Imidazole: Prospect Solvent for Lignocellulosic Biomass Fractionation

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Imidazole – prospect solvent for lignocellulosic biomass fractionation and delignification Ana Morais, Joana Pinto, Daniela Nunes, Luisa Bivar Roseiro, M. Conceição Oliveira, Elvira Fortunato, and Rafal Bogel-Lukasik ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.5b01600 • Publication Date (Web): 29 Dec 2015 Downloaded from http://pubs.acs.org on January 4, 2016

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ACS Sustainable Chemistry & Engineering

Imidazole

–

prospect

solvent

for

lignocellulosic

biomass

fractionation and delignification Ana Rita C. Morais,a,b Joana Vaz Pinto,c Daniela Nunes,c Luísa B. Roseiro,a Maria Conceição Oliveira,d Elvira Fortunatoc and Rafał Bogel-Łukasika*

a

Unidade de Bioenergia, Laboratório Nacional de Energia e Geologia, I.P., Estrada do Paço

do

Lumiar

22,

1649-038

Lisboa,

Portugal.

*e-mail:

[email protected];

Fax:

+351217163636; Phone: +351210924600 ext. 4224 b

LAQV/REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia,

Universidade Nova de Lisboa, 2829-516 Caparica, Portugal c

CENIMAT, I3N, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516

Caparica, Portugal d

Centro Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, 1049-001

Lisboa, Portugal

ACS Paragon Plus Environment


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Abstract The future widespread production of biomass-derived fuels, chemicals and materials requires cost-effective processing of sustainable feedstock. The use of imidazole as solvent for biomass creates a novel approach that helps to accomplish this idea in a green fashion. This work proposes imidazole as a novel solvent for wheat straw pre-treatment, which allowed the production of cellulose- and hemicellulose-rich fractions and added-value products from depolymerisation of lignin. Various temperatures (110, 140 and 170 ºC) and processing times (1, 2 and 4 hours) of pre-treatment were investigated. Both cellulose and hemicellulose recovery were highly dependent on reaction temperature. The best result for the recovery of cellulose-rich material was obtained at 170 ºC during 2 h achieving 62.4% w∙w-1, while native wheat straw is composed by only 38.8% w∙w-1 cellulose. For the same conditions, optimal results were also obtained regarding the enzymatic hydrolysis yield (99.3% w∙w-1 glucan to glucose yield) in cellulose-rich material. This result was possible to be obtained due to morphological and structural changes in cellulose-rich materials accompanied by extensive delignification (up to 92%). The presence of added-value phenolic compounds in recovered imidazole was analysed by capillary electrophoresis and HPLC-MS. Vanillin and other lignin-based products were identified. Finally, the high purity of recovered imidazole was demonstrated by 1H and 13C NMR. Keywords pre-treatment, imidazole, lignocellulose, delignification, enzymatic hydrolysis, crystallinity, biorefinery, green solvent


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Introduction Recently, lignocellulosic biomass started to be recognised as one of the most important environmentally sustainable alternative to fossil resources. To accomplish this, several biorefinery initiatives are being developed, which are mainly focused on sustainable exploitation of all lignocellulosic biomass fractions in terms of their separation and integrated valorisation.1 Conversion of lignocellulosic biomass into carbohydrate-derived fermentable sugars constitutes a remarkable challenge, chiefly due to the complex and recalcitrant architecture of the feedstock.2-3 Up to now, there are several biomass processing approaches capable to overcome these obstacles, resulting in biomass dissolution and release of fermentable sugars. However, such processes are normally characterised by low yields and moderate selectivities, which consequently burden the production of chemicals and fuels at competitive costs. Thus, to exploit the full potential of these lignocellulosics, new strategies for their processing are needed.4 One of the possible solutions is the deconstruction of biomass into its major components with more sustainable and alternative solvents. In the last years, non-aqueous systems such as ionic liquids (ILs) have gained increasing interest for biomass processing due to its capacity to dissolve lignocellulosic biomass.5-11 ILs are novel solvents with melting point below 100 ⁰C and the effect of both anion and cation were extensively studied to comprehend their influence on biomass dissolution as well as on structural modifications of its components.12-13 Imidazolium-based ILs are the most extensively studied ILs and have demonstrated that either the cation or the anion is considerably important in the biomass processing. Special attention must be given to the imidazolium cation that has a potential of π-stacking and possibility to form a hydrogen bond-based interaction with lignin.14-15 Albeit their well-known benefits, the costs associated to synthesis and purification of ILs is normally claimed as decision in the economic inadequateness of ILs-based biorefineries.16 However, Hallett and co-workers demonstrated recently that the synthesis of some ILs containing hydrogen sulphate anion produced from inexpensive starting materials such as simple amines and sulphuric acid, can compete with cheaper pre-treatment methods regarding to efficiency and process economy.17 Notwithstanding, there is still a need for neoteric solvents for biomass processing that can be used at industrial scale and at competitive costs. One of such candidates is imidazole, which up to now is used only as solvent for starch



