Optimising parameters for pilot scale steam explosion and continuous pressurised disc refining of Miscanthus and sugarcane bagasse for xylose and xylo-oligosaccharide release.

The first comparative pre-treatment study of Miscanthus (Mxg) and sugarcane bagasse (SCB) using steam explosion (SE) and pressurised disc refining (PDR) pretreatment to optimise xylose and xylo-oligosaccharide release is described. The current investigation aimed to 1) Develop optimised batch-wise steam explosion parameters for Mxg and SCB, 2) Scale from static batch steam explosion to dynamic continuous pressurised disc refining, 3) Identify, understand, and circumvent scale-up production hurdles. Optimised SE parameters released 82% (Mxg) and 100% (SCB) of the available xylan. Scaling to PDR, Miscanthus yielded 85% xylan, highlighting how robust scouting assessments for boundary process parameters can result in successful technical transfer. In contrast, SCB technical transfer was not straightforward, with significant differences observed between the two processes, 100% (SE) and 58% (PDR). This report underlines the importance of feedstock-specific pretreatment strategies to underpin process development, scale-up, and optimisation of carbohydrate release from biomass.

Production of oligosaccharides and biofuels from Miscanthus using combinatorial steam explosion and ionic liquid pretreatment.

Pretreatment strategies are fundamental to effectively deconstruct lignocellulosic biomass and economically produce biofuels, biomaterials and bio-based chemicals. This study evaluated individual and combinatorial steam explosion (SE) and ionic liquid (IL) pretreatments for production of high-value oligosaccharides from a novel seed-based Miscanthus hybrid (Mx2779). The two ILs used for pretreatment were triethylammonium hydrogen sulphate [TEA][HSO4] and 1-ethyl-3-methylimidazolium acetate [C2mim][OAc]. The results showed that each pretreatment leads to distinct effects on the fragmentation (cellulose and xylan dissolution, delignification, deacetylation) and physicochemical modification (cellulose and lignin properties) of lignocellulose. This, in turn, dictated enzymatic hydrolysis efficiencies of the cellulose pulp to glucose or gluco-oligosaccharides for downstream applications. Our findings suggest that the stand-alone SE or [C2mim][OAc] pretreatments may offer cost advantages over [TEA][HSO4] through the production of oligosaccharides such as xylo- and gluco-oligosaccharides. This study also highlights technical and economic pretreatment process challenges related to the production of oligosaccharides from Miscanthus Mx2779 biomass.

Pilot-scale production of xylo-oligosaccharides and fermentable sugars from Miscanthus using steam explosion pretreatment.

This study investigated pilot-scale production of xylo-oligosaccharides (XOS) and fermentable sugars from Miscanthus using steam explosion (SE) pretreatment. SE conditions (200 °C; 15 bar; 10 min) led to XOS yields up to 52 % (w/w of initial xylan) in the hydrolysate. Liquid chromatography-mass spectrometry demonstrated that the solubilised XOS contained bound acetyl- and hydroxycinnamate residues, physicochemical properties known for high prebiotic effects and anti-oxidant activity in nutraceutical foods. Enzymatic hydrolysis of XOS-rich hydrolysate with commercial endo-xylanases resulted in xylobiose yields of 380 to 500 g/kg of initial xylan in the biomass after only 4 h, equivalent to ~74 to 90 % conversion of XOS into xylobiose. Fermentable glucose yields from enzymatic hydrolysis of solid residues were 8 to 9-fold higher than for untreated material. In view of an integrated biorefinery, we demonstrate the potential for efficient utilisation of Miscanthus for the production of renewable sources, including biochemicals and biofuels.

Draft Genome Assemblies of Xylose-Utilizing Candida tropicalis and Candida boidinii with Potential Application in Biochemical and Biofuel Production.

Non-albicans Candida species are growing in prominence in industrial biotechnology due to their ability to utilize hemicellulose. Here, we present the draft genome sequences of an inhibitor-tolerant Candida tropicalis strain (Y6604) and Candida boidinii NCAIM Y01308T.

Monitoring real-time enzymatic hydrolysis of Distillers Dried Grains with Solubles (DDGS) by dielectric spectroscopy following hydrothermal pre-treatment by steam explosion.

Dielectric spectroscopy (DS) has been used to monitor the simultaneous saccharification and fermentation of lignocellulosic biomass by measuring its dielectric state. However, it is unknown whether following steam explosion (SE) pre-treatment, lignocellulose would still maintain a dielectric state, and, if maintained, whether the dissipation during enzymatic hydrolysis could be monitored. Distillers Dried Grains with Solubles (DDGS), pre-treated by SE, was found to have a capacitance (C = 580 kHz) of approximately 24 pF cm(-1). Following addition of full-strength cellulolytic cocktail A (CC-A; R(2) = 0.97) and 1/3 strength cocktail B (CC-B; R(2) = 0.96), a natural logarithmic decay in capacitance was determined. Furthermore, the DS biomass probes quantified the initial linear rate of dissipation in capacitance during hydrolysis. The rate of CC-B was 34% that of CC-A. These data extend scope and utility of DS biomass probes for monitoring the enzymatic hydrolysis of SE-pre-treated lignocellulosic substrates in real-time.

Breeding for Bio-ethanol Production in Lolium perenne L.: Association of Allelic Variation with High Water-Soluble Carbohydrate Content.

