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Assessment of a protic ionic liquid with respect to fractionation and changes in the structural features of hardwood and softwood
Bioresource Technology Reports 8 (2019) 100334
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Assessment of a protic ionic liquid with respect to fractionation and changes in the structural features of hardwood and softwood
T
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Isık Semercia, , Fatma Gülerb,c, Gulsah Ersana, Kaan Soysala, Onur Ozturka, Hasan Altinisika, Sena Tirpana, Filiz Ozcelikb a
Department of Energy Engineering, Ankara University, 06830 Golbasi, Ankara, Turkey Department of Food Engineering, Ankara University, 06830 Golbasi, Ankara, Turkey c Department of Food Engineering, Bulent Ecevit University, 67900 Caycuma, Zonguldak, Turkey b
A R T I C LE I N FO
A B S T R A C T
Keywords: Lignocellulosic biomass Hornbeam Pine Lignin Pretreatment Protic ionic liquid
In this work, the potential of protic ionic liquid, triethylammonium hydrogen sulfate (TEAHSO4) was shown regarding the pretreatment of the hardwood, hornbeam and softwood, pine at different temperatures and times. TEAHSO4 was effective for lignin extraction and enhancing the enzymatic digestibility of both the biomass. Cellulose content of hornbeam increased to 85% with 87% lignin extraction from the biomass subjected to pretreatment at 150 °C for 3 h which was then readily converted into glucose with 97% yield. On the other hand, pine as a softwood demonstrated more resilience to deconstruction; 76% glucose yield was achieved following the enzymatic hydrolysis of biomass pretreated at 150 °C for 30 min. As put forward in the present work through TEAHSO4 pretreatment, any lignocellulosic feedstock that can be effectively disassembled into cellulose and lignin should be regarded as high value for the production of liquid and gaseous fuels through thermochemical and biochemical routes.
1. Introduction Lignocellulosic biomass as a smart and carbon-neutral resource has a great promise to replace fossil fuels for the future generations. Based on the statistics of International Energy Agency (IEA), bioenergy contributed almost half of the renewable energy with almost 460 MToe in 2017 excluding conventional use of biomass. Besides, its share among other renewable resources is estimated to increase 76 MToe from 2017 to 2023 (IEA Renewables). One of the key criteria that can keep the success of biomass in renewables is to choose the right strategy that makes use of each biomass component effectively. Different strategies can be manifested as thermochemical routes such as direct combustion, gasification and pyrolysis or biological routes involving hydrolysis and fermentation for the valorization of biomass. Today, biorefineries that process biomass into several product streams, fuels, chemicals and materials aim to maximize energy output from lignocellulosic feedstocks. Generation of less waste and reduction of carbon footprint are the other important components integrated into the biorefinery concept. For this reason, implementing a biomass processing strategy that generates the least amount of waste and thereby, enables full biomass use with the highest value return is significant. Processing lignocellulosic biomass with ionic liquids (ILs) has made ⁎
Corresponding author. E-mail address: [email protected] (I. Semerci).
https://doi.org/10.1016/j.biteb.2019.100334 Received 7 October 2019; Accepted 7 October 2019 Available online 23 October 2019 2589-014X/ © 2019 Elsevier Ltd. All rights reserved.
a significant progress since the last decade. Major properties that make ILs more favorable than conventional solvents are their low or negligible vapors and tunable characteristics along with their stability and recyclability. Studies launching with cellulose dissolution in ILs (Swatloski et al., 2002) was followed by biomass pretreatment with ILs that targeted either cellulose decrystallization or lignin removal (Tan et al., 2009; Wu et al., 2011). Meanwhile, successful interaction of ILs with a variety of lignocellulosic feedstocks also revealed the potential of dialkylimidazolium ILs such as 1-ethyl-3-methylimidazolium acetate (EMIMAc) (Sun et al., 2009) and 1-butyl-3 methylimidazolium chloride (BMIMCl) (Xie et al., 2018) in this field. In the last decade, diakylimidazolium ILs also provided reaction media for the production of platform chemicals. Transformation of cellulose to glucose in SO3H-functionalized ILs (Liu et al., 2018; Tao et al., 2011) and conversion of monomeric sugars to 5-HMF, furfural and levulinic acid in ILs have been recent advancements (Eminov et al., 2016; Liu et al., 2018). Despite their steady performance towards different biomass derived components, synthesis of dialkylimidazolium ILs consists deliberate and time-consuming processes that require costly starting chemicals. This creates an impediment for commercial scale-up of IL processing of biomass. Evaluation of lignocellulose interaction with protic ILs (PILs) as
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pine pretreatment. Better findings were found after DMBAHSO4 and HBIMHSO4 pretreatments; more than 70% of saccharification and lignin recovery yields were obtained when pine was pretreated with DMBAHSO4 at 10% biomass loading and 170 °C for 30 min (Gschwend et al., 2019). Herein, we report the comparison of hardwood and softwood species, hornbeam and pine with respect to the structural and compositional changes in the biomass obtained upon TEAHSO4 pretreatment and the potential of the species to be effectively converted into glucose, as well. Effects of pretreatment temperature and time were investigated since the severity of these parameters is the major determinant of the recovery of the components. The recovered solids were characterized with respect to their chemical compositions which were supported by SEM and XRD analysis, as well. On the other hand, XPS, 1H NMR and TGA were employed to provide the structural features of the lignin precipitates.
cheaper and more conveniently synthesized alternatives may render the process scale-up. As initiated and centered by Hallett group at Imperial College London under the name “IonoSolv Process” (Gschwend et al., 2016), referring to lignin and hemicellulose removal from biomass while keeping cellulose intact in the structure, utilization of PILs for biomass pretreatment paved the way for process commercialization. In the recent years, different feedstocks have been subjected to pretreatment with PILs. These comprise bagasse from different sources (Reis et al., 2017; Rocha et al., 2017; Uju et al., 2013), energy crops (Verdía et al., 2014), residues such as cotton stalks (Semerci and Güler, 2018) and pine apple crown (de Miranda et al., 2018), softwoods such as willow (Gschwend et al., 2019; Weigand et al., 2017) and pine (Gschwend et al., 2019). All the mentioned feedstocks pretreated with PILs revealed a common outcome; lignin contents decreased while maintaining a majority of cellulose. Besides, weak carbohydrate-lignin interactions (such as complexes involving ferulic acid) (Brandt et al., 2012) were observed along with broken β-O-4 ether and CeC bonds. In this study, we introduced the potential of PIL, triethylammonium hydrogen sulfate (TEAHSO4) to deconstruct hardwood and softwood species, hornbeam and pine, respectively and present their transformations from structural and product perspectives. The clearest structural distinction between hardwoods and softwoods is the structure of lignin. Softwoods such as pine, cedar and spruce have been recognized to be dominated by G lignin units being harder to be digested during pretreatment in contrast to S-units that are mainly found in the hardwoods such as beech, oak and hornbeam (Li et al., 2016). Besides, hardwood lignins which contain less CeC linkages are less resistant to deconstruction. So, the general conclusion is that biomass deconstruction is a more challenging task in softwoods than in hardwoods (Zhang et al., 2016a). Hornbeam (Carpinus betulus) with cellulose content between 30 and 50% has been explored in terms of its pretreatment through a few traditional techniques such steam explosion (Barbanera et al., 2018). Recently, hornbeam pretreated with aprotic IL, 1-butyl-3-methylimidazolium chloride (BMIMCl) was subjected to simultaneous saccharification and fermentation (SSF). SSF in the presence of R. oryzae, two products were mainly obtained, ethanol (4.7 g/L) and fumaric acid (1.7 g/L) along with 5-fold increase in the glucose yield (Dotsenko et al., 2018). Unlike hornbeam, softwood species pine (Pinus pinaster) has been much extensively studied due being an energy dense, widespread and inexpensive resource. Although it has been mainly processed through conventional techniques, autohydrolysis (Rigual et al., 2018), hydrothermal (Nitsos et al., 2016), and also, acid and SO2 catalyzed steam pretreatments (Huang and Ragauskas, 2012) with the aim of second-generation ethanol production, pine pretreatment in the presence of different aprotic and PILs gave promising findings (L. Zhang et al., 2016a). Considering the expense and elaborate synthesis of aprotic ILs including the pioneer IL, EMIMAc, PILs appealed to many lignocellulosic feedstocks for being cheaper, their simple synthesis and potential towards lignin removal from biomass (George et al., 2014). PILs can remove large portions of lignin from biomass and several mechanisms have been introduced for this interaction. In addition to hydrogen bonding which has been proposed in a few studies, ionic attractions via electrostatic/coulombic forces were also reported to be responsible for the favor of lignin dissolution process (Achinivu, 2018). PIL, 1-methylimidazolium chloride (HMIMCl) successfully converted pine into lignin rich precipitate, liquid fraction containing hemicellulose fragments, and cellulose enriched solid fraction which readily hydrolyzed into glucose (Cox and Ekerdt, 2013). Hallett and co-workers demonstrated the potential of TEAHSO4 for the deconstruction of pine and Miscanthus. Pretreatment of pine at 10% (w/w) biomass loading and 120 °C for 8 h resulted in 5% lignin precipitate and 13% glucose yield (Gschwend et al., 2016). Recently, the same group offered PILs, 1-butylimidazolium hydrogen sulfate (HBIMHSO4) and N,N-dimethylbutylammonium hydrogen sulfate (DMBAHSO4) in addition to TEAHSO4 for
