Adam Elliston

High concentrations of cellulosic ethanol achieved by fed batch semi simultaneous saccharification and fermentation of waste-paper

Highlights ► Batch addition of paper waste in SSSF results in up to 11.6% (v/v) ethanol. ► Low overall enzyme loadings (3.7 FPU/g substrate). ► High cumulative substrate loadings (65% w/v). ► High ethanol concentrations will improve distillation efficiencies.

Characterization of cell wall components of wheat straw following hydrothermal pretreatment and fractionation

Thermophysical pretreatment enhances the enzymatic hydrolysis of lignocellulose. However, its impact on cell wall chemistry is still poorly understood. This paper reports the effects of hydrothermal pretreatment on the degradation and alkali-extractability of wheat straw cell wall polymers. Pretreatment resulted in loss and/or solubilization of arabinoxylans (by 53%), ferulic and diferulic acids which are important cross-linking agents accompanied by concomitant increases in cellulose (up to 43%) and lignin (29%). The remaining water-insoluble hemicelluloses were more readily extractable in alkali and were reduced in molecular weight indicating substantial thermochemical depolymerization. They were also associated with smaller but significant amounts of (cellulose-derived) glucose. The alkali-insoluble residues consisted predominantly of cellulosic glucose and lignin and contained p-coumaric acid. The depolymerization of hemicelluloses, reduction in cinnamic acids and partial degradation of cellulose is likely to contribute significantly to the accessibility of cellulases during subsequent enzymolysis.

Characterization of cell wall components of wheat bran following hydrothermal pretreatment and fractionation.

BackgroundPretreatments are a prerequisite for enzymatic hydrolysis of biomass and production of ethanol. They are considered to open up the plant cell wall structure by altering, moving or solubilizing lignin and hydrolyzing a proportion of hemicellulosic moieties. However, there is little information concerning pretreatment-induced changes on wheat bran cell wall polymers and indeed on changes in cell wall phenolic esters in bran or other lignocellulosic biomass. Here, we evaluate polymeric changes (chemical and physical) as a result of selected hydrothermal pretreatment conditions on destarched wheat bran using controlled polymer extraction methods. Quantification of cell wall components together with soluble oligosaccharides, the insoluble residues and ease of extractability and fractionation of biomass residues were conducted.ResultsPretreatment solubilized selected arabinoxylans and associated cross-linking ferulic and diferulic acids with a concomitant increase in lignin and cellulosic glucose. The remaining insoluble arabinoxylans were more readily extractable in alkali and showed considerable depolymerization. The degree of arabinose substitution was less in xylans released by higher concentrations of alkali. The recalcitrant biomass which remained after pretreatment and alkali extraction contained mostly cellulosic glucose and Klason lignin. Pretreatment generated small but insignificant amounts of yeast-inhibiting compounds such as furfural and hydroxymethyl furfural.As such, simultaneous saccharification and fermentation of the hydrothermally pretreated bran resulted in increased ethanol yields compared to that of the control (97.5% compared to 63% theoretical).ConclusionHydrothermal pretreatment of destarched wheat bran resulted in degradation and depolymerization of the hemicellulosic arabinoxylans together with some breakdown of cellulosic glucose. This was accompanied by a significant reduction in the cross-linking phenolic acids such as ferulic and diferulic acids. The results suggest that hydrothermal pretreatment enhances enzymatic digestibility of the cellulose not only by depolymerization and solubilization of the hemicelluloses but by breakdown of interpolymeric phenolic cross-links between the remaining insoluble polymers. This allows easier access of hydrolytic enzymes by opening or loosening of the cell wall thus resulting in enhanced saccharification of cellulose and subsequent fermentation to ethanol. The reduction in cinnamic acids by selected breeding or biotechnological approaches could provide a useful basis for improved saccharification and fractionation of wheat bran polysaccharides.