Increasing the extractable sugar yield from perennial crops is one strategy to generate renewable fuels such as bio-ethanol. Lolium perenne L. (perennial ryegrass) can contain significant (>30% dry matter) water-soluble sugars in the form of polymeric fructan which is readily extracted, broken down and fermented to bio-ethanol. A population of L. perenne generated from four parents which differed in water-soluble carbohydrate (WSC) content was subjected to multiple rounds of selection and recombination on the basis of early spring WSC content to produce a high WSC, and a low WSC population. A control population was generated by selecting the same number of plants at random. The alleles present at six candidate gene loci were analysed before and after selection and correlated to WSC content. Significant differences in the allele frequency of L. perenne soluble-acid invertase1:4 were observed between the three populations with one haplotype significantly associated with the high WSC C2S+ population (after three rounds of selection and two rounds of recombination). Moreover, WSC content was also associated with biomass accumulation. Thus, in addition to a 2.84-fold increase in WSC yield, the C2S+ population also had 1.48-fold more biomass per plant, resulting in 3.9-fold higher WSC yield per plant than the control population.

Modelling real-time simultaneous saccharification and fermentation of lignocellulosic biomass and organic acid accumulation using dielectric spectroscopy.

Dielectric spectroscopy (DS) is routinely used in yeast and mammalian fermentations to quantitatively monitor viable biomass through the inherent capacitance of live cells; however, the use of DS to monitor the enzymatic break down of lignocellulosic biomass has not been reported. The aim of the current study was to examine the application of DS in monitoring the enzymatic saccharification of high sugar perennial ryegrass (HS-PRG) fibre and to relate the data to changes in chemical composition. DS was capable of both monitoring the on-line decrease in PRG fibre capacitance (C=580 kHz) during enzymatic hydrolysis, together with the subsequent increase in conductivity (G=580 kHz) resulting from the production of organic acids during microbial growth. Analysis of the fibre fractions revealed >50% of HS-PRG lignocellulose had undergone enzymatic hydrolysis. These data demonstrated the utility of DS biomass probes for on-line monitoring of simultaneous saccharification and fermentation (SSF).

Safener responsiveness and multiple herbicide resistance in the weed black-grass (Alopecurus myosuroides).

Safeners enhance the selectivity of graminicidal herbicides such as fenoxaprop ethyl in cereals, by increasing their rates of detoxification in the crop. While studying the selectivity of fenoxaprop ethyl in wheat, we determined that the safeners mefenpyr diethyl and fenchlorazole ethyl also enhanced herbicide tolerance in the competing weed black-grass (Alopecurus myosuroides). Fenoxaprop ethyl was detoxified by conjugation with glutathione in both wheat and black-grass, with the resulting metabolites processed to the respective cysteine derivatives, which were then N-glycosylated. In black-grass, these detoxification pathways were only slightly enhanced by safeners, suggesting that metabolism alone was unlikely to account for increased herbicide tolerance. Instead, it was determined that safening was associated with an accumulation of glutathione and hydroxymethylglutathione and enzymes with antioxidant functions including phi and lambda glutathione transferases, active as glutathione peroxidases and thiol transferases respectively. These safener-induced changes closely mirrored those determined in two independent black-grass populations that had acquired multiple herbicide resistance (MHR) in the field. In addition to enhanced glutathione metabolism, both safener treatment and MHR resulted in elevated levels of flavonoids in the foliage of black-grass plants, notably flavone-C-glycosides and anthocyanins. Our results demonstrate that safening in a grass weed is associated with an inducible activation in antioxidant and secondary metabolism which mirrors the biochemical phenotype exhibited in plants that are resistant to multiple classes of herbicides.

Effects of elevated CO2 on the vasculature and phenolic secondary metabolism of Plantago maritima.

We have examined the effect of elevated CO2 on the vasculature and phenolic secondary metabolism on clones of the maritime plant Plantago maritima (L.). Plants were exposed to either ambient (360 micromol CO2 mol(-1)) or elevated (600 micromol CO2 mol(-1)) atmospheric CO2 within a Solardome facility and harvested after 12 months' growth. Histochemical analysis of the leaves identified increases in the diameter of the minor leaf vein and associated lignified vessels in plants exposed to elevated CO2. In the roots the number of lignified root vessels and stele width were also increased, but overall the lignified vessel-wall thickness was reduced in plants exposed to elevated CO2, compared to those grown under ambient CO2. To investigate whether or not these subtle changes in lignification were associated with perturbations in phenolic metabolism, aromatic natural products were analysed by HPLC-MS after treatment with cellulase to hydrolyse the respective glycosidic conjugates. The phenylpropanoids p-coumaric acid, caffeic acid, ferulic acid and the flavone luteolin were identified, together with the caffeoyl phenylethanoid glycosides, verbascoside and plantamajoside which were resistant to enzymatic digestion. Exposure to enhanced CO2 resulted in subtle changes in the levels of individual metabolites. In the foliage a one-year exposure to enhanced CO2 resulted in an increased accumulation of caffeic acid, whilst in the roots p-coumaric acid and verbascoside were enhanced. Our results suggest that significant changes in the vasculature of P. maritima on exposure to increased CO2 are associated with only minor changes in the leaves of specific lignin-related metabolites.