Background: Expressing microbial polysaccharide-modifying enzymes in plants is an attractive appr... more Background: Expressing microbial polysaccharide-modifying enzymes in plants is an attractive approach to custom tailor plant lignocellulose and to study the importance of wall structures to plant development. Expression of α-glucuronidases in plants to modify the structures of glucuronoxylans has not been yet attempted. Glycoside hydrolase (GH) family 115 α-glucuronidases cleave the internal α-D-(4-O-methyl)glucopyranosyluronic acid ((Me)GlcA) from xylans or xylooligosaccharides. In this work, a GH115 α-glucuronidase from Schizophyllum commune, ScAGU115, was expressed in Arabidopsis thaliana and targeted to apoplast. The transgene effects on native xylans' structures, plant development, and lignocellulose saccharification were evaluated and compared to those of knocked out glucuronyltransferases AtGUX1 and AtGUX2. Results: The ScAGU115 extracted from cell walls of Arabidopsis was active on the internally substituted aldopentaouronic acid (XUXX). The transgenic plants did not show any change in growth or in lignocellulose saccharification. The cell wall (Me)GlcA and other non-cellulosic sugars, as well as the lignin content, remained unchanged. In contrast, the gux1gux2 double mutant showed a 70% decrease in (Me)GlcA to xylose molar ratio, and, interestingly, a 60% increase in the xylose content. Whereas ScAGU115-expressing plants exhibited a decreased signal in native secondary walls from the monoclonal antibody UX1 that recognizes (Me)GlcA on non-acetylated xylan, the signal was not affected after wall deacetylation. In contrast, gux1gux2 mutant was lacking UX1 signals in both native and deacetylated cell walls. This indicates that acetyl substitution on the xylopyranosyl residue carrying (Me)GlcA or on the neighboring xylopyranosyl residues may restrict post-synthetic modification of xylans by ScAGU115 in planta. Conclusions: Active GH115 α-glucuronidase has been produced for the first time in plants. The cell wall-targeted ScAGU115 was shown to affect those glucuronate substitutions of xylan, which are accessible to UX1 antibody and constitute a small fraction in Arabidopsis, whereas majority of (Me)GlcA substitutions were resistant, most likely due to the shielding by acetyl groups. Plants expressing ScAGU115 did not show any defects under laboratory conditions indicating that the UX1 epitope of xylan is not essential under these conditions. Moreover the removal of the UX1 xylan epitope does not affect lignocellulose saccharification.
Supercritical carbon dioxide (scCO 2) has been investigated for the generation of valuable waxy c... more Supercritical carbon dioxide (scCO 2) has been investigated for the generation of valuable waxy compounds and as an added-value technology in a holistic maize stover biorefinery. ScCO 2 extraction and fractionation was carried out prior to hydrolysis and fermentation of maize stover. Fractionation of the crude extracts by scCO 2 resulted in wax extracts having different compositions and melting temperatures, enabling their utilisation in different applications. One of such fraction demonstrated significant potential as a renewable defoaming agent in washing machine detergent formulations. Furthermore, scCO 2 extraction has been shown to have a positive effect on the downstream processing of the maize stover. Fermentation of the scCO 2 extracted maize stover hydrolysates exhibited a higher glucose consumption and greater potential growth for surfactant (in comparison with non-scCO 2 treated stover) and ethanol production (a 40% increase in overall ethanol production after scCO 2 pre-treatment). This work represents an important development in the extraction of high value components from low value wastes and demonstrates the benefits of using scCO 2 extraction as a first-step in biomass processing, including enhancing downstream processing of the biomass for the production of 2 nd generation biofuels as part of an integrated holistic biorefinery.
Ester-linked hydroxycinnamic acids ferulic acid (FA) and para-coumaric acid (p-CA) play important... more Ester-linked hydroxycinnamic acids ferulic acid (FA) and para-coumaric acid (p-CA) play important roles in crosslinking within cell wall arabinoxylans (AX) and between AX and lignin in grass cell walls. The addition of hydroxycinnamates to AX, is mediated by the Mitchell clade of BAHD acyl-coenzyme A-utilizing transferases. Overexpression of OsAT10 (a Mitchell clade BAHD acyl transferase) in rice, has previously been shown to increase p-CA content in AX in leaves and stems, leading to increased cell wall digestibility, potentially associated with a concomitant decrease in FA content. To investigate the physiological role of OsAT10 we established CRISPR/Cas9 rice knock-out mutants devoid of OsAT10. Our analysis of hydroxycinnamic acid content in wild type plants revealed that AX associated p-CA is found almost exclusively in rice husks, with very little found in other tissues. Mutant plants were essentially devoid of ester-linked p-CA associated with AX, indicating that OsAT10 repres...
