Yield and Woody Biomass Traits of Novel Shrub Willow Hybrids at Two Contrasting Sites

Shrub willow has great potential as a dedicated bioenergy crop, but commercialization and adoption by growers and end-users will depend upon the identification and selection of high-yielding cultivars with biomass chemistry and quality amenable to conversion to biofuels and bioenergy. In this study, critical traits for biomass production were evaluated among new genotypes of shrub willow produced through hybrid breeding. This study assessed the variation in yield, pest and disease resistance, biomass composition, and wood density in shrub willow, as well as the impact of genotypic and environmental factors on these particular phenotypes. Analysis of clonal genotypes established on two contrasting sites in New York State, Tully and Belleville, showed statistical differences by site for all of the traits. The greatest yield was observed at Belleville, NY, for two cultivars, ‘Fish Creek’ (41 Mg?ha^?1) and ‘Onondaga’ (40 Mg?ha^?1). Yields of Salix eriocephala genotypes were lowest, and they displayed susceptibility to rust and beetle damage. Variation in cellulose content in the stem biomass was controlled by environmental factors, with the majority of the genotypes displaying greater cellulose content at Belleville compared with Tully. In contrast, wood density was significantly greater at Tully than Belleville, and cellulose content was correlated with wood density. There were no significant correlations between biomass yield and density or any of the composition traits. These trials demonstrate that new genotypes produce improved yield and pest and disease resistance, with diverse compositional traits that can be matched with conversion technologies.     

Correlations of expression of cell wall biosynthesis genes with variation in biomass composition in shrub willow (Salix spp.) biomass crops

We have measured significant genetically determined variation in biomass composition among breeding populations of shrub willow, a biomass feedstock crop. This project was aimed to ask whether patterns of cell wall gene expression can be correlated with genetic variation in biomass composition at harvest, in order to develop assays of early differences in gene expression as indicators of harvestable biomass chemical composition and potentially reduce the time of selection for new willow genotypes. Previous studies have demonstrated that manipulation of expression of cell wall biosynthetic genes results in altered biomass chemical composition. We analyzed genes encoding enzymes involved in lignin biosynthesis and carbohydrate active enzymes selected based on their functional characterization and conservation in Populus trichocarpa and Arabidopsis thaliana. Fragments of 20 genes were cloned from young stem cDNA of Salix sachalinensis and Salix miyabeana. Expression profiling in willow stem apical tissue and developing stem tissue was performed for each isolated gene using probe-based quantitative real-time PCR. Two willow parental genotypes and six progeny within a hybrid family were selected for analysis, and significant differences in expression among the individuals and between tissue types were observed for most of the genes. Significant correlations between patterns of gene expression and variation in the biomass chemical composition of those genotypes provide insight into the genetic regulation of lignocellulosic deposition in this important bioenergy crop and could be utilized as a tool for early selection of new genotypes.     

Heavy Metal Uptake by Willow Clones from Sewage Sludge-Treated Soil: The Potential for Phytoremediation

Willow (Salix spp.) has shown potential for use in the phytoremediation of soil contaminated with heavy metals. In particular, it can be grown in short rotation coppice systems to produce biomass that can be used for energy production. Twenty different species or varieties of willow, grown over 2 years (1995 to 1997) on a soil that was highly contaminated with heavy metals due to long-term sewage sludge disposal, showed considerable variation in survival, biomass production and metal uptake. The willows could be divided into two groups after the first harvest. One group had relatively low Ni and Cu in the bark and high Cd and Zn in the wood, with a good survival rate and biomass production. This group partitioned Cu, Cd, and Zn into the wood tissue from the bark, whereas Ni was excluded. The second group had relatively high Ni and Cu in the bark and low Cd and Zn in the wood and performed poorly in terms of survival and biomass production. Of the 20 types of willow used, 11 showed potential for use in phytoremediation, combining good survival and biomass production with high metal uptake. Of the others, 2 failed to survive until the second harvest and the other 7 had very poor survival rates.     

High throughput screening of hydrolytic enzymes from termites using a natural substrate derived from sugarcane bagasse

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.     

