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Production of xylooligosaccharides and monosaccharides from hydrogen peroxide-acetic acid-pretreated poplar by two-step enzymatic hydrolysis
Journal Pre-proofs Short Communication Production of xylooligosaccharides and monosaccharides from hydrogen peroxide-acetic acid-pretreated poplar by two-step enzymatic hydrolysis Xixun Hao, Peiyao Wen, Jia Wang, Jinye Wang, Jiaxin You, Junhua Zhang PII: DOI: Reference:
S0960-8524(19)31579-2 https://doi.org/10.1016/j.biortech.2019.122349 BITE 122349
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Bioresource Technology
Received Date: Revised Date: Accepted Date:
18 September 2019 27 October 2019 28 October 2019
Please cite this article as: Hao, X., Wen, P., Wang, J., Wang, J., You, J., Zhang, J., Production of xylooligosaccharides and monosaccharides from hydrogen peroxide-acetic acid-pretreated poplar by two-step enzymatic hydrolysis, Bioresource Technology (2019), doi: https://doi.org/10.1016/j.biortech.2019.122349
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Production of xylooligosaccharides and monosaccharides from hydrogen peroxide-acetic acid-pretreated poplar by two-step enzymatic hydrolysis
Xixun Hao1, Peiyao Wen1, Jia Wang1, Jinye Wang1, Jiaxin You2,3, Junhua Zhang1,2,3
1 College 2Key
of Forestry, Nothwest A&F University, Yangling 712100, China
Laboratory of Forestry Genetics & Biotechnology (Nanjing Forestry University), Ministry of
Education, Nanjing 210037, China 3Jiangsu
Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, College of
Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China
Corresponding author: Junhua ZHANG Tel.: +86-13892883052; fax: +86-29-87082213 E-mail address: [email protected] (J. Zhang)
1
Abstract The severe pretreatment of poplar makes xylan difficult to utilize efficiently. In this work, poplar was pretreated by hydrogen peroxide–acetic acid (HPAC) with H2SO4 as catalyst to remove lignin, and the solid residues were used to produce xylooligosaccharides (XOS) and monosaccharides by two-step xylanase and cellulase treatment. The results indicated that higher H2SO4 concentrations in the HPAC pretreatment of poplar afforded stronger lignin removal ability. An increased XOS yield of 19.8% was obtained from 200 mM H2SO4-catalyzed poplar by xylanase and the XOS purity was high, with a very low xylose/XOS ratio of 0.14. Higher glucose (75.2%) and xylose (61.4%) yields were obtained from the HPAC-pretreated poplar using 50 mM H2SO4 as catalyst. Finally, 16.9 g XOS and 296.4 g glucose were produced from 1 kg poplar by xylanase and cellulase. This study provides a method for producing functional XOS and monosaccharides from poplar using a simple reduced-pollution strategy. Keywords: Poplar; Xylooligosaccharides; Monosaccharides; Hydrogen peroxide–acetic acid pretreatment; Xylanase; Cellulase
2
1. Introduction Poplar species are distributed globally, especially in Asia, North America, and parts of Europe. They are considered promising biomass feedstocks for biofuels and high-value-added products owing to their fast growth, short vegetative cycle, drought tolerance, and resistance to pests and insects, among other properties (Sannigrahi et al., 2009; Wen et al., 2019). Pretreatment of poplar is a prerequisite to its highly efficient bioconversion into fermentable sugars, which are substrates for biofuels and biochemicals production. Owing to the complex structure of lignin and high recalcitrance of poplar, various pretreatments have been applied to poplar, including dilute acid/alkali, ammonia fiber expansion, and hydrothermal pretreatments (Esteghlalian et al.,1997; Kumar et al., 2009; Maria et al., 2003., Huang et al., 2016; Huang et al., 2019a). Most of these pretreatments are severe, making xylan in poplar hard to utilize efficiently (Negro et al, 2003; Zhang et al., 2014). Xylan, as the second most abundant polysaccharide on earth, is an important component of lignocelluloses, which can be used to produce xylooligosaccharides (XOS) (Aachary et al., 2011; Amorim et al., 2019). XOS can selectively feed beneficial bacteria, such as bifidobacteria and lactobacilli, within the digestive tract and are emerging as potential novel sources of prebiotics (Aachary et al.; 2011; Huang et al., 2016; Huang et al., 2019b). Coproduction of XOS and monosaccharides from biomass has been developed using hydrothermal and acetic acid pretreatments with cellulase hydrolysis (Chen et al., 2018; Chen et al., 2019; Huang et al., 2018; Wen et al., 2019; Zhang et al., 2017). However, a large amount of xylan is converted into xylose during these pretreatments, which decreases the XOS yield and increases the subsequent purification cost. XOS production by xylanase hydrolysis is a more environmentally friendly approach, with advantages of less treatment step and relatively high purity because of avoiding xylan extraction process and the use of -xylosidae-free/less endoxylanase (Amorim et al., 2019; Bragatto et al., 2013; Kumar and Satyanarayana, 2015). XOS production from isolated xylan by xylanase has been extensively investigated (Aachary et al., 2011; Amorim et al., 2019; Romero-Fernandez et al., 2018). However, the xylan isolation process is usually conducted using chemicals at relatively high temperatures, which can increase the XOS production cost and cause environmental pollution (Jayapal 3
