Sugarcane bagasse derived xylooligosaccharides produced by an arabinofuranosidase/xylobiohydrolase from Bifidobacterium longum in synergism with xylanases.

Arabinoxylan is a major hemicellulose in the sugarcane plant cell wall with arabinose decorations that impose steric restrictions on the activity of xylanases against this substrate. Enzymatic removal of the decorations by arabinofuranosidases can allow a more efficient arabinoxylan degradation by xylanases. Here we produced and characterized a recombinant Bifidobacterium longum arabinofuranosidase from glycoside hydrolase family 43 (BlAbf43) and applied it, together with GH10 and GH11 xylanases, to produce xylooligosaccharides (XOS) from wheat arabinoxylan and alkali pretreated sugarcane bagasse. The enzyme synergistically enhanced XOS production by GH10 and GH11 xylanases, being particularly efficient in combination with the latter family of enzymes, with a degree of synergism of 1.7. We also demonstrated that the enzyme is capable of not only removing arabinose decorations from the arabinoxylan and from the non-reducing end of the oligomeric substrates, but also hydrolyzing the xylan backbone yielding mostly xylobiose and xylose in particular cases. Structural studies of BlAbf43 shed light on the molecular basis of the substrate recognition and allowed hypothesizing on the structural reasons of its multifunctionality.

Design of experiments driven optimization of alkaline pretreatment and saccharification for sugarcane bagasse.

To maximize the sugar release from sugarcane bagasse, a high-resolution Fractional Factorial Design (FFD) was combined with a Central Composite Orthogonal (CCO) design to simultaneously evaluate a wide range of variables for alkaline pretreatment (NaOH: 0.1-1 mol/L, temperature: 100-220 °C, and time: 20-80 min) and enzymatic saccharification (enzyme loading: 2.5-17.5%, and reaction volume: 550-850 µL). A total of 46 experimental conditions were evaluated and the maximum sugar yield (423 mg/g) was obtained after 18 h enzymatic hydrolysis under optimized conditions (0.25 mol/L NaOH at 202 °C for 40 min, with 12.5% of enzyme loading). Biomass compositional analyses showed that the pretreatments strongly removed lignin (up to 70%), silica (up to 80%) and promoted cellulose enrichment (25-110%). This robust design of experiments resulted in maximizing enzymatic hydrolysis efficiency of sugarcane bagasse and further indicated that this combined approach is versatile for other lignocellulosic biomasses.

Biomass composition of the golden tide pelagic seaweeds Sargassum fluitans and S. natans (morphotypes I and VIII) to inform valorisation pathways.

Massive strandings of the pelagic brown algae Sargassum have occurred in the Caribbean, and to a lesser extent, in western Africa, almost every year since 2011. These events have major environmental, health, and economic impacts in the affected countries. Once on the shore, Sargassum is mechanically harvested and disposed of in landfills. Existing commercial applications of other brown algae indicate that the pelagic Sargassum could constitute a valuable feedstock for potential valorisation. However, limited data on the composition of this Sargassum biomass was available to inform on possible application through pyrolysis or enzymatic fractionation of this feedstock. To fill this gap, we conducted a detailed comparative biochemical and elemental analysis of three pelagic Sargassum morphotypes identified so far as forming Atlantic blooms: Sargassum natans I (SnI), S. fluitans III (Sf), and S. natans VIII (SnVIII). Our results showed that SnVIII accumulated a lower quantity of metals and metalloids compared to SnI and Sf, but it contained higher amounts of phenolics and non-cellulosic polysaccharides. SnVIII also had more of the carbon storage compound mannitol. No differences in the content and composition of the cell wall polysaccharide alginate were identified among the three morphotypes. In addition, enzymatic saccharification of SnI produced more sugars compared to SnVIII and Sf. Due to high content of arsenic, the use of pelagic Sargassum is not recommended for nutritional purposes. In addition, low yields of alginate extracted from this biomass, compared with brown algae used for industrial production, limit its use as viable source of commercial alginates. Further work is needed to establish routes for future valorisation of pelagic Sargassum biomass.

Overcoming the trade-off between grain weight and number in wheat by the ectopic expression of expansin in developing seeds leads to increased yield potential.

Wheat is the most widely grown crop globally, providing 20% of all human calories and protein. Achieving step changes in genetic yield potential is crucial to ensure food security, but efforts are thwarted by an apparent trade-off between grain size and number. Expansins are proteins that play important roles in plant growth by enhancing stress relaxation in the cell wall, which constrains cell expansion. Here, we describe how targeted overexpression of an α-expansin in early developing wheat seeds leads to a significant increase in grain size without a negative effect on grain number, resulting in a yield boost under field conditions. The best-performing transgenic line yielded 12.3% higher average grain weight than the control, and this translated to an increase in grain yield of 11.3% in field experiments using an agronomically appropriate plant density. This targeted transgenic approach provides an opportunity to overcome a common bottleneck to yield improvement across many crops.