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dissolution_ENREF_1818 and is broadly applied as precursor of imidazolium-based ILs. Imidazole is characterised by low toxicity and high boiling point and as such, due to the negligible vapour pressure, is easy to handle and to recycle, being an interesting cheap alternative to other more expensive intensively explored solvents (e.g. ILs) in biomass processing. To the best of our knowledge, this is the first research referring to the use of imidazole as solvent for fractionation, delignification and depolymerisation of lignocellulosic material. Insights into the biomass morphology changes, cellulose crystallinity and the effect of imidazole treatment on efficiency of enzymatic hydrolysis of recovered cellulosic materials are demonstrated. For this purpose, the Fourier-Transform Infrared Spectroscopy (FTIR), Xray Diffraction (XRD) and Scanning Electron Microscopy (SEM) analyses were performed. Additionally, examples of value-added products from lignin depolymerisation were identified using capillary electrophoresis (CE) and HPLC-MS2. Results and discussion Wheat straw processing with imidazole Biomass fractionation with imidazole was examined at a temperature range between 110 and 170 ⁰C and reaction time between 1 and 4 h. All experiments produced solids composed mainly of polysaccharides (cellulose and hemicellulose). The chemical characterisation of each of the produced fraction, yield of recovered carbohydrates and respective mass losses were determined and are depicted in Table S1 in Supporting Information (SI). The obtained results show a dominant effect of temperature on the recovery of each fraction. Higher temperature diminishes the amount of recovered cellulose material, but yields cellulose-rich fraction with higher purity. The increase in temperature from 110 ºC to 170 ºC for the same 2 h reaction time endorses the increase in the cellulose content in cellulose-rich fraction by almost 1.5 fold. For the best case scenario (170 ºC, 2 h), the cellulose content in cellulose-rich material was 62.4% w·w-1, while native wheat straw is composed by only 38.8% w·w-1 cellulose. For lower temperatures, the cellulose content is lower, being the lowest (42.2% w·w-1) at 110 ºC for 2 h, as depicted in Figure 1.


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Cellulose-rich material composition/ . -1 % w w dry weight

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60

50

40

30

20

10

0 80 native

110

140

170

Temperature/oC Figure 1. Native wheat straw19 and cellulose-rich material compositions obtained from biomass pre-treated in the presence of imidazole at different temperatures. Black bars represent cellulose, light-grey corresponds to hemicellulose content and dark-grey bars depict the lignin content in the solids analysed.

The analysis of Figure 1 shows that the increase in temperature from 110 ºC to 170 ºC promotes lignin removal from 54.5% w·w-1 to 91.4% w·w-1, when compared to lignin content in native wheat straw. This result can be explained by more favourable biomass dissolution at higher temperatures which, in turn, helps to disrupt the ester bonds linking lignin and hemicellulose, and fades the hydrogen bonds existing between lignin, cellulose and hemicellulose.20 Similar lignin removal results were reported in literature using distinct biomass processing technologies. For example, ILs demonstrated to have capacity to remove lignin. da Costa Lopes et al. reported the use of 1-ethyl-3-methylimidazolium acetate IL in biomass fractionation.9 The fractionation of biomass using this IL allowed to obtain carbohydrates-rich materials and a separated lignin fraction with 87% w·w-1 purity. Brandt et al. tested different 1-butyl-3-H-imidazolium hydrogen sulphate and found that this IL was able to remove up to 93% w·w-1 lignin present in raw material in the process carried out at



