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

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

A study of ionic liquids for dissolution of sugarcane bagasse

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

ATR-FTIR measurement of biomass components in phosphonium ionic liquids

Qualitative and quantitative measurements of biomass components dissolved in the phosphonium ionic liquids (ILs), trihexyltetradecylphosphonium chloride ([P66614]Cl) and tributylmethylphosphonium methylsulphate ([P4441]MeSO 4), are obtained using attenuated total reflectance-FTIR. Absorption bands related to cellulose, hemicelluloses, and lignin dissolution monitored in situ in biomass-IL mixtures indicate lignin dissolution in both ILs and some holocellulose dissolution in the hydrophilic [P4441]MeSO 4. The kinetics of lignin dissolution reported here indicate that while dissolution in the hydrophobic IL [P66614]Cl appears to follow an accepted mechanism of acid catalyzed -aryl ether cleavage, dissolution in the hydrophilic IL [P4441]MeSO 4 does not appear to follow this mechanism and may not be followed by condensation reactions (initiated by reactive ketones). The measurement of lignin dissolution in phosphonium ILs based on absorbance at 1510 cm 1 has demonstrated utility. When coupled with the gravimetric Klason lignin method, ATR-FTIR study of reaction mixtures can lead to a better understanding of the delignification process. © 2012 Copyright Taylor and Francis Group, LLC.

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

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

Ionic liquid pre-treatment and fractionation of sugarcane bagasse for the production of bioethanol

Pretretament is an essential and expensive processing step for the manufacturing of ethanol from lignocellulosic raw materials. Ionic liquids are a new class of solvents that have the potential to be used as pretreatment agents. The attractive characteristics of ionic liquid pretreatment of lignocellulosics such as thermal stability, dissolution properties, fractionation potential, cellulose decrystallisation capacity and saccharification impact are investigated in this thesis. Dissolution of bagasse with 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) at high temperatures (110 �‹C to 160 �‹C) is investigated as a pretreatment process. Material balances are reported and used along with enzymatic saccharification data to identify optimum pretreatment conditions (150 �‹C for 90 min). At these conditions, the dissolved and reprecipitated material is enriched in cellulose, has a low crystallinity and the cellulose component is efficiently hydrolysed (93 %, 3 h, 15 FPU). At pretreatment temperatures < 150 �‹C, the undissolved material has only slightly lower crystallinity than the starting. At pretreatment temperatures . 150 �‹C, the undissolved material has low crystallinity and when combined with the dissolved material has a saccharification rate and extent similar to completely dissolved material (100 %, 3h, 15 FPU). Complete dissolution is not necessary to maximize saccharification efficiency at temperatures . 150 �‹C. Fermentation of [C4mim]Cl-pretreated, enzyme-saccharified bagasse to ethanol is successfully conducted (85 % molar glucose-to-ethanol conversion efficiency). As compared to standard dilute acid pretreatment, the optimised [C4mim]Cl pretreatment achieves substantially higher ethanol yields (79 % cf. 52 %) in less than half the processing time (pretreatment, saccharification, fermentation). Fractionation of bagasse partially dissolved in [C4mim]Cl to a polysaccharide rich and a lignin rich fraction is attempted using aqueous biphasic systems (ABSs) and single phase systems with preferential precipitation. ABSs of ILs and concentrated aqueous inorganic salt solutions are achievable (e.g. [C4mim]Cl with 200 g L-1 NaOH), albeit they exhibit a number of technical problems including phase convergence (which increases with increasing biomass loading) and deprotonation of imidazolium ILs (5 % - 8 % mol). Single phase fractionation systems comprising lignin solvents / cellulose antisolvents, viz. NaOH (2M) and acetone in water (1:1, volume basis), afford solids with, respectively, 40 % mass and 29 % mass less lignin than water precipitated solids. However, this delignification imparts little increase in saccharification rates and extents of these solids. An alternative single phase fractionation system is achieved simply by using water as an antisolvent. Regulating the water : IL ratio results in a solution that precipitates cellulose and maintains lignin in solution (0.5 water : IL mass ratio) in both [C4mim]Cl and 1-ethyl-3-methylimidazolium acetate ([C2mim]OAc)). This water based fractionation is applied in three IL pretreatments on bagasse ([C4mim]Cl, 1-ethyl-3-methyl imidazolium chloride ([C2mim]Cl) and [C2mim]OAc). Lignin removal of 10 %, 50 % and 60 % mass respectively is achieved although only 0.3 %, 1.5 % and 11.7 % is recoverable even after ample water addition (3.5 water : IL mass ratio) and acidification (pH . 1). In addition the recovered lignin fraction contains 70 % mass hemicelluloses. The delignified, cellulose-rich bagasse recovered from these three ILs is exposed to enzyme saccharification. The saccharification (24 h, 15 FPU) of the cellulose mass in starting bagasse, achieved by these pretreatments rank as: [C2mim]OAc (83 %)>>[C2mim]Cl (53 %)=[C4mim]Cl(53%). Mass balance determinations accounted for 97 % of starting bagasse mass for the [C4mim]Cl pretreatment , 81 % for [C2mim]Cl and 79 %for [C2mim]OAc. For all three IL treatments, the remaining bagasse mass (not accounted for by mass balance determinations) is mainly (more than half) lignin that is not recoverable from the liquid fraction. After pretreatment, 100 % mass of both ions of all three ILs were recovered in the liquid fraction. Compositional characteristics of [C2mim]OAc treated solids such as low lignin, low acetyl group content and preservation of arabinosyl groups are opposite to those of chloride IL treated solids. The former biomass characteristics resemble those imparted by aqueous alkali pretreatment while the latter resemble those of aqueous acid pretreatments. The 100 % mass recovery of cellulose in [C2mim]OAc as opposed to 53 % mass recovery in [C2mim]Cl further demonstrates this since the cellulose glycosidic bonds are protected under alkali conditions. The alkyl chain length decrease in the imidazolium cation of these ILs imparts higher rates of dissolution and losses, and increases the severity of the treatment without changing the chemistry involved.