et al., 2013). Therefore, XOS production from pretreated xylan-containing biomass by endoxylanase before cellulase hydrolysis avoids xylan isolation process (Bragatto et al., 2013), showing potential applications in the coproduction of XOS and monosaccharides from lignocelluloses. To our knowledge, the coproduction of XOS and monosaccharides from poplar using xylanase and cellulase has not been reported previously. Previous reports have confirmed that hydrogen peroxide–acetic acid (HPAC) pretreatment is an effective approach to selectively remove lignin in biomass and to retain most of carbohydrate constituents (Bragatto et al., 2013; Tan et al., 2010; Wen et al., 2019), which might benefit to following XOS production and cellulase hydrolysis. In addition, the presences of H2SO4 in HPAC can catalyst the formation of preacetic acid, a strong oxidizing reagent, in situ and consequently enhance the removal of lignin by cleaving -aryl ether bonds (Lawrence et al, 1978; Wi et al., 2015; Zhao et al., 2009). In this study, poplar was pretreated with HPAC to selectively remove lignin, and xylan in the pretreated poplar was hydrolyzed by xylanase to produce XOS. The effects of pretreatment conditions and xylanase dosage on the XOS yields were investigated. The poplar solid residue after xylanase hydrolysis was further hydrolyzed by cellulases to produce monosaccharides. 2. Materials and methods 2.1 Materials Poplar used in this work was collected from Suqian, Jiangsu Province, China, and was generously provided by Prof. Yong Xu from Nanjing Forestry University, China. The raw material was milled and passed through an 80-mesh sieve (≤0.18mm), and had a moisture content of 9.40%. The glucan, xylan, and acid-insoluble lignin contents in the raw poplar were 42.1%, 15.8%, and 25.7%, respectively (Table 1), measured as described in the National Renewable Energy Laboratory protocol (Sluiter et al., 2012). Xylobiose, xylotriose, xylotetraose, xylopentaose, and xylohexaose were purchased from Megazyme (Wicklow, Ireland). 2.2 Enzymes Cellic CTec2, purchased from Novozymes A/S (Bagsvaerd, Denmark), was used as cellulase preparation (CEL). Xylanase (XYL, Pentopan Mono BG) with an activity of 23,000 nkat/mL, 4
according to the method of Bailey et al. (1992), was purchased from Novo Nordisk A/S (Bagsvaerd, Denmark). CTec2 had an activity of 123.0 filter paper units (FPU)/mL (176.2 mg protein/mL), according to the protocol of the International Union of Pure and Applied Chemistry (Ghose, 2009). Protein was quantified by the Lowry method using bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) as the standard (Lowry et al., 1951). 2.3 HPAC pretreatment HPAC pretreatment was performed in a sealed Teflon-lined stainless-steel autoclave (HT-100H-316L, Anhui Kemi Machinery Technology Co., Ltd., Anhui, China) heated by a constant temperature oil bath (HH-SB, Jinhua Wenhua Equipment and Instrument Co., Ltd., Zhejiang, China). Poplar was pretreated with hydrogen peroxide (30%, w/w) and acetic acid (99%, w/w) in a 1:1(v/v) ratio at 60 °C for 2 h, with a solid–liquid ratio of 1:10 (w/v) (Wen et al., 2019). To investigate the effect of sulfuric acid concentration on pretreatment, H2SO4 (50 or 200 mM) was added as catalyst. After pretreatment, the solids were recovered by filtration and washed with distilled water until the washing fluid reached a neutral pH value. The solids were then air-dried and stored at 20°C for chemical composition analysis and enzymatic hydrolysis. 2.4 XOS production by xylanase The hydrolysis of HPAC-pretreated poplar by XYL was performed in 10-mL test tubes (601051-1, Biosharp, Hefei, China) with a working volume of 3 mL containing 0.02% NaN3 in 50 mM sodium citrate buffer (pH 5.0) at 50 °C with shaking at 200 rpm using an orbital shaker. The HPAC-pretreated poplar had a consistency of 5% (w/v) and XYL loadings of 1,000, 5,000, and 10,000 nkat/g dry matter (DM). Two replicate test samples were withdrawn at 6, 12 and 24 h, and boiled for 10 min to stop enzymatic hydrolysis. After cooling, the samples were separated by centrifugation (10,000g, 10 min), and XOS and xylose in the supernatants were analyzed. 2.5 Glucose production by cellulase After hydrolysis of HPAC-pretreated poplar by XYL, the solid residues were further hydrolyzed by CEL in the same hydrolysis system with a working volume of 2 mL and a solid consistency of 5% (w/v). The CEL dosage was 10 mg/g DM. Samples were withdrawn at 6, 24 and 48 h for glucose and 5