2. Materials and methods 2.1. Materials Hornbeam and pine sawdust, which were obtained from a saw mill located in Salihli district of Manisa, Turkey, were sieved to a particle size less than 1 cm. The particles were air dried and stored at room temperature. Ethanol, trisodium citrate dihydrate, citric acid monohydrate, sulfuric acid, calcium carbonate, D-glucose and D-xylose were purchased from Merck (Darmstadt, Germany). Triethylamine and sulfuric acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). The cellulase enzyme, Cellic Ctec2 was kindly provided by Novozymes (Denmark). 2.2. Pretreatment of biomass IL, triethylammonium hydrogen sulfate was synthesized through an acid-base reaction using sulfuric acid and triethylamine according a previously reported procedure (Semerci and Güler, 2018). IL was characterized by 1H NMR (Supplementary material). Before analysis being conducted with Bruker 300 MHz spectrometer, IL sample was dissolved in deuterated dimethyl sulfoxide. The chemical shifts are as follows: TEAHSO4 (300 MHz, DMSO‑d6): δ 3.03 (q, 6H, NCH2CH3), 1.10 (t, 9H, NCH2). Biomass pretreatments were conducted with aqueous solutions of triethylammonium hydrogen sulfate in a similar fashion reported in the Ionosolv protocol (Gschwend et al., 2016). All pretreatments were conducted in 50 mL Pyrex tubes with teflon-lined screw caps. 1 g of airdried biomass was pretreated with 10 g aqueous solution having 4:1 IL:H2O weight ratio. The pretreatment temperature was held at 120, 150 and 170 °C and the pretreatment was performed between 30 min and 8 h. After the pretreatments, the samples were cooled to room temperature and ethanol was added to the pretreated slurries. Solid fraction was washed with ethanol three times for 15 min in a shaker to remove the residual PIL. Finally, the pretreated biomass was recovered by filtration and dried at 70 °C under vacuum for 24 h before it was subjected to enzymatic hydrolysis and characterization. All pretreatments were performed in duplicate. The recovered PIL was used to precipitate lignin through ethanol evaporation in rotary evaporator and water addition. The precipitated lignin was washed with water three times and centrifuged at 6000 rpm for 20 min each time. Finally, the recovered material was dried at 70 °C under vacuum for 24 h before it was subjected to characterization. Percentage solid recovery, SR (%), lignin extracted, LE (%) and lignin precipitate yield, LPY(%) were determined according to the following equations:
SR (%) = (WPRT / WUT ) × 100
(1)
where WPRT is the weight of biomass recovered after pretreatment (g) 2
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Table 1 Chemical compositions of hornbeam (HB) and pine (P) before pretreatment and after pretreatment under certain conditions. Pretreatment conditions
SR (%)
Cellulose (%)
Hemicellulose (%)
Lignin (%)
LE (%)
LPY (%)
UHB UP PHB-170 °C-3 h PHB-170 °C-4 h PP-170 °C-3 h PP-170 °C-4 h PHB-150 °C-3 h PHB-150 °C-4 h PP-150 °C-3 h PP-150 °C-4 h PHB-120 °C-6 h PHB-120 °C-8 h PP-120 °C-6 h PP-120 °C-8 h
– – 39.3 37.3 48.1 47.4 40.3 43.0 58.8 57.8 44.1 45.6 72.7 67.3
34.2 38.5 76.3 70.2 69.5 64.7 84.5 79.8 56.3 59.1 76.6 75.4 42.3 48.4
17.0 ± 1.7 22.6 ± 0.6 0 0 0 0 0 0 11.2 ± 0.1 10.0 ± 0.1 0 0 8.4 ± 1.4 7.2 ± 1.1
26.3 ± 0.0 28.9 ± 0.6 7.8 ± 0.3 7.4 ± 1.3 26.4 ± 0.1 31.5 ± 0.6 8.5 ± 0.2 11.8 ± 2.0 24.1 ± 1.4 25.7 ± 0.1 16.9 ± 1.7 18.0 ± 2.2 29.3 ± 0.2 29.3 ± 0.5
– – 88.3 89.5 51.8 43.2 86.9 80.7 46.1 41.5 71.6 68.8 19.0 25.0
– – 72.7 61.2 58.3 55.7 78.0 71.7 27.0 35.9 19.4 36.5 14.2 15.8
± ± ± ± ± ± ± ± ± ± ± ±
0.2 1.0 0.9 1.6 0.7 0.2 1.8 0.0 2.6 0.5 0.3 0.8
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.6 3.2 1.0 3.1 0.0 0.2 3.5 5.8 4.2 0.4 4.8 1.5 6.6 2.1
(3)
where WP is the weight of the precipitate obtained after IL pretreatment and WUL is the weight of lignin in the untreated biomass subjected to pretreatment.
CrI = [(I002 − IAM )/ I002] × 100 2.3. Enzymatic hydrolysis
3.1 4.0 1.6 0.3 7.9 1.4 2.0 2.4 0.7 7.0 2.5 0.1
(5)
where I002 is the height of the crystalline peak at around 2θ=22.5° and IAM is the height of the amorphous reflection at around 2θ= 18°. X-ray photoelectron spectroscopy (XPS) analysis of lignin samples was performed in a PHI 5000 VersaProbe spectrometer using a monochromatic Al Kα X-ray source. C1s spectra at the binding energy of 285 eV were used as a reference. For proton nuclear magnetic resonance (1H NMR) spectroscopy analysis, lignin was dissolved in deuterated dimethyl sulfoxide and the data was recorded with a Bruker 300 MHz spectrometer. Thermogravimetric analysis (TGA) was carried out in a STD650 Simultaneous DSC/TGA instrument by heating the samples up to 800 °C (heating rate: 10 °C min−1) in a nitrogen atmosphere to monitor the thermal decomposition of lignin samples.
Untreated and pretreated biomass samples at 3% (w/v) and Cellic Ctec2 (210 FPU/ml) at 2% (v/v) were loaded in 0.05 M sodium citrate buffer at pH 4.8. The samples were kept at 50 °C in a shaker incubator for 48 h. Enzymatic reaction was stopped by keeping the samples for 5 min in boiling water. Later, the samples were centrifuged at 6000 rpm for 20 min and the supernatants were filtered through 0.20 μm PTFE membrane filter before their analysis for glucose concentration. Prominence LC-20A Modular HPLC System equipped with refractive index (RI) detector and Transgenomic Carbosep Coregel 87H3 column was operated at 55 °C with 5 mM H2SO4 mobile phase at a flow rate of 0.5 mL/min. The sample volume injected was 20 μL and the retention time was 20 min. Percentage glucose yield, GY(%) was calculated on the basis of the cellulose content of the untreated hornbeam and pine samples according the following formula:
GY (%) = (SR (%) × CG )/(CB × CelluloseU (%) × 1.11) × 100
± ± ± ± ± ± ± ± ± ± ± ±
Scanning electron microscopy (SEM) images were obtained using a QUANTA 400 F Field Emission SEM operated at 20 kV. All samples were sputter coated with gold/palladium (Au/Pd) prior to analysis. SEM images of the samples were taken at different magnifications (250×, 500×, 1000× and 2000×). X-ray diffraction (XRD) analysis of the biomass samples was conducted with Rigaku Ultima-IV Diffractometer between 2θ= 10° and 40° Bragg angles using Cu radiation at a scanning speed of 1°/min. Percentage crystallinity indices (CrI) of biomass were determined based on the peak height method (Park et al., 2010) and calculated according to the following formula;
(2)
where LUT (%) is the lignin content of untreated biomass, SR (%) is the solid recovery after pretreatments and LPRT (%) is the lignin content of the biomass subjected to pretreatment (Haykir et al., 2013).
LPY (%) = [WP / WUL] × 100
0.4 1.9 0.7 1.1 0.1 3.3 1.8 0.3 2.9 4.5 0.1 0.4
2.5. Characterization of biomass
and WUT is the weight of untreated biomass subjected to pretreatment (g).
LE (%) = [(LUT (%) − (SR (%) × LPRT (%)/100))/ LUT (%)] × 100
± ± ± ± ± ± ± ± ± ± ± ±
3. Results and discussion 3.1. Effect of pretreatment on the composition of biomass
(4)
Compositional analysis of biomass is significant since it determines the value of lignocellulosic feedstocks and the possibility of effective valorization of components to fuels, chemicals and materials. Table 1 and Supplementary material showed the composition of untreated biomass samples and solid recovery, composition, lignin extracted and the lignin precipitate yields for the biomass samples pretreated under certain conditions. As shown, lower solid recoveries (37%–95%) obtained for hornbeam compared to pine (47%–95%) under the same conditions indicating the effective deconstruction capacity of IL on the hardwood, hornbeam. As pretreatment temperature decreased, higher solid recoveries were obtained due to the reduction in the process severity. These results were also in correlation with the percentage lignin and hemicellulose removed from the biomass; TEAHSO4 readily removed hemicellulose from hornbeam in all conditions and extracted more than 88% of the lignin from the structure with the pretreatment at
where SR (%) is the percentage solid recovery, CG is the glucose concentration in the hydrolysates (g/L), CB is the initial concentration of the biomass in the hydrolysis buffer that is subjected to enzymatic hydrolysis (g/L) and CelluloseU (%) is the cellulose content of the untreated biomass. 1.11 is the stoichiometric conversion factor of cellulose to equivalent glucose (Haykir and Bakir, 2013).
