Impact of altered cell wall composition on saccharification efficiency in stem tissue of Arabidopsis RABA GTPase-deficient knockout mutants

Use of biomass for second-generation biofuel production is severely hindered by the inherent recalcitrance of the plant cell wall to digestion. Trafficking is crucial for compartmentalisation within the cell. This process is partly regulated by small Rab GTPase proteins. In particular, control of trafficking to the cell wall is regulated through the RABA clade. Manipulation of this regulatory system offers tantalising opportunities for manipulation of cell wall composition and hence recalcitrance. Trafficking-defective rabA mutants have already been shown to impact cell wall composition. To study the impacts of these mutants on cell wall digestion, we developed a saccharification process for Arabidopsis based on the hot water method. We then showed that following pre-treatment, stems from the T-DNA knockouts of the three RABA4 genes expressed in Arabidopsis stem show an increased sugar release on saccharification. These rabA4 mutants have been shown to impact the “hemicellulose-rich” fraction during cell wall fractionation. Furthermore, we go on to show that these mutant lines also show increased sugar release when subjected to saccharification without pre-treatment. Finally, we used X-ray diffraction to show that rabA4 mutants had no impact on cellulose crystallinity, thus supporting the hypothesis that the increases in saccharification were not due to alterations of the cellulose microfibrils but were a direct effect of reduced hemicellulose levels. We also present data to show that the growth characteristics of these plants were unaffected. The data obtained from these lines are most easily explained by the reported alteration in hemicellulose increasing pre-treatment efficiency

Structural reorganisation of cellulose fibrils in hydrothermally deconstructed lignocellulosic biomass and relationships with enzyme digestibility

Background: The investigation of structural organisation in lignocellulose materials is important to understand changes in cellulase accessibility and reactivity resulting from hydrothermal deconstruction, to allow development of strategies to maximise bioethanol process efficiencies. To achieve progress, wheat straw lignocellulose and comparative model wood cellulose were characterised following increasing severity of hydrothermal treatment. Powder and fibre wide-angle X-ray diffraction techniques were employed (WAXD), complemented by enzyme kinetic measurements up to high conversion. Results: Evidence from WAXD indicated that cellulose fibrils are not perfectly crystalline. A reduction in fibril crystallinity occurred due to hydrothermal treatment, although dimensional and orientational data showed that fibril coherency and alignment were largely retained. The hypothetical inter-fibril spacing created by hydrothermal deconstruction of straw was calculated to be insufficient for complete access by cellulases, although total digestion of cellulose in both treated straw and model pulp was observed. Both treated straw and model pulps were subjected to wet mechanical attrition, which caused separation of smaller fibril aggregates and fragments, significantly increasing enzyme hydrolysis rate. No evidence from WAXD measurements was found for preferential hydrolysis of non-crystalline cellulose at intermediate extent of digestion, for both wood pulp and hydrothermally treated straw. Conclusions: The increased efficiency of enzyme digestion of cellulose in the lignocellulosic cell wall following hydrothermal treatment is a consequence of the improved fibril accessibility due to the loss of hemicellulose and disruption of lignin. However, incomplete accessibility of cellulase at the internal surfaces of fibrillar aggregates implies that etching type mechanisms will be important in achieving complete hydrolysis. The reduction in crystalline perfection following hydrothermal treatment may lead to an increase in fibril reactivity, which could amplify the overall improvement in rate of digestion due to accessibility gains. The lack of preferential digestion of non-crystalline cellulose is consistent with the existence of localised conformational disorder, at surfaces and defects, according to proposed semicrystalline fibril models. Cellulases may not interact in a fully selective manner with such disordered environments, so fibril reactivity may be considered as a function of average conformational states

Towards an integrated art music

This thesis consists of a portfolio of compositions in acoustic and electronic media with an accompanying commentary. The central theme of these works is the development of new strategies for the integration of diverse sound sources, styles and performance aesthetics. Key topics include the fusing of dramatic structures from classical and popular music, the blending of acoustic and electronic instruments, performance strategies for electroacoustic music with live musicians, how concert music can engage with the idea of “the mix” from commercial music, the creation of sound worlds that interpolate between conventional ‘musical’ ingredients and environmental sound

Hydro-mechanical processing of brewer's spent grain as a novel route for separation of protein products with differentiated techno-functional properties

