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Micropyrolysis of natural poplar mutants with altered p-hydroxyphenyl lignin content
Accepted Manuscript Title: Micropyrolysis of natural poplar mutants with altered p-hydroxyphenyl lignin content Author: Jop Vercruysse Andries Poupaert Ruben Vanholme Catherine Bastien Bartel Vanholme Wout Boerjan Wolter Prins Frederik Ronsse PII: DOI: Reference:
S0165-2370(16)30095-X http://dx.doi.org/doi:10.1016/j.jaap.2016.08.021 JAAP 3807
To appear in:
J. Anal. Appl. Pyrolysis
Received date: Accepted date:
26-2-2016 23-8-2016
Please cite this article as: Jop Vercruysse, Andries Poupaert, Ruben Vanholme, Catherine Bastien, Bartel Vanholme, Wout Boerjan, Wolter Prins, Frederik Ronsse, Micropyrolysis of natural poplar mutants with altered phydroxyphenyl lignin content, Journal of Analytical and Applied Pyrolysis http://dx.doi.org/10.1016/j.jaap.2016.08.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Micropyrolysis of natural poplar mutants with altered p-hydroxyphenyl lignin content
Corresponding author: Jop Vercruysse1, [email protected], Tel: +32 (0)9 264 6190, Fax: +32 (0)9 264 6235 Authors: Jop Vercruysse1, Andries Poupaert1, Ruben Vanholme2,3, Catherine Bastien4, Bartel Vanholme2,3, Wout Boerjan2,3, Wolter Prins1, Frederik Ronsse1 1
Department of Biosystems Engineering, Bioscience Engineering Faculty, Ghent University, Coupure
links 653, B-9000 Gent, Belgium 2
Department of Plant Systems Biology, VIB, Technologiepark 927, B-9052 Gent, Belgium
3
Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, B-
9052 Gent, Belgium 4
National Institute for Agricultural Research, Centre of Orleans, Laboratory of Breeding, Genetics
and Physiology of Forest Trees, 2163, Avenue de la Pomme de Pin, CS 40001 Ardon, 45166 Olivet Cedex, France.
Highlights
Analytical pyrolysis GC-MS for thermochemical degradation analysis of natural poplar mutants
Shift in lignin-derived compounds for mutants homozygous for the defective PnHCT1-Δ73 allele
Higher production of non-methoxylated products at the expense of methoxylated phenols
Correlation analysis revealing demethoxylation of G and S lignin units into non-methoxylated phenols
Abstract Analytical pyrolysis coupled to GC-MS (Py-GC-MS) was applied for the thermochemical degradation analysis of wood from poplar (Populus nigra) defective in p-HYDROXYCINNAMOYLCoA:SHIKIMATE p-HYDROXYCINNAMOYL TRANSFERASE 1 (HCT1), an enzyme essential in the biosynthesis of guaiacyl and syringyl units in lignin. The aim of this study was to assess the potential of natural poplar trees with characteristic lignin composition to improve the yield of valuable chemicals in biomass fast pyrolysis, with a focus on phenolic product formation during lignin decomposition. Analysis of the fast pyrolysis vapor phase obtained from the lignocellulosic biomass of trees homozygous for the defective PnHCT1-Δ73 allele revealed significant shifts in the spectrum of ligninderived pyrolytic compounds as compared to wild type and heterozygous trees. More particular for the homozygotes, a higher production of the non-methoxylated p-hydroxycinnamic alcohol (p-coumaryl alcohol) and 2,3-dihydrobenzofuran was detected at the expense of mono-methoxylated phenols. Correlation analysis also suggested further decomposition reactions such as demethoxylation, demethylation and alkylation of the mono- and di-methoxylated phenolic moieties during pyrolysis, giving rise to a remarkable amount of non-methoxylated phenols in the pyrolytic spectrum.
Keywords: Lignocellulosic biomass; Lignin; Analytical Pyrolysis; Gas Chromatography; Mass spectrometry
1 Introduction Lignocellulose biomass of plants is a biorenewable resource that can be used in fast pyrolysis systems to produce bio-oil. Fast pyrolysis is an anoxic and fast heating process to temperatures typically in the 400-600°C range which provides rapid and complete conversion of solid organic biomass into 60-75 wt% bio-oil, 15-25 wt% solid biochar and 10-20 wt% non-condensable gases. [1, 2] Upgrading the biooil can either serve to substitute petroleum fuels, whereas additional extraction can lead towards the production of valuable chemicals. [3-5] As such the pyrolysis bio-oil can counteract fossil resource depletion and global climate change associated with rising atmospheric carbon dioxide levels, giving it
a very high ecological and economical potential. [6-8] The robustness of the process is an important advantage of pyrolysis as it overcomes feedstock problems related to biomass recalcitrance. Lignin for example has an important biological role in the secondary cell wall of plants, but it strongly influences industrial processing of biomass, e.g. by requiring extensive pretreatments prior to enzymatic hydrolysis of the polysaccharides (in particular cellulose) into fermentable sugars. [9, 10] The only pretreatment required for fast pyrolysis is biomass drying and grinding to a sufficiently small particle size in order to provide optimal heat transfer. [11-13]
The production of large amounts of high quality bio-oil as a source of energy is the main aim in current fast pyrolysis research. However, fast pyrolysis also yields up to 10 wt% of valuable mono-phenols such as guaiacols, syringols, alkyl phenols and catechols. [3] These compounds have economic potential as substitutes for their petrochemical counterparts and would be used in the synthesis of a variety of products including wood adhesives, bioplastics, pharmaceuticals, fragrances and octane enhancers for transportation fuels. [3, 4] Improving the quantity and quality of valuable pyrolysis products is currently mainly achieved by adjusting process conditions. Equally important is the improvement of upstream processes including feedstock selection. [14, 15] In this regard, there is a large potential for improving plant cell walls by exploiting the available genetic resources and by genetic modification. This domain has been successfully explored for fermentation purposes in recent years, i.e. breeding or engineering plant cell walls that are easier to convert to fermentable sugars. [9, 10, 16, 17] This potential (i.e. of biomass with intentionally modified cell wall composition) has remained largely unexplored in fast pyrolysis, where biomass feedstock with a different lignin amount and/or lignin composition might be very attractive, for instance for the production of phenolic compounds. [18] For this reason there is vast interest in engineering of bioenergy crops to such extent that the derived bio-oil is enriched in addedvalue target components. [11, 12, 19, 20]
Breeding is still the most commonly used method for crop improvement. This involves crossing of genetically diverse parental lines and the subsequent careful screening of the progenies for beneficial phenotypes. Here the quality of the breeding germplasm and hence the genetic variation within a
breeding population is key to success. Using a deep sequencing approach, rare natural variants of lignin biosynthesis genes were searched for in a natural population of Black poplar (Populus nigra). [21] A rare recessive allele was identified, PnHCT1-Δ73, that encodes a truncated version of pHYDROXYCINNAMOYL-CoA:SHIKIMATE
p-HYDROXYCINNAMOYL
TRANSFERASE
(HCT1), a key enzyme of the phenylpropanoid pathway (Figure A in Supplementary Materials), converting first p-coumaroyl-CoA to p-coumaroyl shikimic acid and later on caffeoyl shikimic acid to caffeoyl-CoA. [22, 23] The latter is further converted towards the methoxylated monolignols coniferyl alcohol and sinapyl alcohol, which are incorporated in lignin as guaiacyl (G) and syringyl (S) units, respectively. [24-26] In line with the enzymatic activity of HCT1, poplar trees homozygous for the defective allele have a different lignin composition characterized by an increase in p-hydroxyphenyl (H) units compared to poplars heterozygous for the defective allele and poplars that do not harbor the defective allele. [22] It is to be expected that this shift towards H units will affect the concentration of non-methoxylated phenolic compounds in the pyrolysis vapors. As these target phenolic compounds are considered valuable, the bio-oil obtained from PnHCT1-Δ73 homozygous poplars could potentially be of higher value for further chemical processing. [27]
The objective of this study was to verify whether lignocellulosic biomass of poplar trees with reduced HCT1 activity yields an altered composition of vapor products in fast pyrolysis, with a focus on the production of phenolic compounds. Micropyrolysis at 500°C coupled to GC-MS (i.e. Py-GC-MS) was used since it is a rapid analysis technique that respects the fast pyrolysis conditions at which a maximum yield of pyrolysis vapors and lignin-derived monophenolic products is obtained. [12, 28, 29] Analytical pyrolysis has been proven to be a useful technique for thermochemical degradation analysis of numerous types of feedstock, biomass, product residues, waste streams, etc. [30-34] The results from the analytical pyrolysis experiments in this study brought new insights to the potential of tailored poplar feedstock for fast pyrolysis applications. [4]
2 Material and methods 2.1 Biomass samples In the first stage of the research, Black poplar (Populus nigra) samples were used that originated from natural populations in Europe. [21] In previous work, poplar trees homozygous for a defective PnHCT1Δ73 allele (also designated Δ73/Δ73) were selected, together with heterozygous (Δ73/+) trees and trees without the defective allele (+/+), for convenience also named ‘wild type’ trees throughout the text. Xylem tissue was sampled from debarked woody cuttings of 1-year-old field-grown trees. A total of 7 genotypes were used and these samples will collectively be referred to as the first generation or in short, G1. This first generation consisted of 1 Δ73/Δ73 genotype (71030, 4 biological replicates obtained by vegetative propagation), 3 Δ73/+ genotypes (BSL39, 3 biological replicates; VDL47, 2 biological replicates; BSL01, 2 biological replicates) and of 3 +/+ genotypes without the defective allele (VDL06; 2 biological replicates; BSL12, 2 biological replicates; LOW17, 2 biological replicates). The lignin composition – in terms of H, G and S units – of these lines had already been determined by thioacidolysis. [22] In line with the enzymatic activity of PnHCT1, the Δ73/Δ73 poplar trees have an altered lignin composition characterized by a 12 and 17-fold increase in H units compared to Δ73/+ and +/+ poplars, respectively, together with a decrease in G units of the homozygotes. [22]
In addition, a second generation of poplars was made by crossing the Δ73/Δ73 male parental genotype (clone 71030) with two independent female clones (71104 and SPM12), both heterozygous for the defective PnHCT1-Δ73 allele, generating families EP11F1 and EP11F2 respectively. Progeny of both crosses were genotyped to distinguish homozygotes from heterozygotes at PnHCT1- Δ73. [22] From the 110 plants of the EP11F1 family, 10 genotypes of the homozygous Δ73/Δ73 type and 10 genotypes of the heterozygous Δ73/+ type were randomly selected in each class. Similarly, from the 43 plants of the EP11F2 family, 10 and 9 genotypes of the Δ73/Δ73 and Δ73/+ type were randomly selected for further analysis, respectively. [22] Three months old thirty-centimeter-tall trees were debarked and stem wood
was sampled. The lignin composition of these different samples, as determined by means of thioacidolysis, has been reported before. [22] The abundance of H units in both progenies increased in the Δ73/Δ73 compared with the Δ73/+ genotypes. However the degree of accumulation was considerably different in both families (up to an 18-fold for EP11F1 and 9-fold for EP11F2), indicating a high dependency on the genetic background. [11, 22] For convenience, these second generation samples will be referred to as G2.