This laboratory scale study aims to demonstrate the effectiveness of thermochemical and biologica... more This laboratory scale study aims to demonstrate the effectiveness of thermochemical and biological saccharification of Miscanthus giganteus (MG) for generation of fermentable saccharides and its subsequent fermentation into solvents i.e. acetone, ethanol and butanol (ABE) using Clostridium acetobutylicum ATCC 824. Saccharide hydrolysates were derived from MG by thermochemical (water, acid and alkali at 130 o C) and biological saccharification (Fibrobacter succinogenes S85) processes and were subjected to batch fermentation for 120 hours using C. acetobutylicum ATCC 824. At the end of fermentation of thermochemically-derived hydrolysates, 742 g m-3 of saccharides from water treatment, 9572 g m-3 of saccharides from acid treatment and g m-3 of saccharides from alkali treatment were fermented and yielded 0.045, 0.0069 and 0.01 g g-1 of total solvents, respectively. Similarly, at the end of fermentation of biological hydrolysate (using F. succinogenes), 2504 g m-3 of saccharides was fermented and yielded 0.091 g g-1 of total solvents. The highest yield of total solvents was achieved by water (thermochemical) and biological saccharification of MG using C. acetobutylicum. Whereas, acid and alkali-treated hydrolysates showed lower yields of solvents presumably due to production of inhibitory compounds during saccharification. Compared to thermochemical saccharification, biological saccharification using F. succinogenes is a promising approach since it yielded the highest amount of solvents whilst being eco-friendly. Our future studies will focus on optimisation of biological saccharification (using F. succinogenes) and sequential co-culture fermentation (using C. acetobutylicum). The development of alternative consolidated bioprocessing approach using biological saccharification will contribute towards making lignocellulosic biofuels a reality.
Woody (lignocellulosic) plant biomass is an abundant renewable feedstock, rich in polysaccharides... more Woody (lignocellulosic) plant biomass is an abundant renewable feedstock, rich in polysaccharides that are bound into an insoluble fiber composite with lignin. Marine crustacean woodborers of the genus Limnoria are among the few animals that can survive on a diet of this recalcitrant material without relying on gut resident microbiota. Analysis of fecal pellets revealed that Limnoria targets hexose-containing polysaccharides (mainly cellulose, and also glucomannans), corresponding with the abundance of cellulases in their digestive system, but xylans and lignin are largely unconsumed. We show that the limnoriid respiratory protein, hemocyanin, is abundant in the hindgut where wood is digested, that incubation of wood with hemocyanin markedly enhances its digestibility by cellulases, and that it modifies lignin. We propose that this activity of hemocyanins is instrumental to the ability of Limnoria to feed on wood in the absence of gut symbionts. These findings may hold potential for...
This is a repository copy of Integrated biorefinery approach to valorise Saccharina latissima bio... more This is a repository copy of Integrated biorefinery approach to valorise Saccharina latissima biomass : Combined sustainable processing to produce biologically active fucoxanthin, mannitol, fucoidans and alginates.
Acacia spp. are invasive in Southern Europe, and their high propagation rates produce excessive b... more Acacia spp. are invasive in Southern Europe, and their high propagation rates produce excessive biomass, exacerbating wildfire risk. However, lignocellulosic biomass from Acacia spp. may be utilised for diverse biorefinery applications. In this study, attenuated total reflectance Fourier transform infrared spectroscopy (FTIR-ATR), high-performance anion-exchange chromatography pulsed amperometric detection (HPAEC-PAD) and lignin content determinations were used for a comparative compositional characterisation of A. dealbata, A. longifolia and A. melanoxylon. Additionally, biomass was treated with three white-rot fungi species (Ganoderma lucidum, Pleurotus ostreatus and Trametes versicolor), which preferentially degrade lignin. Our results showed that the pre-treatments do not significantly alter neutral sugar composition while reducing lignin content. Sugar release from enzymatic saccharification was enhanced, in some cases possibly due to a synergy between white-rot fungi and mild ...
Proceedings of the International Society of Sugar Cane Technologists 30 : 661-664. (2019), Sep 1, 2019
Knowledge of cell-wall composition in sugarcane hybrids could be used for the selection of cultiv... more Knowledge of cell-wall composition in sugarcane hybrids could be used for the selection of cultivars with high bagasse digestibility in order to improve sugar release in 2G ethanol production. Cell-wall components of 76 hybrids of a sugarcane cross, their parents and two highfibre genotypes were analyzed. The Van Soest method was used to determine cellulose, hemicellulose and acid detergent lignin contents (ADL). Spectroscopic acetyl bromide lignin (ABS) was also determined. A correlation analysis was performed with major cell-wall polymers and agronomic traits. Despite the minor differences observed between parental lines' cell-wall composition, genotypes with high cellulose and low lignin content were identified in the progeny. Differences among them depended on the method used for lignin measurement. The ADL mean was 43% lower than the ABS mean, in agreement with lignin losses during acid detergent treatment previously reported. There was no correlation between ADL and ABS assays. Bagasse of genotypes with higher stalk weights was associated with higher cellulose content and lower ADL content. Saccharification studies are needed to validate lignin assessment methodologies to be used as bioenergy cultivar selection tools.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Acacia spp. are invasive in Southern Europe, and their high propagation rates produce excessive b... more Acacia spp. are invasive in Southern Europe, and their high propagation rates produce excessive biomass, exacerbating wildfire risk. However, lignocellulosic biomass from Acacia spp. may be utilised for diverse biorefinery applications. In this study, attenuated total reflectance Fourier transform infrared spectroscopy (FTIR-ATR), high-performance anion-exchange chromatography pulsed amperometric detection (HPAEC-PAD) and lignin content determinations were used for a comparative compositional characterisation of A. dealbata, A. longifolia and A. melanoxylon. Additionally, biomass was treated with three white-rot fungi species (Ganoderma lucidum, Pleurotus ostreatus and Trametes versicolor), which preferentially degrade lignin. Our results showed that the pre-treatments do not significantly alter neutral sugar composition while reducing lignin content. Sugar release from enzymatic saccharification was enhanced, in some cases possibly due to a synergy between white-rot fungi and mild alkali pretreatments. For example, in A. dealbata stems treated with alkali and P. ostreatus, saccharification yield was 702.3 nmol mg−1, which is higher than the samples treated only with alkali (608.1 nmol mg−1), and 2.9-fold higher than the non-pretreated controls (243.9 nmol mg−1). By characterising biomass and pretreatments, generated data creates value for unused biomass resources, contributing to the implementation of sustainable biorefining systems. In due course, the generated value will lead to economic incentives for landowners to cut back invasive Acacia spp. more frequently, thus reducing excess biomass, which exacerbates wildfire risk.
