The main aims of exploring SRC willow: development and life cycle, nitrogen mobilisation, biomass composition, genotypic variation in cell wall ultra-structure as well as enhancing the assessment and the definition of biofuel potential were all addressed in this thesis.
The N15 pot experiment (See Chapter 3) allowed an insight into nitrogen fluxes in developing SRC willow. While establishing that the cutting has a surprisingly extended role in initial establishment, allowing early canopy development, the experiment also shed light on later nitrogen partitioning and highlighted some potential conflicts between high biomass yield and post-harvest nitrogen retention.
A crucial finding in this experiment was that NUE was very different between willow genotypes of dissimilar biomass yield. This was the first example of one of the key themes of all the work presented in this thesis; that there is a large degree of variation between varieties of SRC willow for a range of traits vital to a biofuel ideotype. This concept was further supported by enzymatic saccharification of stem biomass from the N15 pot experiment, which also showed that the genotypes assessed varied substantially one from another. A clear result of this study was the absence of any correlation with biomass yield.
If found to be generally true, this is an important result as it implies that enzymatic saccharification traits will have to be selected for independently in breeding programmes. To investigate this further and to determine whether there
was sufficient variation to map the underlying causal genes, enzymatic
saccharification was investigated in the K8 QTL experiment (see Chapter 4). Here, it was firmly established that there is a high degree of variation in cell wall derived glucose yields in different genotypes of SRC willow and that those yields were independent from biomass yields. Like NUE, a picture emerged that there is a vast range of variation even in a population representing a very small proportion of willow diversity as a whole. This shows for both NUE and glucose yield, which are two traits of crucial importance for the environmental and economic feasibility of lignocellulosic biofuel production, that there is large scope for improvement of SRC willow as a dedicated biofuel crop.
The K8 QTL experiment led to the successful identifications of several regions of the genome associated with glucose yields from enzymatic saccharification. This not only confirmed that this trait is under some degree of genetic control, which could potentially be selected for, but also presents opportunities for identifying candidate genes involved in glucose yields. As no Salix species have as yet had their genome sequenced or been successfully transformed, other avenues outside genetic examination and manipulation were considered. Breeding was not possible in the scope of this PhD so modification of glucose yields at a developmental level was contemplated as a possibility.
Tension wood had been identified within samples from the K8 population and not only presented an interesting approach to glucose yield modification per se but also an opportunity to develop compositional analysis techniques for willow, facilitating later investigation into the factors influencing those glucose yields. The tension wood experiment (see Chapter 5) did indeed succeed both in increasing and,
Nick Brereton – Thesis 2010
Page 163
through the use of a cellulose synthesis inhibitor, decreasing glucose yields by inducing extreme phenotypes. This establishes increased tension wood formation in SRC willow as a specific target beneficial to glucose yields. As this physiological characteristic exists naturally in SRC willow, it could potentially be exaggerated through breeding, genetic modification or induction during tree development without impacting biomass yields.
An important finding in the tension wood experiment was that, in this system, the feedstock composition did not exclusively define glucose yields. There were elements of cell wall recalcitrance to enzymatic saccharification that were independent of glucan and lignin content. Also, the separation of cell wall
composition and accessibility, both paramount to total glucose yields per weight of biomass, could be assessed experimentally. The degree to which tension wood formation varies between varieties of SRC willow was not investigated here.
In order to further investigate the links between feedstock composition and accessibility to cell wall degrading enzymes, as well as trying to assess the impact of these elements on the pretreatment process of lignocellulosic biofuel production, a non-optimal pretreatment experiment was devised (see Chapter 6).