xylose analysis. Two replicate tests were conducted and average values are reported. 2.6 Carbohydrate analysis The concentration of XOS (DP 2-6) was analyzed using high performance anion exchange chromatography coupled with pulsed amperometric detection system according to the method reported by Zhang et al. (2017). The glucose and xylose concentrations in the supernatants were determined using an Agilent 1260 infinity HPLC system, as described previously (Yang et al., 2017). 2.7 Calculations Calculation of the solid recovery and removal of poplar components in HPAC pretreatment, and the glucose and xylose yields in the hydrolysates of HPAC-pretreated poplar, were performed according to our previous report (Wen et al., 2019). The XOS (DP 2–6) yield in the hydrolysates of HPAC-pretreated poplar produced by XYL was calculated using equation (1): XOS (DP2 ― 6) in hydrolysate
XOS yield (%) = Total amount of xylan in
pretreated poplar
× 100
(1)
In the hydrolysis of HPAC-pretreated poplar by XYL, the ratio of xylose to XOS (xylose/XOS) was calculated using equation (2): Xylose in hydrolysate by XYL
(2)
Xylose/XOS = XOS (DP2 ― 6) in hydrolysate by XYL
The glucose and xylose yields from the hydrolysis of HPAC-pretreated poplar were calculated by the following equations (3) and (4): Glucose in enzymatic hydrolysate × 0.9
Glucose yield (%) = Theoretical amount of glucan in
pretreated poplar
Xylose in enzymatic hydrolysate × 0.88
Xylose yield (%) = Theoretical amount of xylan in
pretreated poplar
× 100
× 100
(3)
(4)
3. Results and discussion 3.1 Chemical compositions of HPAC-pretreated poplar In our previous work, HPAC pretreatment of poplar with relatively low HAPC concentration 6
(60%) at low temperature (60°C) shows a very good delignifiability with a lignin removal of 70.0% and a low carbohydrate removal (˂5%) (Wen et al., 2019). In this work, the effect of sulfuric acid concentration on HPAC pretreatment of poplar with 60% HPAC at 60°C was investigated (Table 1). The glucan, xylan, and lignin contents in non-pretreated poplar were 42.1%, 15.8%, and 25.7%, respectively. When poplar was pretreated using 60% HPAC with 50 mM or 200 mM H2SO4 as catalyst, most of glucan was retained and the lignin content decreased from 25.7% to 11.8% and 5.2%, respectively. Bragatto et al. (2013) reported that the lignin content of sugarcane bagasse decreased from 20.9% to 2.1% and 0.8% when pretreated with 1:1 (v/v) mixture of acetic acid and hydrogen peroxide, respectively. Wi et al. (2015) reported that the lignin contents of pine wood and oak wood decreased from 32.8% and 26.3% to 0.9% and 1.9%, respectively, when pretreated with 100% HPAC at 80 °C for 2 h. These results indicated strong delignification by HPAC in the poplar pretreatment, which was in good agreement with previous reports. Wen et al. (2019) reported that 70% lignin removal was observed in 60% HPAC-pretreatment of acetic acid-pretreated poplar with 100 mM H2SO4 as catalyst, which was higher than that (65.4%) with 50 mM H2SO4 and lower than that (88.8%) with 200 mM H2SO4 (Table 1) as catalysts. In this study, very low glucan removal (1%–7%) was obtained from 60% HPAC-pretreated poplar with 50–200 mM H2SO4 as catalyst (Table 1). These results suggested that the H2SO4 concentration in HPAC pretreatment played an important role in lignin removal, with a higher H2SO4 concentration showing stronger lignin removal ability. 3.2 XOS production from HPAC-pretreated poplar by xylanase The commercial xylanase preparation (Pentopan Mono BG) was produced from Aspergillus oryzae, which is a typical endoxylanase that might be suitable for XOS production. In this study, the dosage of xylanase in XOS production from HPAC-pretreated poplar was investigated, with the results showing that the XOS yield increased with increasing XYL dosage from 1,00010,000 nkat/g DM (Fig. 1). Furthermore, higher XOS yields were obtained from HPAC-pretreated poplar prepared using 200 mM H2SO4 as catalyst than that with 50 mM H2SO4. Within the tested conditions, the highest XOS yield of 19.8% was obtained from 60% HPAC with 200 mM H2SO4 using 10,000 nkat XYL per gram DM for 24 h (Fig. 1F). Xylobiose, xylotriose and xylotetraose yields were 5.5%, 4.6% and 6.5%, 7
respectively, which were the main oligosaccharides in XOS prepared from poplar using XYL, and the yields of individual XOS components increased with increasing hydrolysis time within 24 h as shown in Supporting Information. Higher yields of xylobiose, xylotriose, and xylotetraose, and lower yields of xylopentaose and xylohexaose, were obtained from HPAC-pretreated poplar using 200 mM H2SO4 as catalyst compared with those using 50 mM H2SO4 as catalyst. As expected, the xylose yields were very low (less than 3%) and the highest ratio of xylose to XOS (xylose/XOS) was only 0.14 after hydrolysis for 24 h (Fig. 1). Bragatto et al. reported (2013) the production of XOS from HPAC-pretreated sugarcane bagasse, with higher amounts of xylose than XOS produced by enzymatic hydrolysis, indicating a xylose/XOS ratio of above 1.0. Huang et al. (2018) reported that acetic acid pretreatment of poplar produced 63.1 g of XOS and 45.8 g of xylose from 1 kg of poplar, with a xylose/XOS ratio of 0.73. Wen et al. (2019) reported that acetic acid pretreatment of poplar produced 69 g of XOS and 46 g of xylose, with a xylose/XOS ratio of 0.67. In this study, although a lower amount of XOS (16.9 g of XOS from 1 