Wood properties of two silver birch clones exposed to elevated CO2 and O3

Effects of elevated carbon dioxide (CO₂) and ozone (O₃) on wood properties of two initially 7‐year‐old silver birch (Betula pendula Roth) clones were studied after a fumigation during three growing seasons. Forty trees, representing two fast‐growing clones (4 and 80), were exposed in open‐top chambers to the following treatments: outside control, chamber control, 2 × ambient [CO₂], 2 × ambient [O₃] and 2 × ambient [CO₂]+2 × ambient [O₃]. After the 3‐year exposure, the trees were felled and wood properties were analyzed. The treatments affected both stem wood structure and chemistry. Elevated [CO₂] increased annual ring width, and concentrations of extractives and starch, and decreased concentrations of cellulose and gravimetric lignin. Elevated O₃ decreased vessel percentage and increased cell wall percentage in clone 80. In vessel percentage, elevated CO₂ ameliorated the O₃‐induced decrease. In clone 4, elevated O₃ decreased nitrogen concentration of wood. The two clones had different wood properties. In clone 4, the concentrations of extractives, starch, soluble sugars and nitrogen were greater than in clone 80, while in clone 80 the concentrations of cellulose and acid‐soluble lignin were higher. Clone 4 also had slightly longer fibres, greater vessel lumen diameter and vessel percentage than clone 80, while in clone 80 cell wall percentage was greater. Our results show that wood properties of young silver birch trees were altered under elevated CO₂ in both clones, whereas the effects of O₃ depended on clone.     

High-Throughput Profiling of the Fiber and Sugar Composition of Sugarcane Biomass

Lignocellulosic biomass from sugarcane (Saccharum spp. hybrids) could potentially be a major feedstock for second-generation biofuel production. Consequently, selecting sugarcane varieties with favorable biomass characteristics, typically less enzymatic recalcitrance and better saccharification yield without sugar-yield penalty, will be important in sugarcane breeding. Economical and high-throughput techniques for profiling the major biomass components of this complex system will facilitate selection of clones with ideal lignocellulosic composition from large numbers of genotypes in breeding programs. We used a combined high-throughput profiling approach to evaluate the biomass composition of samples from a sugarcane germplasm collection. This employed near-infrared (NIR) spectroscopy for fiber characterization and high-performance liquid chromatography (HPLC) for determining the sugar content in juice. The results for 331 samples, from a diverse sugarcane population of 186 genotypes, derived from 143 parents of different genetic backgrounds, showed that high-quality NIR spectroscopic predictions were feasible for cellulose, hemicellulose, lignin, and extractives values in fiber, and sugars in juice were suitably analyzed by HPLC. The analysis of total biomass indicated that this NIR- and HPLC-based high-throughput method allowed a robust phenotypic assessment of a large number of samples for the key biomass traits in the sugarcane system, including total dry biomass, fiber, sugar content, and theoretical ethanol yields, and could potentially become the method of choice for sugarcane germplasm screening in breeding programs targeting the support of biofuel production.     

Genetic improvement of the chemical composition of Scots pine (Pinus sylvestris L.) juvenile wood for bioenergy production

Chemical composition is one of the key characteristics that determines wood quality and in turn its suitability for different end products and applications. The inclusion of chemical compositional traits in forest tree improvement requires high‐throughput techniques capable of rapid, non‐destructive and cost‐efficient assessment of large‐scale breeding experiments. We tested whether Fourier‐transform infrared (FTIR) spectroscopy, coupled with partial least squares regression, could serve as an alternative to traditional wet chemistry protocols for the determination of the chemical composition of juvenile wood in Scots pine for tree improvement purposes. FTIR spectra were acquired for 1,245 trees selected in two Scots pine (Pinus sylvestris L.) full‐sib progeny tests located in northern Sweden. Predictive models were developed using 70 reference samples with known chemical composition (the proportion of lignin, carbohydrates [cellulose, hemicelluloses and their structural monosaccharides glucose, mannose, xylose, galactose, and arabinose] and extractives). Individual‐tree narrow‐sense heritabilities and additive genetic correlations were estimated for all chemical traits as well as for growth (height and stem diameter) and wood quality traits (density and stiffness). Genetic control of the chemical traits was mostly moderate. Of the major chemical components, highest heritabilities were observed for hemicelluloses (0.43–0.47), intermediate for lignin and extractives (0.30–0.39), and lowest for cellulose (0.20–0.25). Additive genetic correlations among chemical traits were, except for extractives, positive while those between chemical and wood quality traits were negative. In both groups (chemical and wood quality traits), correlations with extractives exhibited opposite signs. Correlations of chemical traits with growth traits were near zero. The best strategy for genetic improvement of Scots pine juvenile wood for bioenergy production is to decrease and stabilize the content of extractives among trees and then focus on increasing the cellulose:lignin ratio.     