2.2 Micropyrolysis experiments (Py-GC-MS) Fast pyrolysis experiments were performed on a micropyrolysis unit (FrontierLab Multi-shot pyrolyser EGA/PY-3030D) coupled to a gas chromatograph and mass spectrometer (Thermo Fisher Scientific Trace GC Ultra and Thermo ISQ MS) for detection of the pyrolysis products. Peak identification and integration was performed in the Xcalibur software. The micropyrolysis unit consists of a sampler, a quartz pyrolysis tube that can be furnace heated to the desired temperature, a heated interface and deactivated needle which is directly inserted into the GC injector. Each sample was run in triplicate.
Sample cups were weighed before pyrolysis using a Mettler Toledo XP6 microbalance with a 1 μg sensitivity. The cup (constructed of deactivated stainless steel) contained about 500 μg of finely ground biomass sample. To assure homogeneity for each poplar sample, a stem from debarked trees was manually cut in 1 cm pieces. For the first generation samples, these pieces were further milled and pulverized in a mortar under nitrogen atmosphere. Stem sections of second generation samples were further cut into eight pieces and 3 small metal balls were added to an Eppendorf tube, followed by nitrogen freezing and grinding by 5 minutes shaking with a Retsch mill MM300 with 20 Hz frequency.
The loaded sample cup was dropped into the quartz pyrolysis tube situated inside the furnace preheated at desired temperature (500°C). As such the sample was heated to the pyrolysis temperature in a very short time period of 15-20 ms, ensuring rapid pyrolysis. The pyrolysis vapors were directly swept into the GC using helium as the carrier gas (gas flow 100 ml/min). The interface temperature was 250°C. The pyrolysis vapors were injected into the GC via a split/splitless injection port (split ratio 1:100) at
250°C. The chromatographic separation of pyrolysis products was performed using a Restek capillary column (Rtx-1707, 60 m L x 0.25 mm I.D. x 0.25 μm df) with a stationary phase consisting of a crossbond 14% cyanopropylphenyl and 86% dimethyl polysiloxane and a constant helium carrier gas flow of 1 ml/min. The GC oven temperature program started with a 3 min hold at 40°C followed by heating to 280°C at 5°C/min and the final temperature was held constant for 1 min. The MS transfer line temperature was 280°C, the ion source temperature was kept at 230°C and the electron ionization energy was 70 eV.
2.3 GC-MS data processing Peak areas were obtained from the total ion current (TIC) chromatogram of the MS scanning in the 29300 m/z range every 0.2 seconds. A typical chromatogram for poplar pyrolysis is shown in Figure B in Supplementary Materials. Individual compounds in the spectra were characterized by comparison with the spectral data of the National Institute of Standards and Technology (NIST) MS library. Component concentrations were expressed in relative abundance (TIC area% is the component peak area divided by the total peak area).
In these micropyrolysis experiments, all components (having a relative abundance of > 0.05%) were identified and quantified. This resulted in an identification of minimum 94% of the total peak area for all samples. The different identified compounds were then also grouped according to chemical functionality, i.e. carbon dioxide, aliphatic hydrocarbons, aromatic hydrocarbons, alcohols, ketones, aldehydes, ethers, carboxylic acids, furans, phenolics and sugar-likes (Table A in Supplementary Materials). For the phenolic compounds – mainly degradation products from lignin – a subdivision was made into so-called non-, mono- and di-methoxylated phenolic species (respectively indicated as ‘NON’, ‘MONO’ and ‘DI’ in the figures), according to their similarity with p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol respectively.
Statistical analysis and correlation analysis was performed in Matlab R2014b. Normality (Lilliefors test) and homoscedasticity (two-sample F-test) of the data was verified, and compounds or component groups
were compared between different samples by using normal Student’s t-test (normality and homoscedasticity), Welch’s t-test (normality and heteroscedasticity) and Wilcoxon rank-sum test (not normally distributed data sets). For all statistical tests a significance level of 5% was respected.
Correlation analysis was performed on a data set matrix of the selected poplar samples where rows are the observations (i.e. samples) and columns the variables (i.e. the relative concentrations of either the individual pyrolysis products or grouped products). The Pearson correlation coefficient of two variables is a measure of their linear dependence. The result of the correlation was categorized in three groups: either no correlation, a significant positive or a significant negative correlation (significance level of 5%). Although it is difficult to give a transparent explanation on the reaction mechanisms, a positive correlation between two pyrolysis products A and B (i.e. vapor phase constituents) could be indicative for a non-competing coproduction from a different or common precursor, while a negative correlation could indicate a competitive or sequential reaction mechanism.
3 Results and discussion 3.1 Pyrolysis of first generation poplar samples – PnHCT1-Δ73 homozygotes compared to heterozygotes and wild type poplar The decomposition behavior of the poplar feedstock can be analyzed by classifying the thermal decomposition products into compound classes according to the aforementioned chemical classification in Table A in Supplementary Materials. [35] No significant differences were noticed in the classes for the homozygous poplar compared to either heterozygous or wild type poplar (Figure 1a). This indicates that the studied perturbation of the phenylpropanoid pathway and its potential effect on the cell wall structure/composition in the homozygotes had no clear effect on the concentration of GC-detectable pyrolysis compound classes, or more general on the biomass decomposition behavior (see also [36-38]) in terms of simultaneous cellulose, hemicellulose and lignin degradation. Furthermore, the total amount of phenolic compounds (i.e. phenols and methoxylated benzenes) produced was not significantly different for homozygous compared to either heterozygous (p-value of 0.328) or wild type poplars (pvalue of 0.260). However, differences were observed in the partitioning among the compounds within
the phenolic fraction (Figure 1b). Compared to heterozygotes or wild types, lignocellulosic biomass of trees homozygous for the defective allele produced significantly less mono-methoxylated phenolic decomposition products upon fast pyrolysis (p-value of 0.007 and 0.018, respectively). This is to a minor extent compensated for by a larger fraction of non-methoxylated phenols in pyrolytic decomposition compounds of the homozygous poplar samples. This shift is likely a direct consequence of the higher phydroxyphenyl and lower guaiacyl content in the lignin of the homozygous feedstock. [22]
In Figure 2, a comparison is presented between the yield of phenolic compounds obtained from pyrolysis and the lignin monomeric composition (H/G/S) as investigated before with thioacidolysis. [22] When assuming that H, G and S units of the lignin thermally degrade into non-, mono- and di-methoxylated phenols respectively, the amount of non-methoxylated phenolic compounds formed is larger than expected for all poplar types. Also, the amount of di-methoxylated phenols is lower than expected. [11] This result suggests that additional demethoxylation reactions of G and S units take place under the conditions used, resulting in a larger pool of non-methoxylated phenols than that would be theoretically expected from the H units in lignin. [4, 26]
Compared to both heterozygous and wild type poplars, homozygous poplars showed significant differences in both comparisons for 18 decomposition products when comparisons were made at the individual pyrolysis vapor GC-detectable components level (Table 1). Most of these were only present in a smaller amount in the vapor phase (≤ 0.5 TIC area%), but remarkable differences were present in lignin derived pyrolysis compounds. Homozygous poplar produced significantly less monomethoxylated phenols like creosol, eugenol, isoeugenol, acetovanillone and coniferyl alcohol. In addition to these phenolic compounds that dropped in concentration, a significant increase was observed for the non-methoxylated phenols 2,3-xylenol and p-coumaryl alcohol, together with an increase of 2,3dihydrobenzofuran in the pyrolysis vapors of the homozygotes. According to Lou et al., also 2,3dihydrobenzofuran is produced by a lignin pyrolysis mechanism through an o-quinonemethide intermediate with allylmethyl group which cyclizes followed by hydrogen abstraction. [35, 39] Thus, the increase in compounds 2,3-xylenol, p-coumaryl alcohol and 2,3-dihydrobenzofuran in the pyrolytic
spectrum of the Δ73/Δ73 feedstock, reflects the higher H unit content in these mutants as compared to the heterozygous and wild-type feedstock. [22]
3.2 Pyrolysis of second generation poplar samples – PnHCT1-Δ73 homozygotes in different genetic backgrounds The results in Section 3.1 demonstrate how a genetic perturbation of the phenylpropanoid pathway results in a biomass with altered pyrolysis vapor composition. With the samples of the second generation poplar families, one can analyze to what extent the effect of the homozygous mutation on the pyrolysis behavior differs in different genetic backgrounds; i.e. by comparing the EP11F1 and EP11F2 families. At the level of compound classes, a remarkable shift (p-value of 0.032) was found for the monomethoxylated phenolic fraction when comparing the homozygous EP11F1 with the homozygous EP11F2 trees (Figure 3). Biomass derived from homozygous EP11F1 poplars were described to have a higher H unit and lower G unit content than homozygous EP11F2 trees. [22] In the pyrogram, the monomethoxylated phenolic amount totaled 5.85 ± 0.55 TIC area% for the homozygous EP11F1 and 6.31 ± 0.26 TIC area% for the homozygous EP11F2 trees. This difference was mainly credited to changes in p-creosol, p-ethylguaiacol and p-propylguaiacol (Table 2). Although the H unit content is higher in EP11F1 homozygotes, it are the EP11F2 homozygous poplars yielding a higher amount of 2,3dihydrobenzofuran and the non-methoxylated m-cresol. For this genotypic comparison, it is promising to see that some of the variations in the Populus nigra feedstock are also reflected in the composition of GC-detectable pyrolysis compounds.
Next, the dose effect of the mutation was examined by comparing the fast pyrolysis vapors of homozygous and heterozygous mutants of the second generation poplar samples, similar to the analysis of the first generation samples. Again a smaller amount of mono-methoxylated compounds was observed for homozygous mutants (Figure 3), the difference being significant for the homozygotes (5.85 ± 0.55 TIC area%) compared to heterozygotes (6.46 ± 0.46 TIC area%) of the EP11F1 family (p-value of 0.015). o-Guaiacol and p-vinylguaiacol are the significantly contributing decomposition compounds (Table 3). In contrast to the remarkable difference in H units for the homozygous mutants (9 to 18 times
more H units than in heterozygotes, [22]) no significant increases in non-methoxylated pyrolysis products were detected as compared to the corresponding heterozygotes. The fractions of non-, monoand di-methoxylated compounds in the pyrolysis vapors of the second generation samples are also distinct compared to the fraction of H, G and S units in the lignin of these samples (Figure 4), reinforcing the previous assessment that also mono- and di-methoxylated monolignols are further thermally broken down into non-methoxylated phenols.
3.3 First and second generation – Comparison Comparing the first with the second generation is in fact comparing 1-year-old with 3-months-old poplar trees (G1 vs G2 generation, respectively). The complete GC-detectable thermal decomposition spectrum classifying the products into different chemical groups is given in Figure 5a, again with the subdivision of the phenols into non-, mono- and di-methoxylated phenolic compounds in Figure 5b. For younger trees, considerably higher levels of carbon dioxide, ketones and aldehydes were detected in the pyrolysis vapor phase, which could be partially explained by a larger cellulose and hemicellulose fraction in the cell wall compared to the cell wall of older poplars which have a larger lignin fraction and as such a higher yield in phenolic degradation compounds. [22, 36, 37] The smaller amount of sugar-like compounds and larger amount of mono-aromatics in the pyrolysis vapors of younger G2 trees as compared to older G1 trees could be explained by the raise in carbon dioxide and ketones production, which suggests a further thermal breakdown of the cellulose and hemicellulose derived compounds in younger trees. This more thorough breakdown for younger G2 poplar was also investigated by inspecting the lignin-derived compounds. Not only a smaller fraction of these vapor constituents was observed for the younger trees, also the composition of this class was different since the share in nonmethoxylated phenols was larger for G2 compared to older G1 samples (Figure 5b). This suggests that the lignin in younger trees is more susceptible for demethoxylation reactions as the trends in non-, monoand di-methoxylated phenol products for the older samples relate much more to the lignin composition than for the younger ones. This further breakdown also contributes to the higher yield of low molecular weight carbonyl products (ketones and aldehydes) for younger G2 trees (Figure 5a). [38]
3.4 Correlation analysis To further investigate thermochemical decomposition mechanisms, the Py-GC-MS dataset generated in this study on Populus nigra pyrolysis provides the opportunity to analyze for correlations between compound classes or individual products. The results reported in Tables 4 and 5 show the correlations between selected compound classes and phenolic products over the complete dataset (G1 and G2 samples).