The incomplete pretreatment experiment utilised the broadest range of SRC willow diversity employed in this work by using 35 different genotypes. This final experiment further confirmed the high variation between types of SRC willow, this time in response to pretreatment. The variation in final glucose yields after
pretreatment was clearly determined both by feedstock composition and accessibility. The impact of this work on both the environmental and net energy outputs of the lignocellulosic biofuel conversion process comes primarily from the
finding that equivalent glucose yields could be achieved in some SRC willow genotypes using a pretreatment of reduced severity. Other findings provided evidence that future experimentation could be substantially simplified by not
performing pretreatment in lab scale analysis of cell wall recalcitrance. This is in view of the fact that enzymatic saccharification of untreated biomass strongly correlated with enzymatic saccharification of pretreated biomass. Also that, contrary to popular belief, high lignin content of biomass is not necessarily detrimental to glucose yields and could in some cases be beneficial to the process as a whole.
Large variation in ethanol yields per hectare were projected from this experiment (see Chapter 6). When these are viewed with highly varied NUE (see Chapter 3), as well as the evidence for large variation in genetic regulation of cell wall structure (see Chapter 4) and the scope of naturally occurring metabolic plasticity to factors which strongly influence glucose yields (see Chapter 5), a clear picture emerges. The variety of SRC willow selected is of fundamental importance if biofuel is to be produced economically and sustainably. It is evident from this work that high enzymatic saccharification yield should be added to the ideotype of SRC willow for dedicated biofuel crops. Importantly, not only are there varieties of SRC willow in the UK which will produce ethanol yields far in excess of early estimates, but the potential for improving those yields in the future, through a wide variety of methods, is substantial.
Each of the four major experiments raised numerous interesting possibilities for prospective study. The N15 system, designed to investigate nitrogen cycling (Chapter 3), was successful but would be greatly enhanced through increased
Nick Brereton – Thesis 2010
Page 165
replication. By this, and by extending the analysis through leaf abcission, valuable information concerning nitrogen remobilisation could be gathered. Further work should use the data gathered here to further define appropriate fertilisation
concentrations. This would allow applications of varied fertilizer concentrations that would limit nitrogen in the trees to different degrees, showing how the NUE
variation is maintained in very low input systems. Sources of the clear differences in NUE could also be revealed through experiments using increased numbers of genotypes from the K8 population and subsequent QTL analysis.
The QTL analysis performed on the K8 population (Chapter 4) clearly established the need to address both biomass structure and composition. The analysis would have been greatly improved by including compositional analysis, increasing the information about sugar yields (glucose release from glucan and DM), but also by increasing the number of genotypes to encompass the entire mapping population. However, a clear path forward for this work would be to search for more of SNPs within the QTLs identified in order to increase the QTL resolution. Candidate genes with large impact over sugar yields could then be identified in poplar using the JGI Populus trichocarpa database (as macrosynteny has been shown between this K8 parents and the P. trichocarpa genome (Hanley, Mallott et al. 2006)). Gene function could then be investigated in model organisms such as Arabidopsis.
Further development of tension wood (Chapter 5) understanding is currently limited by the ability to quantitatively assay its abundance in adult trees. However, a potential future investigation could utilise plantations of willow grown under
conditions likely to induce tension wood formation. The Orkney Islands present such an option with both highly hilly land and severe wind conditions. Another vital area
for enquiry is the variation in tension wood formation between genotypes of willow. If the severity of this response varies between genotypes, then the trait could be considered for inclusion in the dedicated lignocellulosic biofuel crop ideotype and potentially be used to direct breeding programmes.
To further advance the understanding of the relationship between cell wall recalcitrance and pretreatment (Chapter 6) optimisation of pretreatment for the least recalcitrant genotype could be performed. This would establish the degree to which pretreatment severity could be reduced for certain feedstocks and allow calculation of the subsequent economic and environmental benefits of that
reduction. However, before elaboration of this work can commence, it is imperative that fermentation of the sugar solutions yielded is performed in order to identify any inhibitors produced by the process. This highly characterised population would also be ideal for investigation into the variation of other potential contributors to cell wall recalcitrance which were not addressed in this study, such as cellulose degree of polymerisation or crystallinity.
Nick Brereton – Thesis 2010
Page 167
References
Abe, H., J. Ohnishi, et al. (2008). "Function of jasmonate in response and tolerance of Arabidopsis to thrip feeding." Plant and Cell Physiology 49(1): 68-80.