kg poplar) was produced from HPAC-pretreated poplar by XYL, a very low xylose/XOS ratio of 0.14 was afforded (Fig. 1C), indicating that the XOS produced was of relatively high purity and that HPAC-pretreated poplar was suitable for XOS production by XYL. 3.3 Glucose production from poplar after HPAC-pretreatment and XYL hydrolysis After XYL hydrolysis of HPAC-pretreated poplar, xylan in poplar was hydrolyzed to XOS and xylose (Fig. 1), and the solid residues were further hydrolyzed by CEL to produce monosaccharides (Fig. 2). Higher glucose (75.2%) and xylose (61.4%) yields were obtained from poplar pretreated using 60% HPAC with 50 mM H2SO4 as catalyst than that (33.3% glucose yield and 43.4% xylose yield) with 200 mM H2SO4 as catalyst using 10 mg CEL/g DM. Furthermore, 1.17 mg/mL and 1.02 mg/mL cellobiose were produced by 60% HPAC with 50 and 200 mM H2SO4 as catalysts, respectively. This cellobiose accumulation indicated that the enzymatic activity of -glucosidase in the CEL preparation was relatively low. As cellobiose is a strong inhibitor of cellobiohydrolase (Haven and Jørgensen, 2013), cellobiose accumulation decreased the cellulolytic hydrolysis capacity. To enhance the glucose yield, it was necessary to add more -glucosidase to convert cellobiose into 8
glucose. Surprisingly, compared with 200 mM H2SO4-catalyzed poplar with a lower lignin content (5.2%), 50 mM H2SO4-catalyzed poplar with a higher lignin content (11.8%) afforded a higher hydrolysis yield (Fig. 2 and Table 1). This phenomenon might be attributed to the more severe acid pretreatment resulting in higher inhibitory effect of lignin in poplar and decreased the accessibility of cellulase to cellulose in poplar, which has been reported previously (Nakagame et al. 2010; Rahikainen et al., 2011). These data suggested that H2SO4 concentrations above 200 mM might not be suitable for monosaccharide production from poplar, despite exhibiting stronger delignification ability (Fig. 2 and Table 1). 3.4 Mass balance Under the tested conditions, the highest XOS yield was 19.8% from xylan in the HPAC-pretreated poplar (60%, 200 mM H2SO4, 10,000 nkat XYL/g DM, Fig. 1), with 14.2 g of XOS produced from 1 kg of poplar by XYL (Fig. 3). For poplar pretreated using 60% HPAC with 50 mM H2SO4 as catalyst, 16.9 g of XOS was produced from 1 kg of poplar by XYL. After enzymatic hydrolysis of poplar by CEL, less monosaccharides (137.5 g of glucose; 31.2 g of xylose) were produced from poplar pretreated using 200 mM H2SO4 as catalyst compared with that pretreated using 50 mM H2SO4 as catalyst (296.4 g of glucose; 78.6 g of xylose). These results indicated that the poplar pretreated using 60% HPAC with 50 mM H2SO4 was more suitable for XOS and monosaccharide production (Fig, 3). Lai et al. (2019) reported that 73 g XOS and 356 g glucose was obtained from 1 kg poplar. Wen et al. (2019) reported that 69 g XOS and 403 g glucose was obtained from 1 kg poplar. In their work, acetic acid pretreatment was used to produce XOS from poplar and high XOS yields with relatively low purity of XOS were obtained (Lai et al., 2019; Wen et al., 2019). Lee et al. (2017) reported that 312 g glucose was produced from 1 kg poplar pretreated by hot compressed water and peracetic acid. In this study, a two-step enzymatic hydrolysis was proposed for the coproduction of XOS and monosaccharides from HPAC-pretreated poplar. Compared with the conventional XOS production process, consisting of xylan extraction, xylan hydrolysis, and cellulose-rich solid hydrolysis, this method showed many advantages, including the high purity of XOS produced, a simple process with 9
easy handling, and reduced pollution. Notably, the XOS yield in the hydrolysis of HPAC-pretreated poplar by 10,000 nkat XYL/g DM was limited (13.6%–19.8%). The data in this study (Fig. 1) indicated that XOS production from poplar could be improved by optimizing the pretreatment, increasing the XYL dosage, extending the hydrolysis time, or selecting an efficient xylanase. Furthermore, the effects of HPAC pretreatment and the role of poplar cellulose and lignin in XOS production need to be further investigated in future. 4. Conclusions HPAC-pretreated poplar showed efficient XOS and glucose production via a two-step enzymatic hydrolysis. Poplar pretreated using 60% HPAC with 50 mM H2SO4 as catalyst was more suitable for this process, producing 16.9 g of XOS and 296.4 g of glucose from 1 kg of poplar using XYL and CEL. Pretreatment with 200 mM H2SO4 resulted in lower amounts of XOS and glucose owing to a low solid recovery (55.7%). XOS produced from HPAC-pretreated poplar by XYL showed a very high purity. This study provides an alternative approach to the production of functional oligosaccharides and fermentable sugars from poplar using a simple and less polluting strategy. Acknowledgements This work was supported by the National Key R&D Program of China (No. 2017YFD0601001) and the National Natural Science Foundation of China (No. 31670598, 31270622). Appendix A. Supplementary data E-supplementary data to this article can be found online. References 1.