The correlations between pyrolysis compound classes in Table 4 reveal that carbon dioxide, ketones, aldehydes, mono-aromatics and non-methoxylated phenols are positively correlated with each other and negatively correlated to the other compound classes, except the alcohols. Alcohols were not correlated with any other compound class. The production of mono-aromatics and non-methoxylated phenols is related to a more thorough decomposition of the lignin fraction (free-radical chain-reactions on the phenolic side-chain of both mono- and di-methoxylated phenols), which also raises the amount of carbon dioxide and light weight oxygenates released like ketones and aldehydes. [27, 38] Methoxylated phenols are simultaneously produced from the lignin decomposition (C = 0.91) (with G and S units being the most abundant fraction) and both mono- and di-methoxylated phenols are further decomposed towards non-methoxylated phenols (C = -0.81 and C = -0.89, respectively).
To further elucidate pyrolysis mediated lignin decomposition, a correlation analysis on the 46 identified phenolic compounds was set up (Table 5). From the non-methoxylated fraction, phenol and 4 alkylated phenols are negatively correlated to most of the mono- and di-methoxylated phenolic compounds. pCresol, m-cresol and chavicol could be directly formed from respectively p-creosol, isocreosol and eugenol by demethoxylation. The formation of phenol and o-cresol implies that besides demethoxylation, also decarboxylation or decarbonylation of the phenolic side chain and alkylation of the aromatic ring take place. [38] Regarding the mono-methoxylated phenols, again two alkylated phenols (p-propylguaiacol and 2-methylresorcinol) show a negative correlation to most of the phenolic products. o-Guaiacol and p-vinylguaiacol are negatively correlated with the dimethoxylated phenols acetosyringone and sinapaldehyde which could mean they are produced by demethoxylation and
decarbonylation. In addition, almost all di-methoxylated phenols appeared to be positively correlated to one-another, confirming that these are made from a common precursor (i.e. S units). From these results, it can be concluded that demethoxylation (from either di- or mono-methoxylated phenols) is a common step in the degradation pathway during pyrolysis. [27, 38] 4 Conclusions Poplar wood with an increase in H lignin was subjected to analytical fast pyrolysis to evaluate the effect of lignin composition on the diversity and concentration of phenolic compounds produced in pyrolysis. In line with the specific shift in lignin composition, an increase in the non-methoxylated products 2,3xylenol, p-coumaryl alcohol and 2,3-dihydrobenzofuran was found for the one-year-old first generation (G1) homozygous mutants at the expense of mono-methoxylated phenols. This shift in monomethoxylated phenols appeared independent of the genetic background and age of the trees upon harvest, as proven by the analysis of the three-months-old second generation (G2) samples. The increase of the aforementioned non-methoxylated products could not be observed in these younger samples. In general the effect of the mutation on the yields of carbon dioxide, light weight oxygenates and sugarlike fraction was not significant, indicating it did not affect cellulose or matrix polysaccharides to such an extent that differences in the spectrum of pyrolytic compounds from these biomass constituents could be detected. An extensive correlation analysis on the fast pyrolysis products of all poplar samples revealed the further decomposition of mono- and di-methoxylated phenolic compounds giving rise to a higher amount of non-methoxylated phenolic compounds, masking the raise of non-methoxylated compounds derived from H units. Reactions like demethoxylation, demethylation, decarboxylation, decarbonylation and alkylation take place in favor of carbon dioxide, ketones and aldehydes production. In conclusion this study proves that the selected variation in the lignin content of the wood feedstock has its outcome in the fast pyrolysis product range. In addition, we found that a substantial amount of the non-methoxylated pyrolysis products appeared to be derived via demethoxylation reactions of monoand di-methoxylated precursors. These results are promising for future research which should aim towards the selection of biomass with a more extreme lignin composition (for example towards one type of building block [40]) that can become a promising feedstock for fast pyrolysis processing.
Acknowledgements The authors acknowledge the UGent Multidisciplinary Research Partnership (MRP) “Ghent Bioeconomy” for its financial support in the analytical pyrolysis-GCMS infrastructure. Ruben Vanholme is indebted to the Research Foundation-Flanders (FWO) for a postdoctoral fellowship.
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Figure 1a. TIC area% response from 500°C pyrolysis of first generation homozygous (G1 Δ73/Δ73 ), heterozygous (G1 Δ73/+) and wild type (G1 +/+) poplar, classified into compound classes according to chemical functionality. Analyses run in triplicate, error bars represent the standard deviation.
40
G1 Δ73/Δ73 G1 Δ73/+
35
G1 +/+ 30
TIC area%
25 20 15 10 5 0
Figure 1b. Subdivision into non-, mono- and di-methoxylated compound classes of the phenolic
TIC area% response of 500°C pyrolysis of first generation homozygous (G1 Δ73/Δ73), heterozygous (G1 Δ73/+) and wild type (G1 +/+) poplar. Analyses run in triplicate, error bars represent the standard deviation. The asterisk indicates a significant difference in percentage of monophenolic products for
TIC area%
homozygous compared to heterozygous / wild type poplar. 30
G1 Δ73/Δ73
20
G1 Δ73/+
10
G1 +/+
*
0 NON MONO
DI
Figure 2. Comparison between the lignin composition of the initial feedstock via thioacidolysis (expressed in percentage H, G, and S units; data obtained from [26]) and the partitioning in the phenolic fraction (percentage non-, mono- and di-methoxylated phenols) after 500°C analytical pyrolysis for first generation homozygous G1 Δ73/Δ73 (a), heterozygous G1 Δ73/+ (b) and wild type poplar G1 +/+ (c). Analyses run in triplicate, error bars represent the standard deviation.
a 70
G1 Δ73/Δ73 lignin composition
60 50
G1 Δ73/Δ73 pyrolytic phenolic fraction composition
%
40 30 20 10 0 H , NON
G , MONO
S , DI
b 70
G1 Δ73/+ lignin composition
60
G1 Δ73/+ pyrolytic phenolic fraction composition
50
%
40 30 20 10 0 H , NON
G , MONO
S , DI
c 70
G1 +/+ lignin composition
60 50
G1 +/+ pyrolytic phenolic fraction composition
%
40 30 20 10 0 H , NON
G , MONO
S , DI
Figure 3. TIC area% response of non-, mono- and di-methoxylated phenolic compound class from 500°C pyrolysis of second generation homo/heterozygotes from the first poplar family (G2 EP11F1 Δ73/Δ73 and G2 EP11F1 Δ73/+) and second family homo/heterozygotes (G2 EP11F2 Δ73/Δ73 and G2 EP11F2 Δ73/+). Analyses run in triplicate, error bars represent the standard deviation. The asterisk indicates a significantly lower percentage of monophenolic products for first family homozygous poplar compared to either the second family homozygotes and compared to the first family heterozygotes.
9
G2 EP11F1 Δ73/Δ73
8
G2 EP11F2 Δ73/Δ73
7
*
TIC area%
6
G2 EP11F1 Δ73/+ G2 EP11F2 Δ73/+
5 4 3 2 1 0 NON
MONO
DI
Figure 4. Comparison between the lignin composition of the initial feedstock via thioacidolysis (expressed in percentage H, G, and S units; data obtained from [26]) and the partitioning in the phenolic fraction (percentage non-, mono- and di-methoxylated phenols) after 500°C analytical pyrolysis for second generation homozygotes (a and b) and heterozygotes (c and d). Analyses run in triplicate, error bars represent the standard deviation.
a
b
70
70
60
60
50
50
G2 EP11F1 Δ73/Δ73 lignin composition
30
G2 EP11F1 Δ73/Δ73 pyrolytic phenolic fraction compostion
40
G2 EP11F2 Δ73/Δ73 lignin composition
30
G2 EP11F2 Δ73/Δ73 pyrolytic phenolic fraction composition
%
%
40
20
20
10
10
0
0
H , NON
G , MONO
S , DI
H , NON
G , MONO
c
S , DI
d
70
70
60
60
50
50
G2 EP11F1 Δ73/+ lignin composition
G2 EP11F2 Δ73/+ lignin composition
40
%
%
40
EP11F1 Δ73/+ pyrolytic phenolic fraction composition
30
20
20
10
10
0
G2 EP11F2 Δ73/+ pyrolytic phenolic fraction composition
30
0
H , NON
G , MONO
S , DI
H , NON
G , MONO
S , DI
Figure 5a. TIC area% response of 500°C pyrolysis products (classified) of homozygous and heterozygous samples of 1-year-old (G1 Δ73/Δ73; G1 Δ73/+) and 3-months-old (G2 EP11F1 and EP11F2 Δ73/Δ73; G2 EP11F1 and EP11F2 Δ73/+) poplar classified into component groups according to chemical functionality. Analyses run in triplicate, error bars represent the standard deviation. 40 35
TIC area%
30 25 20 15 10 5 0
G1 Δ73/Δ73 G1 Δ73/+ G2 Δ73/Δ73 G2 Δ73/+
Figure 5b. Subdivision into non-, mono- and di-methoxylated compound classes of the phenolic TIC area% response of 500°C pyrolysis of homozygous and heterozygous samples of 1-year-old (G1 Δ73/Δ73; G1 Δ73/+) and 3-months-old (G2 EP11F1 and EP11F2 Δ73/Δ73; G2 EP11F1 and EP11F2 Δ73/+) poplar. Analyses run in triplicate, error bars represent the standard deviation. 25
G1 Δ73/Δ73 G1 Δ73/+
TIC area%
20
G2 Δ73/Δ73 G2 Δ73/+
15 10 5 0 NON
MONO
DI
Table 1: TIC area% response of the significantly different 500°C pyrolysis products in both the comparison of first generation homozygous samples G1 Δ73/Δ73 with heterozygous (G1 Δ73/+) poplar and G1 Δ73/Δ73 with wild type (G1 +/+) poplar. Values in bold are the samples with highest response for the according compound.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