Towards a commercial lignocellulosic ethanol industry in Australia : the Mackay Renewable Biocommodities Pilot Plant

In 2007, the Queensland University of Technology (QUT) received funding from the Australian Government through the NCRIS program and from the then Queensland Government Department of State Development to construct a pilot research and development facility for the production of bioethanol and other renewable biocommodities from biomass including sugar cane bagasse. This facility is being constructed adjacent to the Racecourse Sugar Mill in Mackay and is known as the Mackay Renewable Biocommodities Pilot Plant (MRBPP). The MRBPP will be capable of processing biomass through a pressurised pretreatment reactor and includes equipment for enzymatic saccharification, fermentation and distillation to produce ethanol. Lignin and fermentation co-products will also be produced at a pilot scale for product development and testing.

Laboratory and pilot scale pretreatment of sugarcane bagasse by acidified aqueous glycerol solutions

Pretreatment of sugarcane bagasse with acidified aqueous glycerol solution was evaluated at both laboratory and pilot scales. Laboratory scale pretreatment (4.00 g dry mass in 40.00 g liquid) with glycerol solutions containing ≤ 20 wt% water and 1.2 wt% HCl at 130 °C for 60 min resulted in biomass having glucan digestibilities of ≥ 88%. Comparable glucan enzymatic digestibility of 90% was achieved with bagasse pretreated at pilot scale (10 kg dry mass in 60 kg liquid) using a glycerol solution containing 0.4 wt% HCl and 17 wt% water at 130 °C for 15 min. We attribute more efficient pretreatment at pilot scale (despite shorter reaction time and reduced acid content) to improved mixing and heat transfer in a horizontal reactor. Pretreatment of sugarcane bagasse with acid-catalysed glycerol solutions likely produces glycerol-glycosides, which together with hydrolysed lignin are potential substrates for the production of biopolymers.