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Figure legends: Fig. 1 Production of XOS from HPAC-pretreated poplar (5%, w/v) by XYL (1,000-10,000 nkat/g DM) at pH 5.0 and 50 °C for 24 h. HPAC pretreatment was performed by 60% HPAC with 50 and 200 mM H2SO4 as catalyst, respectively. Error bars represent standard errors. Fig. 2 Enzymatic hydrolysis of HPAC-pretreated poplar (5%, w/v) by 10 mg CTec2/g DM at 50 °C for 48 h. Error bars represent standard errors. Fig. 3 Mass balance of XOS and monosaccharide production from HPAC-pretreated poplar by XYL and CEL. Error bars represent the standard errors. Highlights:
Two-step enzymatic hydrolysis was proposed for XOS and monosaccharide production.
H2SO4 concentration in HPAC pretreatment played an important role in lignin removal.
Poplar pretreated with 50 mM H2SO4 improved XOS and monosaccharide production.
1 kg poplar could produce 16.9 g XOS, 296.4 g glucose and 78.6 g xylose.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
13
Fig. 1
5
12 Time (h)
18
0.6 10 0.4 5
Yield (%)
Xylose/XOS
1.0 6 12 Xylose Time (h） XOS Xylose/XOS
24 0.8 0.6
10 0.4
12 Time (h）
(E) 200-60% HPAC;
18
24
1.00
5,000 nkat/g
Xylose XOS Xylose/XOS
0.80 0.60
10 0.40 0.20 1.0
(F) 200-60% HPAC; 10,000 nkat/g
20 0
0.0 18
6
5
0.2 (C) 50-60% HPAC; 10,000 nkat/g
0.0
15
0 Xylose / XOS Yield (%)
Yield (%)
0.8
15
Xylose
6
XOS
12 Time （h)
0.00 18
24
0.8
Xylose/XOS
0.6 10 0.4
14
5
0.2
0
0.0 0
6
12 Time (h)
18
24
Fig. 2
0.2
20
1.0
XOS
Yield (%）
5
24
Xylose
15
0.4
0
Xylose / XOS
6
(B) 50-60% HPAC; 5,000 nkat/g
0
10
0
0.0 0
20 0
0.6
0.2
0
15
0.8 Xylose / XOS
0.4
Xylose XOS Xylose/XOS
15 Yield (%)
10
1.0
(D) 200-60% HPAC; 1,000 nkat/g
Xylose / XOS
0.8 0.6
20
20
5
0.2
0
0.0 0
6
12 Time (h)
18
24
Xylose / XOS
Xylose XOS Xylose/XOS
15 Yield (%)
1.0
(A) 50-60% HPAC; 1,000 nkat/g
Xylose / XOS
20
Fig.
3
1 kg Poplar Glucan 421.4 g
HPAC
H2SO4
Glucan (%)
Xylan 157.9 g Xylan (%)
Lignin (%)
Solid
HPAC pretreatment
HPAC pretreatment
50 mM H2SO4, 60% HPAC, 60 ˚C
200 mM H2SO4, 60% HPAC, 60 ˚C
752.8 g HPAC-pretreated poplar
557.2 g HPAC-pretreated poplar
Glucan 394.2 g
Glucan 413.3 g
Xylan 124 g
Xylan 71.9 g
Lignin 88.9 g
Lignin 28.9 g
Others 145.7 g
Others 43.1 g
Xylanase hydrolysis
Xylanase hydrolysis
10,000 nkat /g DM, 24 h
10,000 nkat /g DM, 24 h
Hydrolysate liquid
Hydrolysate liquid
XOS 16.9 g
XOS 14.2 g
Xylose 2.4 g
Xylose 1.8 g
Xylan
Lignin
Cellulase hydrolysis
Cellulase hydrolysis 10 mg/g DM, 48 h
10 mg/g DM, 48 h
Hydrolysate liquid
Hydrolysate liquid
Glucose 296.4 g
Glucose 137.5 g
Xylose 76.2 g
Xylose 31.2 g
Cellobiose 18.1 g
Cellobiose 14.3 g
Xylose Table 1
Glucan
Lignin 256.6 g
Xylose
Chemical compositions of poplar pretreated by 60%-100% HPAC with 50200 mM H2SO4 as catalyst. The derivations are standard errors of three independent experiment.