RT (min)
Name
9.25 11.51 17.27 23.79 23.94 26.34 28.01 28.19 29.91 31.66 32.44 35.17 36.38 37.75 39.41 43.28 43.34 44.00
Formic acid Toluene 2-Propylfuran 3-Methyl-5H-furan-2-one 2,3-Dimethyl- 2-pentenoic acid Maltol Creosol 2,3-Xylenol 1-Ethylidene-1H-indene 2,3-Dihydrobenzofuran Eugenol (Z)-Isoeugenol 4-(t-Butyl)benzaldehyde Acetovanillone 1-Naphthalenol 1,2-Dimethoxy-4-(3-methoxy-1-propenyl)benzene p-Coumaryl alcohol Coniferyl alcohol
Δ73/Δ73 TIC area% stdev 0,02 0,03 0,23 0,03 0,02 0,00 0,14 0,01 0,15 0,00 0,30 0,00 0,78 0,03 0,11 0,02 0,01 0,00 0,33 0,06 0,39 0,02 0,96 0,04 0,04 0,00 0,37 0,02 0,09 0,01 0,00 0,00 0,35 0,05 2,24 0,20
TIC area% 0,11 0,15 0,04 0,13 0,13 0,28 0,87 0,05 0,06 0,17 0,44 1,19 0,08 0,42 0,03 0,05 0,03 3,22
Δ73/+ stdev 0,02 0,02 0,02 0,01 0,01 0,01 0,06 0,02 0,02 0,11 0,04 0,12 0,04 0,03 0,01 0,01 0,01 0,40
p-value 0,001 0,001 0,017 0,006 0,002 0,007 0,012 0,006 0,000 0,023 0,042 0,005 0,038 0,038 0,000 0,006 0,001 0,002
TIC area% 0,09 0,17 0,06 0,12 0,12 0,27 0,91 0,07 0,05 0,12 0,47 1,23 0,10 0,43 0,05 0,05 0,02 3,23
+/+ stdev 0,04 0,04 0,02 0,01 0,02 0,01 0,10 0,03 0,03 0,13 0,05 0,20 0,07 0,03 0,02 0,01 0,01 0,83
p-value 0,025 0,043 0,004 0,004 0,003 0,004 0,032 0,038 0,018 0,015 0,015 0,022 0,010 0,021 0,019 0,010 0,001 0,033
Table 2: TIC area% response of the significantly different 500°C pyrolysis products after comparison between second generation EP11F1 homozygous (EP11F1 Δ73/Δ73) and EP11F2 homozygous poplar (EP11F2 Δ73/Δ73). Values in bold are the samples with highest response for the according compound. RT (min) Name 9.64 14.81 15.13 20.43 22.12 22.45 22.76 22.88 23.94 26.65 26.87 27.70 30.20 30.29 30.85 31.66 32.66 33.30 40.77 41.64 42.67
3-Penten-2-one Glycol monoacetate 5H-Furan-2-one 2-Hydroxy-2-cyclopenten-1-one 4-Hydroxybutanoic acid 5H-Furan-2-one 3-Methyl-3-cyclohexen-1-one 3,4-Dihydro-2-methoxy-2H-pyran 2,3-Dimethyl- 2-pentenoic acid 2-Methyl-1,3-cyclohexanedione m-Cresol p-Creosol 3-Pyridinol p-Ethylguaiacol 6-Methyl-1,4-dioxaspiro[2.4]heptan-5-one 2,3-Dihydrobenzofuran p-Propylguaiacol 5-Hydroxymethyldihydrofuran-2-one Melezitose Methyl-(2-hydoxy-3-ethoxy-benzyl)ether α-Lactose
G2 EP11F1 Δ73/Δ73 TIC area% stdev 0.07 0.03 0.46 0.31 0.06 0.03 2.01 0.14 0.44 0.11 0.88 0.06 0.36 0.06 0.27 0.05 0.12 0.01 0.12 0.03 0.72 0.09 0.38 0.20 0.28 0.17 0.19 0.12 0.17 0.17 1.19 0.33 0.07 0.01 0.35 0.05 0.24 0.04 0.02 0.01 0.15 0.04
G2 EP11F2 Δ73/Δ73 TIC area% stdev 0.09 0.01 0.29 0.10 0.04 0.02 1.87 0.15 0.34 0.09 0.81 0.06 0.41 0.03 0.24 0.21 0.13 0.02 0.09 0.02 0.81 0.09 0.52 0.04 0.05 0.10 0.32 0.06 0.01 0.03 1.55 0.30 0.10 0.04 0.30 0.03 0.32 0.13 0.03 0.01 0.20 0.05
p-value 0.008 0.023 0.049 0.037 0.049 0.016 0.036 0.041 0.002 0.043 0.031 0.008 0.001 0.017 0.009 0.020 0.006 0.013 0.028 0.040 0.013
Table 3: TIC area% response of the significantly different (homozygous compared to heterozygous samples) 500°C pyrolysis products from second generation EP11F1 (EP11F1 Δ73/Δ73 compared to EP11F1 Δ73/+) and EP11F2 (EP11F2 Δ73/Δ73 compared to EP11F2 Δ73/+, indicated with asterisk) poplar. Double asterisk indicates the significant difference occurring in both families. Values in bold are the samples with highest response for the according compound.
RT (min)
Name
7.76 9.64 23.79 25.85
2,3-Butanedione 3-Penten-2-one* 3-Methyl-5H-furan-2-one o-Guaiacol
31.83
p-Vinylguaiacol**
G2 EP11F1 Δ73/Δ73
G2 EP11F1 Δ73/+
G2 EP11F2 Δ73/Δ73
G2 EP11F2 Δ73/+
TIC area% 0,72 0,07 0,16 1,36
stdev 0,04 0,03 0,02 0,11
TIC area% 0,68 0,08 0,18 1,46
stdev 0,05 0,03 0,03 0,14
p-value 0,037 0,595 0,041 0,038
TIC area% 0,71 0,09 0,17 1,41
stdev 0,06 0,01 0,03 0,11
TIC area% 0,61 0,07 0,17 1,45
stdev 0,24 0,01 0,01 0,07
p-value 0,562 0,008 0,967 0,437
1,78
0,18
2,02
0,17
0,007
1,71
0,12
1,82
0,10
0,043
Table 4: Correlation coefficients between 500°C fast pyrolysis product compound classes for all the poplar samples. + and - indicate a significant positive and negative correlation, while 0 indicates no
+ +
+ -
-
+
DI
+ + +
MONO
+ + + +
Mono-arom.
+ + + + +
NON
+ + + + + +
Sugars
+ + -
Phenols
+ + + -
Furans / Pyran
0 0 0 0 0 0 0 0 0 0 0
Acids/esters
Aldehydes
0 + + + + + + +
Ethers
Ketones
0 + + + + -
Alcohols
Carbon dioxide Aliphatic Alcohols Ketones Aldehydes Ethers Acids/esters Furans / Pyran Phenols Sugars Mono-arom. NON MONO DI
Aliphatic
Carbon dioxide
correlation.
+ -
-
+
DI
+ +
MONO
+ + +
NON
+ + + +
Mono-arom.
+ + + + +
Sugars
+ + + + + +
Phenols
+ + -
Furans / Pyran
+ + + -
Acids/esters
0 0 0 0 0 0 0 0 0 0 0
Ethers
Ketones
0 + + + + + + +
Aldehydes
Alcohols
0 + + + + -
Aliphatic
Carbon dioxide Carbon dioxide Aliphatic Alcohols Ketones Aldehydes Ethers Acids/esters Furans / Pyran Phenols Sugars Mono-arom. NON MONO DI
MONO
NON
Phenol o-Cresol 2,6-Xylenol p-Cresol m-Cresol 2,3-Xylenol Chavicol p-Hydroxybenzylidene acetone p-Formyl phenol Methylparaben p-Coumarylalcohol o-Guaiacol Isocreosol p-Creosol p-Ethylguaiacol p-Vinylguaiacol Eugenol p-Propylguaiacol + 0 + + 0 + 0 0 0 0 0 + + 0 + 0 0 0 0 + 0 + 0 0 0 + 0 + 0 0 0 0 0 + 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + 0 + 0 0 + 0 + 0 + + 0 0 + + 0 0 + 0 0 + 0 0 0 0 0 -
5-tert-Butylpyrogallol
3,5-Dimethoxy-4-hydroxyphenylacetic acid
Sinapaldehyde
2,5-Dimethoxybenzyl acetate
2,5-Dimethoxy-benzenemethanol acetate
1-(2,4,6-Trihydroxyphenyl)-2-pentanone
Acetosyringone
MONO
1,2-Dimethoxy-4-(3-methoxy-1-propenyl)benzene
3,5-Dimethoxy-4-hydroxyphenylacetic acid
Syringaldehyde
Methoxyeugenol
1-(3,4-Dimethoxyphenyl)ethanone
1,2,3-Trimethoxy-5-methylbenzene
1,2,4-Trimethoxybenzene
Syringol
(E)-Coniferol
Methyl-(2-hydoxy-3-ethoxy-benzyl)ether
2-Allyl-1,4-dimethoxy-3-methylbenzene
Coniferol
Acetovanillone
2-Methylresorcinol
Vanillin
Resorcinol
(Z)-Isoeugenol
NON
(E)-Isoeugenol
1,2-Benzenediol
p-Propylguaiacol
Eugenol
p-Vinylguaiacol
p-Creosol
0 0 0 + 0 0
p-Ethylguaiacol
Isocreosol
+ + 0 + 0
o-Guaiacol
p-Coumarylalcohol
Methylparaben
+ + + 0 + + 0 0 + -
p-Formyl phenol
p-Hydroxybenzylidene acetone
Chavicol
2,3-Xylenol
m-Cresol
0 0 0 0 0 0 0 0 0 0 0 0 0
p-Cresol
2,6-Xylenol
o-Cresol
Phenol
Table 5: Correlation coefficients between 500°C fast pyrolysis phenolic products for all the poplar samples. + and - indicate a significant positive and negative correlation, while 0 indicates no correlation. First 11 phenols are non-methoxylated,
followed by 18 mono-methoxylated and 17 di-methoxylated phenols.
DI
DI
1,2-Benzenediol (E)-Isoeugenol (Z)-Isoeugenol Resorcinol Vanillin 2-Methylresorcinol Acetovanillone Coniferol 2-Allyl-1,4-dimethoxy-3-methylbenzene Methyl-(2-hydoxy-3-ethoxybenzyl)ether (E)-Coniferol Syringol 1,2,4-Trimethoxybenzene 1,2,3-Trimethoxy-5-methylbenzene 1-(3,4-Dimethoxyphenyl)ethanone Methoxyeugenol Syringaldehyde 3,5-Dimethoxy-4-hydroxyphenylacetic acid 1,2-Dimethoxy-4-(3-methoxy-1propenyl)benzene Acetosyringone 1-(2,4,6-Trihydroxyphenyl)-2-pentanone 2,5-Dimethoxy-benzenemethanol acetate 2,5-Dimethoxybenzyl acetate Sinapaldehyde 3,5-Dimethoxy-4-hydroxyphenylacetic acid 5-tert-Butylpyrogallol
0 0 + -
0 0 + -
0 0 0 0 0 0 0 0 0
0 0 + -
0 0 + -
0 0 0 0 0 0 0 0
0 0 + -
+ + + 0 0 + + +
+ + + 0 0 + + +
+ + + 0 0 + + +
+ + + 0 0 + + +
0 0 0 0 0 0 0 0 -
+ + + 0 0 + + +
+ + + 0 0 + + +
0 0 0 0 0 + 0 0 0
0 0 0 0 0 0 0 -
+ + + 0 0 + + +
0 0 0 0
+ + 0 0 + + +
+ 0 0 + + +
0 0 + + +
0 0 0 0
0 0 0 0
-
+ +
+
-
-
0 0 0 0 0 0 0 0
-
-
0 0 0 0 0 0 0 0
-
+ + + + + + + +
0 + + + + + + +
+ + + + + + + +
+ + + + + + + +
0 0 0 0 0 0 0 0
+ + + + + + + +
+ + + + + + + +
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
+ + + + + + + +
0 -
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
-
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + + + + +
+ + + + + +
+ + + + +
+ + + +
+ + +
+ +
+
-
-
0
-
-
0
-
+
+
+
+
0
+
+
0
0
+
-
+
+
+
0
0
-
+
+
+
+
+
+
+
+
+
+
+
0 -
0 -
0 0 0
-
0 -
0 0 0
-
+ + +
0 + +
0 + +
0 + +
0 0
+ + +
0 + +
0 0 0
0 0
+ + +
0 -
0 + +
0 + +
+ + +
0 0 0
0 0 0
-
+ + +
+ + +
+ + +
0 + +
+ + +
+ + +
+ + +
+ + +
0 + +
+ + +
+ + +
+ + +
0 +
+
-
-
0 0 0
-
-
0 0 0
-
+ + +
+ + +
+ + +
+ + +
0 0 -
+ + +
+ + +
0 0 0
0 0 -
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S0165-2370(16)30095-X http://dx.doi.org/doi:10.1016/j.jaap.2016.08.021 JAAP 3807
To appear in:
J. Anal. Appl. Pyrolysis
Received date: Accepted date:
26-2-2016 23-8-2016
Please cite this article as: Jop Vercruysse, Andries Poupaert, Ruben Vanholme, Catherine Bastien, Bartel Vanholme, Wout Boerjan, Wolter Prins, Frederik Ronsse, Micropyrolysis of natural poplar mutants with altered phydroxyphenyl lignin content, Journal of Analytical and Applied Pyrolysis http://dx.doi.org/10.1016/j.jaap.2016.08.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Micropyrolysis of natural poplar mutants with altered p-hydroxyphenyl lignin content
Corresponding author: Jop Vercruysse1, [email protected], Tel: +32 (0)9 264 6190, Fax: +32 (0)9 264 6235 Authors: Jop Vercruysse1, Andries Poupaert1, Ruben Vanholme2,3, Catherine Bastien4, Bartel Vanholme2,3, Wout Boerjan2,3, Wolter Prins1, Frederik Ronsse1 1
Department of Biosystems Engineering, Bioscience Engineering Faculty, Ghent University, Coupure
links 653, B-9000 Gent, Belgium 2
Department of Plant Systems Biology, VIB, Technologiepark 927, B-9052 Gent, Belgium
3
Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, B-
9052 Gent, Belgium 4
National Institute for Agricultural Research, Centre of Orleans, Laboratory of Breeding, Genetics
and Physiology of Forest Trees, 2163, Avenue de la Pomme de Pin, CS 40001 Ardon, 45166 Olivet Cedex, France.