2.4. Composition analysis Composition analysis of untreated and pretreated samples was performed according two step acid hydrolysis reported by NREL (Sluiter et al., 2008). Sugar content of the hydrolysates was measured with HPLC using the same conditions as given for the analysis of hydrolysates obtained upon enzymatic hydrolysis. 3
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pretreatment at 150 °C provided the intact structure of cellulose in addition to lignin and hemicellulose removal. Thereby, 97% of glucose yield was obtained being 2-fold higher than the glucose yield of untreated hornbeam (46%). IL pretreatment showed lower capability regarding the enzymatic digestibility of softwood, pine compared to hardwood, hornbeam which was observed in a similar fashion in the previously reported studies for softwood and hardwood feedstocks subjected to different pretreatments including IL pretreatment (Asada et al., 2015; Nitsos et al., 2016; Rigual et al., 2018; X. Zhang et al., 2016b). The degree of lignin and hemicellulose removal and the preservation of cellulose content of the structure were the major determinants of the glucose yields based on the cellulose content of untreated biomass. For pine, the highest glucose yield was found as 76% for pine pretreated at 150 °C for 30 min (Supplementary material). Despite this 1.8-fold increase in glucose yield based on the enzymatic digestibility of untreated pine (42% glucose yield), pretreatment at 150 °C for 30 min was not effective for lignin extraction; approximately 15% lignin removal was observed. Currently, aqueous solutions sulfate-based PILs (with 20 wt% water), TEAHSO4, DMBAHSO4 and HBIMHSO4 were assessed for pine pretreatment at 170 °C for 30 min; promising findings introduced DMBAHSO4 as an ideal candidate for softwoods (Gschwend et al., 2019). Ultra-low cost DMBAHSO4 not only led to satisfactory lignin recovery and hydrolysis yields (70%) but also dominated for modifying the structure of lignin to a dramatic level through effective β-O-4 cleavage. Above all, HBIMHSO4 employed at biomass loadings as high as 50% was still effective in pine deconstruction; lignin recovery and saccharification yields were around 30% and 40%, respectively. While TEAHSO4 pretreatment conducted under identical pretreatment conditions gave 16% lignin extraction in this study (Supplementary material), 30% lignin recovery yield was achieved for pine in the aforementioned study. Additionally, pretreatment of pine with cholinium arginate at 90 °C for 12 h under nitrogen with a biomass/IL ratio of 1/ 15 (w/w) and stirring gave 33% lignin extraction and 31% glucose yield (An et al., 2015). Another PIL, 1-ethylimidazolium chloride (EimCl), which dissolved biomass completely under the following conditions; 2 wt% loading, 110 °C, 18 h, resulted in at least 75% glucose yield for the recovered pine (Hossain et al., 2019).
170 °C for 3 h. Along with lignin and hemicellulose removal, cellulose content of hornbeam increased more than 2-fold from 34% to 84% when pretreated at 150 °C for 3 h. This implies effective conversion of hornbeam samples to a cellulose rich solid fraction with minimal cellulose degradation. Pretreatment at 170 °C for 3 h was also effective for pine; more than 50% lignin was extracted and cellulose content of the biomass increased up to almost 70%. In a previous study, lignin removal for pine pretreated using EMIMAc and EMIMAc-dimethyl sulfoxide (DMSO) solutions at 110 °C for 3 h was found less than 3% (X. Zhang et al., 2016b). In comparison to eucalyptus and switchgrass, pine was found the most resistant to EMIMAc pretreatment at 160 °C for 3 h; lignin removal efficiencies were 49%, 55% and 69% for pine, eucalyptus and switchgrass, respectively (Li et al., 2013). Additionally, food-derived imidazolium acesulfamate IL, 1-butyl-3-methylimidazolium acesulfamate (BMIMAce) selectively extracted lignin from pine with an extraction efficiency of 38% (Pinkert et al., 2011). 3.2. Effect of pretreatment on the digestibility of biomass After pretreatments, pretreated biomass was subjected to enzymatic hydrolysis for 48 h. Glucose yields (%) were given in Fig. 1 and Supplementary material which showed the effect of pretreatment temperature and time on the conversion of the cellulose in both the untreated hornbeam and untreated pine to glucose. The highest glucose yield was roughly 97% which was attained for hornbeam pretreated at 150 °C for 3 h. Pretreatment of hornbeam at 170 °C also resulted in sufficient glucose yields; almost 70% and 85% of cellulose in the untreated biomass was converted to glucose after pretreatments conducted for 3 and 4 h, respectively. These finding were also found consistent with the composition of the biomass. Pretreatment of hornbeam at 170 °C for 3 h and 4 h removed approximately 90% of the lignin and completely the hemicellulose from the structure. Effective transformation of cellulose to glucose was anticipated since lignin has been recognized to set a critical barrier to the enzymatic hydrolysis of biomass (Li et al., 2016). However, due to moderate cellulose degradation, glucose yields were lower than 90% for the other horn- beam samples pretreated at 170 °C. As the degree of cellulose recovered gets lower through degradation at higher temperatures, one gets far away from one of the major goals of PIL pretreatment which is preservation of cellulose. Unlike 170 °C,
100 90
Glucose yield (%)
80 70 60 50 40 30 20 10 0
Fig. 1. Glucose yields obtained at the 48th hour of enzymatic hydrolysis of untreated biomass and biomass subjected to TEAHSO4 pretreatments. 4
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3.3. Effect of pretreatment on the structure of recovered solids Recovered solids were evaluated regarding their morphology and biomass crystallinity using SEM and XRD, respectively. Images taken at magnifications 250×, 500×, 1000× and 2000× were shown in Supplementary material. According to the images, untreated species, both the untreated hornbeam and pine, exhibited intact and rigid structures. However, these structures apparently opened up after TEAHSO4 pretreatment, especially hornbeam pretreated at 170 °C and 150 °C for 3 h. Thin fibrillar structures revealed indicated the cellulose rich biomass and the removal of lignin (at least 87% lignin removed from hornbeam) and hemicellulose completely as correlated with the chemical composition of pretreated hornbeam. Unlike hornbeam, coarse fibrillar structures were observed for pine which was in good agreement with the impact of TEAHSO4 on pine deconstruction; lignin was removed at lower levels (only up to 52%). Interestingly, pine pretreated at 150 °C demonstrated curly macro bundles. For hornbeam pretreated at 120° for 8 h, neat fibrillar structures were still present except for being a little thicker. Lastly, modest interaction of pine with TEAHSO4 at 120° for 8 h did not seem to serve the purpose; morphological similarities were found between the untreated and pretreated biomass. The visualization of the morphology with SEM properly supported the compositional changes in the feedstocks. X-ray diffraction was used to evaluate the percentage crystallinity indices (CrI) of untreated and pretreated samples and observe if there would be a relation between the crystalline structure and digestibility of biomass. As revealed in Fig. 2a and b, crystalline structure of the hornbeam and pine, respectively was represented with three diffraction peaks at around 16.5°, 22.5°, and 35°. For both the biomass, extent of the peaks increased for the pretreated samples compared to untreated samples. Moreover, the peaks in general got more intense and particularly the peak at around 22.5° got narrower as the pretreatment temperature increased. Accordingly, an increase in the CrI (%) of the samples with pretreatment severity was observed (Table 2). CrI (%) of the samples increased due to the effective removal of amorphous fractions of the structure with IL. Regardless of this increase in biomass crystallinity, enhanced glucose yields were obtained (Fig. 1). With respect to these points, interaction of IL with the lignin and hemicellulose dominated over the changes in biomass crystallinity in biomass conversion to glucose. As similarly stated for different lignocellulosic feedstocks (Achinivu et al., 2013; George et al., 2014; Reis et al., 2017), increased crystallinity of the hornbeam did not act adversely on the biomass digestibility. Fig. 2. XRD patterns of hornbeam (a) and pine (a) samples before and after pretreatments with TEAHSO4 under certain pretreatment conditions.