Microwave heating technology has attracted great attention in biorefineries to generate biofuels ... more Microwave heating technology has attracted great attention in biorefineries to generate biofuels and biomass-derived chemicals. NaOH and H 2 SO 4 pretreatments are often applied to improve the process efficiency. Traditional heating pretreatments require long reaction times and contribute to rapid sugar degradation. Although effective, H 2 SO 4 pretreatment suffers from the disadvantages of equipment corrosion, inhibitors generation, and waste water production. Herein, we performed NaOH, H 2 SO 4 , and FeCl 3 pretreatments on sugar cane bagasse under microwave heating, and compared their effects on the biomass physical features, chemical compositions, and sugar release during pretreatment process. Our results showed that high amounts of sugar were obtained, and xylose and glucose were selectively recovered in the pretreatment liquor within 5-10 min of microwave heating by changing pretreatment media. NaOH efficiently fractionated biomass, generated xylose as the major product in pretreatment liquor, and left the solid residue more digestible. H 2 SO 4 and FeCl 3 effectively degraded both the hemicellulose and cellulose, producing glucose as the major product in the pretreatment liquor. Compared with H 2 SO 4 , FeCl 3 pretreatment more efficiently removed lignin and recovered 48% glucose within only 5 min. Biomass chemical composition and morphological characteristics were studied and compared, confirming that microwave assisted FeCl 3 pretreatment is a new and feasible approach to replace H 2 SO 4 for biofuel and biomass-derived chemicals production.
Miscanthus is a major bioenergy crop in Europe and a potential feedstock for second generation bi... more Miscanthus is a major bioenergy crop in Europe and a potential feedstock for second generation biofuels. Thermochemical pretreatment is a significant step in the process of converting lignocellulosic biomass into fermentable sugars. In this work, microwave energy was applied to facilitate NaOH and H 2 SO 4 pretreatments of Miscanthus. This was carried out at 180 °C in a monomode microwave cavity at 300 W. Our results show that H 2 SO 4 pretreatment contributes to the breakdown of hemicelluloses and cellulose, leading to a high glucose yield. The maximum sugar yield from available carbohydrates during pretreatment is 75.3% (0.2 M H 2 SO 4 20 Min), and glucose yield is 46.7% under these conditions. NaOH and water pretreatments tend to break down only hemicellulose in preference to cellulose, contributing to high xylose yield. Compared to conventional heating NaOH/H 2 SO 4 pretreatment, 12 times higher sugar yield was obtained by using microwave assisted pretreatment within half the time. NaOH pretreatments lead to a significantly enhanced digestibility of the residue, because the effective removal of lignin and hemicellulose makes cellulose fibres more accessible to cellulases. Morphological study of biomass shows that the tightly packed fibres in the Miscanthus were dismantled and exposed under NaOH condition. We studied sugar degradation under microwave assisted H 2 SO 4 conditions. The results shows that 6-8% biomass was converted into levulinic acid (LA) during pretreatment, showing the possibility of using microwave technology to produce LA from biomass. The outcome of this work shows great potential for using microwave in the thermo-chemical pretreatment for biomass and also selective production of LA from biomass.