Highlights
Analytical pyrolysis GC-MS for thermochemical degradation analysis of natural poplar mutants
Shift in lignin-derived compounds for mutants homozygous for the defective PnHCT1-Δ73 allele
Higher production of non-methoxylated products at the expense of methoxylated phenols
Correlation analysis revealing demethoxylation of G and S lignin units into non-methoxylated phenols
Abstract Analytical pyrolysis coupled to GC-MS (Py-GC-MS) was applied for the thermochemical degradation analysis of wood from poplar (Populus nigra) defective in p-HYDROXYCINNAMOYLCoA:SHIKIMATE p-HYDROXYCINNAMOYL TRANSFERASE 1 (HCT1), an enzyme essential in the biosynthesis of guaiacyl and syringyl units in lignin. The aim of this study was to assess the potential of natural poplar trees with characteristic lignin composition to improve the yield of valuable chemicals in biomass fast pyrolysis, with a focus on phenolic product formation during lignin decomposition. Analysis of the fast pyrolysis vapor phase obtained from the lignocellulosic biomass of trees homozygous for the defective PnHCT1-Δ73 allele revealed significant shifts in the spectrum of ligninderived pyrolytic compounds as compared to wild type and heterozygous trees. More particular for the homozygotes, a higher production of the non-methoxylated p-hydroxycinnamic alcohol (p-coumaryl alcohol) and 2,3-dihydrobenzofuran was detected at the expense of mono-methoxylated phenols. Correlation analysis also suggested further decomposition reactions such as demethoxylation, demethylation and alkylation of the mono- and di-methoxylated phenolic moieties during pyrolysis, giving rise to a remarkable amount of non-methoxylated phenols in the pyrolytic spectrum.
Keywords: Lignocellulosic biomass; Lignin; Analytical Pyrolysis; Gas Chromatography; Mass spectrometry
1 Introduction Lignocellulose biomass of plants is a biorenewable resource that can be used in fast pyrolysis systems to produce bio-oil. Fast pyrolysis is an anoxic and fast heating process to temperatures typically in the 400-600°C range which provides rapid and complete conversion of solid organic biomass into 60-75 wt% bio-oil, 15-25 wt% solid biochar and 10-20 wt% non-condensable gases. [1, 2] Upgrading the biooil can either serve to substitute petroleum fuels, whereas additional extraction can lead towards the production of valuable chemicals. [3-5] As such the pyrolysis bio-oil can counteract fossil resource depletion and global climate change associated with rising atmospheric carbon dioxide levels, giving it
a very high ecological and economical potential. [6-8] The robustness of the process is an important advantage of pyrolysis as it overcomes feedstock problems related to biomass recalcitrance. Lignin for example has an important biological role in the secondary cell wall of plants, but it strongly influences industrial processing of biomass, e.g. by requiring extensive pretreatments prior to enzymatic hydrolysis of the polysaccharides (in particular cellulose) into fermentable sugars. [9, 10] The only pretreatment required for fast pyrolysis is biomass drying and grinding to a sufficiently small particle size in order to provide optimal heat transfer. [11-13]
The production of large amounts of high quality bio-oil as a source of energy is the main aim in current fast pyrolysis research. However, fast pyrolysis also yields up to 10 wt% of valuable mono-phenols such as guaiacols, syringols, alkyl phenols and catechols. [3] These compounds have economic potential as substitutes for their petrochemical counterparts and would be used in the synthesis of a variety of products including wood adhesives, bioplastics, pharmaceuticals, fragrances and octane enhancers for transportation fuels. [3, 4] Improving the quantity and quality of valuable pyrolysis products is currently mainly achieved by adjusting process conditions. Equally important is the improvement of upstream processes including feedstock selection. [14, 15] In this regard, there is a large potential for improving plant cell walls by exploiting the available genetic resources and by genetic modification. This domain has been successfully explored for fermentation purposes in recent years, i.e. breeding or engineering plant cell walls that are easier to convert to fermentable sugars. [9, 10, 16, 17] This potential (i.e. of biomass with intentionally modified cell wall composition) has remained largely unexplored in fast pyrolysis, where biomass feedstock with a different lignin amount and/or lignin composition might be very attractive, for instance for the production of phenolic compounds. [18] For this reason there is vast interest in engineering of bioenergy crops to such extent that the derived bio-oil is enriched in addedvalue target components. [11, 12, 19, 20]
Breeding is still the most commonly used method for crop improvement. This involves crossing of genetically diverse parental lines and the subsequent careful screening of the progenies for beneficial phenotypes. Here the quality of the breeding germplasm and hence the genetic variation within a
breeding population is key to success. Using a deep sequencing approach, rare natural variants of lignin biosynthesis genes were searched for in a natural population of Black poplar (Populus nigra). [21] A rare recessive allele was identified, PnHCT1-Δ73, that encodes a truncated version of pHYDROXYCINNAMOYL-CoA:SHIKIMATE
p-HYDROXYCINNAMOYL
TRANSFERASE
(HCT1), a key enzyme of the phenylpropanoid pathway (Figure A in Supplementary Materials), converting first p-coumaroyl-CoA to p-coumaroyl shikimic acid and later on caffeoyl shikimic acid to caffeoyl-CoA. [22, 23] The latter is further converted towards the methoxylated monolignols coniferyl alcohol and sinapyl alcohol, which are incorporated in lignin as guaiacyl (G) and syringyl (S) units, respectively. [24-26] In line with the enzymatic activity of HCT1, poplar trees homozygous for the defective allele have a different lignin composition characterized by an increase in p-hydroxyphenyl (H) units compared to poplars heterozygous for the defective allele and poplars that do not harbor the defective allele. [22] It is to be expected that this shift towards H units will affect the concentration of non-methoxylated phenolic compounds in the pyrolysis vapors. As these target phenolic compounds are considered valuable, the bio-oil obtained from PnHCT1-Δ73 homozygous poplars could potentially be of higher value for further chemical processing. [27]
The objective of this study was to verify whether lignocellulosic biomass of poplar trees with reduced HCT1 activity yields an altered composition of vapor products in fast pyrolysis, with a focus on the production of phenolic compounds. Micropyrolysis at 500°C coupled to GC-MS (i.e. Py-GC-MS) was used since it is a rapid analysis technique that respects the fast pyrolysis conditions at which a maximum yield of pyrolysis vapors and lignin-derived monophenolic products is obtained. [12, 28, 29] Analytical pyrolysis has been proven to be a useful technique for thermochemical degradation analysis of numerous types of feedstock, biomass, product residues, waste streams, etc. [30-34] The results from the analytical pyrolysis experiments in this study brought new insights to the potential of tailored poplar feedstock for fast pyrolysis applications. [4]
2 Material and methods 2.1 Biomass samples In the first stage of the research, Black poplar (Populus nigra) samples were used that originated from natural populations in Europe. [21] In previous work, poplar trees homozygous for a defective PnHCT1Δ73 allele (also designated Δ73/Δ73) were selected, together with heterozygous (Δ73/+) trees and trees without the defective allele (+/+), for convenience also named ‘wild type’ trees throughout the text. Xylem tissue was sampled from debarked woody cuttings of 1-year-old field-grown trees. A total of 7 genotypes were used and these samples will collectively be referred to as the first generation or in short, G1. This first generation consisted of 1 Δ73/Δ73 genotype (71030, 4 biological replicates obtained by vegetative propagation), 3 Δ73/+ genotypes (BSL39, 3 biological replicates; VDL47, 2 biological replicates; BSL01, 2 biological replicates) and of 3 +/+ genotypes without the defective allele (VDL06; 2 biological replicates; BSL12, 2 biological replicates; LOW17, 2 biological replicates). The lignin composition – in terms of H, G and S units – of these lines had already been determined by thioacidolysis. [22] In line with the enzymatic activity of PnHCT1, the Δ73/Δ73 poplar trees have an altered lignin composition characterized by a 12 and 17-fold increase in H units compared to Δ73/+ and +/+ poplars, respectively, together with a decrease in G units of the homozygotes. [22]
In addition, a second generation of poplars was made by crossing the Δ73/Δ73 male parental genotype (clone 71030) with two independent female clones (71104 and SPM12), both heterozygous for the defective PnHCT1-Δ73 allele, generating families EP11F1 and EP11F2 respectively. Progeny of both crosses were genotyped to distinguish homozygotes from heterozygotes at PnHCT1- Δ73. [22] From the 110 plants of the EP11F1 family, 10 genotypes of the homozygous Δ73/Δ73 type and 10 genotypes of the heterozygous Δ73/+ type were randomly selected in each class. Similarly, from the 43 plants of the EP11F2 family, 10 and 9 genotypes of the Δ73/Δ73 and Δ73/+ type were randomly selected for further analysis, respectively. [22] Three months old thirty-centimeter-tall trees were debarked and stem wood
was sampled. The lignin composition of these different samples, as determined by means of thioacidolysis, has been reported before. [22] The abundance of H units in both progenies increased in the Δ73/Δ73 compared with the Δ73/+ genotypes. However the degree of accumulation was considerably different in both families (up to an 18-fold for EP11F1 and 9-fold for EP11F2), indicating a high dependency on the genetic background. [11, 22] For convenience, these second generation samples will be referred to as G2.
2.2 Micropyrolysis experiments (Py-GC-MS) Fast pyrolysis experiments were performed on a micropyrolysis unit (FrontierLab Multi-shot pyrolyser EGA/PY-3030D) coupled to a gas chromatograph and mass spectrometer (Thermo Fisher Scientific Trace GC Ultra and Thermo ISQ MS) for detection of the pyrolysis products. Peak identification and integration was performed in the Xcalibur software. The micropyrolysis unit consists of a sampler, a quartz pyrolysis tube that can be furnace heated to the desired temperature, a heated interface and deactivated needle which is directly inserted into the GC injector. Each sample was run in triplicate.
