicals would contribute to the environmental sustainability, as well as the economic viability, of the future biorefineries. Lignocellulosic biomass is relatively inexpensive and abundant, without competing with food sources, and thus it can be considered as the most suitable feedstock for a full-scale biorefinery. The production of highly functionalized intermediates such as 5-(hydroxymethyl)furfural (5-HMF) and 2,5-dimethylfuran (2,5-DMF) from lignocellulose has been established. The conversion of such platform chemicals into other high-value products remains a challenge. A plethora of published studies focused on the transformations of the functional groups rather than using the furan ring chemistry. The overall project objective was the study of novel reaction protocols to produce heterocyclic and aromatic platform chemicals from established biomass intermediates by using ionic liquids as catalysts or co-catalysts. Thiophene synthesis from bio-derived furanics, such as 2,5-DMF, was studied under acidic conditions, focusing on Lewis acidic ionic liquids that promoted the recyclization of bio-derived 2,5-DMF into its thiophene analogue. Moisture-stable chlorometallate ILs were synthesised and characterised separately to elucidate speciation, acidity and thermal stability prior to being applied towards thiophene synthesis in liquid-liquid biphasic reactions. Lewis acidic chlorozincate(II) ionic liquids were also supported on an inexpensive, high surface area silica support, affording supported ionic liquids (SILPs) that were studied for their surface properties and IL/support interactions. The SILP materials were successfully tested as catalysts for the novel, gas-phase synthesis of 2,5-dimethylthiophene (2,5-DMT) in a pressurised fluidised bed reactor that was constructed in-house. The latter experiments aimed for process control where parameters such as temperature and substrate/H2S ratios were controlled with the overall process economics improving as a thin layer of ionic liquid was used instead of a biphasic system. 2,5-DMF was also investigated as an activated diene in acid-catalysed Diels-Alder reactions in the presence of maleic anhydride towards the synthesis of phthalic anhydride derivatives. The domino reactions for the formation of 3,6-dimethylphthalic anhydride (3,6-DMPA), a potential precursor for polymer synthesis, was studied in binary IL/acid systems of different acidity profiles, indicating the potential towards the direct synthesis of 3,6-DMPA from bio-based furanic compounds. Open Access

Lignocellulosic biomass has the potential to be used as feedstock for the sustainable and carbon-neutral production of fuels, materials and chemicals. For the realisation of this potential, cost-effective fractionation of the biomass in different product streams is necessary. The work presented in this thesis focuses on the use of protic ionic liquids for the fractionation of various types of lignocellulosic biomass with the aim of achieving process improvements leading to a potential cost reduction at industrial scale. Firstly, the use of triethylammonium hydrogensulfate as a lower-cost alternative to more commonly used aprotic ionic liquids for the fractionation of the grass Miscanthus x giganteus has been shown. A cellulose enriched pulp giving high enzymatic saccharification yields was recovered after pretreatment under mild conditions. Subsequent process intensification showed that high saccharification yields can be obtained after as little as 15 min of pretreatment time. Secondly, the more easily grown yet more recalcitrant softwood pine was used as a feedstock. N,N-dimethylbutylammonium hydrogensulfate was found to be effective at producing a highly digestible cellulose pulp. Thirdly, waste wood from construction and demolition as well as pre-consumer construction wood containing various heavy metals (as preservatives and contaminants) were successfully fractionated. The biocide copper was quantitatively extracted from copper azole treated wood, allowing for the production of bioethanol via fermentation. The copper was shown to be recoverable via electrodeposition. The use of contaminated waste wood as a feedstock for the production of materials, fuels and chemicals not only eliminates an unresolved waste management problem but also increases the economic viability of the bioeconomy. The ionic liquid was shown to be recyclable at least six times without losing performance. Alongside the pulp, the obtained lignin was analysed using 31P and 2D HSQC NMR, gel permeation chromatography and elemental analysis in order to elucidate the lignin extraction mechanism. Open Access

Lignocellulosic biomass and Algae are feedstocks that hold great potential in production of sustainable biofuels, materials and chemicals. This work focusses on the application of ionic liquid based fractionation of lignocellulosic biomass and algae. Firstly, the pretreatment of Miscanthus x giganteus using different ionic liquid with low concentration was performed. The delignification yield increased gradually as concentration of ionic liquids go higher. Dilute triethylammonium hydrogen sulphate with different cation: anion ratio was used in pretreatment of miscanthis, pine and willow. The results showed increased removal of cellulose, hemicellulose and lignin with lower cation: anion ratio. Liquor analysis on liquid phase after pretreatment using triethylammonium hydrogen sulphate on Miscanthus under varies of conditions indicate that hemicellulose removal rate is better under conditions of higher temperatures and longer reaction time. Secondly, Algae fractionation using ionoSolv process was tested. Among all seaweeds tested, Posidonia oceanica showed high saccharification yield after pretreatment under very mild conditions. Finally, 2-stage pretreatment aiming to remove hemicellulose and lignin in two separate steps was carried out. The results showed promising hemicellulose removal and delignification yield with promising saccharification yield. A further study on using recycled ionic liquid in the 2nd stage pretreatment has demonstrated consistent results of effective pretreatment over 5 cycles. Open Access