Background: Miscanthus is a major bioenergy crop in Europe and a potential feedstock for second g... more Background: Miscanthus is a major bioenergy crop in Europe and a potential feedstock for second generation biofuels. The most efficient and realistic method to produce fermentable sugars from lignocellulosic biomass is by enzymatic hydrolysis, assisted by thermo-chemical pretreatment. Recently, microwave technology has drawn growing attention, because of its unique effects and performance on biomass. Result: In this work, microwave energy was applied to facilitate NaOH and H 2 SO 4 pretreatment for Miscanthus under different temperatures (130-200 °C) for 20 min. The yields of reducing sugars from Miscanthus during the pretreatment process increased up to 180 °C and then declined with increasing temperature. Out results here showed a remarkable sugar yield from available carbohydrate (73 %) at the temperature of 180 °C by using 0.2 M H 2 SO 4. In comparison with conventional heating pretreatment studied at same temperature with same biomass material, the reducing sugar release in this study was 17 times higher within half the time. It was highlighted that the major sugar component could be tuned by changing pretreatment temperature or pretreatment media. Optimally, the glucose and xylose yield from available carbohydrate are 47 and 22 % by using 0.2 M H 2 SO 4 and NaOH respectively when temperature was 180 °C. The digestibility of pretreated Miscanthus was 10 times higher than that of untreated biomass. 68-86 % of the lignin content was removed from biomass by 0.2 M NaOH. Simultaneous saccharification fermentation (SSF) results showed an ethanol production of 143-152 mg/g biomass by using H 2 SO 4 /NaOH microwave assisted pretreatment, which is 7 times higher than that of untreated Miscanthus. Biomass morphology was studied by SEM, showing temperature has a strong influence on lignin removal process, as different lignin deposits were observed. At the temperature of 180 °C, NaOH pretreated biomass presented highly exposed fibres, which is a very important biomass characteristic for improved enzymatic hydrolysis. Conclusion: Compared to conventional pretreatment, microwave assisted pretreatment is more energy efficient and faster, due to its unique heating mechanism leading to direct interaction between the polar part of biomass and electromagnetic field. The results of this work present promising potential for using microwave to assist biomass thermo-chemical pretreatment.
A proof-of-concept project compared extraction of arabinoxylans (AX) from sugarcane bagasse and 1... more A proof-of-concept project compared extraction of arabinoxylans (AX) from sugarcane bagasse and 19 wheat bran via alkaline hydrogen peroxide followed by enzyme-assisted extraction with 20 combinations of feruloyl esterases and a xylanase. Bagasse contains comparable amounts of AX to 21 wheat bran, but with a much lower arabinoxylan substitution on the xylan backbone (A:X ratio of 22 around 0.2 compared with 0.6 for wheat bran), hence offering AX products with distinctive 23 functionality and potential end uses. In the current work, bagasse released its AX more readily than 24 wheat bran, and released a wider range of molecular weights. Use of feruloyl esterase and xylanase 25 enzymes on their own or following alkaline peroxide extraction did not enhance AX release 26 substantially; however, the xylanase appeared to be effective at reducing the size of AX molecules, 27 and there is scope to optimise the effects of enzymes to produce specific AX product fractions. As 28 bagasse frequently arises within the context of bioethanol production, integration of AX extraction 29 with ethanol production could allow economic production of a portfolio of AX products, as has been 30 demonstrated in principle for AX co-production in a wheat ethanol plant.
Thermobia domestica belongs to an ancient group of insects and has a remarkable ability to digest... more Thermobia domestica belongs to an ancient group of insects and has a remarkable ability to digest crystalline cellulose without microbial assistance. By investigating the digestive proteome of Thermobia, we have identified over 20 members of an uncharacterized family of lytic polysaccharide monooxygenases (LPMOs). We show that this LPMO family spans across several clades of the Tree of Life, is of ancient origin, and was recruited by early arthropods with possible roles in remodeling endogenous chitin scaffolds during development and metamorphosis. Based on our in-depth characterization of Thermobia's LPMOs, we propose that diversification of these enzymes toward cellulose digestion might have endowed ancestral insects with an effective biochemical apparatus for biomass degradation, allowing the early colonization of land during the Paleozoic Era. The vital role of LPMOs in modern agricultural pests and disease vectors offers new opportunities to help tackle global challenges in food security and the control of infectious diseases.
Plant cell wall biosynthesis is a complex process that requires proteins and enzymes from glycan ... more Plant cell wall biosynthesis is a complex process that requires proteins and enzymes from glycan synthesis to wall assembly. We show that disruption of At3g50120 (DUF247-1), a member of the DUF247 multigene family containing 28 genes in Arabidopsis, results in alterations to the structure and composition of cell wall polysaccharides and reduced growth and plant size. An ELISA using cell wall antibodies shows that the mutants also exhibit ~50% reductions in xyloglucan (XyG), glucuronoxylan (GX) and heteromannan (HM) epitopes in the NaOH fraction and ~50% increases in homogalacturonan (HG) epitopes in the CDTA fraction. Furthermore, the polymer sizes of XyGs and GXs are reduced with concomitant increases in short-chain polymers, while those of HGs and mHGs are slightly increased. Complementation using 35S:DUF247-1 partially recovers the XyG and HG content, but not those of GX and HM, suggesting that DUF247-1 is more closely associated with XyGs and HGs. DUF247-1 is expressed throughou...
With a high potential to generate biomass, maize stover arises as an outstanding feedstock for bi... more With a high potential to generate biomass, maize stover arises as an outstanding feedstock for biofuel production. Maize stover presents the added advantage of being a multiple exploitation of the crop as a source of food, feed, and energy. In this study, contrasting groups of recombinant inbred lines (RILs) from a maize multiparent advanced generation intercross (MAGIC) population that showed variability for saccharification efficiency were screened by FTIR-ATR spectroscopy to explore compositional differences between high and low saccharification yielders. High and low saccharification efficiency groups differed in cell wall compositional features: high saccharification RILs displayed higher proportions of S subunits, aromatic compounds, and hemicellulose as opposed to low saccharification efficiency groups in which FTIR predicted higher proportions of lignin, more precisely lignin being richer in subunits G, and greater proportions of crystalline cellulose and acetyl methyl ester...