Sample cups were weighed before pyrolysis using a Mettler Toledo XP6 microbalance with a 1 μg sensitivity. The cup (constructed of deactivated stainless steel) contained about 500 μg of finely ground biomass sample. To assure homogeneity for each poplar sample, a stem from debarked trees was manually cut in 1 cm pieces. For the first generation samples, these pieces were further milled and pulverized in a mortar under nitrogen atmosphere. Stem sections of second generation samples were further cut into eight pieces and 3 small metal balls were added to an Eppendorf tube, followed by nitrogen freezing and grinding by 5 minutes shaking with a Retsch mill MM300 with 20 Hz frequency.
The loaded sample cup was dropped into the quartz pyrolysis tube situated inside the furnace preheated at desired temperature (500°C). As such the sample was heated to the pyrolysis temperature in a very short time period of 15-20 ms, ensuring rapid pyrolysis. The pyrolysis vapors were directly swept into the GC using helium as the carrier gas (gas flow 100 ml/min). The interface temperature was 250°C. The pyrolysis vapors were injected into the GC via a split/splitless injection port (split ratio 1:100) at
250°C. The chromatographic separation of pyrolysis products was performed using a Restek capillary column (Rtx-1707, 60 m L x 0.25 mm I.D. x 0.25 μm df) with a stationary phase consisting of a crossbond 14% cyanopropylphenyl and 86% dimethyl polysiloxane and a constant helium carrier gas flow of 1 ml/min. The GC oven temperature program started with a 3 min hold at 40°C followed by heating to 280°C at 5°C/min and the final temperature was held constant for 1 min. The MS transfer line temperature was 280°C, the ion source temperature was kept at 230°C and the electron ionization energy was 70 eV.
2.3 GC-MS data processing Peak areas were obtained from the total ion current (TIC) chromatogram of the MS scanning in the 29300 m/z range every 0.2 seconds. A typical chromatogram for poplar pyrolysis is shown in Figure B in Supplementary Materials. Individual compounds in the spectra were characterized by comparison with the spectral data of the National Institute of Standards and Technology (NIST) MS library. Component concentrations were expressed in relative abundance (TIC area% is the component peak area divided by the total peak area).
In these micropyrolysis experiments, all components (having a relative abundance of > 0.05%) were identified and quantified. This resulted in an identification of minimum 94% of the total peak area for all samples. The different identified compounds were then also grouped according to chemical functionality, i.e. carbon dioxide, aliphatic hydrocarbons, aromatic hydrocarbons, alcohols, ketones, aldehydes, ethers, carboxylic acids, furans, phenolics and sugar-likes (Table A in Supplementary Materials). For the phenolic compounds – mainly degradation products from lignin – a subdivision was made into so-called non-, mono- and di-methoxylated phenolic species (respectively indicated as ‘NON’, ‘MONO’ and ‘DI’ in the figures), according to their similarity with p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol respectively.
Statistical analysis and correlation analysis was performed in Matlab R2014b. Normality (Lilliefors test) and homoscedasticity (two-sample F-test) of the data was verified, and compounds or component groups
were compared between different samples by using normal Student’s t-test (normality and homoscedasticity), Welch’s t-test (normality and heteroscedasticity) and Wilcoxon rank-sum test (not normally distributed data sets). For all statistical tests a significance level of 5% was respected.
Correlation analysis was performed on a data set matrix of the selected poplar samples where rows are the observations (i.e. samples) and columns the variables (i.e. the relative concentrations of either the individual pyrolysis products or grouped products). The Pearson correlation coefficient of two variables is a measure of their linear dependence. The result of the correlation was categorized in three groups: either no correlation, a significant positive or a significant negative correlation (significance level of 5%). Although it is difficult to give a transparent explanation on the reaction mechanisms, a positive correlation between two pyrolysis products A and B (i.e. vapor phase constituents) could be indicative for a non-competing coproduction from a different or common precursor, while a negative correlation could indicate a competitive or sequential reaction mechanism.
3 Results and discussion 3.1 Pyrolysis of first generation poplar samples – PnHCT1-Δ73 homozygotes compared to heterozygotes and wild type poplar The decomposition behavior of the poplar feedstock can be analyzed by classifying the thermal decomposition products into compound classes according to the aforementioned chemical classification in Table A in Supplementary Materials. [35] No significant differences were noticed in the classes for the homozygous poplar compared to either heterozygous or wild type poplar (Figure 1a). This indicates that the studied perturbation of the phenylpropanoid pathway and its potential effect on the cell wall structure/composition in the homozygotes had no clear effect on the concentration of GC-detectable pyrolysis compound classes, or more general on the biomass decomposition behavior (see also [36-38]) in terms of simultaneous cellulose, hemicellulose and lignin degradation. Furthermore, the total amount of phenolic compounds (i.e. phenols and methoxylated benzenes) produced was not significantly different for homozygous compared to either heterozygous (p-value of 0.328) or wild type poplars (pvalue of 0.260). However, differences were observed in the partitioning among the compounds within
the phenolic fraction (Figure 1b). Compared to heterozygotes or wild types, lignocellulosic biomass of trees homozygous for the defective allele produced significantly less mono-methoxylated phenolic decomposition products upon fast pyrolysis (p-value of 0.007 and 0.018, respectively). This is to a minor extent compensated for by a larger fraction of non-methoxylated phenols in pyrolytic decomposition compounds of the homozygous poplar samples. This shift is likely a direct consequence of the higher phydroxyphenyl and lower guaiacyl content in the lignin of the homozygous feedstock. [22]
In Figure 2, a comparison is presented between the yield of phenolic compounds obtained from pyrolysis and the lignin monomeric composition (H/G/S) as investigated before with thioacidolysis. [22] When assuming that H, G and S units of the lignin thermally degrade into non-, mono- and di-methoxylated phenols respectively, the amount of non-methoxylated phenolic compounds formed is larger than expected for all poplar types. Also, the amount of di-methoxylated phenols is lower than expected. [11] This result suggests that additional demethoxylation reactions of G and S units take place under the conditions used, resulting in a larger pool of non-methoxylated phenols than that would be theoretically expected from the H units in lignin. [4, 26]
Compared to both heterozygous and wild type poplars, homozygous poplars showed significant differences in both comparisons for 18 decomposition products when comparisons were made at the individual pyrolysis vapor GC-detectable components level (Table 1). Most of these were only present in a smaller amount in the vapor phase (≤ 0.5 TIC area%), but remarkable differences were present in lignin derived pyrolysis compounds. Homozygous poplar produced significantly less monomethoxylated phenols like creosol, eugenol, isoeugenol, acetovanillone and coniferyl alcohol. In addition to these phenolic compounds that dropped in concentration, a significant increase was observed for the non-methoxylated phenols 2,3-xylenol and p-coumaryl alcohol, together with an increase of 2,3dihydrobenzofuran in the pyrolysis vapors of the homozygotes. According to Lou et al., also 2,3dihydrobenzofuran is produced by a lignin pyrolysis mechanism through an o-quinonemethide intermediate with allylmethyl group which cyclizes followed by hydrogen abstraction. [35, 39] Thus, the increase in compounds 2,3-xylenol, p-coumaryl alcohol and 2,3-dihydrobenzofuran in the pyrolytic
spectrum of the Δ73/Δ73 feedstock, reflects the higher H unit content in these mutants as compared to the heterozygous and wild-type feedstock. [22]
3.2 Pyrolysis of second generation poplar samples – PnHCT1-Δ73 homozygotes in different genetic backgrounds The results in Section 3.1 demonstrate how a genetic perturbation of the phenylpropanoid pathway results in a biomass with altered pyrolysis vapor composition. With the samples of the second generation poplar families, one can analyze to what extent the effect of the homozygous mutation on the pyrolysis behavior differs in different genetic backgrounds; i.e. by comparing the EP11F1 and EP11F2 families. At the level of compound classes, a remarkable shift (p-value of 0.032) was found for the monomethoxylated phenolic fraction when comparing the homozygous EP11F1 with the homozygous EP11F2 trees (Figure 3). Biomass derived from homozygous EP11F1 poplars were described to have a higher H unit and lower G unit content than homozygous EP11F2 trees. [22] In the pyrogram, the monomethoxylated phenolic amount totaled 5.85 ± 0.55 TIC area% for the homozygous EP11F1 and 6.31 ± 0.26 TIC area% for the homozygous EP11F2 trees. This difference was mainly credited to changes in p-creosol, p-ethylguaiacol and p-propylguaiacol (Table 2). Although the H unit content is higher in EP11F1 homozygotes, it are the EP11F2 homozygous poplars yielding a higher amount of 2,3dihydrobenzofuran and the non-methoxylated m-cresol. For this genotypic comparison, it is promising to see that some of the variations in the Populus nigra feedstock are also reflected in the composition of GC-detectable pyrolysis compounds.
Next, the dose effect of the mutation was examined by comparing the fast pyrolysis vapors of homozygous and heterozygous mutants of the second generation poplar samples, similar to the analysis of the first generation samples. Again a smaller amount of mono-methoxylated compounds was observed for homozygous mutants (Figure 3), the difference being significant for the homozygotes (5.85 ± 0.55 TIC area%) compared to heterozygotes (6.46 ± 0.46 TIC area%) of the EP11F1 family (p-value of 0.015). o-Guaiacol and p-vinylguaiacol are the significantly contributing decomposition compounds (Table 3). In contrast to the remarkable difference in H units for the homozygous mutants (9 to 18 times
more H units than in heterozygotes, [22]) no significant increases in non-methoxylated pyrolysis products were detected as compared to the corresponding heterozygotes. The fractions of non-, monoand di-methoxylated compounds in the pyrolysis vapors of the second generation samples are also distinct compared to the fraction of H, G and S units in the lignin of these samples (Figure 4), reinforcing the previous assessment that also mono- and di-methoxylated monolignols are further thermally broken down into non-methoxylated phenols.
3.3 First and second generation – Comparison Comparing the first with the second generation is in fact comparing 1-year-old with 3-months-old poplar trees (G1 vs G2 generation, respectively). The complete GC-detectable thermal decomposition spectrum classifying the products into different chemical groups is given in Figure 5a, again with the subdivision of the phenols into non-, mono- and di-methoxylated phenolic compounds in Figure 5b. For younger trees, considerably higher levels of carbon dioxide, ketones and aldehydes were detected in the pyrolysis vapor phase, which could be partially explained by a larger cellulose and hemicellulose fraction in the cell wall compared to the cell wall of older poplars which have a larger lignin fraction and as such a higher yield in phenolic degradation compounds. [22, 36, 37] The smaller amount of sugar-like compounds and larger amount of mono-aromatics in the pyrolysis vapors of younger G2 trees as compared to older G1 trees could be explained by the raise in carbon dioxide and ketones production, which suggests a further thermal breakdown of the cellulose and hemicellulose derived compounds in younger trees. This more thorough breakdown for younger G2 poplar was also investigated by inspecting the lignin-derived compounds. Not only a smaller fraction of these vapor constituents was observed for the younger trees, also the composition of this class was different since the share in nonmethoxylated phenols was larger for G2 compared to older G1 samples (Figure 5b). This suggests that the lignin in younger trees is more susceptible for demethoxylation reactions as the trends in non-, monoand di-methoxylated phenol products for the older samples relate much more to the lignin composition than for the younger ones. This further breakdown also contributes to the higher yield of low molecular weight carbonyl products (ketones and aldehydes) for younger G2 trees (Figure 5a). [38]
3.4 Correlation analysis To further investigate thermochemical decomposition mechanisms, the Py-GC-MS dataset generated in this study on Populus nigra pyrolysis provides the opportunity to analyze for correlations between compound classes or individual products. The results reported in Tables 4 and 5 show the correlations between selected compound classes and phenolic products over the complete dataset (G1 and G2 samples).
