BACKGROUND:Variation in the reaction wood (RW) response has been shown to be a principle component driving differences in lignocellulosic sugar yield from the bioenergy crop willow. The phenotypic cause(s) behind these differences in sugar yield, beyond their common elicitor, however, remain unclear. Here we use X-ray micro-computed tomography (μCT) to investigate RW-associated alterations in secondary xylem tissue patterning in three dimensions (3D).RESULTS:Major architectural alterations were successfully quantified in 3D and attributed to RW induction. Whilst the frequency of vessels was reduced in tension wood tissue (TW), the total vessel volume was significantly increased. Interestingly, a delay in programmed-cell-death (PCD) associated with TW was also clearly observed and readily quantified by μCT.CONCLUSIONS:The surprising degree to which the volume of vessels was increased illustrates the substantial xylem tissue remodelling involved in reaction wood formation. The remodelling suggests an important physiological compromise between structural and hydraulic architecture necessary for extensive alteration of biomass and helps to demonstrate the power of improving our perspective of cell and tissue architecture. The precise observation of xylem tissue development and quantification of the extent of delay in PCD provides a valuable and exciting insight into this bioenergy crop trait.

The application of the protic ionic liquid 1-butylimidazolium hydrogen sulfate in the deconstruction (aka pretreatment) and fractionation of lignocellulosic biomass has been investigated. A cellulose rich pulp and a lignin fraction were produced. The pulp was subjected to enzymatic saccharification which allowed recovery of up to 90% of the glucan as fermentable glucose. The influence of the solution acidity on the deconstruction of Miscanthus giganteus was examined by varying the 1-butylimidazole to sulfuric acid ratio. Increased acidity led to shorter pretreatment times and resulted in reduced hemicellulose content in the pulp. Addition of water to the ionic liquid resulted in enhanced saccharification yields. The ability to tune acidity through the use of protic ionic liquids offers a significant advantage in flexibility over dialkylimidazolium analogues.

Minimizing nitrogen (N) fertilization inputs during cultivation is essential for sustainable production of bioenergy and biofuels. The biomass crop willow (Salix spp.) is considered to have low N fertilizer requirements due to efficient recycling of nutrients during the perennial cycle. To investigate how successfully different willow genotypes assimilate and allocate N during growth, and remobilize and consequently recycle N before the onset of winter dormancy, N allocation and N remobilization (to and between different organs) were examined in 14 genotypes of a genetic family using elemental analysis and 15N as a label. Cuttings were established in pots in April and sampled in June, August and at onset of senescence in October. Biomass yield of the trees correlated well with yields recorded in the field. Genotype-specific variation was observed for all traits measured and general trends spanning these sampling points were identified when trees were grouped by biomass yield. Nitrogen reserves in the cutting fuelled the entirety of the canopy establishment, yet earlier cessation of this dependency was linked to higher biomass yields. The stem was found to be the major N reserve by autumn, which constitutes a major source of N loss at harvest, typically every 2–3 years. These data contribute to understanding N remobilization in short rotation coppice willow and to the identification of traits that could potentially be selected for in breeding programmes to further improve the sustainability of biomass production.

Background: The recalcitrance of lignocellulosic cell wall biomass to deconstruction varies greatly in angiosperms, yet the source of this variation remains unclear. Here, in eight genotypes of short rotation coppice willow (Salix sp.) variability of the reaction wood (RW) response and the impact of this variation on cell wall recalcitrance to enzymatic saccharification was considered.Results: A pot trial was designed to test if the 'RW response' varies between willow genotypes and contributes to the differences observed in cell wall recalcitrance to enzymatic saccharification in field-grown trees. Biomass composition was measured via wet chemistry and used with glucose release yields from enzymatic saccharification to determine cell wall recalcitrance. The levels of glucose release found for pot-grown control trees showed no significant correlation with glucose release from mature field-grown trees. However, when a RW phenotype was induced in pot-grown trees, glucose release was strongly correlated with that for mature field-grown trees. Field studies revealed a 5-fold increase in glucose release from a genotype grown at a site exposed to high wind speeds (a potentially high RW inducing environment) when compared with the same genotype grown at a more sheltered site.Conclusions: Our findings provide evidence for a new concept concerning variation in the recalcitrance to enzymatic hydrolysis of the stem biomass of different, field-grown willow genotypes (and potentially other angiosperms). Specifically, that genotypic differences in the ability to produce a response to RW inducing conditions (a 'RW response') indicate that this RW response is a primary determinant of the variation observed in cell wall glucan accessibility. The identification of the importance of this RW response trait in willows, is likely to be valuable in selective breeding strategies in willow (and other angiosperm) biofuel crops and, with further work to dissect the nature of RW variation, could provide novel targets for genetic modification for improved biofuel feedstocks.