Lignocellulosic biomass has the potential to partially displace fossil fuels in the production of energy, materials, chemicals and transportation fuels. In order to realize this potential, cost-effective and low-energy fractionation of biomass into different product streams is necessary. This work uses low-cost protic ionic liquids to deconstruct and fractionate lignocellulosic biomass, with a focus on agricultural residues, energy crops and woody biomass. It aims to demonstrate process intensification and scale-up leading to potential cost reductions. Firstly, the use of triethylammonium hydrogen sulfate was investigated as an alternative to expensive, thermally unstable aprotic ionic liquids for pretreatment of high-ash and high-lignin agroresidues. Pretreatment of sugarcane bagasse, rice husk and rice straw was shown to give up to 90% glucose release under mild conditions. 100-fold scale-up of ionoSolv pretreatment from the 10 mL to the 1 L-scale was performed, enabling evaluation of key parameters expected to impact process economics, including solids loading, feedstock particle size and stirring speed. It was shown that excellent performance could be maintained using a biomass to solvent ratio of 1:2 g/g. Use of larger particle sizes up to 10 mm offered significant potential to reduce comminution energy. Attention was then turned to utilization of the lignin stream derived from ionoSolv biorefining. Fine-tuning of the process severity (temperature, time, acidity) enabled control of lignin molecular weight and functionality. Fast pyrolysis of mildly treated lignins gave the highest bio-oil yields with maximal energy recovery in the liquid product. Fractional precipitation produced lignin fractions with narrow polydispersity and low molecular weight of 1000 g mol-1 with yields of up to 14 wt%. Potential applications of lignin were assessed based on HSQC NMR, 31P NMR, gel permeation chromatography and thermal analysis. Lignin condensation and re-precipitation were identified as the most important parameters affecting both glucose yields and valorization potential of lignin. Open Access

In this thesis, mechanistic insights into lignin depolymerisation and more specifically, the hydrolysis of the abundant β-O-4 ether linkage was gained. This was accomplished by exploring the reactivity of different lignin model compounds in acidic ionic liquids. Reported herein are investigations into the reactivity of 2-phenoxyethanol in 1-butylimidazolium hydrogensulfate with varying amounts of sulfuric acid. Ring sulfonation was observed with the ether bond remaining intact. A similar study was then conducted using 1-ethyl-3-methylimidazolium bromide in the presence of hydrobromic acid. A Hammett plot confirmed the mechanism of ether cleavage proceeding via an A2 mechanism, yielding the phenolic product and 2-bromoethanol. The preparation of an array of lignin model compounds bearing the full β-O-4 linkage was also accomplished. Mechanistic studies indicated that the rate determining step involves substrate dehydration followed by hydrolysis in ionic liquid media. This is analogous to the mechanism reported in acidic aqueous media, however was found to proceed 25 times faster. The reactivity of guaiacolglycerol-β-guaiacol ether was compared between hydrogensulfate ionic liquids. An Eyring investigation showed a consistent mechanism in all ionic liquids with full isokinetic compensation observed between the ΔH‡ and ΔS‡ components. The differences in the absolute values of ΔH‡ and ΔS‡ were deduced to be due to the interaction of the hydrogensulfate anion with the protonated substrate, influenced primarily by the associative forces between the cation and anion, namely hydrogen bonding and sterics. A final study explored the oxidative depolymerisation of ionosolv lignin using a polyoxometalate catalyst in 1-butylimidazolium hydrogensulfate. Vanillin and syringaldehyde were identified to be the products of highest abundance and it was observed that shorter pretreatment times led to higher aldehyde yields. Preliminary studies identified that a 5 wt % POM loading with hydrogen peroxide at 100 ° C provided the optimal conditions to yield the aldehydes. Open Access

