Roger Ibbett

Uncovering the genome-wide transcriptional responses of the filamentous fungus Aspergillus niger to lignocellulose using RNA sequencing.

A key challenge in the production of second generation biofuels is the conversion of lignocellulosic substrates into fermentable sugars. Enzymes, particularly those from fungi, are a central part of this process, and many have been isolated and characterised. However, relatively little is known of how fungi respond to lignocellulose and produce the enzymes necessary for dis-assembly of plant biomass. We studied the physiological response of the fungus Aspergillus niger when exposed to wheat straw as a model lignocellulosic substrate. Using RNA sequencing we showed that, 24 hours after exposure to straw, gene expression of known and presumptive plant cell wall-degrading enzymes represents a huge investment for the cells (about 20% of the total mRNA). Our results also uncovered new esterases and surface interacting proteins that might form part of the fungal arsenal of enzymes for the degradation of plant biomass. Using transcription factor deletion mutants (xlnR and creA) to study the response to both lignocellulosic substrates and low carbon source concentrations, we showed that a subset of genes coding for degradative enzymes is induced by starvation. Our data support a model whereby this subset of enzymes plays a scouting role under starvation conditions, testing for available complex polysaccharides and liberating inducing sugars, that triggers the subsequent induction of the majority of hydrolases. We also showed that antisense transcripts are abundant and that their expression can be regulated by growth conditions.

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

Graphical abstract Highlights ► Thermal analysis provides real-time data on hydrothermal reactions under realistic conditions. ► From DSC, hemicellulose hydrolysis has low enthalpy change but xylose degradation is exothermic. ► Hydrothermal exothermic degradation reactions may be similar to early stage biomass pyrolysis. ► DMTA shows that the polymeric structure of lignin in biomass is degraded at high temperatures. ► Hydrothermal reactions are effective at greater than 50% solids content.

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

BackgroundThe 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.ResultsEvidence 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.ConclusionsThe 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.

RNA-sequencing reveals the complexities of the transcriptional response to lignocellulosic biofuel substrates in Aspergillus niger

BackgroundSaprobic fungi are the predominant industrial sources of Carbohydrate Active enZymes (CAZymes) used for the saccharification of lignocellulose during the production of second generation biofuels. The production of more effective enzyme cocktails is a key objective for efficient biofuel production. To achieve this objective, it is crucial to understand the response of fungi to lignocellulose substrates. Our previous study used RNA-seq to identify the genes induced in Aspergillus niger in response to wheat straw, a biofuel feedstock, and showed that the range of genes induced was greater than previously seen with simple inducers.ResultsIn this work we used RNA-seq to identify the genes induced in A. niger in response to short rotation coppice willow and compared this with the response to wheat straw from our previous study, at the same time-point. The response to willow showed a large increase in expression of genes encoding CAZymes. Genes encoding the major activities required to saccharify lignocellulose were induced on willow such as endoglucanases, cellobiohydrolases and xylanases. The transcriptome response to willow had many similarities with the response to straw with some significant differences in the expression levels of individual genes which are discussed in relation to differences in substrate composition or other factors. Differences in transcript levels include higher levels on wheat straw from genes encoding enzymes classified as members of GH62 (an arabinofuranosidase) and CE1 (a feruloyl esterase) CAZy families whereas two genes encoding endoglucanases classified as members of the GH5 family had higher transcript levels when exposed to willow. There were changes in the cocktail of enzymes secreted by A. niger when cultured with willow or straw. Assays for particular enzymes as well as saccharification assays were used to compare the enzyme activities of the cocktails. Wheat straw induced an enzyme cocktail that saccharified wheat straw to a greater extent than willow. Genes not encoding CAZymes were also induced on willow such as hydrophobins as well as genes of unknown function. Several genes were identified as promising targets for future study.ConclusionsBy comparing this first study of the global transcriptional response of a fungus to willow with the response to straw, we have shown that the inducing lignocellulosic substrate has a marked effect upon the range of transcripts and enzymes expressed by A. niger. The use by industry of complex substrates such as wheat straw or willow could benefit efficient biofuel production.

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

BackgroundThe 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.ResultsThe 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.ConclusionsA 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.

Expression of Aspergillus niger CAZymes is determined by compositional changes in wheat straw generated by hydrothermal or ionic liquid pretreatments

BackgroundThe capacity of fungi, such as Aspergillus niger, to degrade lignocellulose is harnessed in biotechnology to generate biofuels and high-value compounds from renewable feedstocks. Most feedstocks are currently pretreated to increase enzymatic digestibility: improving our understanding of the transcriptomic responses of fungi to pretreated lignocellulosic substrates could help to improve the mix of activities and reduce the production costs of commercial lignocellulose saccharifying cocktails.ResultsWe investigated the responses of A. niger to untreated, ionic liquid and hydrothermally pretreated wheat straw over a 5-day time course using RNA-seq and targeted proteomics. The ionic liquid pretreatment altered the cellulose crystallinity while retaining more of the hemicellulosic sugars than the hydrothermal pretreatment. Ionic liquid pretreatment of straw led to a dynamic induction and repression of genes, which was correlated with the higher levels of pentose sugars saccharified from the ionic liquid-pretreated straw. Hydrothermal pretreatment of straw led to reduced levels of transcripts of genes encoding carbohydrate-active enzymes as well as the derived proteins and enzyme activities. Both pretreatments abolished the expression of a large set of genes encoding pectinolytic enzymes. These reduced levels could be explained by the removal of parts of the lignocellulose by the hydrothermal pretreatment. The time course also facilitated identification of temporally limited gene induction patterns.ConclusionsThe presented transcriptomic and biochemical datasets demonstrate that pretreatments caused modifications of the lignocellulose, to both specific structural features as well as the organisation of the overall lignocellulosic structure, that determined A. niger transcript levels. The experimental setup allowed reliable detection of substrate-specific gene expression patterns as well as hitherto non-expressed genes. Our data suggest beneficial effects of using untreated and IL-pretreated straw, but not HT-pretreated straw, as feedstock for CAZyme production.

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.

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.