Adegbidi, H. G., T. A. Volk, et al. (2001). "Biomass and nutrient removal by willow clones in experimental bioenergy plantations in New York State." Biomass & Bioenergy 20(6): 399-411.
Adler, A., A. Karacic, et al. (2008). "Biomass allocation and nutrient use in fast-growing woody and herbaceous perennials used for phytoremediation." Plant and Soil 305(1- 2): 189-206.
Adler, A., T. Verwijst, et al. (2005). "Estimation and relevance of bark proportion in a willow stand." Biomass & Bioenergy 29(2): 102-113.
Alfenore, S., X. Cameleyre, et al. (2004). "Aeration strategy: a need for very high ethanol performance in Saccharomyces cerevisiae fed-batch process." Applied Microbiology and Biotechnology 63(5): 537-542.
Allison, G. G., S. C. Thain, et al. (2009). "Quantification of hydroxycinnamic acids and lignin in perennial forage and energy grasses by Fourier-transform infrared spectroscopy and partial least squares regression." Bioresource Technology 100(3): 1252-1261. Alston, J. M., J. M. Beddow, et al. (2009). "Agricultural Research, Productivity, and Food
Prices in the Long Run." Science 325(5945): 1209-1210.
Anderson, J. R., W. S. Barnes, et al. (2002). "2,6-dichlorobenzonitrile, a cellulose biosynthesis inhibitor, affects morphology and structural integrity of petunia and lily pollen tubes." Journal of Plant Physiology 159(1): 61-67.
Andersson-Gunneras, S., E. J. Mellerowicz, et al. (2006). "Biosynthesis of cellulose-enriched tension wood in Populus tremula: global analysis of transcripts and metabolites identifies biochemical and developmental regulators in secondary wall
biosynthesis.(vol 45, pg 144, 2005)." Plant Journal 46(2): 349-349.
Andersson-Gunneras, S., E. J. Mellerowicz, et al. (2006). "Biosynthesis of cellulose-enriched tension wood in Populus: global analysis of transcripts and metabolites identifies biochemical and developmental regulators in secondary wall biosynthesis." Plant Journal 45(2): 144-165.
Argus, G. (1997). "Infrageneric classification of Salix (Salicaceae) in the new world." Systematic Botany Monographs 52: 1–121.
Armstrong, A., C. Johns, et al. (1999). "Effects of spacing and cutting cycle on the yield of poplar grown as an energy crop." Biomass & Bioenergy 17(4): 305-314.
Aylott, M. J., E. Casella, et al. (2008). "Yield and spatial supply of bioenergy poplar and willow short-rotation coppice in the UK." New Phytologist 178(2): 358-370.
Bergkvist, P. and S. Ledin (1998). "Stem biomass yields at different planting designs and spacings in willow coppice systems." Biomass & Bioenergy 14(2): 149-156.
BioenergyScheme (2007). Best Practice Manual for SRC Willow. Biofuels Policy, Department of Agriculture & Food.
Bollmark, L., L. Sennerby-Forsse, et al. (1999). "Seasonal dynamics and effects of nitrogen supply rate on nitrogen and carbohydrate reserves in cutting-derived Salix viminalis plants." Canadian Journal of Forest Research-Revue Canadienne De Recherche Forestiere 29(1): 85-94.
Bowman, W. D. and R. T. Conant (1994). "Shoot Growth Dynamics and Photosynthetic Response to Increased Nitrogen Availability in the Alpine Willow Salix-Glauca." Oecologia 97(1): 93-99.
Brandt, A., J. P. Hallett, et al. (2010). "The effect of the ionic liquid anion in the pretreatment of pine wood chips." Green Chemistry 12(4): 672-679.
Brauer, E. K. and B. J. Shelp (2010). "Nitrogen use efficiency: re-consideration of the bioengineering approach." Botany 88(2): 103-109.