3.4. Effect of pretreatment on the structure of lignin precipitates Structures of lignin precipitates recovered from TEAHSO4 solutions after pretreatment were analyzed through XPS, 1H NMR, and TGA in order to show the effect of pretreatment temperature on the lignin chemical structure. Lignin precipitate yields (%) were given in Table 1. Lignin precipitate yields (%) were found in accordance with the lignin extraction capability of TEAHSO4 at different pretreatment temperatures and times. The highest lignin precipitate yield (%) was obtained from hornbeam pretreated at 150 °C and for 3 h as 78%. The aforementioned precipitate was compared with the one obtained at 170 °C and for 3 h (73% lignin precipitate yield) structurally. XPS is a useful method to evaluate the surface chemical composition of materials (based on the atomic percentage). Table 3 showed the percentage carbon distribution on the material surface which is obtained by high resolution spectra (Fig. 3). Table 3 showed that there is a decrease in the aliphatic and aromatic carbon bonding as represented by the peak C1 with an increase in temperature from 150 °C to 170 °C. This indicated the effective depolymerization of lignin through disruption of CeC linkages in lignin through protonation by TEAHSO4. The increase in the percentage of C2 peaks from 150 °C to 170 °C implied the preservation of the phenolic hydroxyl, aliphatic hydroxyl and
Table 2 CrI indices of untreated and TEAHSO4 pretreated biomass. Pretreatment conditions
CrI (%)
PHB-170 °C-3 h PP-170 °C-3 h PHB-150 °C-3 h PP-150 °C-3 h PHB-120 °C-8 h PP-120 °C-8 h UHB UP
79 77 76 73 70 71 67 63
ether bonds (β-O-4 bonds). Hardwood lignin was reported to contain 60–62 units of β-O-4 linkages per 100C9 units as the most dominant structure (Ragauskas and Yoo, 2018). Based on the C2 percentages shown in Table 3, these linkages apparently reduced with IL pretreatment. Besides, the slight decrease in the percentages of C3 and C4 represented the disruption of carbonyl and carboxyl groups in lignin,
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showed the disruption of aliphatic carbon bonding. Moreover, the intensity of the signal at around 3.1, signifying the proton in β-1 bond (another CeC bond), decreased with an increase in pretreatment temperature. The chemical shift of the protons in methoxy groups was located at around 3.7 ppm. The intensity of this peak was lower for the lignin obtained during TEAHSO4 pretreatment at 170 °C for 3 h implying the removal of methoxy groups from the structure of lignin. The signals at around 6.7 ppm and 8.8 ppm were assigned to the aromatic region of lignin, aromatic protons in guaiacyl (G) and syringyl (S) units and phenolic protons, respectively. As shown in Fig. 4a, the intensities of these peaks slightly got higher by increasing the pretreatment temperature which was consistent with the percentage increase of C2 peak of XPS spectrum. Lastly, TGA provided the thermal stability of lignin samples extracted from hornbeam as shown in Fig. 5. Accordingly, thermal stability of lignin decreased slightly as the pretreatment temperature increased from 150 °C to 170 °C; the residual weight decreased from 43.8% to 42.3%. This was in accordance with the percentage decrease of C1 peak of XPS spectrum which assigned to the aliphatic CeC linkages.
Table 3 XPS binding energies and assigned carbon species percentages. Peak
C1 C2 C3 C4
Position BE (eV)
283–284 284–285 285.5–287 286–289
Bond type
C-C, C-H C-O, C-O-C O-C-O, C=O COO
Percentage of total carbon (%) PHB-170 °C-3 h
PHB-150 °C-3 h
33.8 57.9 6.1 2.2
40.6 48.3 8.1 3.0
respectively with an increase in pretreatment temperature (BañulsCiscar et al., 2016; Tian et al., 2015). The presence of functional groups, abundance of syringyl and guaiacyl units and the side chains in the lignin structure was also monitored via 1H NMR. As shown in Fig. 4, the major signals were detected at around 1.3 ppm, 3.1 ppm, 3.7 ppm, 6.7 ppm and 8.8 ppm. In order to correspond the signals to the correct assignments, a previously reported study was considered (Rashid et al., n.d.). Fig. 4a and b represent the spectra of lignin precipitates obtained after hornbeam pretreatments conducted at 170 °C and 150 °C for 3 h, respectively. The initial peak at around 1.3 ppm represented the protons in the aliphatic region which was weaker in the spectrum of the lignin obtained after biomass pretreatment at 170 °C for 3 h compared to the one at 150 °C for 3 h. This result was in correlation with XPS finding which also
4. Conclusions TEAHSO4 was found more effective towards the hardwood, hornbeam with respect to the changes in the composition and chemical
Fig. 3. XPS high resolution spectra of C1s region for lignin samples obtained from hornbeam pretreated with TEAHSO4 at 170 °C (a) and 150 °C (b) for 3 h. 6
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Fig. 4. 1H NMR spectra of lignin samples obtained from hornbeam pretreated with TEAHSO4 at 170 °C (a) and 150 °C (b) for 3 h.
structure of the lignocellulosic biomass and its conversion to glucose. Hornbeam was readily fractionated into cellulose rich solid and lignin precipitate when subjected to pretreatment at 150 °C for 3 h. Biorefineries are primarily dependent on getting the most value out of the major fractions of the lignocellulosic feedstocks. For this reason, acknowledging the PIL, TEAHSO4 as a suitable candidate for biomass
pretreatment should be emphasized as an important conclusion of this work. Declaration of competing interest The authors declare that they have no known competing financial 7
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within SSF. Bioresour. Technol. 250, 429–438. https://doi.org/10.1016/j.biortech. 2017.11.064. Eminov, S., Brandt, A., Wilton-Ely, J.D., Hallett, J.P., 2016. The highly selective and nearquantitative conversion of glucose to 5-hydroxymethylfurfural using ionic liquids. PLoS One 11, e0163835. https://doi.org/10.1371/journal.pone.0163835. George, A., Brandt, A., Tran, K., Zahari, S.M., Klein-Marcuschamer, D., Sun, N., Sathitsuksanoh, N., Shi, J., Stavila, V., Parthasarathi, R., Singh, S., Holmes, B.M., Welton, T., Simmons, B.A., Hallett, J.P., 2014. Design of low-cost ionic liquids for lignocellulosic biomass pretreatment. Green Chem. 17, 1728–1734. https://doi.org/ 10.1039/c4gc01208a. Gschwend, F.J., Brandt, A., Chambon, C.L., Tu, W.-C., Weigand, L., Hallett, J.P., 2016. Pretreatment of lignocellulosic biomass with low-cost ionic liquids. J. Vis Exp. Jove. https://doi.org/10.3791/54246. Gschwend, F.J., Chambon, C.L., Biedka, M., Brandt-Talbot, A., Fennell, P.S., Hallett, J.P., 2019. 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Fuel 206, 145–154. https://doi.org/10.1016/ j.fuel.2017.06.014. Semerci, I., Güler, F., 2018. Protic ionic liquids as effective agents for pretreatment of cotton stalks at high biomass loading. Ind. Crop. Prod. 125, 588–595. https://doi. org/10.1016/j.indcrop.2018.09.046. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2008. Determination of Sugars, Byproducts, and Degradation Products in Liquid Fraction Process Samples, Laboratory Analytical Procedure (LAP), Proce. Lab Anal Proced NREL/TP-510-42618. pp. 1–14. Sun, N., Rahman, M., Qin, Y., Maxim, M.L., Rodríguez, H., Rogers, R.D., 2009. Complete dissolution and partial delignification of wood in the ionic liquid 1-ethyl-3-methylimidazolium acetate. Green Chem. 11, 646–655. https://doi.org/10.1039/b822702k. Swatloski, R.P., Spear, S.K., Holbrey, J.D., Rogers, R.D., 2002. Dissolution of cellose with ionic liquids. J. Am. Chem. Soc. 124, 4974–4975. https://doi.org/10.1021/ ja025790m. 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Fig. 5. TGA spectra of lignin samples obtained from hornbeam pretreated with TEAHSO4 at 170 °C and 150 °C for 3 h.
interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors acknowledge the financial support from The Scientific and Technological Research Council of Turkey (TUBITAK) via project 116M444. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biteb.2019.100334. References Achinivu, E.C., 2018. Protic ionic liquids for lignin extraction—a lignin characterization study. Int. J. Mol. Sci. 19, 428. https://doi.org/10.3390/ijms19020428. Achinivu, E.C., Howard, R.M., Li, G., Gracz, H., Henderson, W.A., 2013. Lignin extraction from biomass with protic ionic liquids. Green Chem. 16, 1114–1119. https://doi.org/ 10.1039/c3gc42306a. An, Y.-X., Zong, M.-H., Wu, H., Li, N., 2015. Pretreatment of lignocellulosic biomass with renewable cholinium ionic liquids: biomass fractionation, enzymatic digestion and ionic liquid reuse. Bioresour. Technol. 192, 165–171. https://doi.org/10.1016/j. biortech.2015.05.064. Asada, C., Sasaki, C., Hirano, T., Nakamura, Y., 2015. Chemical characteristics and enzymatic saccharification of lignocellulosic biomass treated using high-temperature saturated steam: comparison of softwood and hardwood. Bioresour. Technol. 182, 245–250. https://doi.org/10.1016/j.biortech.2015.02.005. Bañuls-Ciscar, J., Abel, M.-L., Watts, J.F., 2016. Characterisation of cellulose and hardwood organosolv lignin reference materials by XPS. Surf. Sci. Spectra 23, 1–8. https://doi.org/10.1116/1.4943099. Barbanera, M., Lascaro, E., Foschini, D., Cotana, F., Buratti, C., 2018. Optimization of bioethanol production from steam exploded hornbeam wood (Ostrya carpinifolia) by enzymatic hydrolysis. Renew. Energy 124, 136–143. https://doi.org/10.1016/j. renene.2017.07.022. Brandt, A., Gräsvik, J., Hallett, J.P., Welton, T., 2012. Deconstruction of lignocellulosic biomass with ionic liquids. Green Chem. 15, 550–583. https://doi.org/10.1039/ c2gc36364j. Cox, B.J., Ekerdt, J.G., 2013. Pretreatment of yellow pine in an acidic ionic liquid: extraction of hemicellulose and lignin to facilitate enzymatic digestion. Bioresour. Technol. 134, 59–65. https://doi.org/10.1016/j.biortech.2013.01.081. de Miranda, R., Neta, J., Ferreir, L., Gomes, W., do Nascimento, C., de Gomes, E.B., Mattedi, S., Soares, C., Lima, Á.S., 2018. Pineapple crown delignification using lowcost ionic liquid based on ethanolamine and organic acids. Carbohydr. Polym. 206, 302–308. https://doi.org/10.1016/j.carbpol.2018.10.112. Dotsenko, A.S., Dotsenko, G.S., Senko, O.V., Stepanov, N.A., Lyagin, I.V., Efremenko, E.N., Gusakov, A.V., Zorov, I.N., Rubtsova, E.A., 2018. Complex effect of lignocellulosic biomass pretreatment with 1-butyl-3-methylimidazolium chloride ionic liquid on various aspects of ethanol and fumaric acid production by immobilized cells
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pretreatment. Faraday Discuss. 202, 331–349. https://doi.org/10.1039/c7fd00059f. Wu, H., Mora-Pale, M., Miao, J., Doherty, T.V., Linhardt, R.J., Dordick, J.S., 2011. Facile pretreatment of lignocellulosic biomass at high loadings in room temperature ionic liquids. Biotechnol. Bioeng. 108, 2865–2875. https://doi.org/10.1002/bit.23266. Xie, W., Zhou, D., Ren, Y., Tang, S., Kuang, M., Du, S., 2018. 1-Butyl-3-methylimidazolium chloride pretreatment of cotton stalk and structure characterization. Renew. Energy. https://doi.org/10.1016/j.renene.2018.03.011. Zhang, L., Pu, Y., Cort, J.R., Ragauskas, A.J., Yang, B., 2016a. Revealing the molecular structural transformation of hardwood and softwood in dilute acid flowthrough pretreatment. ACS Sustain. Chem. Eng. 4, 6618–6628. https://doi.org/10.1021/ acssuschemeng.6b01491. Zhang, X., Zhao, W., Li, Y., Li, C., Yuan, Q., Cheng, G., 2016b. Synergistic effect of pretreatment with dimethyl sulfoxide and an ionic liquid on enzymatic digestibility of white poplar and pine. RSC Adv. 6, 62278–62285. https://doi.org/10.1039/ c6ra14206k.
Tao, F., Song, H., Chou, L., 2011. Hydrolysis of cellulose in SO3H-functionalized ionic liquids. Bioresour. Technol. 102, 9000–9006. https://doi.org/10.1016/j.biortech. 2011.06.067. Tian, Z., Zong, L., Niu, R., Wang, X., Li, Y., Ai, S., 2015. Recovery and characterization of lignin from alkaline straw pulping black liquor: as feedstock for bio-oil research. J. Appl. Polym. Sci. 132https://doi.org/10.1002/app.42057. n/a-n/a. Uju, Nakamoto, A., Shoda, Y., Goto, M., Tokuhara, W., Noritake, Y., Katahira, S., Ishida, N., Ogino, C., Kamiya, N., 2013. Low melting point pyridinium ionic liquid pretreatment for enhancing enzymatic saccharification of cellulosic biomass. Bioresour. Technol. 135, 103–108. doi:https://doi.org/10.1016/j.biortech.2012.06.096. Verdía, P., Brandt, A., Hallett, J.P., Ray, M.J., Welton, T., 2014. Fractionation of lignocellulosic biomass with the ionic liquid 1-butylimidazolium hydrogen sulfate. Green Chem. 16, 1617–1627. https://doi.org/10.1039/c3gc41742e. Weigand, L., Mostame, S., Brandt-Talbot, A., Welton, T., Hallett, J.P., 2017. Effect of pretreatment severity on the cellulose and lignin isolated from Salix using ionoSolv
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Assessment of a protic ionic liquid with respect to fractionation and changes in the structural features of hardwood and softwood
T
⁎
Isık Semercia, , Fatma Gülerb,c, Gulsah Ersana, Kaan Soysala, Onur Ozturka, Hasan Altinisika, Sena Tirpana, Filiz Ozcelikb a
Department of Energy Engineering, Ankara University, 06830 Golbasi, Ankara, Turkey Department of Food Engineering, Ankara University, 06830 Golbasi, Ankara, Turkey c Department of Food Engineering, Bulent Ecevit University, 67900 Caycuma, Zonguldak, Turkey b
A R T I C LE I N FO
A B S T R A C T
Keywords: Lignocellulosic biomass Hornbeam Pine Lignin Pretreatment Protic ionic liquid
In this work, the potential of protic ionic liquid, triethylammonium hydrogen sulfate (TEAHSO4) was shown regarding the pretreatment of the hardwood, hornbeam and softwood, pine at different temperatures and times. TEAHSO4 was effective for lignin extraction and enhancing the enzymatic digestibility of both the biomass. Cellulose content of hornbeam increased to 85% with 87% lignin extraction from the biomass subjected to pretreatment at 150 °C for 3 h which was then readily converted into glucose with 97% yield. On the other hand, pine as a softwood demonstrated more resilience to deconstruction; 76% glucose yield was achieved following the enzymatic hydrolysis of biomass pretreated at 150 °C for 30 min. As put forward in the present work through TEAHSO4 pretreatment, any lignocellulosic feedstock that can be effectively disassembled into cellulose and lignin should be regarded as high value for the production of liquid and gaseous fuels through thermochemical and biochemical routes.
1. Introduction Lignocellulosic biomass as a smart and carbon-neutral resource has a great promise to replace fossil fuels for the future generations. Based on the statistics of International Energy Agency (IEA), bioenergy contributed almost half of the renewable energy with almost 460 MToe in 2017 excluding conventional use of biomass. Besides, its share among other renewable resources is estimated to increase 76 MToe from 2017 to 2023 (IEA Renewables). One of the key criteria that can keep the success of biomass in renewables is to choose the right strategy that makes use of each biomass component effectively. Different strategies can be manifested as thermochemical routes such as direct combustion, gasification and pyrolysis or biological routes involving hydrolysis and fermentation for the valorization of biomass. Today, biorefineries that process biomass into several product streams, fuels, chemicals and materials aim to maximize energy output from lignocellulosic feedstocks. Generation of less waste and reduction of carbon footprint are the other important components integrated into the biorefinery concept. For this reason, implementing a biomass processing strategy that generates the least amount of waste and thereby, enables full biomass use with the highest value return is significant. Processing lignocellulosic biomass with ionic liquids (ILs) has made ⁎
Corresponding author. E-mail address: [email protected] (I. Semerci).
https://doi.org/10.1016/j.biteb.2019.100334 Received 7 October 2019; Accepted 7 October 2019 Available online 23 October 2019 2589-014X/ © 2019 Elsevier Ltd. All rights reserved.
a significant progress since the last decade. Major properties that make ILs more favorable than conventional solvents are their low or negligible vapors and tunable characteristics along with their stability and recyclability. Studies launching with cellulose dissolution in ILs (Swatloski et al., 2002) was followed by biomass pretreatment with ILs that targeted either cellulose decrystallization or lignin removal (Tan et al., 2009; Wu et al., 2011). Meanwhile, successful interaction of ILs with a variety of lignocellulosic feedstocks also revealed the potential of dialkylimidazolium ILs such as 1-ethyl-3-methylimidazolium acetate (EMIMAc) (Sun et al., 2009) and 1-butyl-3 methylimidazolium chloride (BMIMCl) (Xie et al., 2018) in this field. In the last decade, diakylimidazolium ILs also provided reaction media for the production of platform chemicals. Transformation of cellulose to glucose in SO3H-functionalized ILs (Liu et al., 2018; Tao et al., 2011) and conversion of monomeric sugars to 5-HMF, furfural and levulinic acid in ILs have been recent advancements (Eminov et al., 2016; Liu et al., 2018). Despite their steady performance towards different biomass derived components, synthesis of dialkylimidazolium ILs consists deliberate and time-consuming processes that require costly starting chemicals. This creates an impediment for commercial scale-up of IL processing of biomass. Evaluation of lignocellulose interaction with protic ILs (PILs) as
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pine pretreatment. Better findings were found after DMBAHSO4 and HBIMHSO4 pretreatments; more than 70% of saccharification and lignin recovery yields were obtained when pine was pretreated with DMBAHSO4 at 10% biomass loading and 170 °C for 30 min (Gschwend et al., 2019). Herein, we report the comparison of hardwood and softwood species, hornbeam and pine with respect to the structural and compositional changes in the biomass obtained upon TEAHSO4 pretreatment and the potential of the species to be effectively converted into glucose, as well. Effects of pretreatment temperature and time were investigated since the severity of these parameters is the major determinant of the recovery of the components. The recovered solids were characterized with respect to their chemical compositions which were supported by SEM and XRD analysis, as well. On the other hand, XPS, 1H NMR and TGA were employed to provide the structural features of the lignin precipitates.
