Rachael Simister

Evaluating the composition and processing potential of novel sources of Brazilian biomass for sustainable biorenewables production

BackgroundThe search for promising and renewable sources of carbohydrates for the production of biofuels and other biorenewables has been stimulated by an increase in global energy demand in the face of growing concern over greenhouse gas emissions and fuel security. In particular, interest has focused on non-food lignocellulosic biomass as a potential source of abundant and sustainable feedstock for biorefineries. Here we investigate the potential of three Brazilian grasses (Panicum maximum, Pennisetum purpureum and Brachiaria brizantha), as well as bark residues from the harvesting of two commercial Eucalyptus clones (E. grandis and E. grandis x urophylla) for biofuel production, and compare these to sugarcane bagasse. The effects of hot water, acid, alkaline and sulfite pretreatments (at increasing temperatures) on the chemical composition, morphology and saccharification yields of these different biomass types were evaluated.ResultsThe average yield (per hectare), availability and general composition of all five biomasses were compared. Compositional analyses indicate a high level of hemicellulose and lignin removal in all grass varieties (including sugarcane bagasse) after acid and alkaline pretreatment with increasing temperatures, whilst the biomasses pretreated with hot water or sulfite showed little variation from the control. For all biomasses, higher cellulose enrichment resulted from treatment with sodium hydroxide at 130°C. At 180°C, a decrease in cellulose content was observed, which is associated with high amorphous cellulose removal and 5-hydroxymethyl-furaldehyde production. Morphological analysis showed the effects of different pretreatments on the biomass surface, revealing a high production of microfibrillated cellulose on grass surfaces, after treatment with 1% sodium hydroxide at 130°C for 30 minutes. This may explain the higher hydrolysis yields resulting from these pretreatments, since these cellulosic nanoparticles can be easily accessed and cleaved by cellulases.ConclusionOur results show the potential of three Brazilian grasses with high productivity yields as valuable sources of carbohydrates for ethanol production and other biomaterials. Sodium hydroxide at 130°C was found to be the most effective pretreatment for enhanced saccharification yields. It was also efficient in the production of microfibrillated cellulose on grass surfaces, thereby revealing their potential as a source of natural fillers used for bionanocomposites production.

A new perspective in bio-refining: levoglucosenone and cleaner lignin from waste biorefinery hydrolysis lignin by selective conversion of residual saccharides

An unexpected opportunity is reported to improve the sustainability of biorefineries whereby 8 wt% levoglucosenone (LGE) can be derived from unconverted saccharides in a lignin-rich biorefinery waste stream in a highly selective fashion (>90%). Additionally, in the process a purer lignin is obtained which can be used for further processing or materials applications. LGE is a valuable and versatile product with a plethora of applications.

An ancient family of lytic polysaccharide monooxygenases with roles in arthropod development and biomass digestion

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.LPMOs catalyze the oxidative breakdown of polysaccharides, thereby facilitating biomass degradation. By analyzing the digestive proteome of firebrats, the authors here identify a yet uncharacterized LPMO family and provide phylogenetic, structural and biochemical insights into its origin and functions.

Exploring tomato Solanum pennellii introgression lines for residual biomass and enzymatic digestibility traits