© 2019 Elsevier Ltd Hydro-mechanical processing using a colloid mill with a large gap setting leads to the preferential breakup of the residual aleurone and endosperm tissues of brewer's spent grain, forming a protein rich fines material with small particle size around 1–10 μm. This fraction can be separated from the coarser husk fraction by centrifugation, giving a protein product with enhanced techno-functional properties. The fines have good stability in aqueous suspensions, with potential for stabilising other particulate materials in food or drink formulations. The fines particles can stabilise oil-water emulsions, possibly through a Pickering mechanism, which may also support use in food applications. Fines suspensions have strong shear-thinning behaviour, which may be beneficial from a textural or transport perspective. Spray drying of fines suspensions is shown to avoid particle coalescence, which is important for effective resuspension on rehydration. The high surface area of the fines also leads to more efficient digestion by proteases. Industrial relevance: A novel hydro-mechanical milling process has been investigated for separation of a protein fine fraction from brewer's spent grain having enhanced techno-functional properties. The small particle size of the fines would be a key attribute for formulation in shake or smoothie products, where sensory attributes of the product would not be compromised and the properties of the fines could confer stability against settling. Applications may be found for the fines material as an ingredient in spreads and sauces or infant purees, in-particular where it might be used to stabilise of products based on oil-water emulsions. The market for protein-rich ingredients for foods and drinks is already established in the fitness and well-being market, as derived from other vegetable or cereal sources such as hemp, pea or rice. This controlled pre-milling step is also shown to lead to greater rate and extent of protease digestion of spent grain, which may be of value for generation of protein and peptide products for well-being and cosmetics applications

Succession of physiological stages hallmarks the transcriptomic response of the fungus Aspergillus niger to lignocellulose.

BackgroundUnderstanding how fungi degrade lignocellulose is a cornerstone of improving renewables-based biotechnology, in particular for the production of hydrolytic enzymes. Considerable progress has been made in investigating fungal degradation during time-points where CAZyme expression peaks. However, a robust understanding of the fungal survival strategies over its life time on lignocellulose is thereby missed. Here we aimed to uncover the physiological responses of the biotechnological workhorse and enzyme producer Aspergillus niger over its life time to six substrates important for biofuel production.ResultsWe analysed the response of A. niger to the feedstock Miscanthus and compared it with our previous study on wheat straw, alone or in combination with hydrothermal or ionic liquid feedstock pretreatments. Conserved (substrate-independent) metabolic responses as well as those affected by pretreatment and feedstock were identified via multivariate analysis of genome-wide transcriptomics combined with targeted transcript and protein analyses and mapping to a metabolic model. Initial exposure to all substrates increased fatty acid beta-oxidation and lipid metabolism transcripts. In a strain carrying a deletion of the ortholog of the Aspergillus nidulans fatty acid beta-oxidation transcriptional regulator farA, there was a reduction in expression of selected lignocellulose degradative CAZyme-encoding genes suggesting that beta-oxidation contributes to adaptation to lignocellulose. Mannan degradation expression was wheat straw feedstock-dependent and pectin degradation was higher on the untreated substrates. In the later life stages, known and novel secondary metabolite gene clusters were activated, which are of high interest due to their potential to synthesize bioactive compounds.ConclusionIn this study, which includes the first transcriptional response of Aspergilli to Miscanthus, we highlighted that life time as well as substrate composition and structure (via variations in pretreatment and feedstock) influence the fungal responses to lignocellulose. We also demonstrated that the fungal response contains physiological stages that are conserved across substrates and are typically found outside of the conditions with high CAZyme expression, as exemplified by the stages that are dominated by lipid and secondary metabolism

Effect of moisture content on thermal and water absorption properties of microfibrillar cellulose with polymeric additives

The aim of this study was 1) to investigate the influence of polymeric additives such as carboxyl methyl cellulose (CMC) and locust bean gum (LBG) added before and after homogenisation on the moisture uptake of microfibrillar cellulose (MFC) in the dry and semi-wet state; and 2) to further understand the thermally induced structural transitions of low moisture MFC in the presence of the polymeric additives. A higher moisture content in the highly dense MFC network maintains the fibrillated network structure, which is lost during the drying process resulting in MFC aggregates. The addition of polymeric additives results in the regaining of the structure upon redispersion of the dry material with CMC being more effective than LBG). Results also indicated that CMC has a high level of compatibility with MFC, whereas LBG appears to have limited distribution in the MFC dense microfibrillar network and probably exists as a separate phase when added after homogenisation, however co-processing of LBG and cellulose significantly changed this behaviour. The presence of low-temperature transitions in MFC/additives/water mixtures indicates the involvement of these semi-flexible polymeric additives in the formation of liquid crystals when added to MFC in low moisture environments (2% and 20% w/w). An insight is offered into the theory of surface interactions between MFC and polymeric additives, which prevents the agglomeration of microfibrils present in the highly fibrillated suspension upon drying