Background: Expressing microbial polysaccharide-modifying enzymes in plants is an attractive appr... more Background: Expressing microbial polysaccharide-modifying enzymes in plants is an attractive approach to custom tailor plant lignocellulose and to study the importance of wall structures to plant development. Expression of α-glucuronidases in plants to modify the structures of glucuronoxylans has not been yet attempted. Glycoside hydrolase (GH) family 115 α-glucuronidases cleave the internal α-D-(4-O-methyl)glucopyranosyluronic acid ((Me)GlcA) from xylans or xylooligosaccharides. In this work, a GH115 α-glucuronidase from Schizophyllum commune, ScAGU115, was expressed in Arabidopsis thaliana and targeted to apoplast. The transgene effects on native xylans' structures, plant development, and lignocellulose saccharification were evaluated and compared to those of knocked out glucuronyltransferases AtGUX1 and AtGUX2. Results: The ScAGU115 extracted from cell walls of Arabidopsis was active on the internally substituted aldopentaouronic acid (XUXX). The transgenic plants did not show any change in growth or in lignocellulose saccharification. The cell wall (Me)GlcA and other non-cellulosic sugars, as well as the lignin content, remained unchanged. In contrast, the gux1gux2 double mutant showed a 70% decrease in (Me)GlcA to xylose molar ratio, and, interestingly, a 60% increase in the xylose content. Whereas ScAGU115-expressing plants exhibited a decreased signal in native secondary walls from the monoclonal antibody UX1 that recognizes (Me)GlcA on non-acetylated xylan, the signal was not affected after wall deacetylation. In contrast, gux1gux2 mutant was lacking UX1 signals in both native and deacetylated cell walls. This indicates that acetyl substitution on the xylopyranosyl residue carrying (Me)GlcA or on the neighboring xylopyranosyl residues may restrict post-synthetic modification of xylans by ScAGU115 in planta. Conclusions: Active GH115 α-glucuronidase has been produced for the first time in plants. The cell wall-targeted ScAGU115 was shown to affect those glucuronate substitutions of xylan, which are accessible to UX1 antibody and constitute a small fraction in Arabidopsis, whereas majority of (Me)GlcA substitutions were resistant, most likely due to the shielding by acetyl groups. Plants expressing ScAGU115 did not show any defects under laboratory conditions indicating that the UX1 epitope of xylan is not essential under these conditions. Moreover the removal of the UX1 xylan epitope does not affect lignocellulose saccharification.
Supercritical carbon dioxide (scCO 2) has been investigated for the generation of valuable waxy c... more Supercritical carbon dioxide (scCO 2) has been investigated for the generation of valuable waxy compounds and as an added-value technology in a holistic maize stover biorefinery. ScCO 2 extraction and fractionation was carried out prior to hydrolysis and fermentation of maize stover. Fractionation of the crude extracts by scCO 2 resulted in wax extracts having different compositions and melting temperatures, enabling their utilisation in different applications. One of such fraction demonstrated significant potential as a renewable defoaming agent in washing machine detergent formulations. Furthermore, scCO 2 extraction has been shown to have a positive effect on the downstream processing of the maize stover. Fermentation of the scCO 2 extracted maize stover hydrolysates exhibited a higher glucose consumption and greater potential growth for surfactant (in comparison with non-scCO 2 treated stover) and ethanol production (a 40% increase in overall ethanol production after scCO 2 pre-treatment). This work represents an important development in the extraction of high value components from low value wastes and demonstrates the benefits of using scCO 2 extraction as a first-step in biomass processing, including enhancing downstream processing of the biomass for the production of 2 nd generation biofuels as part of an integrated holistic biorefinery.
Ester-linked hydroxycinnamic acids ferulic acid (FA) and para-coumaric acid (p-CA) play important... more Ester-linked hydroxycinnamic acids ferulic acid (FA) and para-coumaric acid (p-CA) play important roles in crosslinking within cell wall arabinoxylans (AX) and between AX and lignin in grass cell walls. The addition of hydroxycinnamates to AX, is mediated by the Mitchell clade of BAHD acyl-coenzyme A-utilizing transferases. Overexpression of OsAT10 (a Mitchell clade BAHD acyl transferase) in rice, has previously been shown to increase p-CA content in AX in leaves and stems, leading to increased cell wall digestibility, potentially associated with a concomitant decrease in FA content. To investigate the physiological role of OsAT10 we established CRISPR/Cas9 rice knock-out mutants devoid of OsAT10. Our analysis of hydroxycinnamic acid content in wild type plants revealed that AX associated p-CA is found almost exclusively in rice husks, with very little found in other tissues. Mutant plants were essentially devoid of ester-linked p-CA associated with AX, indicating that OsAT10 repres...