cheaper and more conveniently synthesized alternatives may render the process scale-up. As initiated and centered by Hallett group at Imperial College London under the name “IonoSolv Process” (Gschwend et al., 2016), referring to lignin and hemicellulose removal from biomass while keeping cellulose intact in the structure, utilization of PILs for biomass pretreatment paved the way for process commercialization. In the recent years, different feedstocks have been subjected to pretreatment with PILs. These comprise bagasse from different sources (Reis et al., 2017; Rocha et al., 2017; Uju et al., 2013), energy crops (Verdía et al., 2014), residues such as cotton stalks (Semerci and Güler, 2018) and pine apple crown (de Miranda et al., 2018), softwoods such as willow (Gschwend et al., 2019; Weigand et al., 2017) and pine (Gschwend et al., 2019). All the mentioned feedstocks pretreated with PILs revealed a common outcome; lignin contents decreased while maintaining a majority of cellulose. Besides, weak carbohydrate-lignin interactions (such as complexes involving ferulic acid) (Brandt et al., 2012) were observed along with broken β-O-4 ether and CeC bonds. In this study, we introduced the potential of PIL, triethylammonium hydrogen sulfate (TEAHSO4) to deconstruct hardwood and softwood species, hornbeam and pine, respectively and present their transformations from structural and product perspectives. The clearest structural distinction between hardwoods and softwoods is the structure of lignin. Softwoods such as pine, cedar and spruce have been recognized to be dominated by G lignin units being harder to be digested during pretreatment in contrast to S-units that are mainly found in the hardwoods such as beech, oak and hornbeam (Li et al., 2016). Besides, hardwood lignins which contain less CeC linkages are less resistant to deconstruction. So, the general conclusion is that biomass deconstruction is a more challenging task in softwoods than in hardwoods (Zhang et al., 2016a). Hornbeam (Carpinus betulus) with cellulose content between 30 and 50% has been explored in terms of its pretreatment through a few traditional techniques such steam explosion (Barbanera et al., 2018). Recently, hornbeam pretreated with aprotic IL, 1-butyl-3-methylimidazolium chloride (BMIMCl) was subjected to simultaneous saccharification and fermentation (SSF). SSF in the presence of R. oryzae, two products were mainly obtained, ethanol (4.7 g/L) and fumaric acid (1.7 g/L) along with 5-fold increase in the glucose yield (Dotsenko et al., 2018). Unlike hornbeam, softwood species pine (Pinus pinaster) has been much extensively studied due being an energy dense, widespread and inexpensive resource. Although it has been mainly processed through conventional techniques, autohydrolysis (Rigual et al., 2018), hydrothermal (Nitsos et al., 2016), and also, acid and SO2 catalyzed steam pretreatments (Huang and Ragauskas, 2012) with the aim of second-generation ethanol production, pine pretreatment in the presence of different aprotic and PILs gave promising findings (L. Zhang et al., 2016a). Considering the expense and elaborate synthesis of aprotic ILs including the pioneer IL, EMIMAc, PILs appealed to many lignocellulosic feedstocks for being cheaper, their simple synthesis and potential towards lignin removal from biomass (George et al., 2014). PILs can remove large portions of lignin from biomass and several mechanisms have been introduced for this interaction. In addition to hydrogen bonding which has been proposed in a few studies, ionic attractions via electrostatic/coulombic forces were also reported to be responsible for the favor of lignin dissolution process (Achinivu, 2018). PIL, 1-methylimidazolium chloride (HMIMCl) successfully converted pine into lignin rich precipitate, liquid fraction containing hemicellulose fragments, and cellulose enriched solid fraction which readily hydrolyzed into glucose (Cox and Ekerdt, 2013). Hallett and co-workers demonstrated the potential of TEAHSO4 for the deconstruction of pine and Miscanthus. Pretreatment of pine at 10% (w/w) biomass loading and 120 °C for 8 h resulted in 5% lignin precipitate and 13% glucose yield (Gschwend et al., 2016). Recently, the same group offered PILs, 1-butylimidazolium hydrogen sulfate (HBIMHSO4) and N,N-dimethylbutylammonium hydrogen sulfate (DMBAHSO4) in addition to TEAHSO4 for
2. Materials and methods 2.1. Materials Hornbeam and pine sawdust, which were obtained from a saw mill located in Salihli district of Manisa, Turkey, were sieved to a particle size less than 1 cm. The particles were air dried and stored at room temperature. Ethanol, trisodium citrate dihydrate, citric acid monohydrate, sulfuric acid, calcium carbonate, D-glucose and D-xylose were purchased from Merck (Darmstadt, Germany). Triethylamine and sulfuric acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). The cellulase enzyme, Cellic Ctec2 was kindly provided by Novozymes (Denmark). 2.2. Pretreatment of biomass IL, triethylammonium hydrogen sulfate was synthesized through an acid-base reaction using sulfuric acid and triethylamine according a previously reported procedure (Semerci and Güler, 2018). IL was characterized by 1H NMR (Supplementary material). Before analysis being conducted with Bruker 300 MHz spectrometer, IL sample was dissolved in deuterated dimethyl sulfoxide. The chemical shifts are as follows: TEAHSO4 (300 MHz, DMSO‑d6): δ 3.03 (q, 6H, NCH2CH3), 1.10 (t, 9H, NCH2). Biomass pretreatments were conducted with aqueous solutions of triethylammonium hydrogen sulfate in a similar fashion reported in the Ionosolv protocol (Gschwend et al., 2016). All pretreatments were conducted in 50 mL Pyrex tubes with teflon-lined screw caps. 1 g of airdried biomass was pretreated with 10 g aqueous solution having 4:1 IL:H2O weight ratio. The pretreatment temperature was held at 120, 150 and 170 °C and the pretreatment was performed between 30 min and 8 h. After the pretreatments, the samples were cooled to room temperature and ethanol was added to the pretreated slurries. Solid fraction was washed with ethanol three times for 15 min in a shaker to remove the residual PIL. Finally, the pretreated biomass was recovered by filtration and dried at 70 °C under vacuum for 24 h before it was subjected to enzymatic hydrolysis and characterization. All pretreatments were performed in duplicate. The recovered PIL was used to precipitate lignin through ethanol evaporation in rotary evaporator and water addition. The precipitated lignin was washed with water three times and centrifuged at 6000 rpm for 20 min each time. Finally, the recovered material was dried at 70 °C under vacuum for 24 h before it was subjected to characterization. Percentage solid recovery, SR (%), lignin extracted, LE (%) and lignin precipitate yield, LPY(%) were determined according to the following equations:
SR (%) = (WPRT / WUT ) × 100
(1)
where WPRT is the weight of biomass recovered after pretreatment (g) 2
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Table 1 Chemical compositions of hornbeam (HB) and pine (P) before pretreatment and after pretreatment under certain conditions. Pretreatment conditions
SR (%)
Cellulose (%)
Hemicellulose (%)
Lignin (%)
LE (%)
LPY (%)
UHB UP PHB-170 °C-3 h PHB-170 °C-4 h PP-170 °C-3 h PP-170 °C-4 h PHB-150 °C-3 h PHB-150 °C-4 h PP-150 °C-3 h PP-150 °C-4 h PHB-120 °C-6 h PHB-120 °C-8 h PP-120 °C-6 h PP-120 °C-8 h
– – 39.3 37.3 48.1 47.4 40.3 43.0 58.8 57.8 44.1 45.6 72.7 67.3
34.2 38.5 76.3 70.2 69.5 64.7 84.5 79.8 56.3 59.1 76.6 75.4 42.3 48.4
17.0 ± 1.7 22.6 ± 0.6 0 0 0 0 0 0 11.2 ± 0.1 10.0 ± 0.1 0 0 8.4 ± 1.4 7.2 ± 1.1
26.3 ± 0.0 28.9 ± 0.6 7.8 ± 0.3 7.4 ± 1.3 26.4 ± 0.1 31.5 ± 0.6 8.5 ± 0.2 11.8 ± 2.0 24.1 ± 1.4 25.7 ± 0.1 16.9 ± 1.7 18.0 ± 2.2 29.3 ± 0.2 29.3 ± 0.5
– – 88.3 89.5 51.8 43.2 86.9 80.7 46.1 41.5 71.6 68.8 19.0 25.0
– – 72.7 61.2 58.3 55.7 78.0 71.7 27.0 35.9 19.4 36.5 14.2 15.8
± ± ± ± ± ± ± ± ± ± ± ±
0.2 1.0 0.9 1.6 0.7 0.2 1.8 0.0 2.6 0.5 0.3 0.8
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.6 3.2 1.0 3.1 0.0 0.2 3.5 5.8 4.2 0.4 4.8 1.5 6.6 2.1
(3)
where WP is the weight of the precipitate obtained after IL pretreatment and WUL is the weight of lignin in the untreated biomass subjected to pretreatment.