Understanding the mechanisms of cooperative physico-chemical treatment and mechanical disintegration of biomass as a route for enhancing enzyme saccharification

A novel chemico-kinetic disintegration model has been applied to study the cooperative relationship between physico-chemical treatment and supplementary wet-state milling of biomass, as an efficient process route to achieve high enzyme accessibility. Wheat straw, Miscanthus and short-rotation willow were studied as three contrasting biomass species, which were subjected to controlled hydrothermal pretreatment using a microwave reactor, followed by controlled wet-state ball-milling. Comparative particle disintegration behaviour and related enzyme digestibilities have been interpreted on the basis of model parameters and with evaluation of textural and chemical differences in tissue structures, aided by the application of specific material characterisation techniques. Supplementary milling led to a 1.3×, 1.6× and 3× enhancement in glucose saccharification yield after 24 h for straw, Miscanthus and willow, respectively, following a standardised 10-min hydrothermal treatment, with corresponding milling energy savings of 98, 97 and 91% predicted from the model, compared to the unmilled case. The results confirm the viability of pretreatment combined with supplementary wet-milling as an efficient process route. The results will be valuable in understanding the key parameters for process design and optimisation and also the key phenotypical parameters for feedstock breeding and selection for highest saccharification yield

The kinetics of inhibitor production resulting from hydrothermal deconstruction of wheat straw studied using a pressurised microwave reactor

BACKGROUND: The use of a microwave synthesis reactor has allowed kinetic data for the hydrothermal reactions of straw biomass to be established from short times, avoiding corrections required for slow heating in conventional reactors, or two-step heating. Access to realistic kinetic data is important for predictions of optimal reaction conditions for the pretreatment of biomass for bioethanol processes, which is required to minimise production of inhibitory compounds and to maximise sugar and ethanol yields. RESULTS: The gravimetric loss through solubilisation of straw provided a global measure of the extent of hydrothermal deconstruction. The kinetic profiles of furan and lignin-derived inhibitors were determined in the hydrothermal hydrolysates by UV analysis, with concentrations of formic and acetic acid determined by HPLC. Kinetic analyses were either carried out by direct fitting to simple first order equations or by numerical integration of sequential reactions. CONCLUSIONS: A classical Arrhenius activation energy of 148 kJmol(−1) has been determined for primary solubilisation, which is higher than the activation energy associated with historical measures of reaction severity. The gravimetric loss is primarily due to depolymerisation of the hemicellulose component of straw, but a minor proportion of lignin is solubilised at the same rate and hence may be associated with the more hydrophilic lignin-hemicellulose interface. Acetic acid is liberated primarily from hydrolysis of pendant acetate groups on hemicellulose, although this occurs at a rate that is too slow to provide catalytic enhancement to the primary solubilisation reactions. However, the increase in protons may enhance secondary reactions leading to the production of furans and formic acid. The work has suggested that formic acid may be formed under these hydrothermal conditions via direct reaction of sugar end groups rather than furan breakdown. However, furan degradation is found to be significant, which may limit ultimate quantities generated in hydrolysate liquors

Chloroplast-rich material from the physical fractionation of pea vine ( Pisum sativum ) postharvest field residue (Haulm)

An innovative procedure for plant chloroplasts isolation has been proposed, which consists of juice extraction by physical fractionation from plant material and recovery of its chloroplast-rich fraction (CRF) by centrifugation. This simple method has been applied to pea vine haulm subjected to different post-harvest treatments: blanching, storage at different relative humidity values and fermentation. Additionally, freeze storage of the extracted juice was carried out. The macronutrient (total lipids, proteins, ash and carbohydrates) and micronutrient (fatty acids, chlorophylls, β-carotene, α-tocopherol and ascorbic acid) content and composition of the CRF have been determined. The CRF isolated from fresh pea vine haulm is a potential source of essential micronutrients (α-linolenic acid, β-carotene, α-tocopherol) and carbohydrates, whereas the post-harvest treatments trialled have a detrimental effect on the nutritional content. Industrial applications for the recovered nutritionally rich fraction, such as food supplement ingredient or animal feeding, are likely envisaged, while optimising the use of green haulm