This laboratory scale study aims to demonstrate the effectiveness of thermochemical and biologica... more This laboratory scale study aims to demonstrate the effectiveness of thermochemical and biological saccharification of Miscanthus giganteus (MG) for generation of fermentable saccharides and its subsequent fermentation into solvents i.e. acetone, ethanol and butanol (ABE) using Clostridium acetobutylicum ATCC 824. Saccharide hydrolysates were derived from MG by thermochemical (water, acid and alkali at 130 o C) and biological saccharification (Fibrobacter succinogenes S85) processes and were subjected to batch fermentation for 120 hours using C. acetobutylicum ATCC 824. At the end of fermentation of thermochemically-derived hydrolysates, 742 g m-3 of saccharides from water treatment, 9572 g m-3 of saccharides from acid treatment and g m-3 of saccharides from alkali treatment were fermented and yielded 0.045, 0.0069 and 0.01 g g-1 of total solvents, respectively. Similarly, at the end of fermentation of biological hydrolysate (using F. succinogenes), 2504 g m-3 of saccharides was fermented and yielded 0.091 g g-1 of total solvents. The highest yield of total solvents was achieved by water (thermochemical) and biological saccharification of MG using C. acetobutylicum. Whereas, acid and alkali-treated hydrolysates showed lower yields of solvents presumably due to production of inhibitory compounds during saccharification. Compared to thermochemical saccharification, biological saccharification using F. succinogenes is a promising approach since it yielded the highest amount of solvents whilst being eco-friendly. Our future studies will focus on optimisation of biological saccharification (using F. succinogenes) and sequential co-culture fermentation (using C. acetobutylicum). The development of alternative consolidated bioprocessing approach using biological saccharification will contribute towards making lignocellulosic biofuels a reality.
Woody (lignocellulosic) plant biomass is an abundant renewable feedstock, rich in polysaccharides... more Woody (lignocellulosic) plant biomass is an abundant renewable feedstock, rich in polysaccharides that are bound into an insoluble fiber composite with lignin. Marine crustacean woodborers of the genus Limnoria are among the few animals that can survive on a diet of this recalcitrant material without relying on gut resident microbiota. Analysis of fecal pellets revealed that Limnoria targets hexose-containing polysaccharides (mainly cellulose, and also glucomannans), corresponding with the abundance of cellulases in their digestive system, but xylans and lignin are largely unconsumed. We show that the limnoriid respiratory protein, hemocyanin, is abundant in the hindgut where wood is digested, that incubation of wood with hemocyanin markedly enhances its digestibility by cellulases, and that it modifies lignin. We propose that this activity of hemocyanins is instrumental to the ability of Limnoria to feed on wood in the absence of gut symbionts. These findings may hold potential for...
This is a repository copy of Integrated biorefinery approach to valorise Saccharina latissima bio... more This is a repository copy of Integrated biorefinery approach to valorise Saccharina latissima biomass : Combined sustainable processing to produce biologically active fucoxanthin, mannitol, fucoidans and alginates.
Acacia spp. are invasive in Southern Europe, and their high propagation rates produce excessive b... more Acacia spp. are invasive in Southern Europe, and their high propagation rates produce excessive biomass, exacerbating wildfire risk. However, lignocellulosic biomass from Acacia spp. may be utilised for diverse biorefinery applications. In this study, attenuated total reflectance Fourier transform infrared spectroscopy (FTIR-ATR), high-performance anion-exchange chromatography pulsed amperometric detection (HPAEC-PAD) and lignin content determinations were used for a comparative compositional characterisation of A. dealbata, A. longifolia and A. melanoxylon. Additionally, biomass was treated with three white-rot fungi species (Ganoderma lucidum, Pleurotus ostreatus and Trametes versicolor), which preferentially degrade lignin. Our results showed that the pre-treatments do not significantly alter neutral sugar composition while reducing lignin content. Sugar release from enzymatic saccharification was enhanced, in some cases possibly due to a synergy between white-rot fungi and mild ...
Proceedings of the International Society of Sugar Cane Technologists 30 : 661-664. (2019), Sep 1, 2019
Knowledge of cell-wall composition in sugarcane hybrids could be used for the selection of cultiv... more Knowledge of cell-wall composition in sugarcane hybrids could be used for the selection of cultivars with high bagasse digestibility in order to improve sugar release in 2G ethanol production. Cell-wall components of 76 hybrids of a sugarcane cross, their parents and two highfibre genotypes were analyzed. The Van Soest method was used to determine cellulose, hemicellulose and acid detergent lignin contents (ADL). Spectroscopic acetyl bromide lignin (ABS) was also determined. A correlation analysis was performed with major cell-wall polymers and agronomic traits. Despite the minor differences observed between parental lines' cell-wall composition, genotypes with high cellulose and low lignin content were identified in the progeny. Differences among them depended on the method used for lignin measurement. The ADL mean was 43% lower than the ABS mean, in agreement with lignin losses during acid detergent treatment previously reported. There was no correlation between ADL and ABS assays. Bagasse of genotypes with higher stalk weights was associated with higher cellulose content and lower ADL content. Saccharification studies are needed to validate lignin assessment methodologies to be used as bioenergy cultivar selection tools.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Acacia spp. are invasive in Southern Europe, and their high propagation rates produce excessive b... more Acacia spp. are invasive in Southern Europe, and their high propagation rates produce excessive biomass, exacerbating wildfire risk. However, lignocellulosic biomass from Acacia spp. may be utilised for diverse biorefinery applications. In this study, attenuated total reflectance Fourier transform infrared spectroscopy (FTIR-ATR), high-performance anion-exchange chromatography pulsed amperometric detection (HPAEC-PAD) and lignin content determinations were used for a comparative compositional characterisation of A. dealbata, A. longifolia and A. melanoxylon. Additionally, biomass was treated with three white-rot fungi species (Ganoderma lucidum, Pleurotus ostreatus and Trametes versicolor), which preferentially degrade lignin. Our results showed that the pre-treatments do not significantly alter neutral sugar composition while reducing lignin content. Sugar release from enzymatic saccharification was enhanced, in some cases possibly due to a synergy between white-rot fungi and mild alkali pretreatments. For example, in A. dealbata stems treated with alkali and P. ostreatus, saccharification yield was 702.3 nmol mg−1, which is higher than the samples treated only with alkali (608.1 nmol mg−1), and 2.9-fold higher than the non-pretreated controls (243.9 nmol mg−1). By characterising biomass and pretreatments, generated data creates value for unused biomass resources, contributing to the implementation of sustainable biorefining systems. In due course, the generated value will lead to economic incentives for landowners to cut back invasive Acacia spp. more frequently, thus reducing excess biomass, which exacerbates wildfire risk.