CrI = [(I002 − IAM )/ I002] × 100 2.3. Enzymatic hydrolysis
3.1 4.0 1.6 0.3 7.9 1.4 2.0 2.4 0.7 7.0 2.5 0.1
(5)
where I002 is the height of the crystalline peak at around 2θ=22.5° and IAM is the height of the amorphous reflection at around 2θ= 18°. X-ray photoelectron spectroscopy (XPS) analysis of lignin samples was performed in a PHI 5000 VersaProbe spectrometer using a monochromatic Al Kα X-ray source. C1s spectra at the binding energy of 285 eV were used as a reference. For proton nuclear magnetic resonance (1H NMR) spectroscopy analysis, lignin was dissolved in deuterated dimethyl sulfoxide and the data was recorded with a Bruker 300 MHz spectrometer. Thermogravimetric analysis (TGA) was carried out in a STD650 Simultaneous DSC/TGA instrument by heating the samples up to 800 °C (heating rate: 10 °C min−1) in a nitrogen atmosphere to monitor the thermal decomposition of lignin samples.
Untreated and pretreated biomass samples at 3% (w/v) and Cellic Ctec2 (210 FPU/ml) at 2% (v/v) were loaded in 0.05 M sodium citrate buffer at pH 4.8. The samples were kept at 50 °C in a shaker incubator for 48 h. Enzymatic reaction was stopped by keeping the samples for 5 min in boiling water. Later, the samples were centrifuged at 6000 rpm for 20 min and the supernatants were filtered through 0.20 μm PTFE membrane filter before their analysis for glucose concentration. Prominence LC-20A Modular HPLC System equipped with refractive index (RI) detector and Transgenomic Carbosep Coregel 87H3 column was operated at 55 °C with 5 mM H2SO4 mobile phase at a flow rate of 0.5 mL/min. The sample volume injected was 20 μL and the retention time was 20 min. Percentage glucose yield, GY(%) was calculated on the basis of the cellulose content of the untreated hornbeam and pine samples according the following formula:
GY (%) = (SR (%) × CG )/(CB × CelluloseU (%) × 1.11) × 100
± ± ± ± ± ± ± ± ± ± ± ±
Scanning electron microscopy (SEM) images were obtained using a QUANTA 400 F Field Emission SEM operated at 20 kV. All samples were sputter coated with gold/palladium (Au/Pd) prior to analysis. SEM images of the samples were taken at different magnifications (250×, 500×, 1000× and 2000×). X-ray diffraction (XRD) analysis of the biomass samples was conducted with Rigaku Ultima-IV Diffractometer between 2θ= 10° and 40° Bragg angles using Cu radiation at a scanning speed of 1°/min. Percentage crystallinity indices (CrI) of biomass were determined based on the peak height method (Park et al., 2010) and calculated according to the following formula;
(2)
where LUT (%) is the lignin content of untreated biomass, SR (%) is the solid recovery after pretreatments and LPRT (%) is the lignin content of the biomass subjected to pretreatment (Haykir et al., 2013).
LPY (%) = [WP / WUL] × 100
0.4 1.9 0.7 1.1 0.1 3.3 1.8 0.3 2.9 4.5 0.1 0.4
2.5. Characterization of biomass
and WUT is the weight of untreated biomass subjected to pretreatment (g).
LE (%) = [(LUT (%) − (SR (%) × LPRT (%)/100))/ LUT (%)] × 100
± ± ± ± ± ± ± ± ± ± ± ±
3. Results and discussion 3.1. Effect of pretreatment on the composition of biomass
(4)
Compositional analysis of biomass is significant since it determines the value of lignocellulosic feedstocks and the possibility of effective valorization of components to fuels, chemicals and materials. Table 1 and Supplementary material showed the composition of untreated biomass samples and solid recovery, composition, lignin extracted and the lignin precipitate yields for the biomass samples pretreated under certain conditions. As shown, lower solid recoveries (37%–95%) obtained for hornbeam compared to pine (47%–95%) under the same conditions indicating the effective deconstruction capacity of IL on the hardwood, hornbeam. As pretreatment temperature decreased, higher solid recoveries were obtained due to the reduction in the process severity. These results were also in correlation with the percentage lignin and hemicellulose removed from the biomass; TEAHSO4 readily removed hemicellulose from hornbeam in all conditions and extracted more than 88% of the lignin from the structure with the pretreatment at
where SR (%) is the percentage solid recovery, CG is the glucose concentration in the hydrolysates (g/L), CB is the initial concentration of the biomass in the hydrolysis buffer that is subjected to enzymatic hydrolysis (g/L) and CelluloseU (%) is the cellulose content of the untreated biomass. 1.11 is the stoichiometric conversion factor of cellulose to equivalent glucose (Haykir and Bakir, 2013).
2.4. Composition analysis Composition analysis of untreated and pretreated samples was performed according two step acid hydrolysis reported by NREL (Sluiter et al., 2008). Sugar content of the hydrolysates was measured with HPLC using the same conditions as given for the analysis of hydrolysates obtained upon enzymatic hydrolysis. 3
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pretreatment at 150 °C provided the intact structure of cellulose in addition to lignin and hemicellulose removal. Thereby, 97% of glucose yield was obtained being 2-fold higher than the glucose yield of untreated hornbeam (46%). IL pretreatment showed lower capability regarding the enzymatic digestibility of softwood, pine compared to hardwood, hornbeam which was observed in a similar fashion in the previously reported studies for softwood and hardwood feedstocks subjected to different pretreatments including IL pretreatment (Asada et al., 2015; Nitsos et al., 2016; Rigual et al., 2018; X. Zhang et al., 2016b). The degree of lignin and hemicellulose removal and the preservation of cellulose content of the structure were the major determinants of the glucose yields based on the cellulose content of untreated biomass. For pine, the highest glucose yield was found as 76% for pine pretreated at 150 °C for 30 min (Supplementary material). Despite this 1.8-fold increase in glucose yield based on the enzymatic digestibility of untreated pine (42% glucose yield), pretreatment at 150 °C for 30 min was not effective for lignin extraction; approximately 15% lignin removal was observed. Currently, aqueous solutions sulfate-based PILs (with 20 wt% water), TEAHSO4, DMBAHSO4 and HBIMHSO4 were assessed for pine pretreatment at 170 °C for 30 min; promising findings introduced DMBAHSO4 as an ideal candidate for softwoods (Gschwend et al., 2019). Ultra-low cost DMBAHSO4 not only led to satisfactory lignin recovery and hydrolysis yields (70%) but also dominated for modifying the structure of lignin to a dramatic level through effective β-O-4 cleavage. Above all, HBIMHSO4 employed at biomass loadings as high as 50% was still effective in pine deconstruction; lignin recovery and saccharification yields were around 30% and 40%, respectively. While TEAHSO4 pretreatment conducted under identical pretreatment conditions gave 16% lignin extraction in this study (Supplementary material), 30% lignin recovery yield was achieved for pine in the aforementioned study. Additionally, pretreatment of pine with cholinium arginate at 90 °C for 12 h under nitrogen with a biomass/IL ratio of 1/ 15 (w/w) and stirring gave 33% lignin extraction and 31% glucose yield (An et al., 2015). Another PIL, 1-ethylimidazolium chloride (EimCl), which dissolved biomass completely under the following conditions; 2 wt% loading, 110 °C, 18 h, resulted in at least 75% glucose yield for the recovered pine (Hossain et al., 2019).