Analysis of Crystallinity Index and Hydrolysis Rates in the Bioenergy Crop Sorghum bicolor

Maximum yield from any cellulosic bioenergy crop is largely dependent upon total dry weight at harvest and process-specific bioconversion rates. Using enzymatic hydrolysis rate as a bioconversion metric, we have investigated the relationship between the biomass crystallinity index (CI) and hydrolysis yield potential (HYP) among ??20 Sorghum bicolor varieties grown in two environments. The comparison of HYP to CI revealed a significant negative correlation in both environments indicating that high cellulose crystallinity in sorghum can have an impact on conversion yield. Interestingly, no correlation was seen between CI and HYP after pretreatment. Compositional analysis revealed a significant positive correlation between lignin content and CI, as well as a significant negative correlation between lignin content and HYP. Additionally, CI and HYP were found to be significantly correlated only after 24?h of hydrolysis. These results suggest that when a sorghum cultivar is being considered for industrial scale production, the inclusion of cellulose crystallinity should be factored into the decision along with total biomass yield and lignin composition.     

Lignin Degradation in Foliar Litter of Two Shrub Species from the Gap Center to the Closed Canopy in an Alpine Fir Forest

To understand the effects of forest gaps on lignin degradation during shrub foliar litter decomposition, a field litterbag experiment was conducted in an alpine fir (Abies faxoniana) forest of the eastern Tibet Plateau. Dwarf bamboo (Fargesia nitida) and willow (Salix paraplesia) foliar litterbags were placed on the forest floor from the gap center to the closed canopy. The litterbags were sampled during snow formation, snow coverage, snow melting and the growing season from October 2010 to October 2012. The lignin concentrations and loss in the litter were measured. Over 2 years, lignin loss was lower in the bamboo litter (34.64–43.89%) than in the willow litter (38.91–55.10%). In the bamboo litter, lignin loss mainly occurred during the first decomposition year, whereas it occurred during the second decomposition year in the willow litter. Both bamboo and willow litter lignin loss decreased from the gap center to the closed canopy during the first year and over the entire 2-year decomposition period. Compared with the closed canopy, the gap center showed higher lignin loss for both bamboo and willow litter during the two winters, but lower lignin loss during the early growing period. Additionally, the dynamics of microbial biomass carbon during litter decomposition followed the same trend as litter lignin loss during the two winters and growing period. These results indicated that alpine forest gaps had significant effects on shrub litter lignin loss and that reduced snow cover during winter warming would inhibit shrub lignin degradation in this alpine forest.     

Screening of willow species for resistance to heavy metals: comparison of performance in a hydroponics system and field trials

The aim of this study was to ascertain whether metal resistance in willow (Salix) clones grown in a hydroponics screening test correlated with data from the same clones grown independently in a field trial. If so, results from a short-term, glasshouse-based system could be extrapolated to the field, allowing rapid identification of willows suitable for planting in metal-contaminated substrates without necessitating longterm field trials. Principal Components Analysis was used to show groups of clones and to assess the relative importance of the parameters measured in both the hydroponics system and the field; including plant response factors such as increase in stem height, as well as metal concentrations in plant tissues. The clones tested fell into two distinct groups. Salix viminalis clones and the basket willow Black Maul (S. triandra) were less resistant to elevated concentrations of heavy metals than a group of hardier clones, including S. burjatica 'Germany,' S.x dasyclados, S. candida and S. spaethii. The more resistant clones produced more biomass in the glasshouse and field, and had higher metal concentrations in the wood. The less resistant clones had greater concentrations of Cu and Ni in the bark, and produced less biomass in the glasshouse and field. Significant relationships were found between the response of the same clones grown the in short-term glasshouse hydroponics system and in the field.     