Microwave heating technology has attracted great attention in biorefineries to generate biofuels ... more Microwave heating technology has attracted great attention in biorefineries to generate biofuels and biomass-derived chemicals. NaOH and H 2 SO 4 pretreatments are often applied to improve the process efficiency. Traditional heating pretreatments require long reaction times and contribute to rapid sugar degradation. Although effective, H 2 SO 4 pretreatment suffers from the disadvantages of equipment corrosion, inhibitors generation, and waste water production. Herein, we performed NaOH, H 2 SO 4 , and FeCl 3 pretreatments on sugar cane bagasse under microwave heating, and compared their effects on the biomass physical features, chemical compositions, and sugar release during pretreatment process. Our results showed that high amounts of sugar were obtained, and xylose and glucose were selectively recovered in the pretreatment liquor within 5-10 min of microwave heating by changing pretreatment media. NaOH efficiently fractionated biomass, generated xylose as the major product in pretreatment liquor, and left the solid residue more digestible. H 2 SO 4 and FeCl 3 effectively degraded both the hemicellulose and cellulose, producing glucose as the major product in the pretreatment liquor. Compared with H 2 SO 4 , FeCl 3 pretreatment more efficiently removed lignin and recovered 48% glucose within only 5 min. Biomass chemical composition and morphological characteristics were studied and compared, confirming that microwave assisted FeCl 3 pretreatment is a new and feasible approach to replace H 2 SO 4 for biofuel and biomass-derived chemicals production.
Miscanthus is a major bioenergy crop in Europe and a potential feedstock for second generation bi... more Miscanthus is a major bioenergy crop in Europe and a potential feedstock for second generation biofuels. Thermochemical pretreatment is a significant step in the process of converting lignocellulosic biomass into fermentable sugars. In this work, microwave energy was applied to facilitate NaOH and H 2 SO 4 pretreatments of Miscanthus. This was carried out at 180 °C in a monomode microwave cavity at 300 W. Our results show that H 2 SO 4 pretreatment contributes to the breakdown of hemicelluloses and cellulose, leading to a high glucose yield. The maximum sugar yield from available carbohydrates during pretreatment is 75.3% (0.2 M H 2 SO 4 20 Min), and glucose yield is 46.7% under these conditions. NaOH and water pretreatments tend to break down only hemicellulose in preference to cellulose, contributing to high xylose yield. Compared to conventional heating NaOH/H 2 SO 4 pretreatment, 12 times higher sugar yield was obtained by using microwave assisted pretreatment within half the time. NaOH pretreatments lead to a significantly enhanced digestibility of the residue, because the effective removal of lignin and hemicellulose makes cellulose fibres more accessible to cellulases. Morphological study of biomass shows that the tightly packed fibres in the Miscanthus were dismantled and exposed under NaOH condition. We studied sugar degradation under microwave assisted H 2 SO 4 conditions. The results shows that 6-8% biomass was converted into levulinic acid (LA) during pretreatment, showing the possibility of using microwave technology to produce LA from biomass. The outcome of this work shows great potential for using microwave in the thermo-chemical pretreatment for biomass and also selective production of LA from biomass.