Low temperature pretreatment of sugarcane bagasse at atmospheric pressure using mixtures of ethylene carbonate and ethylene glycol

A low temperature lignocellulose pretreatment process was developed using acid-catalysed mixtures of alkylene carbonate and alkylene glycol. Pretreatment of sugarcane bagasse with mixtures of ethylene carbonate (EC) and ethylene glycol (EG) was more effective than that with mixtures of propylene carbonate (PC) and propylene glycol (PG). These mixtures were more effective than the individual components in making bagasse cellulose more amenable to cellulase digestion. Glucan digestibilities of ≥87% could be achieved with a wide range of EC to EG ratios from 9:1 to 1:1 (w/w). Pretreatment of bagasse by the EC/EG mixture with a ratio of 4:1 in the presence of 1.2% H2SO4 at 90 °C for 30 min led to the highest glucan enzymatic digestibility of 93%. The high glucan digestibilities obtained under these acidic conditions were due to (a) the ability of alkylene carbonate to cause significant biomass size reduction, (b) the ability of alkylene glycol to cause biomass defibrillation, (c) the ability of alkylene carbonate and alkylene glycol to remove xylan and lignin, and (d) the magnified above attributes in the mixtures of alkylene carbonate and alkylene glycol.

Pretreatment of sugarcane bagasse by acidified aqueous polyol solutions

Pretreatments of sugarcane bagasse by three high boiling-point polyol solutions were compared in acid-catalysed processes. Pretreatments by ethylene glycol (EG) and propylene glycol solutions containing 1.2 % H2SO4 and 10 % water at 130 °C for 30 min removed 89 % lignin from bagasse resulting in a glucan digestibility of 95 % with a cellulase loading of ~20 FPU/g glucan. Pretreatment by glycerol solution under the same conditions removed 57 % lignin with a glucan digestibility of 77 %. Further investigations with EG solutions showed that increases in acid content, pretreatment temperature and time, and decrease in water content improved pretreatment effectiveness. A good linear correlation of glucan digestibility with delignification was observed with R2 = 0.984. Bagasse samples pretreated with EG solutions were characterised by scanning electron microscopy, Fourier transform infrared spectroscopy and X-ray diffraction, which confirmed that improved glucan enzymatic digestibility is mainly due to delignification and defibrillation of bagasse. Pretreatment by acidified EG solutions likely led to the formation of EG-glycosides. Up to 36 % of the total lignin was recovered from pretreatment hydrolysate, which may improve the pretreatment efficiency of recycled EG solution.

Glycerol carbonate as green solvent for pretreatment of sugarcane bagasse

Background Pretreatment of lignocellulosic biomass is a prerequisite for effective saccharification to produce fermentable sugars. We have previously reported an effective low temperature (90 °C) process at atmospheric pressure for pretreatment of sugarcane bagasse with acidified mixtures of ethylene carbonate (EC) and ethylene glycol (EG). In this study, “greener” solvent systems based on acidified mixtures of glycerol carbonate (GC) and glycerol were used to treat sugarcane bagasse and the roles of each solvent in deconstructing biomass were determined. Results Pretreatment of sugarcane bagasse at 90 °C for only 30 min with acidified GC produced a solid residue having a glucan digestibility of 90% and a glucose yield of 80%, which were significantly higher than a glucan digestibility of 16% and a glucose yield of 15% obtained for bagasse pretreated with acidified EC. Biomass compositional analyses showed that GC pretreatment removed more lignin than EC pretreatment (84% vs 54%). Scanning electron microscopy (SEM) showed that fluffy and size-reduced fibres were produced from GC pretreatment whereas EC pretreatment produced compact particles of reduced size. The maximal glucan digestibility and glucose yield of GC/glycerol systems were about 7% lower than those of EC/ethylene glycol (EG) systems. Replacing up to 50 wt% of GC with glycerol did not negatively affect glucan digestibility and glucose yield. The results from pretreatment of microcrystalline cellulose (MCC) showed that (1) pretreatment with acidified alkylene glycol (AG) alone increased enzymatic digestibility compared to pretreatments with acidified alkylene carbonate (AC) alone and acidified mixtures of AC and AG, (2) pretreatment with acidified GC alone slightly increased, but with acidified EC alone significantly decreased, enzymatic digestibility compared to untreated MCC, and (3) there was a good positive linear correlation of enzymatic digestibility of treated and untreated MCC samples with congo red (CR) adsorption capacity. Conclusions Acidified GC alone was a more effective solvent for pretreatment of sugarcane bagasse than acidified EC alone. The higher glucose yield obtained with GC-pretreated bagasse is possibly due to the presence of one hydroxyl group in the GC molecular structure, resulting in more significant biomass delignification and defibrillation, though both solvent pretreatments reduced bagasse particles to a similar extent. The maximum glucan digestibility of GC/glycerol systems was less than that of EC/EG systems, which is likely attributed to glycerol being less effective than EG in biomass delignification and defibrillation. Acidified AC/AG solvent systems were more effective for pretreatment of lignin-containing biomass than MCC.