Rapid Assessment of Lignin Content and Structure in Switchgrass (Panicum virgatum L.) Grown Under Different Environmental Conditions

Switchgrass (Panicum virgatum L.) is a candidate feedstock in bioenergy, and plant breeding and molecular genetic strategies are being used to improve germplasm. In order to assess these subsequent modifications, baseline biomass compositional data are needed in a relevant variety of environments. In this study, switchgrass cv. Alamo was grown in the field, greenhouse, and growth chamber and harvested into individual leaf and stem tissue components. These components were analyzed with pyrolysis vapor analysis using molecular beam mass spectrometry, Fourier transform infrared, and standard wet chemistry methods to characterize and compare the composition among the different growth environments. The details of lignin content, S/G ratios, and degree of cross-linked lignin are discussed. Multivariate approaches such as projection to latent structures regression found a very strong correlation between the lignin content obtained by standard wet chemistry methods and the two high throughput techniques employed to rapidly assess lignin in potential switchgrass candidates. The models were tested on unknown samples and verified by wet chemistry. The similar lignin content found by the two methods shows that both approaches are capable of determining lignin content in biomass in a matter of minutes.     

Wood decay activity and cellulase production by freshwater lignicolous fungi

Wood decay activity and coupled cellulase production were examined for freshwater lignicolous Ascomycetes, Deuteromycetes and an Oomycete. Wood decay ability was assessed by weight changes in wood and bark blocks of ash and cottonwood colonized by test fungi. Changes in wood components were also measured. Production of coupled cellulases was determined by measurement of activity of culture filtrates. Except for early successional species, most fungi caused weight loss in sapwood blocks; all species caused weight loss in bark blocks. Bark blocks were decayed more rapidly than sapwood blocks and cottonwood blocks were decayed more rapidly than those of ash. For four species examined, cellulose and lignin disappeared simultaneously, with cellulose disappearing more rapidly than lignin. All species produced extracellular exoglucanase, endoglucanase and glucosidase when grown in liquid media containing crystalline cellulose. Enzyme production by most of the species was increased by the addition of glucose.     

A review of biomass quality research relevant to the use of poplar and willow for energy conversion

It has been recognized that the chemical and physical properties of biomass feedstocks can play an important role in the efficiency of most energy conversion processes. These properties include the ratio of bark to wood, moisture content, specific gravity, calorific or heating value, and the relative content of extractives, α-cellulose, hemicellulose, and lignin.A review of the literature dealing with the quality of poplar and willow biomass feedstock for energy conversion revealed that considerable variation existed in many of these traits. This variation may make it possible to improve the quality of feedstock through breeding and selection.Little information exists with respect to heritability (both in the broad sense and narrow sense), the genetic correlation between characters, and the presence of genotype-environment interaction. A better understanding of these parameters is essential if the apparent variability is to be used to improve biomass quality.     

Solid-State NMR Investigation of Bio-chars Produced from Biomass Components and Whole Biomasses

