Background
The potential for production of biofuels from biological material has been highly acknowledged around the world during the last decade. The advantages are several, such as the high abundance of the raw material, sustainability, independence from fossil fuels and the positive impact on the global CO2 emissions. In addition to ethanol, several other chemicals can be simultaneously obtained in biorefineries in an analogous manner to the variety of products obtained from petroleum in oil refineries. A lot of the interest has focused on production of ethanol from feedstock such as sugarcane and corn. However, use of these feedstocks is somewhat problematic because it can sometimes compete with the production of food and the agricultural land use. Therefore, it is important to identify alternative or additional sources of biological material for production of biofuels. Lignocellulosic biomass has the potential to serve as feedstock for sustainable production of biofuels. In our latitudes, the obvious choice is to produce so-called second generation biofuels from the lignocellulose of the forest trees. Examples of the use of lignocellulose in biofuel production exist in Sweden even today (www.domsjoe.com) and new approaches are being developed (www.sekab.com). However, the use of the lignocellulose is challenging due to its recalcitrance i.e. resistance to the chemical and enzymatic hydrolytic treatments to deconstruct the lignocellulose into the constituent monomeric sugar units. Also, the forest industry sometimes considers biofuel production from the lignocellulose problematic due to the competition with other end uses of the woody biomass. We intend in the BioImprove programme to reduce some of the obstacles inherent in the biological material for the use of lignocellulose in bioenergy production.
Cellulose is the major biopolymer in the cell wall and makes up between 40 to 50% of wood. It is a homopolymer consisting of β-1,4-glucan chains, which participate in inter- and intra-hydrogen bonding to crystallize into cellulose microfibrils. The cellulose fibril network provides strength and rigidity to the plant cell wall and represents the supramolecular scaffold with which other wall components such as pectins, hemicelluloses and lignin are associated. Regardless of whether thermochemical or biotechnical processes, such as enzymatic hydrolysis, are used in the biorefinery, the processability of cellulose depends on whether it is crystalline or amorphous. Very little is known about the factors regulating cellulose crystallinity even though the activity of cell wall-residing cellulases, interaction of the cellulose microfibrils with the cortical microtubules, the polymerization degree of cellulose and the length of the cellulose microfibrils have been implicated. We know that cellulose is synthesized in plants by rosette-like complexes in the plasma membrane, but how the function of these complexes is controlled and how it affects the structure of cellulose is not known. With very few exceptions, analysis of mutants affected in cellulose content has suffered from inappropriate analytical tools and analyses in complex tissues instead of focusing specifically on the secondary cell walls. In this programme we will utilize the best tools available to analyse cellulose structure and crystallinity in hybrid aspen secondary cell walls in order to gain information about the control of cellulose structure, such as crystallinity, and how it affects bioprocessing properties of the wood.
Hemicelluloses, the second main type of cell wall polysaccharides, constitute about one fourth of wood biomass and are required for correct assembly of the secondary cell wall. Xylan, which is the main hemicellulose in hardwoods, is considered problematic from the biorefinery perspective and pulping, as it forms complex linkages with other cell wall components, notably lignin. Therefore, improving the separation of xylan from lignin and cellulose is one of main goals for lignocellulose improvement. This will be possible by manipulating xylan side chains, since the side chains are mainly responsible for these interactions.
In addition to the polysaccharides, the cell wall contains lignin. Lignin is a complex polymer that is highly resistant to the hydrolytic conditions during cell wall deconstruction. One of the most curious questions about lignin chemistry is the polymerisation phase, which is still largely unclear. In this programme, both biochemical and molecular approaches are taken to elucidate and characterise some of the enzymatic steps in lignin biosynthesis.
