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. 2020 Sep 21;5(38):24780–24789. doi: 10.1021/acsomega.0c03419

Directional Structure Modification of Poplar Biomass-Inspired High Efficacy of Enzymatic Hydrolysis by Sequential Dilute Acid–Alkali Treatment

Fuxi Shi †, Yajun Wang ‡, Maryam Davaritouchaee §, Yiqing Yao †,*, Kang Kang ∥
PMCID: PMC7528282  PMID: 33015496

Abstract

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A major challenge in converting lignocellulose to biofuel is overcoming the resistance of the biomass structure. Herein, sequential dilute acid–alkali/aqueous ammonia treatment was evaluated to enhance enzymatic hydrolysis of poplar biomass by removing hemicellulose first and then removing lignin with acid and base, respectively. The results show that glucose release in sequential dilute acid–alkali treatments (61.4–71.4 mg/g) was 7.3–24.8% higher than sequential dilute acid–aqueous ammonia treatments (57.2–61.8 mg/g) and 283.8–346.3% higher than control (16.0 mg/g), respectively. Dilute acid treatment removed most hemicellulose (84.9%) of the biomass, followed by alkaline treatment with 27.5% removal of lignin. Roughness, surface area, and micropore volume of the biomass were crucial for the enzymatic hydrolysis. Furthermore, the ultrastructure changes observed using crystallinity, Fourier transform infrared spectroscopy, thermogravimetric analysis, and pyrolysis gas chromatography/mass spectrometry support the effects of sequential dilute acid–alkali treatment. The results provide an efficient approach to facilitate a better enzymatic hydrolysis of the poplar samples.

1. Introduction

Lignocellulosic biomass is an abundant and renewable feedstock for the production of biofuels and bioproducts.1,2 Enzymatic hydrolysis is the key step in the biochemical conversion of the biomass because it can convert cellulose and hemicellulose of the biomass into fermentable sugars.3 The recalcitrance of lignocellulose is the main challenge for efficient enzymatic scarification.

Chemical characteristics of lignocellulose in terms of cellulose, hemicellulose, and lignin lead to the recalcitrance of the biomass. Cellulose microfibrils, acting as a cell wall scaffold, result in a rigid and compact mesh-like structure.4,5 Hemicelluloses-lignin matrix that is covalently interwoven with nanoscale structural heterogeneities is a main factor, that limits the penetration and action of enzymes.6,7 Lignin, as a polymer, is composed of three canonical monolignols (p-hydroxycinnamyl alcohols) including sinapyl, coniferyl, and p-coumaryl alcohols which form the guaiacyl (G), sinapyl (S), and p-hydroxyphenyl (H) units.8 The linkages between monolignols can be formed through ether bonds such as the β-O-4 and 5-O-4 linkages, C–C bonds such as 5–5 linkages, or a combination of C–C and ether linkages as in β–5 + α-O-4 and β–β + γ-O-α + α-O-γ among others.9 Plant taxa, plant developmental stage, and plant tissue determine the total content and relative abundance of monolignols, types of linkages, and degree of cross-linking with polysaccharides.10−12

To effectively break down the lignocellulosic matrix, it is necessary to pretreat the biomass prior to the enzymatic hydrolysis. Pretreatment can change the physicochemical features and improve the accessibility of cellulose to enzymes by changing proportion and distribution of lignin, hemicellulose, and cellulose, altering structural morphology, and disrupting the crystalline of cellulose. Among the conventional pretreatment methods, dilute acid and alkali pretreatment are extensively studied for industrial applications and have been used for various biomass treatment because of various advantages such as high efficiency, simplicity, and low cost.13,14 Acid treatment can change the structure of lignin, cellulose, and hemicellulose, which leads to reduction in biomass recalcitrance.15 Acid pretreatment selectively hydrolyzes hemicelluloses within the lignocellulosic biomass because the glycosidic bonds contained in hemicelluloses are susceptible to acid.16 The removal of hemicellulose leads to the increase of the pore size of the biomass and therefore the enhanced accessibility and digestibility of cellulose.17 The drawback of severe acid pretreatment is the generation of certain byproduct compounds that can reduce the yield of enzymatic hydrolysis. On the other hand, alkali pretreatment has the advantage of removing lignin significantly and thus increases the accessibility of cellulose to enzyme in the modified biomass structure. The main drawback of alkali treatment is its mild effect on hemicellulose.14 Naturally, combining acid and alkali pretreatments has the potential to capture the advantages of both methods and thus has attracted increasing attentions recently. For example, rice straw pretreatment with sequential alkali and acid methods led to improved sugar yield by the systematic removal of lignin and hemicellulose.18 Additionally, sequential acid and alkali pretreatments showed better sugar recovery compared to each did alone.19 Acid or alkali pretreatment has also been performed at high temperature or pressure.20,21