Bio-char is a by-product from thermochemical treatment of biomass and has been identified as an energy condensed product with a comparable heating value as commercial coal. However, the combustion of such solid product as an energy resource is only a preliminary application. It is highly possible to convert bio-char, which always has a condensed aromatic and porous structure to various high-value products. The investigations of the structures and formation pathways for the bio-char are very important to any future applications. In this study, six different biomass components, including cellulose, lignin, and tannin, and three whole biomasses—pine wood, pine residue, and pine bark—have been used to produce bio-char at 400, 500, and 600 °C. Solid-state NMR and FT-IR have been employed in this study to characterize the structures for the bio-chars. The results indicated that the bio-chars produced from lignin contained some methoxyl groups, and the bio-chars produced from tannin contained significantly higher amount of phenolic hydroxyl groups. Compared to the bio-chars produced from pine wood and residue, the bio-chars produced from pine bark contained more aromatic C–O bonds, and aliphatic C–O and C–C bonds, which may be due to the significantly higher amount of lignin and tannin in the pine bark. However, the elevated amounts of aromatic C–O and aliphatic C–O and C–C bonds in the bio-chars from pine bark appeared to be completely decomposed at 600 °C.     

An NIRS-based assay of chemical composition and biomass digestibility for rapid selection of Jerusalem artichoke clones

Background

High-throughput evaluation of lignocellulosic biomass feedstock quality is the key to the successful commercialization of bioethanol production. Currently, wet chemical methods for the determination of chemical composition and biomass digestibility are expensive and time-consuming, thus hindering comprehensive feedstock quality assessments based on these biomass specifications. To find the ideal bioethanol feedstock, we perform a near-infrared spectroscopic (NIRS) assay to rapidly and comprehensively analyze the chemical composition and biomass digestibility of 59 Jerusalem artichoke (Helianthus tuberosus L., abbreviated JA) clones collected from 24 provinces in six regions of China.

Results

The distinct geographical distribution of JA accessions generated varied chemical composition as well as related biomass digestibility (after soluble sugars extraction and mild alkali pretreatment). Notably, the soluble sugars, cellulose, hemicellulose, lignin, ash, and released hexoses, pentoses, and total carbohydrates were rapidly and perfectly predicted by partial least squares regression coupled with model population analyses (MPA), which exhibited significantly higher predictive performance than controls. Subsequently, grey relational grade analysis was employed to correlate chemical composition and biomass digestibility with feedstock quality score (FQS), resulting in the assignment of tested JA clones to five feedstock quality grades (FQGs). Ultimately, the FQGs of JA clones were successfully classified using partial least squares-discriminant analysis model coupled with MPA, attaining a significantly higher correct rate of 97.8% in the calibration subset and 91.1% in the validation subset.

Conclusions

Based on the diversity of JA clones, the present study has not only rapidly and precisely examined the biomass composition and digestibility with MPA-optimized NIRS models but has also selected the ideal JA clones according to FQS. This method provides a new insight into the selection of ideal bioethanol feedstock for high-efficiency bioethanol production.

     

Wood ontogeny during the first year of hybrid poplar development

During the first year of hybrid poplar development, we assessed radial growth dynamics quantified by the proportion of secondary xylem tissue within the stem area, the vessel area percentage, the content of both lignin and cellulose, the lignin monomeric composition, and the macromolecular properties of cellulose. The intraannual radial growth dynamics in the proportion of secondary xylem tissue was fitted by the Gompertz regression line whereas changes in the vessel area percentage were fitted maximally by a cubic regression line. Under constant temperature and photoperiod, this study revealed that nonlinear patterns of radial growth dynamics are the result of a developmental programme which drives cambial activity and ageing. The increased proportion of guaiacyl units found may be important for the greater stability of the lignin structure in the first year of hybrid poplar development. The tensile strength of juvenile wood was ensured by the trade-off between a slight increase in the degree of polymerization of cellulose and a slight decrease in the content of cellulose during ageing.     

Degradation of polysaccharides and lignin in wood ozonation

Ozonation has been considered as a method for the pretreatment of plant biomass to obtain cellulose and monosaccharides. Ozone consumption by aspen wood with various moisture contents has been investigated. We have considered the gradual transformation of the substrate: wood to ozonated wood to cellulose-containing product (CP) to holocelluloze (HC) and to cellulose. Yields of ozonated wood (OW), the (CP), water-soluble ozonation products, HC, and cellulose have been determined. The lignin content in the CP has been estimated. Both HC and cellulose samples have been studied by IR spectroscopy. The degree of polymerization and molecular mass distribution of cellulose obtained from ozonated wood have been determined. It has been shown that wood destruction by ozone is accompanied by degradation of lignin, hemicelluloses, and cellulose.It has been found that physicochemical properties of cellulose obtained from ozonated wood can be regulated by the variation of the initial moisture content in the substrate. Both molecular ozone and radical species, which are generated in the course of ozone reactions with water present in the substrate structure, participate in wood destruction.     