120 ⁰C during 20 h reaction.21 A major benefit from the use of imidazole is that imidazole is cheaper than ILs. One of the major differences between ILs and imidazole presented herein is that ILs permit to precipitate, and thus, to recover lignin in a form relatively unchanged when compared to the raw material. Contrastingly, imidazole promotes the total dissolution of lignin, making its recovery in the native form impossible. Kang et al. studied the use of 1methylimidazole on the pre-treatment of diverse biomasses. At the experimental conditions (25 ºC and 5 min), the use of 1-methylimidazole on the pre-treatment of loblolly pine did not show any visual changes. Only when phosphoric acid swollen or steam exploded biomasses were employed, the removal of lignin between 50% and 90% depending on type of steam exploded biomass and concentration.22

Hemicellulose-rich material composition/ . -1 % w w dry weight

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50

40

30

20

10

0 80 native

110

140

170

o

Temperature/ C Figure 2. Native wheat straw19 and hemicellulose-rich material compositions obtained from biomass pre-treated in the presence of imidazole at different temperatures. Black bars represent cellulose, light-grey corresponds to hemicellulose content and dark-grey bars depict the lignin in the solids.


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A second recovered fraction was the hemicellulose-rich material and the composition of this fraction is demonstrated in Figure 2. Observing the obtained results, it can be concluded that the increase in temperature from 110 ºC to 170 ºC guides to an increase in hemicellulose-rich material recovery. Additionally, the hemicellulose content in these materials is increased, however, this effect is less dominant (14.6% w·w-1) than in the case of cellulose material (20.2% w·w-1). The hemicellulose-rich material with the highest purity of 54.5% w·w-1 was obtained for the harshest conditions examined (170 ºC, 4 h). This effect is caused by the promotion of more efficient fractionation, hence, by a progressive disappearance of cellulose (from 35.9% w·w-1 at 110 ºC, 2 h to 17.2% w·w-1 at 170 ºC, 4 h) from hemicelluloserich fraction counterbalanced by the aforementioned increase in cellulose content in cellulose-rich fraction. Analogously to cellulose content in cellulose-rich fraction, the hemicellulose content (sum of xylan and arabinan present in raw material) in hemicelluloserich material increased up to 52.0% w·w-1 while native wheat straw contains only 22.4% w·w-1. Analysing the effect of reaction time, it can be stated that at 170 ºC it had negligible effect on both polysaccharide-rich materials recovery and delignification, as depicted in Table S1 in SI. Morphological analysis of recovered materials Another relevant aspect of enhancing the process sustainability is the need of carbohydrate recovery from the fractionated materials. Since imidazole demonstrated a significant delignification performance, this kind of biomass pre-treatment is an interesting technology to be studied in terms of potential structural and morphological changes in the recovered cellulose-rich materials. The characterisation of these changes would allow to understand and to explore the possibility of converting the recovered materials into sugars by enzymatic hydrolysis. SEM was used to observe the physical changes, namely size reduction and destruction of fibres, in the materials produced at different experimental conditions. The SEM images together with the pictures of the native wheat straw and produced samples are depicted in Figure 3.



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Figure 3. Scanning electron microscopy images and pictures of native wheat straw (a, e) and regenerated cellulose samples produced at 110 ºC (b, f), 140 ºC (c, g) and 170 ºC (d, h) for 2 h reaction.