As a form of alkali pretreatment, aqueous ammonia is often used for pretreating lignocellulosic biomass to remove lignin.22 Reported ammonia-based processes include ammonia fiber expansion for treating sugarcane bagasse, leaves,23 and municipal solid waste;24 aqueous ammonia percolation for treating hybrid poplar;25 soaking aqueous ammonia for treating barley hull;26 and ammonia recycled percolation for treating corn straw.27 In the case of pretreatment of energy cane bagasse with ammonium hydroxide (Aita et al., 2010), it was found that the morphology of the sample changed from compact and rigid to swollen and loose. As a selective solvent for lignin, ammonia cleaved the ether and ester bonds within lignin and between lignin and carbohydrates via ammonolysis, thereby increased accessibility of the remaining holocellulose fraction to cellulases.28 Reductions in lignin, hemicellulose, and cellulose after pretreatment were 55%, 30%, and 9%, respectively; conversions of glucan and xylan increased significantly, leading to high yield of ethanol.

Although extensive studies had been carried out on dilute acid, alkali, or ammonia pretreatment, the interests were mainly focused on evaluating the efficiency of enzyme hydrolysis.29,30 It is desirable also to understand the mechanisms of lignocellulose deconstruction including the role of lignin and hemicellulose degradation in sugar yield and their multiscale structural changes during the sequential treatment process.

In fact, ultrastructure changes of lignin, hemicellulose, and cellulose can vary significantly depending on the type of lignocellulosic biomass, reactor setup, and scale of pretreatment.31 The mechanisms for each treatment, particularly how they alter the physicochemical compositions and structures of biomass, remain not fully understood.32 Among different lignocellulosic biomass, poplar was considered as a good choice for woody energy crops31 as this type of biomass displays various advantageous agronomic properties.31,33−35 As one of the ideal energy crops, poplar is widely used for processing of pallets, cheap plywoods, and paper pulp for the characteristics of fast growing, short rotation, and richer in cellulose and hemicellulose.36 Further, as biomass materials, poplar processing residues could be converted to ethanol or biohydrogen.37,38 Various pretreatment methods have been applied for enhancing energy conversion of poplar wood, such as acid and alkali, which have been widely applied individually. However, studies of combination of acid and alkali for poplar wood pretreatment are little, and their combined effect on the structural alternation of the poplar wood and the mechanism of their potential interactions have not been systematically investigated. In this study, in order to increase the composition of enzyme-digestible cellulosic materials, a dilute acid treatment as the first step was conducted to remove hemicellulose from the poplar wood sample and to expose lignin. Then, an alkali/aqueous ammonia treatment as the second step was utilized to efficiently eliminate the exposed lignin. The pretreatments were evaluated for their co-effect on the enzymatic hydrolysis efficiency of poplar wood. Acid and alkali/aqueous ammonia treatments were applied sequentially to remove hemicellulose and lignin fraction from the raw material, respectively. In this research, the effects of dilute acid treatment, dilute acid–alkali treatments, and the dilute acid–aqueous ammonia treatment on the sample composition have been studied by compositional analysis as well as enzymatic hydrolysis. The influence of pretreatment methods on biomass ultrastructure was studied using various techniques including scanning electron microscopy (SEM), X-ray diffraction (XRD), pyrolysis gas chromatography/mass spectrometry (Py-GC/MS), Fourier transform infrared (FTIR) spectroscopy, N2 adsorption, and thermogravimetric analysis (TGA). It is anticipated that the results presented in this paper will facilitate understanding the poplar wood structural change under different pretreatments and advance the utilization of this biomass resource.