Brereton, N. J. B., F. E. Pitre, et al. (2010). "QTL Mapping of Enzymatic Saccharification in Short Rotation Coppice Willow and Its Independence from Biomass Yield." BioEnergy Research 3(3): 251-261.
BritishSugar©. (2010). Retrieved 24/10/10, from http://www.britishsugar.co.uk/. Brosse, N., P. Sannigrahi, et al. (2009). "Pretreatment of Miscanthus x giganteus Using the
Ethanol Organosolv Process for Ethanol Production." Industrial & Engineering Chemistry Research 48(18): 8328-8334.
Bullard, M. J., S. J. Mustill, et al. (2002). "Yield improvements through modification of planting density and harvest frequency in short rotation coppice Salix spp. - 2. Resource capture and used in two morphologically diverse varieties." Biomass & Bioenergy 22(1): 27-39.
Cano-Delgado, A., S. Penfield, et al. (2003). "Reduced cellulose synthesis invokes lignification and defense responses in Arabidopsis thaliana." Plant Journal 34(3): 351-362. Cantarella, M., L. Cantarella, et al. (2004). "Effect of inhibitors released during steam-
explosion treatment of poplar wood on subsequent enzymatic hydrolysis and SSF." Biotechnology Progress 20(1): 200-206.
Chauvet, E. (1987). "Changes in the Chemical-Composition of Alder, Poplar and Willow Leaves during Decomposition in a River." Hydrobiologia 148(1): 35-44.
Chen, F. and R. A. Dixon (2007). "Lignin modification improves fermentable sugar yields for biofuel production." Nature Biotechnology 25(7): 759-761.
Christersson, L. (1987). "Biomass Production by Irrigated and Fertilized Salix Clones." Biomass 12(2): 83-95.
Christian, D. G., A. B. Riche, et al. (2008). "Growth, yield and mineral content of Miscanthus x giganteus grown as a biofuel for 14 successive harvests." Industrial Crops and Products 28(3): 320-327.
Cochard, H., E. Casella, et al. (2007). "Xylem vulnerability to cavitation varies among poplar and willow clones and correlates with yield." Tree Physiology 27(12): 1761-1767. Cohen, J. (1988). Statistical power analysis for the behavioral sciences, New Jersey: Lawrence
Erlbaum.
Cooke, J. E. K. and M. Weih (2005). "Nitrogen storage and seasonal nitrogen cycling in Populus: bridging molecular physiology and ecophysiology." New Phytologist 167(1): 19-30.
Cote, B. and C. Camire (1985). "Nitrogen Cycling in Dense Plantings of Hybrid Poplar and Black Alder." Plant and Soil 87(1): 195-208.
Dadi, A. P., S. Varanasi, et al. (2006). "Enhancement of cellulose saccharification kinetics using an ionic liquid pretreatment step." Biotechnology and Bioengineering 95(5): 904-910.
De Klein, C., R. S. A. Novoa, et al. (2006). N2O Emissions from managed soils, and CO2
Emissions from Lime and Urea Application. 2006 IPCC Guidelines for National Greenhouse Gas Inventories. Volume 4: Agriculture, Forestry and Other Land Use: 11.11.
Debell, D. S. and M. A. Radwan (1979). "Growth and Nitrogen Relations of Coppiced Black Cottonwood and Red Alder in Pure and Mixed Plantings." Botanical Gazette 140: S97-S101.
DeBolt, S., R. Gutierrez, et al. (2007). "Nonmotile cellulose synthase subunits repeatedly accumulate within localized regions at the plasma membrane in Arabidopsis hypocotyl cells following 2,6-dichlorobenzonitrile treatment." Plant Physiology
145(2): 334-338.
Nick Brereton – Thesis 2010
Page 169
Dekker, R. F. H. and A. F. A. Wallis (1983). "Enzymic Saccharification of Sugarcane Bagasse Pretreated by Autohydrolysis Steam Explosion." Biotechnology and Bioengineering
25(12): 3027-3048.