15
(mM)
recovery
removal
removal
removal
(%)
(%)
(%)
(%)
None
42.1±0.6
15.8±0.2
25.7±0.4
－
－
－
60%
50
52.4±1.6
16.5±0.9
11.8±0.7
75.3
6.5
21.5
65.4
60%
200
74.2±1.4
12.9±0.1
5.2±0.3
55.7
1.9
54.4
88.8
16
－
S0960-8524(19)31579-2 https://doi.org/10.1016/j.biortech.2019.122349 BITE 122349
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
18 September 2019 27 October 2019 28 October 2019
Please cite this article as: Hao, X., Wen, P., Wang, J., Wang, J., You, J., Zhang, J., Production of xylooligosaccharides and monosaccharides from hydrogen peroxide-acetic acid-pretreated poplar by two-step enzymatic hydrolysis, Bioresource Technology (2019), doi: https://doi.org/10.1016/j.biortech.2019.122349
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© 2019 Published by Elsevier Ltd.
Production of xylooligosaccharides and monosaccharides from hydrogen peroxide-acetic acid-pretreated poplar by two-step enzymatic hydrolysis
Xixun Hao1, Peiyao Wen1, Jia Wang1, Jinye Wang1, Jiaxin You2,3, Junhua Zhang1,2,3
1 College 2Key
of Forestry, Nothwest A&F University, Yangling 712100, China
Laboratory of Forestry Genetics & Biotechnology (Nanjing Forestry University), Ministry of
Education, Nanjing 210037, China 3Jiangsu
Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, College of
Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China
Corresponding author: Junhua ZHANG Tel.: +86-13892883052; fax: +86-29-87082213 E-mail address: [email protected] (J. Zhang)
1
Abstract The severe pretreatment of poplar makes xylan difficult to utilize efficiently. In this work, poplar was pretreated by hydrogen peroxide–acetic acid (HPAC) with H2SO4 as catalyst to remove lignin, and the solid residues were used to produce xylooligosaccharides (XOS) and monosaccharides by two-step xylanase and cellulase treatment. The results indicated that higher H2SO4 concentrations in the HPAC pretreatment of poplar afforded stronger lignin removal ability. An increased XOS yield of 19.8% was obtained from 200 mM H2SO4-catalyzed poplar by xylanase and the XOS purity was high, with a very low xylose/XOS ratio of 0.14. Higher glucose (75.2%) and xylose (61.4%) yields were obtained from the HPAC-pretreated poplar using 50 mM H2SO4 as catalyst. Finally, 16.9 g XOS and 296.4 g glucose were produced from 1 kg poplar by xylanase and cellulase. This study provides a method for producing functional XOS and monosaccharides from poplar using a simple reduced-pollution strategy. Keywords: Poplar; Xylooligosaccharides; Monosaccharides; Hydrogen peroxide–acetic acid pretreatment; Xylanase; Cellulase
2
1. Introduction Poplar species are distributed globally, especially in Asia, North America, and parts of Europe. They are considered promising biomass feedstocks for biofuels and high-value-added products owing to their fast growth, short vegetative cycle, drought tolerance, and resistance to pests and insects, among other properties (Sannigrahi et al., 2009; Wen et al., 2019). Pretreatment of poplar is a prerequisite to its highly efficient bioconversion into fermentable sugars, which are substrates for biofuels and biochemicals production. Owing to the complex structure of lignin and high recalcitrance of poplar, various pretreatments have been applied to poplar, including dilute acid/alkali, ammonia fiber expansion, and hydrothermal pretreatments (Esteghlalian et al.,1997; Kumar et al., 2009; Maria et al., 2003., Huang et al., 2016; Huang et al., 2019a). Most of these pretreatments are severe, making xylan in poplar hard to utilize efficiently (Negro et al, 2003; Zhang et al., 2014). Xylan, as the second most abundant polysaccharide on earth, is an important component of lignocelluloses, which can be used to produce xylooligosaccharides (XOS) (Aachary et al., 2011; Amorim et al., 2019). XOS can selectively feed beneficial bacteria, such as bifidobacteria and lactobacilli, within the digestive tract and are emerging as potential novel sources of prebiotics (Aachary et al.; 2011; Huang et al., 2016; Huang et al., 2019b). Coproduction of XOS and monosaccharides from biomass has been developed using hydrothermal and acetic acid pretreatments with cellulase hydrolysis (Chen et al., 2018; Chen et al., 2019; Huang et al., 2018; Wen et al., 2019; Zhang et al., 2017). However, a large amount of xylan is converted into xylose during these pretreatments, which decreases the XOS yield and increases the subsequent purification cost. XOS production by xylanase hydrolysis is a more environmentally friendly approach, with advantages of less treatment step and relatively high purity because of avoiding xylan extraction process and the use of -xylosidae-free/less endoxylanase (Amorim et al., 2019; Bragatto et al., 2013; Kumar and Satyanarayana, 2015). XOS production from isolated xylan by xylanase has been extensively investigated (Aachary et al., 2011; Amorim et al., 2019; Romero-Fernandez et al., 2018). However, the xylan isolation process is usually conducted using chemicals at relatively high temperatures, which can increase the XOS production cost and cause environmental pollution (Jayapal 3