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Observation of SEM images illustrates that the native wheat straw exhibits a highly dense, rigid and intact surface, very resistant to enzymatic attack.23 On the other hand, regenerated cellulose-rich samples show an increasing surface roughness and disruption of lamellar structure effects, which increases with the increase in pre-treatment temperature from 110 ºC to 170 ºC for 2 h. These physical changes are especially visible, either by SEM or by unarmed eye for the regenerated cellulose material obtained in reactions carried out at 170 ºC (Figure 3 c and h). The cellulosic material shows anomalous porosity with pores over several length dimensions and the surface of this material became coarser and looser. Furthermore, its morphology is consistent with a conglomerate texture in which fibres appeared fused into homogeneous macrostructure, resembling to cellulose recovered from ILs fractionation treatment, mainly due to the lignin extraction.6-7, 11 Crystallinity Crystallinity has been reported as one of the most influential and important factors of enzymatic hydrolysis efficiency, because higher cellulose crystallinity makes biomass inaccessible to enzymatic attack.24-25 The cellulose crystallinity results from inter- and intramolecular hydrogen bonds between cellulose chains and can be modified by employing biomass pre-treatment technologies.26-27 However, the determination of cellulose crystallinity of regenerated solids constitutes a challenge, since lignocellulosic biomass is composed by diverse amorphous compounds, such as hemicellulose and lignin, in addition to disordered cellulose.28 As imidazole was found to be highly selective for the delignification of lignocellulosic biomass, and because lignin hampers the efficient saccharification process, it can be expected that the pre-treatment with imidazole reduces the crystallinity of cellulose-rich material. Hence, these materials as well as pure cellulose were subject to XRD diffraction and FTIR to examine this phenomenon. XRD diffraction permits to measure the crystallinity of the material as a whole or, in other words, it considers disordered cellulose, hemicellulose and lignin,29 while FTIR analysis determines the relative crystallinity of cellulose in the cellulose-rich material.30 This differentiating nuance of both techniques has a significant importance on the interpretation of the results, as it is discussed below. The XRD patterns of native wheat straw and regenerated cellulose-rich materials for various temperatures are shown in Figure 4. The XRD diffractogram of native biomass exhibits



prominent signals of 2θ at 16.7º and 22.2º which are typical to crystalline cellulose I. The signal of 2θ at 16.7º is the overlapping signals of (101) and (10-1) planes, while very intense signal at 22.2º corresponds to the (002) crystalline plane. The “valley” at 18.1º is associated to amorphous region which include disordered cellulose, hemicellulose and lignin.

20000

16000

Intensity

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12000

8000

4000

0 10

15

20

25

30

35

40

2θ

Figure 4. XRD diffractograms of native wheat straw (red line) and recovered cellulose-rich materials from 110 ⁰C (blue line), 140 ⁰C (green line) and 170 ⁰C (black line) for 2h residence time.

Comparing the XRD diffractogram of native sample to those obtained after imidazole treatment, it can be easily seen that there are significant changes in the diffraction pattern. Therefore, based on the XRD diffractograms, the CrI were calculated according to equation presented in the Experimental Section and results are compiled in Table S1 in SI.

Surprisingly, an increase in temperature promotes the increase in CrI, which can also be confirmed by the increasing intensity of 2θ signal found at 22.2º. The CrI for native wheat straw is as high as 49.4%, while the CrI for regenerated cellulose materials increases from


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59.4% to 64.8% and 66.4% at 110 ºC, 140 ºC and 170 ºC for 2 h, respectively. Therefore, it can be concluded that crystallinity index of produced material analysed by XRD is increasing with the increase in the severity of the reaction. This seems to be obvious, considering the relative increase of cellulose amount in recovered materials from 42.2% w·w-1 to 62.4% w·w1

at 110 ºC and 170 ºC, respectively, and a predominant more than 90% lignin removal from

cellulose-rich material.31-34 Observation of this data shows an almost perfect linear relationship between the CrI and cellulose content in the analysed material. Hence, it can be concluded that imidazole does not interfere directly with the material crystallinity. To confirm this, additional experiments were performed and the microcrystalline cellulose, either pure or pre-treated with imidazole at 110 °C and 140 °C for 2 h, were analysed. The XRD analysis data shown in Table S2 in SI confirms that CrI for these 3 samples are identical (81.6 ± 1.3%), which again confirms that imidazole does not change the crystallinity of cellulose-rich materials. Analysing the CrI results for samples for different reaction times, it is clear that time has negligible effect on Crl value. This result can be expected as CrI values are directly correlated to the chemical characterisation of the recovered material, (Table S3 in SI), and show no significant changes between samples produced at 170 ºC at 1, 2 and 4 h. Comparing the obtained data to those presented in literature, it can be stated that, in general, CrI value does not change for longer reaction times and, consequently, the cellulose crystallinity is insignificantly affected. The inter- and intramolecular hydrogen bond network limits a breaking down of glycosidic bonds in the imidazole pre-treatment, similarly, to what was found for hot-compressed water biomass processing.31 Therefore, the results obtained in the present work leads to the conclusion that imidazole-based fractionation change the crystallinity of cellulose materials, but exclusively by the great lignin removal, because delignification contributes to the enhancement of cellulose content in the recovered materials. FTIR spectra observed in the range of 800-4000 cm-1 were used to characterise the crystallinity of native biomass and regenerated cellulose-rich materials, as well as their qualitative chemical composition from imidazole fractionation experiments. The absorption band at 1437 cm-1 assigned to the CH2 scissoring motion is an intense signal characterised for cellulose I, while absorption band at 898 cm-1 assigned to C-O-C stretching at β-(1,4)glycosidic linkage is an intense and sharp signal, mostly associated to cellulose II and