Steam explosion of oilseed rape straw: Establishing key determinants of saccharification efficiency

Oilseed rape straw was steam exploded into hot water at a range of severities. The residues were fractionated into solid and liquid phases and chemically characterised. The effect of steam explosion on enzymatic hydrolysis of the water-insoluble fractions was investigated by studying initial cellulase binding and hydrolysis yields for different cellulase doses. Time-course data was modelled to establish rate-dependent differences in saccharification as a function of pretreatment severity and associated chemical composition. The study concluded: (1) the initial hydrolysis rate was limited by the amount of (pectic) uronic acid remaining in the substrate; (2) the proportion of rapidly hydrolysable carbohydrate was most closely and positively related to lignin abundance and (3) the final sugar yield most closely related to xylan removal from the substrate. Comparisons between milled and un-milled steam exploded straw highlighted the influence that physical structure has on hydrolysis rates and yields, particularly at low severities.

Simultaneous saccharification and fermentation of steam exploded duckweed: improvement of the ethanol yield by increasing yeast titre.

Highlights • Steam explosion of duckweed enhances SSF at low (2% w/v) substrate concentrations.• High substrate concentrations (20% w/v) result in much lower ethanol yields.• Ethanol yields can be considerably increased with higher yeast inoculum.• Or by preconditioning yeasts in steam explosion liquor containing inhibitors.• The extra/preconditioned yeast metabolise fermentation inhibitors.

Effect of steam explosion on waste copier paper alone and in a mixed lignocellulosic substrate on saccharification and fermentation.

Highlights • Steam explosion of copier paper reduces xylose and produces inhibitors.• Steam explosion at SF 3.6 and 3.9 increased initial rates of saccharification.• Steam explosion at moderate severities may reduce processing times.• Co-steam explosion of waste paper and wheat straw reduces inhibitor production.

Methodology for enabling high-throughput simultaneous saccharification and fermentation screening of yeast using solid biomass as a substrate.

BackgroundHigh-throughput (HTP) screening is becoming an increasingly useful tool for collating biological data which would otherwise require the employment of excessive resources. Second generation biofuel production is one such process. HTP screening allows the investigation of large sample sets to be undertaken with increased speed and cost effectiveness. This paper outlines a methodology that will enable solid lignocellulosic substrates to be hydrolyzed and fermented at a 96-well plate scale, facilitating HTP screening of ethanol production, whilst maintaining repeatability similar to that achieved at a larger scale.ResultsThe results showed that utilizing sheets of biomass of consistent density (handbills), for paper, and slurries of pretreated biomass that could be pipetted allowed standardized and accurate transfers to 96-well plates to be achieved (±3.1 and 1.7%, respectively). Processing these substrates by simultaneous saccharification and fermentation (SSF) at various volumes showed no significant difference on final ethanol yields, either at standard shake flask (200 mL), universal bottle (10 mL) or 96-well plate (1 mL) scales. Substrate concentrations of up to 10% (w/v) were trialed successfully for SSFs at 1 mL volume. The methodology was successfully tested by showing the effects of steam explosion pretreatment on both oilseed rape and wheat straws.ConclusionsThis methodology could be used to replace large shake flask reactions with comparatively fast 96-well plate SSF assays allowing for HTP experimentation. Additionally this method is compatible with a number of standardized assay techniques such as simple colorimetric, High-performance liquid chromatography (HPLC) and Nuclear magnetic resonance (NMR) spectroscopy. Furthermore this research has practical uses in the biorefining of biomass substrates for second generation biofuels and novel biobased chemicals by allowing HTP SSF screening, which should allow selected samples to be scaled up or studied in more detail.

Effect of Brassica napus cultivar on cellulosic ethanol yield

BackgroundIntraspecific variations in biomass composition are likely to influence their suitability for biorefining. This may be particularly important in species such as Brassica napus, which contain many different crop types bred for different purposes. Here, straw derived from 17 B. napus cultivars, of varying crop types, were steam exploded, saccharified and fermented to establish differences in biomass composition relevant to cellulosic ethanol production.ResultsDespite being grown and processed in the same manner, straw from the various cultivars produced different saccharification and fermentation yields after processing. Fermentation inhibitor abundances released by steam explosion also varied between genotypes. Cultivars with glucan-rich straw did not necessarily produce higher saccharification or ethanol yields after processing. Instead, the compositions of non-cellulosic components were more reliable indicators of substrate quality. The abundance of pectins and arabinogalactans had the greatest influence on saccharification efficiency between straw genotypes.ConclusionsIn dicotyledonous species, such as B. napus, variations in the abundance of pectins between crop cultivars are likely to influence processing efficiency for bioethanol production. Knowledge of these genotypic variants provides targets for plant breeding and could aid in the development of improved cellulase cocktails.