2. Results and Discussion

2.1. Degradations of Poplar Wood after Different Treatments

It was expected that the sequential treatment should improve the relative proportion of cellulose by reducing the hemicellulose and lignin contents of poplar biomass. The changes of major constituents are shown in Table 1. The cellulose, hemicellulose, and lignin contents decreased to different degrees, and the degradations of cellulose, hemicellulose, and lignin are 5.0–32.0%, 84.9–95.5%, and 2.9–27.5%, respectively. Notably, dilute acid treatment removed most of the hemicellulose, while only 8.6–10.6% increase was achieved by subsequent alkali treatment, and much lower increase of 0.4–2.9% was achieved by subsequent aqueous ammonia treatment. Dilute acid was verified to be able to specially remove hemicellulose efficiently, which was in agreement with the results of other studies.39 However, after the dilute acid treatment, most of the hemicellulose was removed and the amount of lignin in the solids was relatively high and even higher than that of raw biomass. The similar result was reported previously for which the cellulose content increased after removing most of the lignin by NaOH.45 For the subsequent treatments following dilute acid treatment, alkali treatments mainly removed lignin and cellulose, while aqueous ammonia treatments had a little effect on the removal of lignin and cellulose. Lignin removal increased with the increase in aqueous ammonia loading from 1 to 3% but dropped to 2.9% at 5% loading. This might be associated with the formation of pseudolignin with the presence of ammonia reagents.46−48 The formation of pseudolignin at sequential dilute acid–aqueous ammonia treatments could inhibit further degradation of cellulose and hemicellulose. In this case, the highest aqueous ammonia (5%)/sequential dilute acid resulted in the lowest degradation of cellulose and hemicellulose. Overall, these results implied higher cellulose exposure to the enzyme during the hydrolysis after sequential dilute acid–alkali treatments, compared with that of sequential dilute acid–aqueous ammonia treatments. The data in Table 1 explained the results of enzymatic saccharification performance.

Table 1. Composition Changes of the Poplar Sample during Sequential Dilute Acid–Alkali/Aqueous Ammonia Treatmentsa.

  dry matter
 cellulose
 hemicellulose
 lignin
treatments TS (g) mass loss (%) after treatment [g] decrease [ %] after treatment [g] decrease [ %] after treatment [g] decrease [ %]
dilute acid treatment 7.0 ± 0.0 29.8 ± 0.5 3.7 ± 0.0 5.1 ± 0.0 0.2 ± 0.0 84.9 ± 1.4 2.4 ± 0.1  
dilute acid–1% alkali treatment 5.3 ± 0.1 47.0 ± 0.8 3.1 ± 0.2 22.5 ± 1.5 0.1 ± 0.0 93.5 ± 1.4 1.8 ± 0.0 17.9 ± 0.6
dilute acid–3% alkali treatment 4.7 ± 0.2 52.7 ± 0.4 2.7 ± 0.2 30.7 ± 1.0 0.1 ± 0.0 94.9 ± 1.0 1.6 ± 0.0 25.6 ± 0.4
dilute acid–5% alkali treatment 4.6 ± 0.1 53.5 ± 0.2 2.7 ± 0.2 32.0 ± 2.1 0.1 ± 0.0 95.5 ± 1.0 1.7 ± 0.0 27.5 ± 0.2
dilute acid–1% aqueous ammonia treatment 6.8 ± 0.4 32.3 ± 1.0 3.7 ± 0.2 5.0 ± 1.1 0.2 ± 0.0 86.3 ± 0.7 2.1 ± 0.0 5.4 ± 1.5
dilute acid–3% aqueous ammonia treatment 6.7 ± 0.2 32.9 ± 0.5 3.7 ± 0.1 6.1 ± 1.2 0.2 ± 0.0 87.8 ± 0.3 2.1 ± 0.0 5.6 ± 0.2
dilute acid–5% aqueous ammonia treatment 6.7 ± 0.1 33.0 ± 0.2 3.7 ± 0.0 5.0 ± 0.9 0.2 ± 0.0 85.3 ± 0.9 2.1 ± 0.0 2.9 ± 1.1
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a

Note: before treatment, the dry mass (TS) was 10 g, the cellulose was 3.9 g, the hemicellulose was 1.6 g, and the lignin was 2.2 g.