Small‐scale and automatable high‐throughput compositional analysis of biomass

Conventional wet chemistry methods to determine biomass composition are labor‐ and time‐intensive and require larger amounts of biomass (300 mg) than is often available. To overcome these limitations and to support a high‐throughput pretreatment and hydrolysis (HTPH) screening system, this article reports on the development of a downscaled biomass compositional analysis that is based on conventional wet chemistry techniques but is scaled down by a factor of 100 to use significantly less material. The procedure is performed in readily available high‐performance liquid chromatography vials and can be automated to reduce operator input and increase throughput. Comparison of the compositional analyses of three biomasses determined by the downscaled approach to those obtained by conventional methods showed that the downscaled method measured statistically identical carbohydrate compositions as standard procedures and also can provide reasonable estimates of lignin and ash contents. These results demonstrate the validity of the downscaled procedure for measuring biomass composition to enable the calculation of sugar yields and determination of trends in sugar release behavior in HTPH screening studies. Biotechnol. Bioeng. 2011;108: 306–312. © 2010 Wiley Periodicals, Inc.     

Consequences of Sphaeropsis tip blight disease for the phytohormone profile and antioxidative metabolism of its pine host

Phytopathogenic fungi infections induce plant defence responses that mediate changes in metabolic and signalling processes with severe consequences for plant growth and development. Sphaeropsis tip blight, induced by the endophytic fungus Sphaeropsis sapinea that spreads from stem tissues to the needles, is the most widespread disease of conifer forests causing dramatic economic losses. However, metabolic consequences of this disease on bark and wood tissues of its host are largely unexplored. Here, we show that diseased host pines experience tissue dehydration in both bark and wood. Increased cytokinin and declined indole‐3‐acetic acid levels were observed in both tissues and increased jasmonic acid and abscisic acid levels exclusively in the wood. Increased lignin contents at the expense of holo‐cellulose with declined structural biomass of the wood reflect cell wall fortification by S. sapinea infection. These changes are consistent with H₂O₂ accumulation in the wood, required for lignin polymerization. Accumulation of H₂O₂ was associated with more oxidized redox states of glutathione and ascorbate pools. These findings indicate that S. sapinea affects both phytohormone signalling and the antioxidative defence system in stem tissues of its pine host during the infection process.     

Improving the Profitability of Willow Crops��Identifying Opportunities with a Crop Budget Model

Short-rotation woody crops like shrub willow are a potential source of biomass for energy generation and bioproducts. However, since willow crops are not widely grown in North America, the economics of this crop and the impacts of key crop production and management components are not well understood. We developed a budget model, EcoWillow v1.4 (Beta), that allows users to analyze the entire production-chain for willow systems from the establishment to the delivery of wood chips to the end-user. EcoWillow was used to analyze how yield, crop management options, land rent, fuel, labor, and other costs influence the Internal Rate of Return (IRR) of willow crop systems in upstate New York. We further identified cost variables with the greatest potential for reducing production and transport costs of willow biomass. Productivity of 12 oven-dried tons (odt) ha^?1 year^?1 and a biomass price of $ (US dollars) 60 odt^?1 results in an IRR of 5.5%. Establishment, harvesting, and transportation operations account for 71% of total costs. Increases in willow yield, rotation length, and truck capacity as well as a reduction in harvester down time, land costs, planting material costs, and planting densities can improve the profitability of the system. Results indicate that planting speed and fuel and labor costs have a minimal effect on the profitability of willow biomass crops. To improve profitability, efforts should concentrate on (1) reducing planting stock costs, (2) increasing yields, (3) optimizing harvesting operations, and (4) co-development of plantation designs with new high-yielding clones to reduce planting density.