Short rotation coppice (SRC) willow is currently emerging as an important dedicated lignocellulosic energy crop in the UK. However, investigation into the variation between species and genotypes in their suitability for liquid transport biofuel processing has been limited. To address this, four traits relevant to biofuel processing (composition, enzymatic saccharification, response to pretreatment and projected ethanol yields) were studied in 35 genotypes of willow including Europe’s leading SRC willow cultivars. Large, genotype-specific variation was observed for all four traits. Significant positive correlations were identified between the accessibility of glucan to enzymatic saccharification before and after pretreatment as well as glucose release and xylose release via acid hydrolysis during pretreatment. Of particular interest is that the lignin content of the biomass did not correlate with accessibility of glucan to enzymatic saccharification. The genotype-specific variations identified have implications for SRC willow breeding and for potential reductions in both the net energy expenditure and environmental impact of the lignocellulosic biofuel process chain. The large range of projected ethanol yields demonstrate the importance of feedstock selection based on an ideotype encompassing the performance of both field biomass growth and ease of conversion.

Ionic liquids are of great interest as potential solvents/catalysts for the production of fuels and chemicals from lignocellulosic biomass. Attention has focused particularly on the pretreatment of lignocellulose to make the cellulose more accessible to enzymatic hydrolysis. Any biomass processing requires a reduction in the size of the harvested biomass by chipping and/or grinding to make it more amenable to chemical and biological treatments. This paper demonstrates that significant energy savings can be achieved in the grinding of pine wood chips when the ionic liquid is added before the grinding operation. We show that this is due to the lubricating properties of the ionic liquids and not to physico-chemical modifications of the biomass. A brief impregnation of the chipped biomass results in higher savings than a longer treatment.

Background Short rotation coppice willow is a potential lignocellulosic feedstock in the United Kingdom and elsewhere; however, research on optimising willow specifically for bioethanol production has started developing only recently. We have used the feedstock Salix viminalis × Salix schwerinii cultivar 'Olof' in a three-month pot experiment with the aim of modifying cell wall composition and structure within the stem to the benefit of bioethanol production. Trees were treated for 26 or 43 days with tension wood induction and/or with an application of the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile that is specific to secondary cell walls. Reaction wood (tension and opposite wood) was isolated from material that had received the 43-day tension wood induction treatment. Results Glucan content, lignin content and enzymatically released glucose were assayed. All measured parameters were altered without loss of total stem biomass yield, indicating that enzymatic saccharification yield can be enhanced by both alterations to cell wall structure and alterations to absolute contents of either glucan or lignin. Conclusions Final glucose yields can be improved by the induction of tension wood without a detrimental impact on biomass yield. The increase in glucan accessibility to cell wall degrading enzymes could help contribute to reducing the energy and environmental impacts of the lignocellulosic bioethanol production process.

Ground lignocellulosic biomass (Miscanthus giganteus, pine (Pinus sylvestris) and willow (Salix viminalis)) was pretreated with ionic liquid-water mixtures of 1-butyl-3-methylimidazolium methyl sulfate and 1-butyl-3-methylimidazolium hydrogen sulfate. A solid fraction enriched in cellulose was recovered, which was subjected to enzymatic hydrolysis. Up to 90% of the glucose and 25% of the hemicellulose contained in the original biomass were released by the combined ionic liquid pretreatment and the enzymatic hydrolysis. After the pretreatment, the ionic liquid liquor contained the majority of the lignin and the hemicellulose. The lignin portion was partially precipitated from the liquor upon dilution with water. The amount of hemicellulose monomers in the ionic liquid liquor and their conversion into furfurals was also examined. The performance of ionic liquid-water mixtures containing 1,3-dialkylimidazolium ionic liquids with acetate, methanesulfonate, trifluoromethanesulfonate and chloride anions was investigated. The applicability of the ionic liquid 1-butylimidazolium hydrogensulfate for lignocellulose pretreatment was also examined. It was found that ionic liquid liquors containing methyl sulfate, hydrogen sulfate and methanesulfonate anions were most effective in terms of lignin/cellulose fractionation and enhancement of cellulose digestibility.

Ethanol organosolv pretreatment was performed on Buddleja davidii to evaluate this bioresource as a potential feedstock for bioethanol production. B. davidii was pretreated and delignified, while 85% of the glucose content of the untreated material was retained in the pretreated solid fraction. The enzymatic hydrolysis showed that organosolv pretreatment produced solid substrates that were readily digestible by cellulases. Gel-permeation chromatography was used to determine the degree of polymerization (DP) of cellulose, and solid-state cross polarization/magic angle spinning C-13 NMR experiments were conducted to study the changes in crystallinity and ultrastructure of cellulose. The results showed a decrease in DP along with an increase in the relative proportions of para-crystalline and amorphous cellulose and a decrease in cellulose I-alpha and I-beta. Removal of lignin and hemicellulose, reduction in DP, and decrease in the crystalline allomorphs (I-alpha and I-beta) increased the amenability of the biomass to enzymatic degradation.