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disordered amorphous cellulose.35 As shown in Figure 5, the absorption band at 1437 cm-1 was faintly more intense for native wheat straw than for regenerated celluloses. The absorption band at 898 cm-1 was weak for native material but strong for regenerated cellulose materials. According to the aforementioned findings, the spectra illustrated in Figure 5 indicate that temperature plays an important role in the increase of absorption band corresponding to amorphous cellulose. High temperatures promote the conversion of cellulose I to cellulose II or amorphous cellulose, enhancing the increase in intensity of absorption band at 898 cm-1. This is in good agreement with what was reported for other recovered cellulose-rich materials for different pre-treatment processes.36 Nevertheless, to prove the existence of cellulose I and its conversion to cellulose II and amorphous cellulose, the LOI was calculated according to the equation presented in the Experimental Section. As depicted in Table S2 in SI, the native biomass has LOI as high as 0.560, while regenerated cellulose-rich samples have lower LOI values ranging from 0.469 to 0.478. This means that, in general, the increase in temperature promotes the reduction of cellulose crystallinity. In particular, the regenerated cellulosic fractions treated at 170 ºC for 2h have 15% lower LOI than original wheat straw. Although the decrease in cellulose crystallinity is relatively low, this can be explained by the fact that pre-treatments performed at high pH, as in the case of imidazole, have normally lesser effect on reduction of cellulose crystallinity than e.g. acidic treatments.37 Concluding, the analyses of the morphological and structural changes of cellulose-rich materials showed that both techniques used provide important complementary and concomitant data. The XRD allows understanding the crystallinity of cellulose-rich fraction as a whole, while FTIR provides information about the crystallinity of cellulose enveloped in the cellulose-rich material.


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0.3

Intensity

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0.2

0.1

0.0 900

1200

1500

1800

Wavelenght/cm-1 Figure 5. FTIR spectra of native wheat straw (red line) and regenerated cellulose materials from imidazole treatments performed at 110ºC (blue line), 140ºC (green line) and 170ºC (black line) for 2h. Dashed lines are presented to better observe the bands for LOI calculations. Enzymatic hydrolysis The efficiency of enzymatic hydrolysis might be affected by several factors, such as hemicellulose and lignin presence, cellulose-rich material crystallinity and degree of polymerisation, as previously discussed. All of these factors are strongly related to the type of biomass and the choice of pre-treatment method.38 To survey the influence of pretreatment method on the fermentable sugars production, the native wheat straw and recovered cellulose-rich materials were subject to enzymatic hydrolysis. Table 1 depicts glucan to glucose yields (% w∙w-1), xylan to xylose yield (% w∙w-1) and total reducing sugars (% w∙w-1) of enzymatic hydrolysis experiments. These results were calculated based on the

glucan and xylan content determined by the composition analysis presented in Table S1 in SI.



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Table 1. Glucan and xylan conversion yields, and respective total reducing sugars yields (%w∙w-1), obtained from enzymatic hydrolysis of native wheat straw and regenerated cellulose-rich materials produced in imidazole-based pre-treatments at different reaction conditions. Pre-treatment reaction conditions

Glucan conversion

Xylan conversion

Time (h)

yield (% w∙w )

yield (% w∙w )

yield (% w∙w-1)

110

2

55.3±2.3

40.3±3.1

49.7±2.6

140

2

81.9±2.4

68.9±3.3

77.8±2.6

170

1

99.8±1.5

80.3±2.8

94.5±1.8

170

2

99.3±1.7

80.9±3.8

94.4±2.2

170

4

92.8±1.3

67.3±1.6

84.7±1.4

34.3±2.1a

12.9±1.9b

27.1±2.1b

a

data taken from literature23 for 96h of enzymatic hydrolysis;

-1

Total reducing sugars

Temperature (ºC)

Native wheat straw

-1

b

unpublished results for 96h of enzymatic

hydrolysis.