BackgroundResidual biomass production for fuel conversion represents a unique opportunity to avoid concerns about compromising food supply by using dedicated feedstock crops. Developing tomato varieties suitable for both food consumption and fuel conversion requires the establishment of new selection methods.ResultsA tomato Solanum pennellii introgression population was assessed for fruit yield, biomass phenotypic diversity, and for saccharification potential. Introgression lines 2–5, 2–6, 6–3, 7–2, 10–2 and 12–4 showed the best combination of fruit and residual biomass production. Lignin, cellulose, hemicellulose content and saccharification rate showed a wide variation in the tested lines. Within hemicellulose, xylose value was high in IL 6–3, IL 7–2 and IL 6–2, whereas arabinose showed a low content in IL 10–2, IL 6–3 and IL 2–6. The latter line showed also the highest ethanol potential production. Alkali pre-treatment resulted in the highest values of saccharification in most of lines tested, suggesting that chemical pretreatment is an important factor for improving biomass processability. Interestingly, extreme genotypes for more than one single trait were found, allowing the identification of better genotypes. Cell wall related genes mapping in genomic regions involved into tomato biomass production and digestibility variation highlighted potential candidate genes. Molecular expression profile of few of them provided useful information about challenged pathways.ConclusionsThe screening of S. pennellii introgression population resulted very useful for delving into complex traits such as biomass production and digestibility. The extreme genotypes identified could be fruitfully employed for both genetic studies and breeding.

Side by side comparison of chemical compounds generated by aqueous pretreatments of maize stover, miscanthus and sugarcane bagasse.

In order to examine the potential for coproduct generation, we have characterised chemical compounds released by a range of alkaline and acidic aqueous pretreatments as well as the effect of these pretreatments on the saccharification ability of the lignocellulosic material. Comparative experiments were performed using three biomass types chosen for their potential as second-generation biofuel feedstocks: maize stover, miscanthus and sugarcane bagasse. The release of lignin from the feedstock correlated with the residual biomass saccharification potential, which was consistently higher after alkaline pretreament for all three feedstock types. Alkaline pretreatment released more complex mixtures of pentose and hexose sugars into the pretreatment liquor than did acid pretreatment. In addition, complex mixtures of aromatic and aliphatic compounds were released into pretreatment liquors under alkaline conditions, in a temperature-dependent manner, but far less so under acidic conditions. We show that the three feedstocks characterised interact with the pretreatment conditions in a specific manner to generate different ranges of products, highlighting the need to tailor pretreatments to both the starting feedstock and desired outcomes.

Characterization of the cellulolytic secretome of Trichoderma harzianum during growth on sugarcane bagasse and analysis of the activity boosting effects of swollenin

This study demonstrates the production of an active enzyme cocktail produced by growing Trichoderma harzianum on sugarcane bagasse. The component enzymes were identified by LCMS‐MS. Glycosyl hydrolases were the most abundant class of proteins, representing 67% of total secreted protein. Other carbohydrate active enzymes involved in cell wall deconstruction included lytic polysaccharide mono‐oxygenases (AA9), carbohydrate‐binding modules, carbohydrate esterases and swollenin, all present at levels of 1%. In total, proteases and lipases represented 5 and 1% of the total secretome, respectively, with the rest of the secretome being made up of proteins of unknown or putative function. This enzyme cocktail was efficient in catalysing the hydrolysis of sugarcane bagasse cellulolignin to fermentable sugars for potential use in ethanol production. Apart from mapping the secretome of T. harzianum, which is a very important tool to understand the catalytic performance of enzyme cocktails, the gene coding for T. harzianum swollenin was expressed in Aspergillus niger. This novel aspect in this work, allowed increasing the swollenin concentration by 95 fold. This is the first report about the heterologous expression of swollenin from T. harzianum, and the findings are of interest in enriching enzyme cocktail with this important accessory protein which takes part in the cellulose amorphogenesis. Despite lacking detectable glycoside activity, the addition of swollenin of T. harzianum increased by two‐fold the hydrolysis efficiency of a commercial cellulase cocktail.

Linkage Mapping of Stem Saccharification Digestibility in Rice.