Dluzniewska, P., A. Gessler, et al. (2007). "Nitrogen uptake and metabolism in Populus x canescens as affected by salinity." New Phytologist 173(2): 279-293.
Eklund, R., M. Galbe, et al. (1988). "2-Stage Steam Pretreatment of Willow for Increased Pentose Yield." Journal of Wood Chemistry and Technology 8(3): 379-392.
Eklund, R., M. Galbe, et al. (1990). "Optimization of Temperature and Enzyme Concentration in the Enzymatic Saccharification of Steam-Pretreated Willow." Enzyme and
Microbial Technology 12(3): 225-228.
Eklund, R., M. Galbe, et al. (1995). "The Influence of So2 and H2so4 Impregnation of Willow Prior to Steam Pretreatment." Bioresource Technology 52(3): 225-229.
Eklund, R. and G. Zacchi (1995). "Simultaneous Saccharification and Fermentation of Steam- Pretreated Willow." Enzyme and Microbial Technology 17(3): 255-259.
Ericsson, T. (1981). "Effects of Varied Nitrogen Stress on Growth and Nutrition in 3 Salix Clones." Physiologia Plantarum 51(4): 423-429.
Ericsson, T. (1981). "Growth and Nutrition of 3 Salix Clones in Low Conductivity Solutions." Physiologia Plantarum 52(2): 239-244.
Finch, J. W., A. Karp, et al. (2009). Miscanthus, short-rotation coppice and the historic environment. English Heritage. English Heritage, Centre for Ecology & Hydrology and Rothamsted Research.
Forster, P., V. Ramaswamy, et al. (2007). Changes in Atmospheric Constituents and in Radiative Forcing. Climate Change 2007: The Physical Science Basis. , Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change: 212.
Garcia-Cubero, M. T., G. Gonzalez-Benito, et al. (2009). "Effect of ozonolysis pretreatment on enzymatic digestibility of wheat and rye straw." Bioresource Technology 100(4): 1608-1613.
Gebauer, G. and E. D. Schulze (1991). "Carbon and Nitrogen Isotope Ratios in Different Compartments of a Healthy and a Declining Picea-Abies Forest in the Fichtelgebirge, Ne Bavaria." Oecologia 87(2): 198-207.
Geisler-Lee, J., M. Geisler, et al. (2006). "Poplar carbohydrate-active enzymes. Gene identification and expression analyses." Plant Physiology 140(3): 946-962.
GenStat® (2008). © Lawes Agricultural Trust Rothamsted Research, VSN International Ltd, UK.
Gessler, A., S. Kopriva, et al. (2004). "Regulation of nitrate uptake at the whole-tree level: interaction between nitrogen compounds, cytokinins and carbon metabolism." Tree Physiology 24(12): 1313-1321.
Gomez, L. D., C. G. Steele-King, et al. (2008). "Sustainable liquid biofuels from biomass: the writing's on the walls." New Phytologist 178(3): 473-485.
Good, A. G., A. K. Shrawat, et al. (2004). "Can less yield more? Is reducing nutrient input into the environment compatible with maintaining crop production?" Trends in Plant Science 9(12): 597-605.
Grous, W. R., A. O. Converse, et al. (1986). "Effect of Steam Explosion Pretreatment on Pore- Size and Enzymatic-Hydrolysis of Poplar." Enzyme and Microbial Technology 8(5): 274-280.
Guidi, W., E. Piccioni, et al. (2008). "Bark content estimation in poplar (Populus deltoides L.) short-rotation coppice in Central Italy." Biomass & Bioenergy 32(6): 518-524. Guidi, W., C. Tozzini, et al. (2009). "Estimation of chemical traits in poplar short-rotation
Gullberg, U. (1993). "Towards Making Willows Pilot Species for Coppicing Production." Forestry Chronicle 69(6): 721-726.
Hallac, B. B., P. Sannigrahi, et al. (2010). "Effect of Ethanol Organosolv Pretreatment on Enzymatic Hydrolysis of Buddleja davidii Stem Biomass." Industrial & Engineering Chemistry Research 49(4): 1467-1472.