Short rotation coppice (SRC) willows (Salix spp.) are fast-growing woody plants which can achieve high biomass yields over short growth cycles with low agrochemical inputs. Biomass from SRC willow is already used for heat and power, but its potential as a source of lignocellulose for liquid transport biofuels has still to be assessed. In bioethanol production from lignocellulose, enzymatic saccharification is used as an approach to release glucose from cellulose in the plant cell walls. In this study, 138 genotypes of a willow mapping population were used to examine variation in enzymatic glucose release from stem biomass to study relationships between this trait and biomass yield traits and to identify quantitative trait loci (QTL) associated with enzymatic saccharification yield. Significant natural variation was found in glucose yields from willow stem biomass. This trait was independent of biomass yield traits. Four enzyme-derived glucose QTL were mapped onto chromosomes V, X, XI, and XVI, indicating that enzymatic saccharification yields are under significant genetic influence. Our results show that SRC willow has strong potential as a source of bioethanol and that there may be opportunities to improve the breeding programs for willows for increasing enzymatic saccharification yields and biofuel production.

A current barrier to the large-scale production of lignocellulosic biofuels is the cost associated with the energy and chemical inputs required for feedstock pretreatment and hydrolysis. The use of controlled partial biological degradation to replace elements of the current pretreatment technologies would offer tangible energy and cost benefits to the whole biofuel process. It has been known for some time from studies of wood decay that, in the early stages of growth in wood, brown rot fungi utilise a mechanism that causes rapid and extensive depolymerisation of the carbohydrate polymers of the wood cell wall. The brown rot hyphae act as delivery vectors to the plant cell wall for what is thought to be a combination of a localised acid pretreatment and a hydroxyl radical based depolymerisation of the cell wall carbohydrate polymers. It is this quality that we have exploited in the present work to enhance the saccharification potential of softwood forest residues for biofuel production. Here we show that after restricted exposure of pine sapwood to brown rot fungi, glucose yields following enzymatic saccharification are significantly increased. Our results demonstrate the potential of using brown rot fungi as a biological pretreatment for biofuel production.

Buddleja davidii is a unique biomass that has many attractive agroenergy features, especially its wide range of growth habitat. The anatomical characteristics of B. davidii were investigated before and after ethanol organosolv pretreatment (one of the leading pretreatment technologies) in order to further understand the alterations that occur to the cellular structure of the biomass which can then be correlated with its enzymatic digestibility. Results showed that the ethanol organosolv pretreatment of B. davidii selectively removes lignin from the middle lamella (ML), which does not significantly disrupt the crystalline structure of cellulose. The removal of ML lignin is a major factor in enhancing enzymatic cellulose‐to‐glucose hydrolysis. The pretreatment also causes cell deformation, resulting in cracks and breaks in the cell wall. These observations, together with characterization analysis of the cell wall polymer material, lend support to the hypothesis that the physical distribution of lignin in the biomass matrix is an important structural feature affecting biomass enzymatic digestibility. Biotechnol. Bioeng. 2010;107: 795–801. © 2010 Wiley Periodicals, Inc.

A novel three-stage bioprocess achieved 75% volatile solids (VS) removal at an organic loading rate (OLR) of 4g VSL(-1)day, a solids retention time (SRT) of 66days, a hydraulic retention time (HRT) of 20days, at a temperature of 35 degrees C. The bioprocess consisted of an anaerobic hydrolytic reactor (HR) where the solids and liquid fractions of the Organic Fraction of the Municipal Solid Waste (OFMSW) were separated with a mesh. The leachate was pumped to a Submerged Anaerobic Membrane Bioreactor (SAMBR) and the treated permeate was polished in an Aerobic Membrane Bioreactor (AMBR). Denaturing Gradient Gel Electrophoresis (DGGE) and DNA sequencing analyses indicated that the increase in methane content in the HR caused by the excess sludge recycle from the SAMBR was associated with an increase in the number of hydrogenotrophic species, mainly Methanobrevibacter sp., Methanobacterium formicicum and Methanosarcina sp. At 20 degrees C VS removal dropped to 50% in the HR and some DGGE bands disappeared when compared to 35 degrees C samples, while some bands such as the one corresponding to Ruminococcus flavefaciens were reduced in intensity. The species associated with the COD-polishing properties of the AMBR correspond to the genera Pseudomonas, Hyphomonas and Hyphomicrobiaceae. These results highlight the positive effect of recycling the excess sludge from the SAMBR to re-inoculate the HR with hydrogenotrophic species.

A compositional analysis was performed on Buddleja davidii to determine its general biomass characteristics and provide detailed analysis of the chemical structures of its cellulose and lignin using NMR. B. davidii is a new potential lignocellulosic bioresource for producing bioethanol because it has several attractive agroenergy features. The biomass composition of B. davidii is 30% lignin, 35% cellulose, and 34% hemicellulose. Solid-state CP/MAS 13C NMR showed that 33% of the cellulose is para-crystalline and 41% is at inaccessible surfaces. Both quantitative 13C and 31P NMR were used to examine the structure of lignin. The lignin was determined to be guaiacyl and syringyl with an h:g:s ratio of 0:81:19.