170 °C for 3 h. Along with lignin and hemicellulose removal, cellulose content of hornbeam increased more than 2-fold from 34% to 84% when pretreated at 150 °C for 3 h. This implies effective conversion of hornbeam samples to a cellulose rich solid fraction with minimal cellulose degradation. Pretreatment at 170 °C for 3 h was also effective for pine; more than 50% lignin was extracted and cellulose content of the biomass increased up to almost 70%. In a previous study, lignin removal for pine pretreated using EMIMAc and EMIMAc-dimethyl sulfoxide (DMSO) solutions at 110 °C for 3 h was found less than 3% (X. Zhang et al., 2016b). In comparison to eucalyptus and switchgrass, pine was found the most resistant to EMIMAc pretreatment at 160 °C for 3 h; lignin removal efficiencies were 49%, 55% and 69% for pine, eucalyptus and switchgrass, respectively (Li et al., 2013). Additionally, food-derived imidazolium acesulfamate IL, 1-butyl-3-methylimidazolium acesulfamate (BMIMAce) selectively extracted lignin from pine with an extraction efficiency of 38% (Pinkert et al., 2011). 3.2. Effect of pretreatment on the digestibility of biomass After pretreatments, pretreated biomass was subjected to enzymatic hydrolysis for 48 h. Glucose yields (%) were given in Fig. 1 and Supplementary material which showed the effect of pretreatment temperature and time on the conversion of the cellulose in both the untreated hornbeam and untreated pine to glucose. The highest glucose yield was roughly 97% which was attained for hornbeam pretreated at 150 °C for 3 h. Pretreatment of hornbeam at 170 °C also resulted in sufficient glucose yields; almost 70% and 85% of cellulose in the untreated biomass was converted to glucose after pretreatments conducted for 3 and 4 h, respectively. These finding were also found consistent with the composition of the biomass. Pretreatment of hornbeam at 170 °C for 3 h and 4 h removed approximately 90% of the lignin and completely the hemicellulose from the structure. Effective transformation of cellulose to glucose was anticipated since lignin has been recognized to set a critical barrier to the enzymatic hydrolysis of biomass (Li et al., 2016). However, due to moderate cellulose degradation, glucose yields were lower than 90% for the other horn- beam samples pretreated at 170 °C. As the degree of cellulose recovered gets lower through degradation at higher temperatures, one gets far away from one of the major goals of PIL pretreatment which is preservation of cellulose. Unlike 170 °C,
100 90
Glucose yield (%)
80 70 60 50 40 30 20 10 0
Fig. 1. Glucose yields obtained at the 48th hour of enzymatic hydrolysis of untreated biomass and biomass subjected to TEAHSO4 pretreatments. 4
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3.3. Effect of pretreatment on the structure of recovered solids Recovered solids were evaluated regarding their morphology and biomass crystallinity using SEM and XRD, respectively. Images taken at magnifications 250×, 500×, 1000× and 2000× were shown in Supplementary material. According to the images, untreated species, both the untreated hornbeam and pine, exhibited intact and rigid structures. However, these structures apparently opened up after TEAHSO4 pretreatment, especially hornbeam pretreated at 170 °C and 150 °C for 3 h. Thin fibrillar structures revealed indicated the cellulose rich biomass and the removal of lignin (at least 87% lignin removed from hornbeam) and hemicellulose completely as correlated with the chemical composition of pretreated hornbeam. Unlike hornbeam, coarse fibrillar structures were observed for pine which was in good agreement with the impact of TEAHSO4 on pine deconstruction; lignin was removed at lower levels (only up to 52%). Interestingly, pine pretreated at 150 °C demonstrated curly macro bundles. For hornbeam pretreated at 120° for 8 h, neat fibrillar structures were still present except for being a little thicker. Lastly, modest interaction of pine with TEAHSO4 at 120° for 8 h did not seem to serve the purpose; morphological similarities were found between the untreated and pretreated biomass. The visualization of the morphology with SEM properly supported the compositional changes in the feedstocks. X-ray diffraction was used to evaluate the percentage crystallinity indices (CrI) of untreated and pretreated samples and observe if there would be a relation between the crystalline structure and digestibility of biomass. As revealed in Fig. 2a and b, crystalline structure of the hornbeam and pine, respectively was represented with three diffraction peaks at around 16.5°, 22.5°, and 35°. For both the biomass, extent of the peaks increased for the pretreated samples compared to untreated samples. Moreover, the peaks in general got more intense and particularly the peak at around 22.5° got narrower as the pretreatment temperature increased. Accordingly, an increase in the CrI (%) of the samples with pretreatment severity was observed (Table 2). CrI (%) of the samples increased due to the effective removal of amorphous fractions of the structure with IL. Regardless of this increase in biomass crystallinity, enhanced glucose yields were obtained (Fig. 1). With respect to these points, interaction of IL with the lignin and hemicellulose dominated over the changes in biomass crystallinity in biomass conversion to glucose. As similarly stated for different lignocellulosic feedstocks (Achinivu et al., 2013; George et al., 2014; Reis et al., 2017), increased crystallinity of the hornbeam did not act adversely on the biomass digestibility. Fig. 2. XRD patterns of hornbeam (a) and pine (a) samples before and after pretreatments with TEAHSO4 under certain pretreatment conditions.
3.4. Effect of pretreatment on the structure of lignin precipitates Structures of lignin precipitates recovered from TEAHSO4 solutions after pretreatment were analyzed through XPS, 1H NMR, and TGA in order to show the effect of pretreatment temperature on the lignin chemical structure. Lignin precipitate yields (%) were given in Table 1. Lignin precipitate yields (%) were found in accordance with the lignin extraction capability of TEAHSO4 at different pretreatment temperatures and times. The highest lignin precipitate yield (%) was obtained from hornbeam pretreated at 150 °C and for 3 h as 78%. The aforementioned precipitate was compared with the one obtained at 170 °C and for 3 h (73% lignin precipitate yield) structurally. XPS is a useful method to evaluate the surface chemical composition of materials (based on the atomic percentage). Table 3 showed the percentage carbon distribution on the material surface which is obtained by high resolution spectra (Fig. 3). Table 3 showed that there is a decrease in the aliphatic and aromatic carbon bonding as represented by the peak C1 with an increase in temperature from 150 °C to 170 °C. This indicated the effective depolymerization of lignin through disruption of CeC linkages in lignin through protonation by TEAHSO4. The increase in the percentage of C2 peaks from 150 °C to 170 °C implied the preservation of the phenolic hydroxyl, aliphatic hydroxyl and
Table 2 CrI indices of untreated and TEAHSO4 pretreated biomass. Pretreatment conditions
CrI (%)
PHB-170 °C-3 h PP-170 °C-3 h PHB-150 °C-3 h PP-150 °C-3 h PHB-120 °C-8 h PP-120 °C-8 h UHB UP
79 77 76 73 70 71 67 63
ether bonds (β-O-4 bonds). Hardwood lignin was reported to contain 60–62 units of β-O-4 linkages per 100C9 units as the most dominant structure (Ragauskas and Yoo, 2018). Based on the C2 percentages shown in Table 3, these linkages apparently reduced with IL pretreatment. Besides, the slight decrease in the percentages of C3 and C4 represented the disruption of carbonyl and carboxyl groups in lignin,
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showed the disruption of aliphatic carbon bonding. Moreover, the intensity of the signal at around 3.1, signifying the proton in β-1 bond (another CeC bond), decreased with an increase in pretreatment temperature. The chemical shift of the protons in methoxy groups was located at around 3.7 ppm. The intensity of this peak was lower for the lignin obtained during TEAHSO4 pretreatment at 170 °C for 3 h implying the removal of methoxy groups from the structure of lignin. The signals at around 6.7 ppm and 8.8 ppm were assigned to the aromatic region of lignin, aromatic protons in guaiacyl (G) and syringyl (S) units and phenolic protons, respectively. As shown in Fig. 4a, the intensities of these peaks slightly got higher by increasing the pretreatment temperature which was consistent with the percentage increase of C2 peak of XPS spectrum. Lastly, TGA provided the thermal stability of lignin samples extracted from hornbeam as shown in Fig. 5. Accordingly, thermal stability of lignin decreased slightly as the pretreatment temperature increased from 150 °C to 170 °C; the residual weight decreased from 43.8% to 42.3%. This was in accordance with the percentage decrease of C1 peak of XPS spectrum which assigned to the aliphatic CeC linkages.
Table 3 XPS binding energies and assigned carbon species percentages. Peak
C1 C2 C3 C4
Position BE (eV)
283–284 284–285 285.5–287 286–289
Bond type
C-C, C-H C-O, C-O-C O-C-O, C=O COO
Percentage of total carbon (%) PHB-170 °C-3 h
PHB-150 °C-3 h
33.8 57.9 6.1 2.2
40.6 48.3 8.1 3.0
respectively with an increase in pretreatment temperature (BañulsCiscar et al., 2016; Tian et al., 2015). The presence of functional groups, abundance of syringyl and guaiacyl units and the side chains in the lignin structure was also monitored via 1H NMR. As shown in Fig. 4, the major signals were detected at around 1.3 ppm, 3.1 ppm, 3.7 ppm, 6.7 ppm and 8.8 ppm. In order to correspond the signals to the correct assignments, a previously reported study was considered (Rashid et al., n.d.). Fig. 4a and b represent the spectra of lignin precipitates obtained after hornbeam pretreatments conducted at 170 °C and 150 °C for 3 h, respectively. The initial peak at around 1.3 ppm represented the protons in the aliphatic region which was weaker in the spectrum of the lignin obtained after biomass pretreatment at 170 °C for 3 h compared to the one at 150 °C for 3 h. This result was in correlation with XPS finding which also
4. Conclusions TEAHSO4 was found more effective towards the hardwood, hornbeam with respect to the changes in the composition and chemical
Fig. 3. XPS high resolution spectra of C1s region for lignin samples obtained from hornbeam pretreated with TEAHSO4 at 170 °C (a) and 150 °C (b) for 3 h. 6
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Fig. 4. 1H NMR spectra of lignin samples obtained from hornbeam pretreated with TEAHSO4 at 170 °C (a) and 150 °C (b) for 3 h.
structure of the lignocellulosic biomass and its conversion to glucose. Hornbeam was readily fractionated into cellulose rich solid and lignin precipitate when subjected to pretreatment at 150 °C for 3 h. Biorefineries are primarily dependent on getting the most value out of the major fractions of the lignocellulosic feedstocks. For this reason, acknowledging the PIL, TEAHSO4 as a suitable candidate for biomass
pretreatment should be emphasized as an important conclusion of this work. Declaration of competing interest The authors declare that they have no known competing financial 7
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Fig. 5. TGA spectra of lignin samples obtained from hornbeam pretreated with TEAHSO4 at 170 °C and 150 °C for 3 h.
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