Cell wall remodeling under salt stress: Insights into changes in polysaccharides, feruloylation, lignification, and phenolic metabolism in maize.

Although cell wall polymers play important roles in the tolerance of plants to abiotic stress, the effects of salinity on cell wall composition and metabolism in grasses remain largely unexplored. Here, we conducted an in-depth study of changes in cell wall composition and phenolic metabolism induced upon salinity in maize seedlings and plants. Cell wall characterization revealed that salt stress modulated the deposition of cellulose, matrix polysaccharides and lignin in seedling roots, plant roots and stems. The extraction and analysis of arabinoxylans by size-exclusion chromatography, 2D-NMR spectroscopy and carbohydrate gel electrophoresis showed a reduction of arabinoxylan content in salt-stressed roots. Saponification and mild acid hydrolysis revealed that salinity also reduced the feruloylation of arabinoxylans in roots of seedlings and plants. Determination of lignin content and composition by nitrobenzene oxidation and 2D-NMR confirmed the increased incorporation of syringyl units in lignin of maize roots. Salt stress also induced the expression of genes and the activity of enzymes enrolled in phenylpropanoid biosynthesis. The UHPLC-MS-based metabolite profiling confirmed the modulation of phenolic profiling by salinity and the accumulation of ferulate and its derivatives 3- and 4-O-feruloyl quinate. In conclusion, we present a model for explaining cell wall remodeling in response to salinity.

Cell wall hydrolases act in concert during aerenchyma development in sugarcane roots.

BACKGROUND AND AIMS: Cell wall disassembly occurs naturally in plants by the action of several glycosyl-hydrolases during different developmental processes such as lysigenous and constitutive aerenchyma formation in sugarcane roots. Wall degradation has been reported in aerenchyma development in different species, but little is known about the action of glycosyl-hydrolases in this process. METHODS: In this work, gene expression, protein levels and enzymatic activity of cell wall hydrolases were assessed. Since aerenchyma formation is constitutive in sugarcane roots, they were assessed in segments corresponding to the first 5 cm from the root tip where aerenchyma develops. KEY RESULTS: Our results indicate that the wall degradation starts with a partial attack on pectins (by acetyl esterases, endopolygalacturonases, ß-galactosidases and α-arabinofuranosidases) followed by the action of ß-glucan-/callose-hydrolysing enzymes. At the same time, there are modifications in arabinoxylan (by α-arabinofuranosidases), xyloglucan (by XTH), xyloglucan-cellulose interactions (by expansins) and partial hydrolysis of cellulose. Saccharification revealed that access to the cell wall varies among segments, consistent with an increase in recalcitrance and composite formation during aerenchyma development. CONCLUSION: Our findings corroborate the hypothesis that hydrolases are synchronically synthesized, leading to cell wall modifications that are modulated by the fine structure of cell wall polymers during aerenchyma formation in the cortex of sugarcane roots.

Sudangrass, an alternative lignocellulosic feedstock for bioenergy in Argentina.

Sudangrass, Sorghum sudanense (Piper) Stapf, is a vigorous forage crop that has also been used for biogas, paper, and electricity production. Due to the large biomass yields achieved by sudangrass and the large area of potential growth in Argentina seven sudangrass accessions from a collection of S. sudanense were analyzed to evaluate their potential as feedstocks for lignocellulosic bioethanol production, and to assess whether there is an association between the response to biotic and abiotic stresses and the composition of the biomass. The biomass composition was analyzed for major cell wall polymers, monosaccharides, and elemental composition. On average, 68% of stem lignocellulosic biomass was comprised of matrix polysaccharides and crystalline cellulose, representing a potential source of sugars for bioethanol production. Xylose was the predominant matrix polysaccharide monosaccharide comprising, on average, 45% of the total sugars, followed by arabinose, glucose, galactose, galacturonic acid, mannose, glucuronic acid, and fucose. Rhamnose was not detected in any of the biomasses analyzed. Silica was the most abundant element in sudangrass stem, followed by chloride, calcium, phosphorus and sulfur. We performed saccharification analyses after pretreatments. Alkaline pretreatment was more effective than water pretreatment. Sodium hydroxide pretreatment exposed different levels of recalcitrance among sudangrass accessions, whereas the water pretreatment did not. Phenological traits were also evaluated, showing significant variability among accessions. The comparison of major cell wall polymers and monosaccharide composition between tolerant and susceptible accessions to abiotic and biotic stresses suggests an association between the composition of the biomass and the response to stress.