2.2. Enzymatic Hydrolysis

Cellulose ultrastructural changes are usually evaluated by using enzymatic sugar release assays. As shown in Figure 1, glucose release during dilute acid treatment was 208.1% higher than that of the control. Glucose release during the sequential dilute acid–alkali treatments was 7.3–24.8% higher than those of sequential dilute acid–aqueous ammonia treatments, 24.5–44.8% higher than that of dilute acid treatment, and 283.8–346.3% higher than that of control, respectively; for sequential dilute acid–alkali treatments, the highest glucose release (71.4 mg/g) was obtained for the sequential dilute acid–5% alkali treatment, which was 16.3% higher than that of sequential dilute acid–1% alkali treatment (p < 0.05) and 2.6% higher than that of sequential dilute acid–3% alkali treatment (p > 0.05). For sequential dilute acid–aqueous ammonia treatments, the glucose releases were 16.0–25.4% higher than that of dilute acid treatment and 257.5–286.3% higher than that of control; the highest glucose release (61.8 mg/g) in the sequential dilute acid–ammonia treatment obtained when ammonia loading was 3%. It was 4.0% higher than that of sequential dilute acid–1% ammonia treatment (p > 0.05) and 8.0% higher than that of sequential dilute acid–5% ammonia treatment (p > 0.05). Xylose releases were 4.8, 6.2, 2.9, 3.4, 1.8, 5.4, 5.4, and 5.3 mg/g for the control, dilute acid treatment, sequential dilute acid–1, 3, and 5% alkali treatments, and sequential dilute acid–1, 3, and 5% aqueous ammonia treatments, respectively. The highest value was observed with the dilute acid treatment, and the second highest was obtained for sequential dilute acid–aqueous ammonia treatments, followed by the control, and the lowest value was obtained from the sequential dilute acid–alkali treatments.

Figure 1.

Figure 1

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Glucose and xylose yield (mg per g dry biomass) after 48 h of enzymatic hydrolysis of control and poplar wood after different treatments.

Sugars released from poplar wood sample after different treatments were in accordance with its compositional analysis data such as cellulose, hemicellulose, and lignin contents. The results in Table 1 and Figure 1 suggested that lower degradation of cellulose resulted in lower glucose production. Compared with the sequential dilute acid–alkali treatments, dilute acid–aqueous ammonia treatments resulted in higher xylose production because of the relatively higher hemicellulose retained in solid residues. Accordingly, after enzymatic saccharification, the highest xylose production was obtained with dilute acid treatment because of the lowest removal of hemicellulose among all the treatments. Another factor that affects sugar release has been reported to be the accessible surface area of cellulose because the prerequisite for enzymatic hydrolysis is the direct contact between cellulase and the cellulose surface.49 Therefore, the higher glucose release for the sequential dilute acid–alkali treatments compared with other treatments might be due to the increasing of the exposure of cellulose of the samples.17 In addition, the behavior of xylan release is accompanied by deacetylation, which is favorable for providing more sites for enzyme attack and reduce the recalcitrance through the linearization of hemicelluloses, which is beneficial for the subsequent enzymatic hydrolysis with higher glucose release.50,51 Furthermore, sequential dilute acid–alkali treatments and sequential dilute acid–aqueous ammonia treatments can be used for removing lignin.39 This leads to increased exposure of the cellulose in acid-treated poplar biomass and enhanced the glucose release. In this case, the extent of hemicellulose removal from the biomass was also related to their xylose releases.

As a result, an accurate interpretation of the change in cellulose accessibility upon biomass treatment along with the results about composition and structural changes could provide a better understanding of the effect of the lignin/hemicellulose removal efficiency on the cellulose accessibility and the enzymatic hydrolysis.

2.3. Apparent Structure Changes

2.3.1. SEM Analysis

The SEM technique was used for analyzing the changes of the apparent structure of poplar samples before and after treatment. It was shown in Figure 2 that the apparent structure of poplar wood for the control group was compact [A(I) and A(II)]. After different treatments, the compact structure was destroyed to varying degrees. After the treatment with dilute acid, the fiber became rough with a rugged and partially broken sample surface [B(I) and B(II)]. The outer surface of waxes was removed and some fibers connected on the surface were partly disrupted. Consequently, the inner part of the sample was exposed. The available surface area is an important factor for the accessibility of enzymes in subsequent enzymolysis.52 The increased surface area and the loose structure of solids after treatment can facilitate enzymes to penetrate the biomass more easily, engage the substrate faster, and hydrolyze lignocellulosic materials more effectively.53 Aqueous ammonia treatment made the dilute acid-treated solids looser and softer [D(I) and D(II)]. However, there were no apparent pores formed on the surface, which is different from that obtained from the sequential dilute acid–alkali treatment [C(I) and C(II)].

Figure 2.

Figure 2

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SEM photographs of (A) control, (B) dilute acid-treated poplar wood, (C) dilute acid–5% alkali-treated poplar wood, and (D) dilute acid–3% ammonia-treated poplar wood. (I) 1k×, (II) 3k×.