The results of enzymatic hydrolysis obtained for native wheat straw showed a maximum glucose yield of 34.3%, and 27.1% of total reducing sugars yield. As expected, the complex and intricate structure of wheat straw is a common obstacle challenging the access of enzymes to cellulosic substrates, resulting normally in low hydrolysis efficiency.39 Analysing the samples produced in the pre-treatment processes, it can be observed that the glucose yield increases with the pre-treatment temperature, confirming the potential of extensive enzymatic digestibility of cellulose materials. The glucose yield achieved a maximum of 55.3, 81.9 and 99.3% at 110, 140 and 170 ºC, respectively, after 72 h enzymatic hydrolysis of recovered cellulose materials produced in 2 h reaction. The biomass fractionation with imidazole (at 110 ºC) and the increase in process temperature to 170 ºC, allowed achieving an increase in glucose yield by 75% and 213%, respectively, in comparison to native biomass. This proves that the pre-treatment with imidazole itself and the reaction temperature play important roles in improving the enzymatic hydrolysis efficiency. Lignin depolymerisation Due to the alkaline character of imidazole and because of the difficulty in lignin recovery, the possibility of lignin depolymerisation was scrutinised. Figure 6 shows an electropherogram


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recorded at 320 nm for the phenolic profile of the recovered liquid fraction after processing at 170 ⁰C during 2h. As previously reported in literature, vanillin, vanillic and rosmarinic acids are examples of lignin derived compounds found in the obtained liquid streams.40-41

Figure 6. Electropherogram recorded at 320 nm showing the CE separations of methanolic SPE fraction. Matching percentages with authentic standards are indicated.

Other unidentified lignin-derived compounds with characteristic phenolic spectra42 were also found. As a proof of concept the same sample analysed by CE was subject to HPLC-MS2 analysis. The total ionic chromatogram recorded in the ESI negative ion mode showed several signals indicating the presence of specific deprotonated oligomers, and the structural elucidation of the most abundant species was obtained by CID-MS/MS experiments on the selected precursor ions using a quadrupole ion trap (Figure 7). The TIC displayed a group of peaks at retention time 18.5, 17.5 and 15.6 min yielding deprotonated molecules with m/z 339, 325 and 311, respectively, whose product ion mass spectra afford similar fragmentation patterns. The main fragments are similar to those reported by Banoub et al.,43 who have shown that the presence of an ion at m/z 183 confirms the presence of the coniferylglycerol



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or guaiacylglycerol unit. Based on these results, the anions m/z 339, 325 and 311 were attributed to the species [C20H19O5]-, [C19H17O5]- and [C18H15O5]-, respectively. Tentatively molecular structures formed from lignin structural unit combinations43 are also depicted in Figure 7. The signal at 9.2 min displayed a deprotonated molecule with m/z 507 and dimeric species with m/z 1015. The precursor m/z 507 was isolated and the MS2 mass spectrum recorded (Figure 7e). Loss of a radical methyl (●CH3), elimination of formaldehyde (CH2O) and a CO2 molecule lead to product ions at m/z 492, 477 and 463, respectively. Elimination of 166 Da leading to m/z 341, and a fragment with m/z 163 indicate the presence of a coniferyl unit. Based on these results it was proposed that species m/z 507 can be attributed to a deprotonated trimer [C28H27O9]- containing the (8→5´)-(3´-methoxyl coumaryl) unit. 43-44 Signals found at 7.5 and 7.9 min gave deprotonated molecules with m/z 383 and 353, respectively. Both deprotonated molecules lose a methyl radical, to produce the product ion

m/z 368 and 338, respectively. The precursors may also eliminate formaldehyde or two molecules of formaldehyde to give products at m/z 323 and 393, and m/z 353 and 323, respectively. Both deprotonated molecules follow similar fragmentation behavior indicating analogous structures.