The development of a pure and thus efficient fuel is of increasing importance in the use of a finite resource to be environmentally responsible and economically viable. Trace amounts of dissolved sodium has been identified as a suspect leading to the formation of internal diesel injector deposits (IDIDs) which proves problematic by reducing fuel efficiency in high pressure diesel injectors. Zinc contaminants have been identified as suspects leading to nozzle deposit formation and copper contaminants quickly reduce the oxidation stability of diesel fuel. I demonstrate in this research the successful extraction of ppm levels of Na+, Zn2+ and Cu2+ from model diesel fuels. A range of ionic liquids (ILs) with a variety of cations and anions were examined for their effectiveness at different loadings of IL relative to the fuel. Results provide several clear trends with some exceptional capabilities of the ILs in the extractions. The tunable properties of the ILs ions allow the ‘design’ to meet the requirements for a particular target and here provide several potential candidates for the extractions 23Na NMR was used in the determination of donor number (DN) and Kamlet-Taft parameters were gathered for each IL providing information of possible hydrogen-bond acidity / basicity (α / β) and dipolarity/polarizability solvent strength (𝜋*). In addition the non-random two liquid model (NRTL) was applied to correlate the experimental extraction results and determine τ parameters for each of the ILs. Donor number (DN), τ parameters and β values in addition to interfacial tension and viscosity provide information of the extraction mechanisms and predict performance, enabling a chemical design of ILs that are ideal for fuel purification. Open Access

The reactivity of cellulose in alkylimidazolium hydrogen sulfate-water ([CnCmim][HSO4]/H2O) mixtures during a biomass deconstruction process at 120 °C was investigated. Two types of sample, Miscanthus and a model polymer cellulose, Microcrystalline Sigmacell-Cellulose (MCC), were used. The studied variables included: [HSO4]-ionic liquids with different acidities, 1-butylimidazolium hydrogen sulfate, [HC4im][HSO4], and 1-butyl-3-methylimidazolium hydrogen sulfate, [C4C1im][HSO4]; acid-to-water ([CnCmim][HSO4]/H2O) ratio, and incubation period. A number of analysis tools and chemical methods were employed to characterise the resultant cellulose products: Scanning Electron Microscopy and Energy Dispersed X-Ray (SEM-EDX), Infrared Spectroscopy, Matrix Assisted Laser Desorption/Ionisation with Time of Flight (MALDI-TOF) Mass Spectroscopy, CHNS elemental analysis, viscosity measurement, compositional analysis and enzymatic saccharification. Deconstruction of Miscanthus in a [HC4im][HSO4]/H2O mixture at 120 °C for 22 h successfully separated cellulose, hemicellulose and lignin. A study on the purification of cellulose sample found that inadequate washing allowed the [HC4im][HSO4] traces to be physically adsorbed. After an extensive washing, indirect evidence, indicating that [HSO4]- anions had chemically adsorbed, was revealed. An investigation involving incubation of MCC in [CnCmim][HSO4]/H2O mixtures at 120 °C was conducted, replicating the deconstruction process. MALDI-TOF analysis demonstrated that the “[HSO4]” anion had chemically adsorbed on the surface of cellulose, forming sulfur-containing oligosaccharides. However, the type of bond responsible for chemisorption could not be identified. The [HSO4]- anion was the active species for chemisorption, regardless of different acidities of ionic liquids. Incubating MCC in [CnCmim][HSO4]/H2O mixtures at 120 °C also exhibited an interesting interplay between chemisorption and depolymerisation. A positive relationship was predominant in the presence of lower water content. Increasing water content displayed a negative relationship. Open Access

There has been much recent interest in using ionic liquids for processing lignocellulosic biomass. While cellulose has an acknowledged application in generating biofuels, it would be valuable to use the abundant lignin present as well. Rhodococcus has been reported previously to degrade lignin. Therefore, it is attractive to consider a scheme in which an ionic liquid is also used to enhance the microbial breakdown of lignin (bioligninolysis). By using vanillic acid as model compound (Chapter 3), results showed that Rhodococcus UKMP-5M is able to degrade vanillic acid as a sole carbon source at 10mM concentration to give the highest growth rate. An oxygen-dependent reaction degrades vanillic acid into protocatechuic acid and formate in a previously undescribed metabolic pathway. In Chapter 4, GC-MS demonstrated guaiacol as the major product of lignin degradation and the lignin degradation assay indicates that the treatment of lignin with ionic liquids assist the lignin degradation despite some ionic liquids showing a toxicity effect on the cells. Toxicological studies (Chapter 5) demonstrated different ionic liquids show varying toxicity to the bacteria. By using classical disk diffusion test in screening 16 different ionic liquids, it was revealed that the toxicity is correlated with the size of the ionic alkyl chain; however, the carbon atom count, not the structure or the distribution of those atoms in the cation, correlates directly with the toxicity. The strongest link was discovered with pH effects rather than with structure in the toxicity of acidic ionic liquids. We also propose that the octanol-water partition coefficient (Kow) has the controlling impact on the toxicity of ionic liquids. Bacterial growth curves exhibited three different trends: a complete inhibition, an increase in toxicity with an increase of ionic liquid concentration and the extension of the lag phase due to bacterial adaptation. This study established that Rhodococcus UKMP-5M could adapt and grow in the presence of ionic liquids with 1-ethyl-3-methylimidazolium acetate, [Emim][OAc] as the most promising candidate in designing an ionic liquid-facilitated system of bioligninolysis. Open Access