General outline of the programme
The first part of the programme includes identification of key genes that have potential in improving wood bioprocessing properties in our model tree species, hybrid aspen (Populus tremula x tremuloides) and that have been modified in the context of ongoing transformation initiatives at STT and UPSC. A set of transgenic lines with modifications in 44 different genes have been selected from these initiatives on the basis of their growth potential and wood chemistry. This set has for the most part been analysed in detail for wood chemistry with pyrolysis mass spectrometry (PyMS) and fourier transform infrared (FT-IR) within the ongoing FORMAS-funded FuncFiber programme (www.funcfiber.se). We will analyse the same set of trees (herein denoted as Bioimprove collection) in this programme for wood chemistry if not already analysed, for the metabolome and proteome, and for the saccharification potential. Based on the results of the saccharification potential, the most interesting lines will be selected for the analysis of the bioprocessing properties and ethanol production. In addition, novel genes will be identified from research initiatives, which elucidate processes, such as xylan acetylation, cellulose crystallinity and lignin polymerisation. Transgenic hybrid aspen lines will be created, and analysed for wood chemistry when applicable and for saccharification potential at the later stages of the programme.
The second part of the programme includes identification of key genes that have potential in improving biomass production. Since the FuncFiber databank contains information mainly about transgenic hybrid aspen lines with altered wood chemistry, we will select 10-15 lines that show different levels of increase in biomass production among the existing transgenic hybrid aspen collections of SweTree Technologies, and test them for growth performance in the field. In addition, the individual research projects aim to identify novel factors regulating biomass accumulation and study the underlying molecular mechanisms.
Technical platforms
1. Analyses of wood chemistry
The Bioimprove collection and selected, new lines will be screened for cell wall chemistry using both high throughput fourier transform infrared (FT-IR) and pyrolysis mass spectrometry combined with multivariate PCA and OPLS analyses. Lines with clear wood phenotypes will be analysed by two dimensional nuclear magnetic resonance (2D-NMR) of dissolved cell walls, combined with multivariate analysis. This is a novel breakthrough technology developed in the FuncFiber program that allows complete semi high throughput analysis of complete cell walls.
Structure of cellulose will be analysed in selected lines for the degree of cellulose polymerization, cellulose crystallinity and microfibril angle. The degree of cellulose polymerization will be measured by SEC setup with a quadruple detector array for multi angle laser light scattering (SEC/RI/MALLS) at the UPSC laboratory. The crystallinity will be measured by determining the degree of amorphous/crystalline domains by CP/MAS 13C NMR spectroscopy in collaboration with Matthias Hedenström (Umeå University). Microfibril angle and mechanical analysis will be measured using x-ray diffraction in collaboration with Dr Ingo Burgert at Max Planck Institute, Potsdam.
Several additional technologies are available at the cell wall and carbohydrate laboratory at UPSC for instance for the analyses of the cell wall component contents (cellulose, hemicellulose, lignin and extractives) by conventional wet chemistry, the monosaccharide composition and the cell wall linkage structure by GC/MS based methods, the carbohydrate structures by MSn capable ion-trap instrument, as well as the analyses of polymers by SEC/RI/MALLS, viscometry, refractive index (RI) and UV absorbance. A technology for chemical imaging with FT-IR microspectroscopy has also been developed to identify and predict wood properties as well as to analyse cell wall chemistry in situ in a qualitative and semi-quantitative manner.
2. Metabolomics and proteomics
The bioimprove collection and selected, new lines will be analysed for their metabolome and proteome utilizing the UPSC platforms (http://www.upsc.se/metabolomics-facility, www.chem.umu.se/upa). Wood samples will be collected from the stem, and analysed by the personnel employed by facility. The metabolomic analyses include both GC- and LC-MS based techniques, facilitating analysis of approximately 600 metabolites in Populus. Besides the general metabolite profiling approaches, the projects have access to analysis of specific compounds or compound classes, such as amines and amino acids, lipids and plant hormones.
The proteomic analyses include a label-free method developed at UPSC. The Populus protein database, with almost 6000 accessions from different subsamples of the xylem, allows identification of most of the proteins in the hybrid aspen material used in this programme. For more targeted approaches in the individual research projects, it is possible to analyse subsamples such as plasma membrane, cell walls or whole extracts from the wood, or to focus on selected classes of proteins such as the cellulose synthases, peroxidises and glycoproteins.