2.3.2. BET Surface Area and Micropore Volume Analysis

The increase in cellulose accessibility to cellulases caused by pretreatment is mainly due to the increase of a specific surface area, and the expansion of the pore size and volume, which is beneficial for improving cellulases adsorption on cellulose surface.54,55 The changes in Brunauer–Emmett–Teller (BET) surface area and micropore volume after pretreatment confirm the effect of treatments on the samples (Table 1) and the ruptured structure in poplar wood shown on SEM images (Figure 2). The treated samples showed higher surface areas (44.13–56.78 m2/g) and micropore volume (0.0177–0.0288 cm3/g) as compared to those of the control sample (43.97 m2/g and 0.0176 cm3/g) (Table 2). In combination with SEM results, this increasing trend was well correlated with the degree of decomposition of the microstructures of the poplar wood. As expected, the removal of most lignin and hemicelluloses after treatments led to a higher surface area and micropore volume compared to the control sample. Therefore, the substrates with higher surface areas were beneficial to form a more intimate contact between cellulase and cellulose in the samples. This resulted in enhanced concentration of glucose by enzymatic saccharification. It has been reported that effective treatment leads to a higher surface area,56,57 while high pore volume leads to a higher enzyme accessibility.58 Considering the sequential dilute acid–alkali treatments for instance, many holes were formed on the solids surface. This is due to the more complete hemicellulose and lignin removal compared with that of sequential dilute acid–aqueous ammonia treatments (Table 1). Therefore, the highest glucose release was observed for sequential dilute acid–alkali treatments (5% alkali loading).

Table 2. BET Surface Area (m2/g) and Micropore Volume (cm3/g) Obtained from Control and Different Treatments.
treatments surface area (m2/g) micropore volume (cm3/g)
control 43.97 0.0176
dilute acid treatment 44.13 0.0177
dilute acid–5% alkali treatment 56.78 0.0288
dilute acid–3% aqueous ammonia treatment 46.09 0.0185
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2.4. Ultrastructure Changes

2.4.1. Crystallinity Analysis

The crystallinity index (CrI) is the ratio of crystalline to amorphous fraction of lignocellulose. It is an important parameter of enzymatic hydrolysis and is directly related to the accessibility of enzyme to cellulose. Hemicellulose and lignin are amorphous while cellulose is mainly crystalline.59 Crystallinity has often been regarded as a major factor that influences the efficiency of enzymatic hydrolysis.60,61 The measured values of CrI % for control, dilute acid treatment, sequential dilute acid–1, 3, and 5% alkali treatments, and sequential dilute acid–1, 3, and 5% aqueous ammonia treatments were 28.7%, 31.5%, 34.7%, 31.6%, 35.1%, 34.0%, 36.0%, and 33.9%, respectively. The correlation between CrI and the removal of lignin and hemicellulose is positive (Table 1).

The increase in CrI is mostly resulted from the removal or degradation of amorphous components such as hemicellulose, lignin, starch, and protein.61 As shown in Figure 3, for the pretreated samples, the cellulose I structure remained for all samples. After treatments, for sequential dilute acid–3% alkali treatment with the lowest CrI % among all treatments, the pattern is different from those of other treatments. This means that the crystalline cellulose was broken down and hydrolyzed with solution of alkali penetrating into the crystalline cellulose and converted into amorphous which contributed to enzymatic hydrolysis. This study showed that CrI % was proportional to enzymatic hydrolysis, as confirmed by previous studies.60,62 This suggests that CrI % is not the only factor that affects enzymatic hydrolysis.63

Figure 3.

Figure 3

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XRD patterns of control and poplar wood after different treatments.

2.4.2. Py-GC/MS Analysis

For the lignin structure, guaiacyl (G)-, syringyl (S)-, and p-hydroxyphenylpropane (H)- type units are interconnected and constitute a complex three-dimensional polymer.64 Py-GC/MS was used in this study to determine the effect of treatment method on different types of units contained in lignin, as shown in Figure 4 and Table S1. It can be determined from the control pyrolysis data that G and S units mainly constitute the poplar lignin. The ratios of the S, G, and H units contain structural information about lignin. The S/G ratio is usually used as a criterion to investigate deconstruction of lignin.65−67 S/G ratios for control, dilute acid treatment, sequential dilute acid–5% alkali treatment, and sequential dilute acid–3% aqueous ammonia were 2.2, 3.6, 1.9, and 5.1, respectively. Dilute acid treatment led to an increase of the S/G ratios, meaning dilute acid selectively removed of G-lignin. However, sequential dilute acid–alkali treatment tended to remove S-lignin, which was in agreement with previous studies.64 This is the same as that of sequential dilute acid–aqueous ammonia treatment. The S/G ratio of the residual lignin after treatments was negatively proportional to the extent of lignin degradation.67 Therefore, sequential dilute acid–alkali treatments led to higher lignin degradation compared with that of sequential dilute acid–aqueous ammonia treatments. This result explained the mechanism involved in the lignin removal as aforementioned from the level of lignin composition.