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. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:327-336, 2016.

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

BACKGROUND: The 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. RESULTS: The 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. CONCLUSION: Our 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.

Effects of pretreatment on morphology, chemical composition and enzymatic digestibility of eucalyptus bark: a potentially valuable source of fermentable sugars for biofuel production - part 1.

BACKGROUND: In recent years, the growing demand for biofuels has encouraged the search for different sources of underutilized lignocellulosic feedstocks that are available in sufficient abundance to be used for sustainable biofuel production. Much attention has been focused on biomass from grass. However, large amounts of timber residues such as eucalyptus bark are available and represent a potential source for conversion to bioethanol. In the present paper, we investigate the effects of a delignification process with increasing sodium hydroxide concentrations, preceded or not by diluted acid, on the bark of two eucalyptus clones: Eucalyptus grandis (EG) and the hybrid, E. grandis x urophylla (HGU). The enzymatic digestibility and total cellulose conversion were measured, along with the effect on the composition of the solid and the liquor fractions. Barks were also assessed using Fourier-transform infrared spectroscopy (FTIR), solid-state nuclear magnetic resonance (NMR), X-Ray diffraction, and scanning electron microscopy (SEM). RESULTS: Compositional analysis revealed an increase in the cellulose content, reaching around 81% and 76% of glucose for HGU and EG, respectively, using a two-step treatment with HCl 1%, followed by 4% NaOH. Lignin removal was 84% (HGU) and 79% (EG), while the hemicellulose removal was 95% and 97% for HGU and EG, respectively. However, when we applied a one-step treatment, with 4% NaOH, higher hydrolysis efficiencies were found after 48 h for both clones, reaching almost 100% for HGU and 80% for EG, in spite of the lower lignin and hemicellulose removal. Total cellulose conversion increased from 5% and 7% to around 65% for HGU and 59% for EG. NMR and FTIR provided important insight into the lignin and hemicellulose removal and SEM studies shed light on the cell-wall unstructuring after pretreatment and lignin migration and precipitation on the fibers surface, which explain the different hydrolysis rates found for the clones. CONCLUSION: Our results show that the single step alkaline pretreatment improves the enzymatic digestibility of Eucalyptus bark. Furthermore, the chemical and physical methods combined in this study provide a better comprehension of the pretreatment effects on cell-wall and the factors that influence enzymatic digestibility of this forest residue.

Expansins expression is associated with grain size dynamics in wheat (Triticum aestivum L.).

Grain weight is one of the most important components of cereal yield and quality. A clearer understanding of the physiological and molecular determinants of this complex trait would provide an insight into the potential benefits for plant breeding. In the present study, the dynamics of dry matter accumulation, water uptake, and grain size in parallel with the expression of expansins during grain growth in wheat were analysed. The stabilized water content of grains showed a strong association with final grain weight (r(2)=0.88, P <0.01). Grain length was found to be the trait that best correlated with final grain weight (r(2)=0.98, P <0.01) and volume (r(2)=0.94, P <0.01). The main events that defined final grain weight occurred during the first third of grain-filling when maternal tissues (the pericarp of grains) undergo considerable expansion. Eight expansin coding sequences were isolated from pericarp RNA and the temporal profiles of accumulation of these transcripts were monitored. Sequences showing high homology with TaExpA6 were notably abundant during early grain expansion and declined as maturity was reached. RNA in situ hybridization studies revealed that the transcript for TaExpA6 was principally found in the pericarp during early growth in grain development and, subsequently, in both the endosperm and pericarp. The signal in these images is likely to be the sum of the transcript levels of all three sequences with high similarity to the TaExpA6 gene. The early part of the expression profile of this putative expansin gene correlates well with the critical periods of early grain expansion, suggesting it as a possible factor in the final determination of grain size.

Involvement of active oxygen species and peroxidases in phytoalexin production induced in soybean hypocotyls by an elicitor from a saprophytic fungus

The involvement of active oxygen species and apoplastic peroxidases in the mechanism of phytoalexin induction were evaluated in soybean hypocotyls using an eliciting extract from the saprophytic fungus Mucor ramosissimus. Hydrogen peroxide and ferric chloride-ascorbic acid a .OH radical generating system, promoted glyceollin production and increase in peroxidase activity similar to those observed with the fungal elicitor. Addition of catalase or potassium ferrocyanide (an inhibitor of peroxidase) inhibited both processes. It is suggested that there is a cause-effect relationship between peroxidase activity and phytoalexin induction both triggered by oxidative processes.