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

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

Shear rheology and filament stretching behaviour of xanthan gum and carboxymethyl cellulose solution in presence of saliva

The objective of the work reported in this paper is to determine if saliva addition has an effect on the rheology of xanthan gum solutions. The reasons for the interest was that it has been previously reported that flavour release from high viscosity xanthan thickened foods is not reduced in the same way as foods thickened by other hydrocolloids at comparable viscosities. It was previously postulated that this could be due to an interaction between saliva and xanthan that could change the microstructure and rheology of xanthan solutions. In this work the effect of saliva on the rheology of CMC and xanthan solutions was compared. Solutions of molecularly dissolved xanthan gum and CMC mixed with water or human whole saliva at a ratio of 5:1 showed little impact of the presence of saliva on steady shear or dynamic viscosity for the two hydrocolloids. In filament thinning experiments saliva addition significantly increased filament break-up time for xanthan gum while it had little effect on the break-up time of the CMC filament. Also, filament thinning appeared a lot less even and was not as reproducible in the case of xanthan gum. Addition of CMC and hydroxypropyl methylcellulose (HPMC) to xanthan gum solutions showed a similar increase in break-up time to saliva, but to see this effect the viscosity of the added CMC or HPMC solution had to be very much higher than the viscosity of saliva. The results are discussed in the context of the structure of xanthan gum and the reported extensional rheology of saliva

Concertacion y seguridad social : de la legitimidad social a la legitimidad tecnocrática

El estudio aborda la concertación, un análisis de las políticas de seguridad social constatando su evolución desde una legitimidad social a una legitimidad tecnocrática en la crisis del welfare state y en la crisis económica con particular referencia al sistema español. Desde la normalización de la acción política de los sindicatos y agentes sociales en sus diversas manifestaciones, como la participación institucional y la "legislación negociada" en la búsqueda del consenso y el negociado de intercambios en el marco político frente a la imposición unilateral. La frustración de la concertación social se produce cuando el gobierno interpreta la democracia como algo puramente aritmético de mayorías que ejercen sin más el poder, en épocas de debilidad sindical, de debilidad del estado nación, de dificultades para las políticas redistributivas, de reforzamiento de los planteamientos neoliberales. La búsqueda de legitimidad en el pacto social no se ve en estas ocasiones como necesaria sino que la legitimidad se traslada al discurso economicista y tecnocrático, no político. Se vuelve a la búsqueda de una legitimidad a través del mercado y del intercambio entre particulares.L'estudi aborda la concertació, una anàlisi de les polítiques de seguretat social constatant la seva evolució des d'una legitimitat social a una legitimitat tecnocràtica en la crisi del welfare state i en la crisi econòmica amb particular referència al sistema espanyol. Des de la normalització de l'acció política dels sindicats i agents socials en les seves diverses manifestacions, com la participació institucional i la "legislació negociada" en la cerca del consens i el negociat d'intercanvis en el marc polític enfront de la imposició unilateral. La frustració de la concertació social es produeix quan el govern interpreta la democràcia com alguna cosa purament aritmètic de majories que exerceixen sense més el poder, en èpoques de feblesa sindical, de feblesa de l'estat nació, de dificultats per a les polítiques redistributives, de reforçament dels plantejaments neoliberals. La cerca de legitimitat en el pacte social no es veu en aquestes ocasions com a necessària sinó que la legitimitat es trasllada al discurs economicista i tecnocràtic, no polític. Es torna a la cerca d'una legitimitat a través del mercat i de l'intercanvi entre particulars.The study addresses the conclusion an analysis of policies of social security noting exposing its evolution from a social legitimacy to a technocratic legitimacy in the crisis of the welfare state and the economic crisis with particular reference to the Spanish system. Since the normalization of the political action of the trade unions and social partners in its various manifestations, such as institutional participation and negotiated "legislation" in the search for consensus and the Bureau of exchanges in the political framework against the unilateral imposition. The frustration of the social agreement occurs when the Government interprets the democracy as something purely arithmetic of majorities that exercise without more power, in times of Union weakness, weakness of the State nation, of difficulties for redistributive policies, strengthening of neoliberal approaches. The search for legitimacy in the social pact is not on these occasions as necessary but that legitimacy is moved to the economistic and technocratic, non-political speech. Turns to the search for legitimacy through the market and the exchange between individuals

Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria

<p>Abstract</p> <p>Background</p> <p>The Gateway recombinatorial cloning system allows easy and rapid joining of DNA fragments. Here we report the construction and evaluation of three different Gram-positive vectors that can be used with the Multisite Gateway cloning system to rapidly produce new gene arrangements in plasmid constructs for use in a variety of Gram-positive bacteria.</p> <p>Results</p> <p>Comparison of patterns of reporter gene expression with conventionally constructed clones show that the presence of residual recombination (att) sites does not have an effect on patterns of gene expression, although overall levels of gene expression may vary. Rapid construction of these new vectors allowed vector/gene combinations to be optimized following evaluation of plasmid constructs in different bacterial cells and demonstrates the benefits of plasmid construction using Gateway cloning.</p> <p>Conclusion</p> <p>The residual <it>att </it>sites present after Gateway cloning did not affect patterns of promoter induction in Gram-positive bacteria and there was no evidence of differences in mRNA stability of transcripts. However overall levels of gene expression may be reduced, possibly due to some post-transcriptional event. The new vectors described here allow faster, more efficient cloning in range of Gram-positive bacteria.</p

Succession of physiological stages hallmarks the transcriptomic response of the&nbsp;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

The mechanisms of hydrothermal deconstruction of lignocellulose: New insights from thermal–analytical and complementary studies

Differential Scanning Calorimetry, Dynamic Mechanical Thermal Analysis, gravimetric and chemical techniques have been used to study hydrothermal reactions of straw biomass. Exothermic degradation initiates above 195 °C, due to breakdown of the xylose ring from hemicellulose, which may be similar to reactions occurring during the early stage pyrolysis of dry biomass, though activated at lower temperature through water mediation. The temperature and magnitude of the exotherm reduce with increasing acid concentration, suggesting a reduction in activation energy and a change in the balance of reaction pathways. The presence of xylan oligomers in auto-catalytic hydrolysates is believed to be due to a low rate constant rather than a specific reaction mechanism. The loss of the lignin glass transition indicates that the lignin phase is reorganised under high temperature auto-catalytic conditions, but remains partially intact under lower temperature acid-catalytic conditions. This shows that lignin degradation reactions are activated thermally but are not effectively catalysed by aqueous acid

A comparison of the sensory and rheological properties of molecular and particulate forms of xanthan gum

A particulate form of xanthan gum was prepared by extrusion cooking. The temperature dependence of the viscosity of this form shows similarities to starch with an increase in viscosity to a maximum with increasing temperature as a result of the swelling of the particles. The rheology and mixing behaviour with water of the particulate and conventional molecular forms of xanthan were compared with a modified starch. The particulate xanthan products mixed rapidly with water in a similar way to ungelatinised starch, whereas conventional molecular xanthan systems mixed poorly. Using an experienced sensory panel, model tomato products thickened with the three systems were compared at equal shear viscosities. The panel could not discriminate between the tomato flavour of the three products, but found that the xanthan products were perceived as being significantly thicker. These observations were consistent with previous work. Salt perception for both xanthan products was poorer than for the starch thickened systems. A hypothesis to explain why xanthan does not fit into the previously postulated link between mixing and perception is presented

Levels of Cellulose rich and hemicellulose rich fractions in stem tissue of wild type and <i>rab</i>A knockout lines.

<p>The residue from the pectin extraction was fractionated using KOH into (A) The cellulose rich fraction and (B) The hemicellulose rich fraction. Each fraction was dried and weighed. The statistical values are as follows; “cellulose rich” fraction p<0.001 (d.f,32 v.r,6.8); “hemicellulose rich” fraction p<0.001 (d.f,32 v.r,15.04) with letters annotating significant difference between means.</p

Null Mutants of Individual <i>RABA</i> Genes Impact the Proportion of Different Cell Wall Components in Stem Tissue of <i>Arabidopsis thaliana</i>

<div><p>In <i>Arabidopsis,</i> and other plants, the RABA GTPases (orthologous to the Rab11a of mammals) have expanded in number and diversity and have been shown to belong to eight sub clades, some of which have been implicated in controlling vesicles that traffic cell wall polymers and enzymes that synthesise or modify them to the cell wall. In order to investigate this, we have investigated whether T-DNA insertion knockouts of individual <i>RABA</i> genes belonging to different sub clades, impact on the composition of the plant cell wall. Single gene knockouts of the <i>RABA1</i>, <i>RABA2</i> and <i>RABA4</i> sub clades primarily affected the percentage composition of pectin, cellulose and hemicellulose within the cell wall, respectively, despite having no obvious phenotype in the whole plant. We hypothesise that vesicles carrying specific types of cargoes from the Golgi to the cell surface may be regulated by particular sub types of RABA proteins, a finding that could have wider implications for how trafficking systems work and could be a useful tool in cell wall research and other fields of plant biology.</p></div