The correlations between pyrolysis compound classes in Table 4 reveal that carbon dioxide, ketones, aldehydes, mono-aromatics and non-methoxylated phenols are positively correlated with each other and negatively correlated to the other compound classes, except the alcohols. Alcohols were not correlated with any other compound class. The production of mono-aromatics and non-methoxylated phenols is related to a more thorough decomposition of the lignin fraction (free-radical chain-reactions on the phenolic side-chain of both mono- and di-methoxylated phenols), which also raises the amount of carbon dioxide and light weight oxygenates released like ketones and aldehydes. [27, 38] Methoxylated phenols are simultaneously produced from the lignin decomposition (C = 0.91) (with G and S units being the most abundant fraction) and both mono- and di-methoxylated phenols are further decomposed towards non-methoxylated phenols (C = -0.81 and C = -0.89, respectively).
To further elucidate pyrolysis mediated lignin decomposition, a correlation analysis on the 46 identified phenolic compounds was set up (Table 5). From the non-methoxylated fraction, phenol and 4 alkylated phenols are negatively correlated to most of the mono- and di-methoxylated phenolic compounds. pCresol, m-cresol and chavicol could be directly formed from respectively p-creosol, isocreosol and eugenol by demethoxylation. The formation of phenol and o-cresol implies that besides demethoxylation, also decarboxylation or decarbonylation of the phenolic side chain and alkylation of the aromatic ring take place. [38] Regarding the mono-methoxylated phenols, again two alkylated phenols (p-propylguaiacol and 2-methylresorcinol) show a negative correlation to most of the phenolic products. o-Guaiacol and p-vinylguaiacol are negatively correlated with the dimethoxylated phenols acetosyringone and sinapaldehyde which could mean they are produced by demethoxylation and
decarbonylation. In addition, almost all di-methoxylated phenols appeared to be positively correlated to one-another, confirming that these are made from a common precursor (i.e. S units). From these results, it can be concluded that demethoxylation (from either di- or mono-methoxylated phenols) is a common step in the degradation pathway during pyrolysis. [27, 38] 4 Conclusions Poplar wood with an increase in H lignin was subjected to analytical fast pyrolysis to evaluate the effect of lignin composition on the diversity and concentration of phenolic compounds produced in pyrolysis. In line with the specific shift in lignin composition, an increase in the non-methoxylated products 2,3xylenol, p-coumaryl alcohol and 2,3-dihydrobenzofuran was found for the one-year-old first generation (G1) homozygous mutants at the expense of mono-methoxylated phenols. This shift in monomethoxylated phenols appeared independent of the genetic background and age of the trees upon harvest, as proven by the analysis of the three-months-old second generation (G2) samples. The increase of the aforementioned non-methoxylated products could not be observed in these younger samples. In general the effect of the mutation on the yields of carbon dioxide, light weight oxygenates and sugarlike fraction was not significant, indicating it did not affect cellulose or matrix polysaccharides to such an extent that differences in the spectrum of pyrolytic compounds from these biomass constituents could be detected. An extensive correlation analysis on the fast pyrolysis products of all poplar samples revealed the further decomposition of mono- and di-methoxylated phenolic compounds giving rise to a higher amount of non-methoxylated phenolic compounds, masking the raise of non-methoxylated compounds derived from H units. Reactions like demethoxylation, demethylation, decarboxylation, decarbonylation and alkylation take place in favor of carbon dioxide, ketones and aldehydes production. In conclusion this study proves that the selected variation in the lignin content of the wood feedstock has its outcome in the fast pyrolysis product range. In addition, we found that a substantial amount of the non-methoxylated pyrolysis products appeared to be derived via demethoxylation reactions of monoand di-methoxylated precursors. These results are promising for future research which should aim towards the selection of biomass with a more extreme lignin composition (for example towards one type of building block [40]) that can become a promising feedstock for fast pyrolysis processing.
Acknowledgements The authors acknowledge the UGent Multidisciplinary Research Partnership (MRP) “Ghent Bioeconomy” for its financial support in the analytical pyrolysis-GCMS infrastructure. Ruben Vanholme is indebted to the Research Foundation-Flanders (FWO) for a postdoctoral fellowship.
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Figure 1a. TIC area% response from 500°C pyrolysis of first generation homozygous (G1 Δ73/Δ73 ), heterozygous (G1 Δ73/+) and wild type (G1 +/+) poplar, classified into compound classes according to chemical functionality. Analyses run in triplicate, error bars represent the standard deviation.
40
G1 Δ73/Δ73 G1 Δ73/+
35
G1 +/+ 30
TIC area%
25 20 15 10 5 0
Figure 1b. Subdivision into non-, mono- and di-methoxylated compound classes of the phenolic
TIC area% response of 500°C pyrolysis of first generation homozygous (G1 Δ73/Δ73), heterozygous (G1 Δ73/+) and wild type (G1 +/+) poplar. Analyses run in triplicate, error bars represent the standard deviation. The asterisk indicates a significant difference in percentage of monophenolic products for
TIC area%
homozygous compared to heterozygous / wild type poplar. 30
G1 Δ73/Δ73
20
G1 Δ73/+
10
G1 +/+
*
0 NON MONO
DI
Figure 2. Comparison between the lignin composition of the initial feedstock via thioacidolysis (expressed in percentage H, G, and S units; data obtained from [26]) and the partitioning in the phenolic fraction (percentage non-, mono- and di-methoxylated phenols) after 500°C analytical pyrolysis for first generation homozygous G1 Δ73/Δ73 (a), heterozygous G1 Δ73/+ (b) and wild type poplar G1 +/+ (c). Analyses run in triplicate, error bars represent the standard deviation.
a 70
G1 Δ73/Δ73 lignin composition
60 50
G1 Δ73/Δ73 pyrolytic phenolic fraction composition
%
40 30 20 10 0 H , NON
G , MONO
S , DI
b 70
G1 Δ73/+ lignin composition
60
G1 Δ73/+ pyrolytic phenolic fraction composition
50
%
40 30 20 10 0 H , NON
G , MONO
S , DI
c 70
G1 +/+ lignin composition
60 50
G1 +/+ pyrolytic phenolic fraction composition
%
40 30 20 10 0 H , NON
G , MONO
S , DI
Figure 3. TIC area% response of non-, mono- and di-methoxylated phenolic compound class from 500°C pyrolysis of second generation homo/heterozygotes from the first poplar family (G2 EP11F1 Δ73/Δ73 and G2 EP11F1 Δ73/+) and second family homo/heterozygotes (G2 EP11F2 Δ73/Δ73 and G2 EP11F2 Δ73/+). Analyses run in triplicate, error bars represent the standard deviation. The asterisk indicates a significantly lower percentage of monophenolic products for first family homozygous poplar compared to either the second family homozygotes and compared to the first family heterozygotes.
9
G2 EP11F1 Δ73/Δ73
8
G2 EP11F2 Δ73/Δ73
7
*
TIC area%
6
G2 EP11F1 Δ73/+ G2 EP11F2 Δ73/+
5 4 3 2 1 0 NON
MONO
DI
Figure 4. Comparison between the lignin composition of the initial feedstock via thioacidolysis (expressed in percentage H, G, and S units; data obtained from [26]) and the partitioning in the phenolic fraction (percentage non-, mono- and di-methoxylated phenols) after 500°C analytical pyrolysis for second generation homozygotes (a and b) and heterozygotes (c and d). Analyses run in triplicate, error bars represent the standard deviation.
a
b
70
70
60
60
50
50
G2 EP11F1 Δ73/Δ73 lignin composition
30
G2 EP11F1 Δ73/Δ73 pyrolytic phenolic fraction compostion
40
G2 EP11F2 Δ73/Δ73 lignin composition
30
G2 EP11F2 Δ73/Δ73 pyrolytic phenolic fraction composition
%
%
40
20
20
10
10
0
0
H , NON
G , MONO
S , DI
H , NON
G , MONO
c
S , DI
d
70
70
60
60
50
50
G2 EP11F1 Δ73/+ lignin composition
G2 EP11F2 Δ73/+ lignin composition
40
%
%
40
EP11F1 Δ73/+ pyrolytic phenolic fraction composition
30
20
20
10
10
0
G2 EP11F2 Δ73/+ pyrolytic phenolic fraction composition
30
0
H , NON
G , MONO
S , DI
H , NON
G , MONO
S , DI
Figure 5a. TIC area% response of 500°C pyrolysis products (classified) of homozygous and heterozygous samples of 1-year-old (G1 Δ73/Δ73; G1 Δ73/+) and 3-months-old (G2 EP11F1 and EP11F2 Δ73/Δ73; G2 EP11F1 and EP11F2 Δ73/+) poplar classified into component groups according to chemical functionality. Analyses run in triplicate, error bars represent the standard deviation. 40 35
TIC area%
30 25 20 15 10 5 0