Rice is the staple food of almost half of the world population, and in excess 90% of it is grown and consumed in Asia, but the disposal of rice straw poses a problem for farmers, who often burn it in the fields, causing health and environmental problems. However, with increased focus on the development of sustainable biofuel production, rice straw has been recognized as a potential feedstock for non-food derived biofuel production. Currently, the commercial realization of rice as a biofuel feedstock is constrained by the high cost of industrial saccharification processes needed to release sugar for fermentation. This study is focused on the alteration of lignin content, and cell wall chemotypes and structures, and their effects on the saccharification potential of rice lignocellulosic biomass. A recombinant inbred lines (RILs) population derived from a cross between the lowland rice variety IR1552 and the upland rice variety Azucena with 271 molecular markers for quantitative trait SNP (QTS) analyses was used. After association analysis of 271 markers for saccharification potential, 1 locus and 4 pairs of epistatic loci were found to contribute to the enzymatic digestibility phenotype, and an inverse relationship between reducing sugar and lignin content in these recombinant inbred lines was identified. As a result of QTS analyses, several cell-wall associated candidate genes are proposed that may be useful for marker-assisted breeding and may aid breeders to produce potential high saccharification rice varieties.

A glycosyl transferase family 43 protein involved in xylan biosynthesis is associated with straw digestibility in Brachypodium distachyon

Summary The recalcitrance of secondary plant cell walls to digestion constrains biomass use for the production of sustainable bioproducts and for animal feed. We screened a population of Brachypodium recombinant inbred lines (RILs) for cell wall digestibility using commercial cellulases and detected a quantitative trait locus (QTL) associated with this trait. Examination of the chromosomal region associated with this QTL revealed a candidate gene that encodes a putative glycosyl transferase family (GT) 43 protein, orthologue of IRX14 in Arabidopsis, and hence predicted to be involved in the biosynthesis of xylan. Arabinoxylans form the major matrix polysaccharides in cell walls of grasses, such as Brachypodium. The parental lines of the RIL population carry alternative nonsynonymous polymorphisms in the Bd GT43A gene, which were inherited in the RIL progeny in a manner compatible with a causative role in the variation in straw digestibility. In order to validate the implied role of our candidate gene in affecting straw digestibility, we used RNA interference to lower the expression levels of the Bd GT43A gene in Brachypodium. The biomass of the silenced lines showed higher digestibility supporting a causative role of the Bd GT43A gene, suggesting that it might form a good target for improving straw digestibility in crops.

Nutrient and drought stress : implications for phenology and biomass quality in miscanthus

Abstract Background and Aims The cultivation of dedicated biomass crops, including miscanthus, on marginal land provides a promising approach to the reduction of dependency on fossil fuels. However, little is known about the impact of environmental stresses often experienced on lower-grade agricultural land on cell-wall quality traits in miscanthus biomass crops. In this study, three different miscanthus genotypes were exposed to drought stress and nutrient stress, both separately and in combination, with the aim of evaluating their impact on plant growth and cell-wall properties. Methods Automated imaging facilities at the National Plant Phenomics Centre (NPPC-Aberystwyth) were used for dynamic phenotyping to identify plant responses to separate and combinatorial stresses. Harvested leaf and stem samples of the three miscanthus genotypes (Miscanthus sinensis, Miscanthus sacchariflorus and Miscanthus × giganteus) were separately subjected to saccharification assays, to measure sugar release, and cell-wall composition analyses. Key Results Phenotyping showed that the M. sacchariflorus genotype Sac-5 and particularly the M. sinensis genotype Sin-11 coped better than the M. × giganteus genotype Gig-311 with drought stress when grown in nutrient-poor compost. Sugar release by enzymatic hydrolysis, used as a biomass quality measure, was significantly affected by the different environmental conditions in a stress-, genotype- and organ-dependent manner. A combination of abundant water and low nutrients resulted in the highest sugar release from leaves, while for stems this was generally associated with the combination of drought and nutrient-rich conditions. Cell-wall composition analyses suggest that changes in fine structure of cell-wall polysaccharides, including heteroxylans and pectins, possibly in association with lignin, contribute to the observed differences in cell-wall biomass sugar release. Conclusions The results highlight the importance of the assessment of miscanthus biomass quality measures in addition to biomass yield determinations and the requirement for selecting suitable miscanthus genotypes for different environmental conditions.