Transcript and metabolite profiling for the evaluation of tobacco tree and poplar as feedstock for the bio-based industry

The global demand for food, feed, energy and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Greenhouse gas emissions in livestock production systems

Agriculture is responsible for a significant proportion of total anthropogenic greenhouse gas emissions (perhaps 18% globally), and therefore has the potential to contribute to efforts to reduce emissions as a means of minimising the risk of dangerous climate change. The largest contributions to emissions are attributed to ruminant methane production and nitrous oxide from animal waste and fertilised soils. Further, livestock, including ruminants, are an important component of global and Australian food production and there is a growing demand for animal protein sources. At the same time as governments and the community strengthen objectives to reduce greenhouse gas emissions, there are growing concerns about global food security. This paper provides an overview of a number of options for reducing methane and nitrous oxide emissions from ruminant production systems in Australia, while maintaining productivity to contribute to both objectives. Options include strategies for feed modification, animal breeding and herd management, rumen manipulation and animal waste and fertiliser management. Using currently available strategies, some reductions in emissions can be achieved, but practical commercially available techniques for significant reductions in methane emissions, particularly from extensive livestock production systems, will require greater time and resource investment. Decreases in the levels of emissions from these ruminant systems (i.e., the amount of emissions per unit of product such as meat) have already been achieved. However, the technology has not yet been developed for eliminating production of methane from the rumen of cattle and sheep digesting the cellulose and lignin-rich grasses that make up a large part of the diet of animals grazing natural pastures, particularly in arid and semi-arid grazing lands. Nevertheless, the abatement that can be achieved will contribute significantly towards reaching greenhouse gas emissions reduction targets and research will achieve further advances.

A staining protocol for identifying secondary compounds in Myrtaceae

• Premise of the study: Here we propose a staining protocol using TBO and Ruthenium red in order to reliably identify secondary compounds in the leaves of some species of Myrtaceae. • Methods and results: Leaves of 10 species representing 10 different genera of Myrtaceae were processed and stained using five different combinations of Ruthenium red and TBO. Optimal staining conditions were determined as 1 min of Ruthenium red (0.05% aqueous) and 45 sec of TBO (0.1% aqueous). Secondary compounds clearly identified under this treatment include mucilage in mesophyll, polyphenols in cuticle, lignin in fibers and xylem, tannins and carboxylated polysaccharides in epidermis and pectic substances in primary cell walls. • Conclusions: Potential applications of this protocol include systematic, phytochemical and ecological investigations in Myrtaceae. It might be applicable to other plant families rich in secondary compounds and could be used as preliminary screening method for extraction of these elements.

Perturbed Lignification Impacts Tree Growth in Hybrid Poplar-A Function of Sink Strength, Vascular Integrity, and Photosynthetic Assimilation

The effects of reductions in cell wall lignin content, manifested by RNA interference suppression of coumaroyl 3'-hydroxylase, on plant growth, water transport, gas exchange, and photosynthesis were evaluated in hybrid poplar trees (Populus alba 3 grandidentata). The growth characteristics of the reduced lignin trees were significantly impaired, resulting in smaller stems and reduced root biomass when compared to wild-type trees, as well as altered leaf morphology and architecture. The severe inhibition of cell wall lignification produced trees with a collapsed xylem phenotype, resulting in compromised vascular integrity, and displayed reduced hydraulic conductivity and a greater susceptibility to wall failure and cavitation. In the reduced lignin trees, photosynthetic carbon assimilation and stomatal conductance were also greatly reduced, however, shoot xylem pressure potential and carbon isotope discrimination were higher and water-use efficiency was lower, inconsistent with water stress. Reductions in assimilation rate could not be ascribed to increased stomatal limitation. Starch and soluble sugars analysis of leaves revealed that photosynthate was accumulating to high levels, suggesting that the trees with substantially reduced cell wall lignin were not carbon limited and that reductions in sink strength were, instead, limiting photosynthesis.

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.