Michael J. Ray

Biomass Characterization of Buddleja davidii: A Potential Feedstock for Biofuel Production

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 (13)C NMR showed that 33% of the cellulose is para-crystalline and 41% is at inaccessible surfaces. Both quantitative (13)C and (31)P 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.

Fractionation of lignocellulosic biomass with the ionic liquid 1-butylimidazolium hydrogen sulfate

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.

QTL Mapping of Enzymatic Saccharification in Short Rotation Coppice Willow and Its Independence from Biomass Yield

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.

Variation in Cell Wall Composition and Accessibility in Relation to Biofuel Potential of Short Rotation Coppice Willows

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.

Reaction wood – a key cause of variation in cell wall recalcitrance in willow

BackgroundThe 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.ResultsA 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.ConclusionsOur 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.

Soaking of pine wood chips with ionic liquids for reduced energy input during grinding

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.

Correlation between anatomical characteristics of ethanol organosolv pretreated Buddleja davidii and its enzymatic conversion to glucose

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.

Insights into nitrogen allocation and recycling from nitrogen elemental analysis and 15N isotope labelling in 14 genotypes of willow.

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.

X-ray micro-computed tomography in willow reveals tissue patterning of reaction wood and delay in programmed cell death

BackgroundVariation 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).ResultsMajor 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.ConclusionsThe 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.