Figure 4.

Figure 4

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Py-GC/MS chromatograms of control and poplar wood after different treatments.

2.4.3. FTIR Spectroscopy Analysis

The changes in functional groups affect the properties of biomass and also play a vital role in enzymatic hydrolysis. In order to analyze and determine the changes of functional groups before and after sample treatment, FTIR analysis was conducted. FTIR spectroscopy has been widely used to investigate lignocellulosic functional group alterations caused by various pretreatments.68 The FTIR spectra of control and dilute acid-treated samples are shown in Figure 5. The dilute acid-treated samples showed a decrease in a peak near 1732 cm–1 associated with acetyl groups,69 indicating that the acetyl groups were partially cleaved. These results mean that dilute acid treatment mainly degraded the hemicellulose contained in poplar with little effect on cellulose and lignin. The presence of acetyl groups has long been reported to inhibit enzymatic hydrolysis, and the deacetylation that occurred during dilute acid treatment suggested hemicelluloses hydrolysis that would facilitate the cellulose hydrolysis to sugar conversion.50,51

Figure 5.

Figure 5

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FTIR spectra of control and poplar wood after different treatments.

Sequential dilute acid–alkali and sequential dilute acid–aqueous ammonia were used in this study for further decomposing the biomass structure based on dilute acid-treated poplar. Bands occurring from 1600 and 1500 cm–1 can be attributed to aromatic skeleton vibrations,70 and the increase in intensity in this range occurred in dilute acid-treated sample, meaning that the removal of hemicellulose led to more exposure of the lignin structure. The intensity decreased for sequential dilute acid–alkali-treated samples indicates partial removal of lignin. However, an increase in the intensity occurred for the aqueous ammonia-treated samples. This contradiction can be attributed to the formation of pseudolignin droplets, which show spherical structures and are a lignin-like compound that can be observed on the surface of pretreated biomass.71 It has been extensively reported that the spherical droplets can be formed under severe treatment condition with exiting of ammonia reagents.46−48 This phenomenon is possible when some treatments generate products such as furfural repolymerize and/or lignin and form a lignin-like material named pseudolignin.72 Lignin is a complex three-dimensional polymer, in which guaiacyl-, syringyl-, and p-hydroxyphenylpropane-type units are interconnected.64 The band at 1200 cm–1, associated with guaiacyl in lignin’s chemical structure, decreased especially at sequential dilute acid–3% alkali treatment,70 which means that the lignin was decomposed. More exposure of cellulose resulted from the removal of hemicellulose and lignin through both dilute acid treatment and sequential dilute acid–alkali/aqueous ammonia treatment. This can be confirmed by the decrease in lignin-associated peaks and increase in peak intensity near 1060 cm–1 (CH2 wag.) and 1317 cm–1 (C–O str. mainly of C3–O3H secondary alcohols) that represent the characteristics of cellulose.73,74

2.4.4. Thermogravimetric (TG/DTG) Analysis

As shown in Figure 6, TG and DTG curves showed different patterns. Compared with the control, the initial temperature of degradation for all treatments was higher because hemicellulose was mainly removed from the poplar wood by dilute acid treatment (Table 1). Thermal decomposition temperatures for cellulose, hemicellulose, and lignin were reported to be 315–400 °C, 220–315 °C, and 160–900 °C, respectively.75 For sequential dilute acid–3% alkali treatment, the rate of degradation started increasing from 270 °C, faster than that of dilute acid treatment, which was also faster than control from 310 °C. This is mainly due to the alkali reagent, which can be used for efficiently removing lignin.36,48 The thermal decomposition for lignin started at about 160 °C,75 this is in accordance with the lowest temperature for the thermal decomposition peak for sequential dilute acid–3% alkali treatment. The rest of residue content for sequential dilute acid–3% alkali treatments was the highest, which might be due to the metal elements left in the sequential dilute acid–alkali-treated samples. However, for sequential dilute acid–3% aqueous ammonia treatment, the thermal decomposition temperature was the highest, which is in accordance with the highest temperature for the thermal decomposition peak. CrI % for sequential dilute acid–3% aqueous ammonia-treated poplar was the highest among control, dilute acid treatment, sequential dilute acid–alkali treatments, and sequential dilute acid–aqueous ammonia treatments. This result indicates that samples with high CrI % have high thermal decomposition temperature. For the control, a shoulder appeared on the left side of the thermal decomposition peak at 291–320 °C. This is because there was no dilute acid treatment, so the hemicellulose remained in poplar wood. The temperature range for the shoulder peak (291–320 °C) was generally in agreement with the thermal decomposition temperature range of hemicellulose as reported.75 Therefore, it can be concluded that the existence of hemicellulose led to the appearance of the shoulder peak.