Integration of the metabolomic and proteomic data will be done using multivariate methods, such as O2PLS (bidirectional orthogonal projections to latent structures) that has been developed at UPSC. Furthermore we propose to infer co-expression network analysis, which will provide a systems biology platform for experimental tree biology that will have a profound effect on e.g. experimental planning and the ability to seek underlying molecular explanations for phenotypic data.
3. Analysis of saccharification potential
A semi-high throughput platform will be set up to analyse saccharification potential in the Bioimprove collection and in all additional lines/genotypes that are being produced in the programme. Saccharification potential defines the degree of sugar release from the lignocellulose during hydrolytic conditions that mimic to a certain degree those present in reactors. Normally the procedure includes two stages: a pretreatment and an enzymatic hydrolysis of small wood particles followed by analysis of the monosaccharide release from the lignocellulose. While the conditions in the reactors usually include high temperatures (~200°C) and high pressure, we intend to set up a protocol where the temperature is 90°C in non-pressurized conditions during the pretreatment. The type of pretreatment is critical for the saccharification efficiency, and we will test the most suitable pretreatment in our conditions and for our material. The advantages of this type of analysis are the small scale of the analysis, rapidity of the analysis and a larger number of samples that can be analysed compared to a reactor. The disadvantage is that the mild hydrolytic conditions do not allow complete release of the sugars, which is not really a problem as long as the main interest is to compare saccharification potential between a set of samples and not the absolute amounts of sugar release. We consider semi-high throughput saccharification analyses important for this programme in order to analyse the impact of the various modifications in large numbers of samples. This analysis is also a screening tool for selecting the most interesting lines for the analysis of bioprocessing properties.
4. Bioprocessing properties
In this programme, selected lines will be evaluated with regard to their chemical properties and processability. With methods commonly used for lignocellulosic materials, wood samples will be pretreated in the presence of an acid catalyst under high pressure and temperature (~200°C). The pretreatment will result in a hemicellulose hydrolysate and a solid residue, which will be analyzed with regard to their chemical composition. The analyses will include the formation of by-products, such as aliphatic acids, furan aldehydes and phenolic compounds. These by-products may affect the fermentability of lignocellulose hydrolysates. Mass spectrometry will be used to investigate the distribution of various phenolic compounds, which can be expected to be very complex, and dependent on the feedstock the lignin properties, catalyst and pretreatment conditions. The susceptibility of the pretreated lignocellulose to enzymatic hydrolysis by cellulases will be investigated. The chemical composition and the fermentability of the resulting hydrolysate will be studied as well as the chemical properties of the lignin-rich residue obtained after the enzymatic hydrolysis. Multivariate data analysis will be employed to study differences between the investigated lines, the chemical composition of the various fractions, the fermentability, and the overall yield of the conversion process.
The fermentation process that will be used in the evaluation will be based on the yeast Saccharomyces cerevisiae. This is the favoured microorganism in industrial production of ethanol and is currently being considered also for production of other biofuels, such as biobutanol. The evaluation process will offer valuable information about conversion of the feedstock not only for the production of cellulosic ethanol, but also for other biofuels and chemicals.
It will be of special interest to investigate the lignin-rich residue obtained after the hydrolysis. It is possible to utilise the lignin as a solid biofuel, which is the most commonly considered option. However, novel products from lignin would be of great interest for the biorefinery industry. The susceptibility of the lignin fraction to thermochemical and biochemical deconstruction will therefore also be studied.
The work on bioprocessing properties will in part be performed in laboratories in Örnsköldsvik and in collaboration with MoRe Research AB; an Örnsköldsvik-based company with equipment and analytical resources useful for pretreatment, separation, and analysis of lignocellulosic materials.
The outline of the chemical and enzymatic conversions of the woody raw material in a small-scale biorefinery. |
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Research