Hamann, T., M. Bennett, et al. (2009). "Identification of cell-wall stress as a hexose- dependent and osmosensitive regulator of plant responses." Plant Journal 57(6): 1015-1026.
Hames, B., R. Ruiz, et al. (2008). Preparation of Samples for Compositional Analysis.
Laboratory Analytical Procedure (LAP), National Renewable Energy Laboratory NREL. Hanley, S. J. (2003). Improving Willow Breeding Efficiency. Rothamsted Research, Long
Ashton Research Station, University of Bristol: 236.
Hanley, S. J., M. D. Mallott, et al. (2006). "Alignment of a Salix linkage map to the Populus genomic sequence reveals macrosynteny between willow and poplar genomes." Tree Genetics & Genomes 3(1): 35-48.
Hanley, S. J., M. D. Mallott, et al. (2007). "Alignment of a Salix linkage map to the Populus genomic sequence reveals macrosynteny between willow and poplar genomes." Tree Genetics & Genomes 3(1): 35-48.
Hatakka, A. I. (1983). "Pretreatment of Wheat Straw by White-Rot Fungi for Enzymic Saccharification of Cellulose." European Journal of Applied Microbiology and Biotechnology 18(6): 350-357.
Hatfield, R. and R. S. Fukushima (2005). "Can lignin be accurately measured?" Crop Science
45(3): 832-839.
Heller, M. C., G. A. Keoleian, et al. (2004). "Life cycle energy and environmental benefits of generating electricity from willow biomass." Renewable Energy 29(7): 1023-1042. Himmel, M. E. (2007). "Biomass recalcitrance: engineering plants and enzymes for biofuels
production (vol 315, pg 804, 2007)." Science 316(5827): 982-982.
Hirel, B., J. Le Gouis, et al. (2007). "The challenge of improving nitrogen use efficiency in crop plants: towards a more central role for genetic variability and quantitative genetics within integrated approaches." Journal of Experimental Botany 58(9): 2369-2387. Hoch, G. (2007). "Cell wall hemicelluloses as mobile carbon stores in non-reproductive plant
tissues." Functional Ecology 21(5): 823-834.
Hodson, R. W., F. M. Slater, et al. (1994). "Effects of digested sewage sludge on short rotation coppice in the UK." Proceedings of the Workshop Swedish University of Agricultural Sciences: 113-118.
Hogetsu, T., H. Shibaoka, et al. (1974). "Involvement of Cellulose Synthesis in Actions of Gibberellin and Kinetin on Cell Expansion - 2,6-Dichlorobenzonitrile as a New Cellulose-Synthesis Inhibitor." Plant and Cell Physiology 15(2): 389-393.
Hu, W. J., S. A. Harding, et al. (1999). "Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees." Nature Biotechnology 17(8): 808-812. IEA (2006). World Energy Outlook, International Energy Agency.
IEA (2010). Key World Energy Statistics. H. o. C. a. I. Office. 9 rue de la Fédération, 75739 Paris Cedex 15, Fance, Internation Energy Agency: 28.
Iiyama, K. and A. F. A. Wallis (1990). "Dissolution of Wood with Acetyl Bromide Solutions - Reactions of Lignin Model Compounds." Journal of Wood Chemistry and Technology
10(1): 39-58.
Ingersent, K. A. (2003). "World agriculture: Towards 2015/2030 - An FAO perspective." Journal of Agricultural Economics 54(3): 513-515.
Jourez, B. and T. Avella-Shaw (2003). "Effect of gravitational stimulus duration on tension wood formation in young stems of poplar (P-euramericana ev 'Ghoy')." Annals of Forest Science 60(1): 31-41.
Nick Brereton – Thesis 2010
Page 171
Jourez, B., A. Riboux, et al. (2001). "Anatomical characteristics of tension wood and opposite wood in young inclined stems of poplar (Populus euramericana cv 'Ghoy')." Iawa