Diverse lineages of Candida albicans live on old oaks

The human pathogen, Candida albicans, is considered an obligate commensal of animals, yet it is occasionally isolated from trees, shrubs and grass. We generated deep genome sequence data for three strains of C. albicans that we isolated from oak trees in an ancient wood-pasture, and compared these to the genomes of the type strain and 21 other clinical strains. C. albicans strains from oak are similar to clinical C. albicans in that they are predominantly diploid and can become naturally homozygous at the mating locus through whole-chromosome loss of heterozygosity (LOH). LOH regions in all genomes arose recently suggesting that LOH mutations usually occur transiently in C. albicans populations. Oak strains differed from clinical strains in showing less LOH, and higher levels of heterozygosity genome-wide. Using phylogenomic analyses, in silico chromosome painting, and comparisons with thousands more C. albicans strains at seven loci, we show that each oak strain is more closely related to strains from humans and other animals than to strains from other oaks. Therefore, the isolation of C. albicans from oak is not easily explained as contamination from a single animal source. The high heterozygosity of oak strains could arise as a result of reduced mitotic recombination in asexual lineages, recent parasexual reproduction or because of natural selection. Regardless of mechanism, the diversity of C. albicans on oaks implies that they have lived in this environment long enough for genetic differences from clinical strains to arise.

Optimising conditions for bioethanol production from rice husk and rice straw: effects of pre-treatment on liquor composition and fermentation inhibitors

BackgroundRice straw and husk are globally significant sources of cellulose-rich biomass and there is great interest in converting them to bioethanol. However, rice husk is reportedly much more recalcitrant than rice straw and produces larger quantities of fermentation inhibitors. The aim of this study was to explore the underlying differences between rice straw and rice husk with reference to the composition of the pre-treatment liquors and their impacts on saccharification and fermentation. This has been carried out by developing quantitative NMR screening methods.ResultsAir-dried rice husk and rice straw from the same cultivar were used as substrates. Carbohydrate compositions were similar, whereas lignin contents differed significantly (husk: 35.3% w/w of raw material; straw 22.1% w/w of raw material). Substrates were hydrothermally pre-treated with high-pressure microwave processing across a wide range of severities. 25 compounds were identified from the liquors of both pre-treated rice husk and rice straw. However, the quantities of compounds differed between the two substrates. Fermentation inhibitors such as 5-HMF and 2-FA were highest in husk liquors, and formic acid was higher in straw liquors. At a pre-treatment severity of 3.65, twice as much ethanol was produced from rice straw (14.22% dry weight of substrate) compared with the yield from rice husk (7.55% dry weight of substrate). Above severities of 5, fermentation was inhibited in both straw and husk. In addition to inhibitors, high levels of cellulase-inhibiting xylo-oligomers and xylose were found and at much higher concentrations in rice husk liquor. At low severities, organic acids and related intracellular metabolites were released into the liquor.ConclusionsRice husk recalcitrance to saccharification is probably due to the much higher levels of lignin and, from other studies, likely high levels of silica. Therefore, if highly polluting chemical pre-treatments and multi-step biorefining processes are to be avoided, rice husk may need to be improved through selective breeding strategies, although more careful control of pre-treatment may be sufficient to reduce the levels of fermentation inhibitors, e.g. through steam explosion-induced volatilisation. For rice straw, pre-treating at severities of between 3.65 and 4.25 would give a glucose yield of between 37.5 and 40% (w/DW, dry weight of the substrate) close to the theoretical yield of 44.1% w/DW, and an insignificant yield of total inhibitors.