Background: Miscanthus is a major bioenergy crop in Europe and a potential feedstock for second g... more Background: Miscanthus is a major bioenergy crop in Europe and a potential feedstock for second generation biofuels. The most efficient and realistic method to produce fermentable sugars from lignocellulosic biomass is by enzymatic hydrolysis, assisted by thermo-chemical pretreatment. Recently, microwave technology has drawn growing attention, because of its unique effects and performance on biomass. Result: In this work, microwave energy was applied to facilitate NaOH and H 2 SO 4 pretreatment for Miscanthus under different temperatures (130-200 °C) for 20 min. The yields of reducing sugars from Miscanthus during the pretreatment process increased up to 180 °C and then declined with increasing temperature. Out results here showed a remarkable sugar yield from available carbohydrate (73 %) at the temperature of 180 °C by using 0.2 M H 2 SO 4. In comparison with conventional heating pretreatment studied at same temperature with same biomass material, the reducing sugar release in this study was 17 times higher within half the time. It was highlighted that the major sugar component could be tuned by changing pretreatment temperature or pretreatment media. Optimally, the glucose and xylose yield from available carbohydrate are 47 and 22 % by using 0.2 M H 2 SO 4 and NaOH respectively when temperature was 180 °C. The digestibility of pretreated Miscanthus was 10 times higher than that of untreated biomass. 68-86 % of the lignin content was removed from biomass by 0.2 M NaOH. Simultaneous saccharification fermentation (SSF) results showed an ethanol production of 143-152 mg/g biomass by using H 2 SO 4 /NaOH microwave assisted pretreatment, which is 7 times higher than that of untreated Miscanthus. Biomass morphology was studied by SEM, showing temperature has a strong influence on lignin removal process, as different lignin deposits were observed. At the temperature of 180 °C, NaOH pretreated biomass presented highly exposed fibres, which is a very important biomass characteristic for improved enzymatic hydrolysis. Conclusion: Compared to conventional pretreatment, microwave assisted pretreatment is more energy efficient and faster, due to its unique heating mechanism leading to direct interaction between the polar part of biomass and electromagnetic field. The results of this work present promising potential for using microwave to assist biomass thermo-chemical pretreatment.
A proof-of-concept project compared extraction of arabinoxylans (AX) from sugarcane bagasse and 1... more A proof-of-concept project compared extraction of arabinoxylans (AX) from sugarcane bagasse and 19 wheat bran via alkaline hydrogen peroxide followed by enzyme-assisted extraction with 20 combinations of feruloyl esterases and a xylanase. Bagasse contains comparable amounts of AX to 21 wheat bran, but with a much lower arabinoxylan substitution on the xylan backbone (A:X ratio of 22 around 0.2 compared with 0.6 for wheat bran), hence offering AX products with distinctive 23 functionality and potential end uses. In the current work, bagasse released its AX more readily than 24 wheat bran, and released a wider range of molecular weights. Use of feruloyl esterase and xylanase 25 enzymes on their own or following alkaline peroxide extraction did not enhance AX release 26 substantially; however, the xylanase appeared to be effective at reducing the size of AX molecules, 27 and there is scope to optimise the effects of enzymes to produce specific AX product fractions. As 28 bagasse frequently arises within the context of bioethanol production, integration of AX extraction 29 with ethanol production could allow economic production of a portfolio of AX products, as has been 30 demonstrated in principle for AX co-production in a wheat ethanol plant.
Thermobia domestica belongs to an ancient group of insects and has a remarkable ability to digest... more Thermobia domestica belongs to an ancient group of insects and has a remarkable ability to digest crystalline cellulose without microbial assistance. By investigating the digestive proteome of Thermobia, we have identified over 20 members of an uncharacterized family of lytic polysaccharide monooxygenases (LPMOs). We show that this LPMO family spans across several clades of the Tree of Life, is of ancient origin, and was recruited by early arthropods with possible roles in remodeling endogenous chitin scaffolds during development and metamorphosis. Based on our in-depth characterization of Thermobia's LPMOs, we propose that diversification of these enzymes toward cellulose digestion might have endowed ancestral insects with an effective biochemical apparatus for biomass degradation, allowing the early colonization of land during the Paleozoic Era. The vital role of LPMOs in modern agricultural pests and disease vectors offers new opportunities to help tackle global challenges in food security and the control of infectious diseases.
Plant cell wall biosynthesis is a complex process that requires proteins and enzymes from glycan ... more Plant cell wall biosynthesis is a complex process that requires proteins and enzymes from glycan synthesis to wall assembly. We show that disruption of At3g50120 (DUF247-1), a member of the DUF247 multigene family containing 28 genes in Arabidopsis, results in alterations to the structure and composition of cell wall polysaccharides and reduced growth and plant size. An ELISA using cell wall antibodies shows that the mutants also exhibit ~50% reductions in xyloglucan (XyG), glucuronoxylan (GX) and heteromannan (HM) epitopes in the NaOH fraction and ~50% increases in homogalacturonan (HG) epitopes in the CDTA fraction. Furthermore, the polymer sizes of XyGs and GXs are reduced with concomitant increases in short-chain polymers, while those of HGs and mHGs are slightly increased. Complementation using 35S:DUF247-1 partially recovers the XyG and HG content, but not those of GX and HM, suggesting that DUF247-1 is more closely associated with XyGs and HGs. DUF247-1 is expressed throughou...
With a high potential to generate biomass, maize stover arises as an outstanding feedstock for bi... more With a high potential to generate biomass, maize stover arises as an outstanding feedstock for biofuel production. Maize stover presents the added advantage of being a multiple exploitation of the crop as a source of food, feed, and energy. In this study, contrasting groups of recombinant inbred lines (RILs) from a maize multiparent advanced generation intercross (MAGIC) population that showed variability for saccharification efficiency were screened by FTIR-ATR spectroscopy to explore compositional differences between high and low saccharification yielders. High and low saccharification efficiency groups differed in cell wall compositional features: high saccharification RILs displayed higher proportions of S subunits, aromatic compounds, and hemicellulose as opposed to low saccharification efficiency groups in which FTIR predicted higher proportions of lignin, more precisely lignin being richer in subunits G, and greater proportions of crystalline cellulose and acetyl methyl ester...
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Papers by Leonardo Gomez