G1 Δ73/Δ73 G1 Δ73/+ G2 Δ73/Δ73 G2 Δ73/+
Figure 5b. Subdivision into non-, mono- and di-methoxylated compound classes of the phenolic TIC area% response of 500°C pyrolysis of homozygous and heterozygous samples of 1-year-old (G1 Δ73/Δ73; G1 Δ73/+) and 3-months-old (G2 EP11F1 and EP11F2 Δ73/Δ73; G2 EP11F1 and EP11F2 Δ73/+) poplar. Analyses run in triplicate, error bars represent the standard deviation. 25
G1 Δ73/Δ73 G1 Δ73/+
TIC area%
20
G2 Δ73/Δ73 G2 Δ73/+
15 10 5 0 NON
MONO
DI
Table 1: TIC area% response of the significantly different 500°C pyrolysis products in both the comparison of first generation homozygous samples G1 Δ73/Δ73 with heterozygous (G1 Δ73/+) poplar and G1 Δ73/Δ73 with wild type (G1 +/+) poplar. Values in bold are the samples with highest response for the according compound.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
RT (min)
Name
9.25 11.51 17.27 23.79 23.94 26.34 28.01 28.19 29.91 31.66 32.44 35.17 36.38 37.75 39.41 43.28 43.34 44.00
Formic acid Toluene 2-Propylfuran 3-Methyl-5H-furan-2-one 2,3-Dimethyl- 2-pentenoic acid Maltol Creosol 2,3-Xylenol 1-Ethylidene-1H-indene 2,3-Dihydrobenzofuran Eugenol (Z)-Isoeugenol 4-(t-Butyl)benzaldehyde Acetovanillone 1-Naphthalenol 1,2-Dimethoxy-4-(3-methoxy-1-propenyl)benzene p-Coumaryl alcohol Coniferyl alcohol
Δ73/Δ73 TIC area% stdev 0,02 0,03 0,23 0,03 0,02 0,00 0,14 0,01 0,15 0,00 0,30 0,00 0,78 0,03 0,11 0,02 0,01 0,00 0,33 0,06 0,39 0,02 0,96 0,04 0,04 0,00 0,37 0,02 0,09 0,01 0,00 0,00 0,35 0,05 2,24 0,20
TIC area% 0,11 0,15 0,04 0,13 0,13 0,28 0,87 0,05 0,06 0,17 0,44 1,19 0,08 0,42 0,03 0,05 0,03 3,22
Δ73/+ stdev 0,02 0,02 0,02 0,01 0,01 0,01 0,06 0,02 0,02 0,11 0,04 0,12 0,04 0,03 0,01 0,01 0,01 0,40
p-value 0,001 0,001 0,017 0,006 0,002 0,007 0,012 0,006 0,000 0,023 0,042 0,005 0,038 0,038 0,000 0,006 0,001 0,002
TIC area% 0,09 0,17 0,06 0,12 0,12 0,27 0,91 0,07 0,05 0,12 0,47 1,23 0,10 0,43 0,05 0,05 0,02 3,23
+/+ stdev 0,04 0,04 0,02 0,01 0,02 0,01 0,10 0,03 0,03 0,13 0,05 0,20 0,07 0,03 0,02 0,01 0,01 0,83
p-value 0,025 0,043 0,004 0,004 0,003 0,004 0,032 0,038 0,018 0,015 0,015 0,022 0,010 0,021 0,019 0,010 0,001 0,033
Table 2: TIC area% response of the significantly different 500°C pyrolysis products after comparison between second generation EP11F1 homozygous (EP11F1 Δ73/Δ73) and EP11F2 homozygous poplar (EP11F2 Δ73/Δ73). Values in bold are the samples with highest response for the according compound. RT (min) Name 9.64 14.81 15.13 20.43 22.12 22.45 22.76 22.88 23.94 26.65 26.87 27.70 30.20 30.29 30.85 31.66 32.66 33.30 40.77 41.64 42.67
3-Penten-2-one Glycol monoacetate 5H-Furan-2-one 2-Hydroxy-2-cyclopenten-1-one 4-Hydroxybutanoic acid 5H-Furan-2-one 3-Methyl-3-cyclohexen-1-one 3,4-Dihydro-2-methoxy-2H-pyran 2,3-Dimethyl- 2-pentenoic acid 2-Methyl-1,3-cyclohexanedione m-Cresol p-Creosol 3-Pyridinol p-Ethylguaiacol 6-Methyl-1,4-dioxaspiro[2.4]heptan-5-one 2,3-Dihydrobenzofuran p-Propylguaiacol 5-Hydroxymethyldihydrofuran-2-one Melezitose Methyl-(2-hydoxy-3-ethoxy-benzyl)ether α-Lactose
G2 EP11F1 Δ73/Δ73 TIC area% stdev 0.07 0.03 0.46 0.31 0.06 0.03 2.01 0.14 0.44 0.11 0.88 0.06 0.36 0.06 0.27 0.05 0.12 0.01 0.12 0.03 0.72 0.09 0.38 0.20 0.28 0.17 0.19 0.12 0.17 0.17 1.19 0.33 0.07 0.01 0.35 0.05 0.24 0.04 0.02 0.01 0.15 0.04
G2 EP11F2 Δ73/Δ73 TIC area% stdev 0.09 0.01 0.29 0.10 0.04 0.02 1.87 0.15 0.34 0.09 0.81 0.06 0.41 0.03 0.24 0.21 0.13 0.02 0.09 0.02 0.81 0.09 0.52 0.04 0.05 0.10 0.32 0.06 0.01 0.03 1.55 0.30 0.10 0.04 0.30 0.03 0.32 0.13 0.03 0.01 0.20 0.05
p-value 0.008 0.023 0.049 0.037 0.049 0.016 0.036 0.041 0.002 0.043 0.031 0.008 0.001 0.017 0.009 0.020 0.006 0.013 0.028 0.040 0.013
Table 3: TIC area% response of the significantly different (homozygous compared to heterozygous samples) 500°C pyrolysis products from second generation EP11F1 (EP11F1 Δ73/Δ73 compared to EP11F1 Δ73/+) and EP11F2 (EP11F2 Δ73/Δ73 compared to EP11F2 Δ73/+, indicated with asterisk) poplar. Double asterisk indicates the significant difference occurring in both families. Values in bold are the samples with highest response for the according compound.
RT (min)
Name
7.76 9.64 23.79 25.85
2,3-Butanedione 3-Penten-2-one* 3-Methyl-5H-furan-2-one o-Guaiacol
31.83
p-Vinylguaiacol**
G2 EP11F1 Δ73/Δ73
G2 EP11F1 Δ73/+
G2 EP11F2 Δ73/Δ73
G2 EP11F2 Δ73/+
TIC area% 0,72 0,07 0,16 1,36
stdev 0,04 0,03 0,02 0,11
TIC area% 0,68 0,08 0,18 1,46
stdev 0,05 0,03 0,03 0,14
p-value 0,037 0,595 0,041 0,038
TIC area% 0,71 0,09 0,17 1,41
stdev 0,06 0,01 0,03 0,11
TIC area% 0,61 0,07 0,17 1,45
stdev 0,24 0,01 0,01 0,07
p-value 0,562 0,008 0,967 0,437
1,78
0,18
2,02
0,17
0,007
1,71
0,12
1,82
0,10
0,043
Table 4: Correlation coefficients between 500°C fast pyrolysis product compound classes for all the poplar samples. + and - indicate a significant positive and negative correlation, while 0 indicates no
+ +
+ -
-
+
DI
+ + +
MONO
+ + + +
Mono-arom.
+ + + + +
NON
+ + + + + +
Sugars
+ + -
Phenols
+ + + -
Furans / Pyran
0 0 0 0 0 0 0 0 0 0 0
Acids/esters
Aldehydes
0 + + + + + + +
Ethers
Ketones
0 + + + + -
Alcohols
Carbon dioxide Aliphatic Alcohols Ketones Aldehydes Ethers Acids/esters Furans / Pyran Phenols Sugars Mono-arom. NON MONO DI
Aliphatic
Carbon dioxide
correlation.
+ -
-
+
DI
+ +
MONO
+ + +
NON
+ + + +
Mono-arom.
+ + + + +
Sugars
+ + + + + +
Phenols
+ + -
Furans / Pyran
+ + + -
Acids/esters
0 0 0 0 0 0 0 0 0 0 0
Ethers
Ketones
0 + + + + + + +
Aldehydes
Alcohols
0 + + + + -
Aliphatic
Carbon dioxide Carbon dioxide Aliphatic Alcohols Ketones Aldehydes Ethers Acids/esters Furans / Pyran Phenols Sugars Mono-arom. NON MONO DI
MONO
NON
Phenol o-Cresol 2,6-Xylenol p-Cresol m-Cresol 2,3-Xylenol Chavicol p-Hydroxybenzylidene acetone p-Formyl phenol Methylparaben p-Coumarylalcohol o-Guaiacol Isocreosol p-Creosol p-Ethylguaiacol p-Vinylguaiacol Eugenol p-Propylguaiacol + 0 + + 0 + 0 0 0 0 0 + + 0 + 0 0 0 0 + 0 + 0 0 0 + 0 + 0 0 0 0 0 + 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + 0 + 0 0 + 0 + 0 + + 0 0 + + 0 0 + 0 0 + 0 0 0 0 0 -
5-tert-Butylpyrogallol
3,5-Dimethoxy-4-hydroxyphenylacetic acid
Sinapaldehyde
2,5-Dimethoxybenzyl acetate
2,5-Dimethoxy-benzenemethanol acetate
1-(2,4,6-Trihydroxyphenyl)-2-pentanone
Acetosyringone
MONO
1,2-Dimethoxy-4-(3-methoxy-1-propenyl)benzene
3,5-Dimethoxy-4-hydroxyphenylacetic acid
Syringaldehyde
Methoxyeugenol
1-(3,4-Dimethoxyphenyl)ethanone
1,2,3-Trimethoxy-5-methylbenzene
1,2,4-Trimethoxybenzene
Syringol
(E)-Coniferol
Methyl-(2-hydoxy-3-ethoxy-benzyl)ether
2-Allyl-1,4-dimethoxy-3-methylbenzene
Coniferol
Acetovanillone
2-Methylresorcinol
Vanillin
Resorcinol
(Z)-Isoeugenol
NON
(E)-Isoeugenol
1,2-Benzenediol
p-Propylguaiacol
Eugenol
p-Vinylguaiacol
p-Creosol
0 0 0 + 0 0
p-Ethylguaiacol
Isocreosol
+ + 0 + 0
o-Guaiacol
p-Coumarylalcohol
Methylparaben
+ + + 0 + + 0 0 + -
p-Formyl phenol
p-Hydroxybenzylidene acetone
Chavicol
2,3-Xylenol
m-Cresol
0 0 0 0 0 0 0 0 0 0 0 0 0
p-Cresol
2,6-Xylenol
o-Cresol
Phenol
Table 5: Correlation coefficients between 500°C fast pyrolysis phenolic products for all the poplar samples. + and - indicate a significant positive and negative correlation, while 0 indicates no correlation. First 11 phenols are non-methoxylated,
followed by 18 mono-methoxylated and 17 di-methoxylated phenols.
DI
DI
1,2-Benzenediol (E)-Isoeugenol (Z)-Isoeugenol Resorcinol Vanillin 2-Methylresorcinol Acetovanillone Coniferol 2-Allyl-1,4-dimethoxy-3-methylbenzene Methyl-(2-hydoxy-3-ethoxybenzyl)ether (E)-Coniferol Syringol 1,2,4-Trimethoxybenzene 1,2,3-Trimethoxy-5-methylbenzene 1-(3,4-Dimethoxyphenyl)ethanone Methoxyeugenol Syringaldehyde 3,5-Dimethoxy-4-hydroxyphenylacetic acid 1,2-Dimethoxy-4-(3-methoxy-1propenyl)benzene Acetosyringone 1-(2,4,6-Trihydroxyphenyl)-2-pentanone 2,5-Dimethoxy-benzenemethanol acetate 2,5-Dimethoxybenzyl acetate Sinapaldehyde 3,5-Dimethoxy-4-hydroxyphenylacetic acid 5-tert-Butylpyrogallol
0 0 + -
0 0 + -
0 0 0 0 0 0 0 0 0
0 0 + -
0 0 + -
0 0 0 0 0 0 0 0
0 0 + -
+ + + 0 0 + + +
+ + + 0 0 + + +
+ + + 0 0 + + +
+ + + 0 0 + + +
0 0 0 0 0 0 0 0 -
+ + + 0 0 + + +
+ + + 0 0 + + +
0 0 0 0 0 + 0 0 0
0 0 0 0 0 0 0 -
+ + + 0 0 + + +
0 0 0 0
+ + 0 0 + + +
+ 0 0 + + +
0 0 + + +
0 0 0 0
0 0 0 0
-
+ +
+
-
-
0 0 0 0 0 0 0 0
-
-
0 0 0 0 0 0 0 0
-
+ + + + + + + +
0 + + + + + + +
+ + + + + + + +
+ + + + + + + +
0 0 0 0 0 0 0 0
+ + + + + + + +
+ + + + + + + +
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
+ + + + + + + +
0 -
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
-
+ + + + + + + +
+ + + + + + + +
+ + + + + + + +
+ + + + + + +
+ + + + + +
+ + + + +
+ + + +
+ + +
+ +
+
-
-
0
-
-
0
-
+
+
+
+
0
+
+
0
0
+
-
+
+
+
0
0
-
+
+
+
+
+
+
+
+
+
+
+
0 -
0 -
0 0 0
-
0 -
0 0 0
-
+ + +
0 + +
0 + +
0 + +
0 0
+ + +
0 + +
0 0 0
0 0
+ + +
0 -
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