et al., 2013). Therefore, XOS production from pretreated xylan-containing biomass by endoxylanase before cellulase hydrolysis avoids xylan isolation process (Bragatto et al., 2013), showing potential applications in the coproduction of XOS and monosaccharides from lignocelluloses. To our knowledge, the coproduction of XOS and monosaccharides from poplar using xylanase and cellulase has not been reported previously. Previous reports have confirmed that hydrogen peroxide–acetic acid (HPAC) pretreatment is an effective approach to selectively remove lignin in biomass and to retain most of carbohydrate constituents (Bragatto et al., 2013; Tan et al., 2010; Wen et al., 2019), which might benefit to following XOS production and cellulase hydrolysis. In addition, the presences of H2SO4 in HPAC can catalyst the formation of preacetic acid, a strong oxidizing reagent, in situ and consequently enhance the removal of lignin by cleaving -aryl ether bonds (Lawrence et al, 1978; Wi et al., 2015; Zhao et al., 2009). In this study, poplar was pretreated with HPAC to selectively remove lignin, and xylan in the pretreated poplar was hydrolyzed by xylanase to produce XOS. The effects of pretreatment conditions and xylanase dosage on the XOS yields were investigated. The poplar solid residue after xylanase hydrolysis was further hydrolyzed by cellulases to produce monosaccharides. 2. Materials and methods 2.1 Materials Poplar used in this work was collected from Suqian, Jiangsu Province, China, and was generously provided by Prof. Yong Xu from Nanjing Forestry University, China. The raw material was milled and passed through an 80-mesh sieve (≤0.18mm), and had a moisture content of 9.40%. The glucan, xylan, and acid-insoluble lignin contents in the raw poplar were 42.1%, 15.8%, and 25.7%, respectively (Table 1), measured as described in the National Renewable Energy Laboratory protocol (Sluiter et al., 2012). Xylobiose, xylotriose, xylotetraose, xylopentaose, and xylohexaose were purchased from Megazyme (Wicklow, Ireland). 2.2 Enzymes Cellic CTec2, purchased from Novozymes A/S (Bagsvaerd, Denmark), was used as cellulase preparation (CEL). Xylanase (XYL, Pentopan Mono BG) with an activity of 23,000 nkat/mL, 4
according to the method of Bailey et al. (1992), was purchased from Novo Nordisk A/S (Bagsvaerd, Denmark). CTec2 had an activity of 123.0 filter paper units (FPU)/mL (176.2 mg protein/mL), according to the protocol of the International Union of Pure and Applied Chemistry (Ghose, 2009). Protein was quantified by the Lowry method using bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) as the standard (Lowry et al., 1951). 2.3 HPAC pretreatment HPAC pretreatment was performed in a sealed Teflon-lined stainless-steel autoclave (HT-100H-316L, Anhui Kemi Machinery Technology Co., Ltd., Anhui, China) heated by a constant temperature oil bath (HH-SB, Jinhua Wenhua Equipment and Instrument Co., Ltd., Zhejiang, China). Poplar was pretreated with hydrogen peroxide (30%, w/w) and acetic acid (99%, w/w) in a 1:1(v/v) ratio at 60 °C for 2 h, with a solid–liquid ratio of 1:10 (w/v) (Wen et al., 2019). To investigate the effect of sulfuric acid concentration on pretreatment, H2SO4 (50 or 200 mM) was added as catalyst. After pretreatment, the solids were recovered by filtration and washed with distilled water until the washing fluid reached a neutral pH value. The solids were then air-dried and stored at 20°C for chemical composition analysis and enzymatic hydrolysis. 2.4 XOS production by xylanase The hydrolysis of HPAC-pretreated poplar by XYL was performed in 10-mL test tubes (601051-1, Biosharp, Hefei, China) with a working volume of 3 mL containing 0.02% NaN3 in 50 mM sodium citrate buffer (pH 5.0) at 50 °C with shaking at 200 rpm using an orbital shaker. The HPAC-pretreated poplar had a consistency of 5% (w/v) and XYL loadings of 1,000, 5,000, and 10,000 nkat/g dry matter (DM). Two replicate test samples were withdrawn at 6, 12 and 24 h, and boiled for 10 min to stop enzymatic hydrolysis. After cooling, the samples were separated by centrifugation (10,000g, 10 min), and XOS and xylose in the supernatants were analyzed. 2.5 Glucose production by cellulase After hydrolysis of HPAC-pretreated poplar by XYL, the solid residues were further hydrolyzed by CEL in the same hydrolysis system with a working volume of 2 mL and a solid consistency of 5% (w/v). The CEL dosage was 10 mg/g DM. Samples were withdrawn at 6, 24 and 48 h for glucose and 5