Figure 6.

Figure 6

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TGA and DTG curves of control and poplar wood after different treatments.

The peaks appeared on the figure correspond to the components contained in biomass including water, hemicellulose, cellulose, and lignin, which have been demonstrated previously.76,77 However, in this study, only one peak for each treatment can be observed between 300 and 400 °C, which represent cellulose because the samples had been dried prior to TG analysis, followed by the removal of most of the hemicellulose by dilute acid and that of the lignin by alkaline/ammonia treatment. As a result, it is hard to observe the peaks for hemicellulose and lignin on the figure. For the sequential dilute acid–3% aqueous ammonia treatment, the peak was different from others and appeared at higher temperature. It indicated that this treatment condition impacted the structure of cellulose or reconstruction and led to the highest CrI %.

3. Conclusions

The sequential dilute acid–alkali treatment was more effective in decomposing poplar samples and enhancing its enzymatic hydrolysis compared to dilute acid treatment and dilute acid–aqueous ammonia treatment. The dilute acid treatment as the first step was used to remove 84.9% of hemicellulose to facilitate better lignin access from the poplar sample via the second treatment method. The alkali treatment as the following step was used to remove lignin up to 27.5% that was much better than that of aqueous ammonia treatment (at best 5.6% removal). These results provide insights into multiscale structural changes of poplar biomass under different pretreatments.

4. Materials and Methods

4.1. Substrate and Enzymes

Poplar processing residues as woody biomass were collected from a pilot-scale plant located in the campus of Washington State University in Pullman, WA, USA. The poplar samples were dried at 105 °C in an oven for 48 h and cut, ground, and sieved through 60 mesh size sieving. The resultant poplar samples were stored at 4 °C prior to use. Enzymes of Cellic CTec2 and Cellic HTec2 were obtained from Sigma (cellulase from Aspergillus niger; catalogue no. C1184). The total cellulase activity of commercial cellulase was 100 FPU g–1 based on the filter paper assay.

4.2. Chemical Pretreatments and Enzymatic Hydrolysis of Poplar Samples

Poplar wood was soaked in 7.8% H2SO4 solution (w/v) and heated in an autoclave at 121 °C, 15 psi for 1 h.39 The dilute acid-treated poplar samples were centrifuged at 4000 rpm for 20 min (BIOBASE, TG-16w, CHN). After separation, the samples were soaked in the same volume of deionized water, and occasionally mixed for 1 h. The washed dilute acid-treated poplar samples were dried at 55 °C to constant weight, and some of the samples were reserved for enzymatic hydrolysis. The dried dilute acid-treated poplar samples were then soaked in alkali (NaOH) solution and aqueous ammonia (NH4OH) solution, respectively. The doses of reagent for both solutions were 1%, 3%, and 5% (w/v), which were heated in an autoclave at 121 °C, 15 psi for 30 min; this pretreatment was carried out in a high pressure reactor.40,41 The sequential dilute acid–alkali/aqueous ammonia-treated poplar samples were centrifuged at 4000 rpm for 20 min. After separation, the samples were also soaked in the same volume of deionized water and stirred occasionally for 1 h to remove alkali/aqueous ammonia on the surface of the solids. The washed dilute acid–alkali/aqueous ammonia-treated poplar samples were dried at 55 °C to constant weight and kept under anhydrous conditions for subsequent enzymatic hydrolysis.

The raw and treated poplar samples were subjected to enzymatic hydrolysis at 50 °C for 48 h and at a consistency of 10% (w/v) in 50 mM sodium citrate buffer (pH 4.8).

Next, appropriate amounts of Cellic CTec2 (35 FPU g–1) and similar amount of Cellic HTec2 were added. The flasks were then put in a shaking incubator at 50 °C for 48 h.42 After hydrolysis, supernatants were collected and used for the sugar analysis. Poplar samples without treatment were set as control. Each experiment was repeated three times.