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Figure 7. HPLC-MS/MS analysis of a sample of lignocellulosic biomass degradation products: a) Total ion chromatogram obtained in the ESI negative mode. Extracted ion chromatogram, MS2 spectrum and proposed structure for the precursor ion b) m/z 339; c) m/z 325; d) m/z 311; e) m/z 507. f) m/z 383 and g) m/z 353.

For a detailed characterization of the deprotonated molecule m/z 353, low-energy collisioninduced dissociation tandem mass spectrometric analysis were performed with a QqTOF mass spectrometry, which enabled to provide dissociation patterns and accurate molecular structures for the precursor and its fragment ions. The high resolution tandem mass



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spectrum and the tentative fragmentation routes of the product ion scan are shown in Figure 1 and Scheme 1 (both in SI). The accurate mass measurements (Table S4 of SI) enabled to propose a molecular formula of [C20H17O6]- for the deprotonated molecule m/z 353, that fits the proposed structures of the fragmentation patterns obtaining by ESIMS/MS. Based on this result, for the above mentioned deprotonated molecule with m/z 383 a molecular structure of [C21H19O7]- can be proposed and it is in accordance with published data.43 However, an arrangement of monolignol units different from those proposed by Banoub et al. was considering in order to support the observed fragmentation patterns obtained in the product ion scan of m/z 383 (Figure 7f).

Imidazole recovery Imidazole is a high-boiling compound (530.2 K)45 and even at reduced pressure (0.1 bar) the evaporation of imidazole is negligible, thus, the recovery of pre-treatment solvent was performed according to the method developed for IL presented elsewhere.9 After the trial of lignin precipitation was examined using NMR. Samples of pure imidazole and imidazole obtained directly after lignin removal (before addition of NaOH) were analysed using 1H and 13

C NMR to verify the efficiency of the employed imidazole recovery step. Figure 8 presents

spectra of both samples. The only difference is the shift observed in imidazole samples obtained after the lignin removal trial. This was caused by the pH of the solution, as at acidic pH, imidazole is deprotonated, and that is observed on 1H and 13C NMR spectra.46


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Figure 8. 1H (a and b) and 13C (c and d) NMR spectra of pure imidazole (a and c) and sample of imidazole after lignin removal (b and d). Conclusions Imidazole showed to be a successful solvent for biomass processing. It was able to selectively separate wheat straw components into cellulose- and hemicellulose-rich materials, without noteworthy degradation of polysaccharides. Additionally, imidazole causes the delignification of the recovered materials, enhancing the purity of these fractions. Biomass fractionation with imidazole promotes lignin removal, favours structural and morphological changes and enhances the performance of enzymatic digestibility of the produced cellulose-rich samples. Additionally, lignin-derived value-added phenolic compounds (e.g. vanillin) make the employment of imidazole an attractive and environmentally benign alternative for biomass processing, within the green biorefinery concept. Supporting Information



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Supporting Information (SI) contains the experimental section and additional information regarding the composition and crystallinity of recovered fractions. Acknowledgments This work was supported by the Fundação para a Ciência e Tecnologia (FCT, Portugal) through the Bilateral Cooperation project FCT/CAPES 2014/2015 (FCT/1909/27/2/2014/S), strategic

projects

UID/CTM/50025/2013,

UId/QUI/5006/2013,

and

grants

SFRH/BD/94297/2013 (ARCM) and IF/00424/2013 (RBL). The NMR spectrometers are part of The National NMR Facility, supported by Fundação para a Ciência e a Tecnologia (RECI/BBBBQB/0230/2012). The work was also supported by CAPES (Brazil) through the Pesquisador Visitante Especial 155/2012 project. The work presented in this paper was carried out in the frame of the EUBis COST Action TD1203 Food Waste Valorisation for Sustainable Chemicals, Materials and Fuels.


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For Table of Contents Use Only

Imidazole – prospect solvent for lignocellulosic biomass fractionation and delignification Ana Rita C. Morais, Joana Vaz Pinto, Daniela Nunes, Luísa B. Roseiro, Maria Conceição Oliveira, Elvira Fortunato and Rafał Bogel-Łukasik

Synopsis Imidazole as a novel solvent with “green” features for one-pot fractionation, delignification and lignin depolymerisation of lignocellulosic biomass


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cellulose HN N

lignin hemicellulose


Imidazole: Prospect Solvent for Lignocellulosic Biomass Fractionation

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