4.3. Analytical Procedures

4.3.1. Chemical Composition Analysis

Monosugars were analyzed using a Dionex ICS-3000 ion chromatography system equipped with a CarboPac TM PA 20 (4 × 50 mm) analytical column and CarboPac TM PA 20 (3 × 30 mm) guard column (Dionex Corporation, CA). According to a previous procedure, the contents of cellulose, hemicellulose, and lignin were determined,39 and the recovery of glucan and xylan was calculated.43

4.3.2. SEM Analysis

The microscope photographs of untreated and pretreated biomass were taken by a SEM (FE-SEM, FEI, 200F) after the samples were sputter-coated with a thin layer of gold. SEM HV was set as 30.0 kV, and the view field was less than 150 μm.

4.3.3. BET Surface Area and Micropore Volume Measurement

BET surface area and porosity were measured with micromeritics TriStar II PLUS (Norcross, GA, USA) using carbon dioxide and nitrogen adsorption isotherms at 273 K and 77.30 K, respectively.

4.3.4. Crystallinity Measurements

Biomass crystallinity was measured using Powder X-ray Diffractometer Siemens D-500 and a copper X-ray source (λ = 0.154 nm).44 The X-ray generator was operated at 35 kV and 25 mA. Scans were collected at 1° per minute from 4 to 45 2θ. The Segal method was used to calculate the sample crystallinity. The degree of crystallinity was calculated as

4.3.4. 1

where Icrys is the overall intensity of the peak at 2θ about 22.9° and Iamor is the intensity at 2θ about 16.9°.

4.3.5. Py-GC/MS Analysis

The pyrolysis processes for both line probe assay (LPA) mutated and the corresponding wild type barley straw were individually performed with a CDS 5000 pyrolysis autosampler (CDS Analytical, Inc., Oxford, PA, USA) attached to a Thermo Trace GC 6890N/MSD 5975B gas chromatography/mass spectrometry system (Agilent Technologies, Inc., Santa Clara, CA, USA). Samples were subjected to the Py-GC/MS. The samples were initially kept briefly in the oven (210 °C) to ensure adequate removal of oxygen prior to pyrolysis and were pyrolyzed by heating nearly instantaneously to 500 °C for 1.0 min. The inlet temperature was maintained at 250 °C. The resulting pyrolysis vapors were separated by means of a 30 m × 0.25 μm inner diameter (5%-phenyl)-methylpolysiloxane nonpolar column, with a split ratio of 50:1. The gas flow rate was 1 mL min–1. Linear heating (3 °C min–1) from 40 to 280 °C was designated for the oven program, and to ensure that no residuals were retained, the oven was held at 280 °C for 10 min. The gas was then sent into a mass spectrometer (Agilent Technologies Inert XL MSD) for analysis.

4.3.6. FTIR Spectroscopy Analysis

After drying, the chemical changes in the structures of untreated and treated samples were measured using a ThermoNicolet Avatar 370 Fourier transform infrared (FTIR) spectrometer in the attenuated total reflectance (SmartPerformer Thermo Electron Corporation Waltham MA, ZnSe crystal). Sixty-four scans were taken for each sample from 4000 to 400 cm–1 at the resolution of 2 cm–1.

4.3.7. Thermogravimetric (TG/DTG) Analysis

The thermogravimetric experiments were conducted using a Mettler-Toledo TGA/SDTA851e (Mettler-Toledo, Inc., Columbus, OH). Five milligrams of samples was put into an alumina pan and vaporized (from 25 to 600 °C, at a heating rate of 10 °C min–1) under a nitrogen atmosphere with a flow rate of 20 mL min–1.

4.4. Statistical Analysis

SPSS 19.0 software was used to determine the standard deviations and whether the observed differences between two or more groups of experimental results were significant. The differences were compared with a p-value of 0.05.

Acknowledgments

This work was partially supported by USDA/NIFA through Hatch Project # WNP0002, Shaanxi Youth Thousand Talents Project (A279021901), Northwest A & F University Young Talent Project (Z111021902), and China Postdoctoral Science Foundation (2015M572601), Research on synergism mechanism of methanogens & carbon carrier in biogas ex-situ purification process.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c03419.

  • Py-GC/MS analysis (PDF)

Author Contributions

F.S., Y.W., and M.D. have equal contribution to this work.

The authors declare no competing financial interest.

Supplementary Material

ao0c03419_si_001.pdf (85.2KB, pdf)

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