xylose analysis. Two replicate tests were conducted and average values are reported. 2.6 Carbohydrate analysis The concentration of XOS (DP 2-6) was analyzed using high performance anion exchange chromatography coupled with pulsed amperometric detection system according to the method reported by Zhang et al. (2017). The glucose and xylose concentrations in the supernatants were determined using an Agilent 1260 infinity HPLC system, as described previously (Yang et al., 2017). 2.7 Calculations Calculation of the solid recovery and removal of poplar components in HPAC pretreatment, and the glucose and xylose yields in the hydrolysates of HPAC-pretreated poplar, were performed according to our previous report (Wen et al., 2019). The XOS (DP 2–6) yield in the hydrolysates of HPAC-pretreated poplar produced by XYL was calculated using equation (1): XOS (DP2 ― 6) in hydrolysate
XOS yield (%) = Total amount of xylan in
pretreated poplar
× 100
(1)
In the hydrolysis of HPAC-pretreated poplar by XYL, the ratio of xylose to XOS (xylose/XOS) was calculated using equation (2): Xylose in hydrolysate by XYL
(2)
Xylose/XOS = XOS (DP2 ― 6) in hydrolysate by XYL
The glucose and xylose yields from the hydrolysis of HPAC-pretreated poplar were calculated by the following equations (3) and (4): Glucose in enzymatic hydrolysate × 0.9
Glucose yield (%) = Theoretical amount of glucan in
pretreated poplar
Xylose in enzymatic hydrolysate × 0.88
Xylose yield (%) = Theoretical amount of xylan in
pretreated poplar
× 100
× 100
(3)
(4)
3. Results and discussion 3.1 Chemical compositions of HPAC-pretreated poplar In our previous work, HPAC pretreatment of poplar with relatively low HAPC concentration 6
(60%) at low temperature (60°C) shows a very good delignifiability with a lignin removal of 70.0% and a low carbohydrate removal (˂5%) (Wen et al., 2019). In this work, the effect of sulfuric acid concentration on HPAC pretreatment of poplar with 60% HPAC at 60°C was investigated (Table 1). The glucan, xylan, and lignin contents in non-pretreated poplar were 42.1%, 15.8%, and 25.7%, respectively. When poplar was pretreated using 60% HPAC with 50 mM or 200 mM H2SO4 as catalyst, most of glucan was retained and the lignin content decreased from 25.7% to 11.8% and 5.2%, respectively. Bragatto et al. (2013) reported that the lignin content of sugarcane bagasse decreased from 20.9% to 2.1% and 0.8% when pretreated with 1:1 (v/v) mixture of acetic acid and hydrogen peroxide, respectively. Wi et al. (2015) reported that the lignin contents of pine wood and oak wood decreased from 32.8% and 26.3% to 0.9% and 1.9%, respectively, when pretreated with 100% HPAC at 80 °C for 2 h. These results indicated strong delignification by HPAC in the poplar pretreatment, which was in good agreement with previous reports. Wen et al. (2019) reported that 70% lignin removal was observed in 60% HPAC-pretreatment of acetic acid-pretreated poplar with 100 mM H2SO4 as catalyst, which was higher than that (65.4%) with 50 mM H2SO4 and lower than that (88.8%) with 200 mM H2SO4 (Table 1) as catalysts. In this study, very low glucan removal (1%–7%) was obtained from 60% HPAC-pretreated poplar with 50–200 mM H2SO4 as catalyst (Table 1). These results suggested that the H2SO4 concentration in HPAC pretreatment played an important role in lignin removal, with a higher H2SO4 concentration showing stronger lignin removal ability. 3.2 XOS production from HPAC-pretreated poplar by xylanase The commercial xylanase preparation (Pentopan Mono BG) was produced from Aspergillus oryzae, which is a typical endoxylanase that might be suitable for XOS production. In this study, the dosage of xylanase in XOS production from HPAC-pretreated poplar was investigated, with the results showing that the XOS yield increased with increasing XYL dosage from 1,00010,000 nkat/g DM (Fig. 1). Furthermore, higher XOS yields were obtained from HPAC-pretreated poplar prepared using 200 mM H2SO4 as catalyst than that with 50 mM H2SO4. Within the tested conditions, the highest XOS yield of 19.8% was obtained from 60% HPAC with 200 mM H2SO4 using 10,000 nkat XYL per gram DM for 24 h (Fig. 1F). Xylobiose, xylotriose and xylotetraose yields were 5.5%, 4.6% and 6.5%, 7
respectively, which were the main oligosaccharides in XOS prepared from poplar using XYL, and the yields of individual XOS components increased with increasing hydrolysis time within 24 h as shown in Supporting Information. Higher yields of xylobiose, xylotriose, and xylotetraose, and lower yields of xylopentaose and xylohexaose, were obtained from HPAC-pretreated poplar using 200 mM H2SO4 as catalyst compared with those using 50 mM H2SO4 as catalyst. As expected, the xylose yields were very low (less than 3%) and the highest ratio of xylose to XOS (xylose/XOS) was only 0.14 after hydrolysis for 24 h (Fig. 1). Bragatto et al. reported (2013) the production of XOS from HPAC-pretreated sugarcane bagasse, with higher amounts of xylose than XOS produced by enzymatic hydrolysis, indicating a xylose/XOS ratio of above 1.0. Huang et al. (2018) reported that acetic acid pretreatment of poplar produced 63.1 g of XOS and 45.8 g of xylose from 1 kg of poplar, with a xylose/XOS ratio of 0.73. Wen et al. (2019) reported that acetic acid pretreatment of poplar produced 69 g of XOS and 46 g of xylose, with a xylose/XOS ratio of 0.67. In this study, although a lower amount of XOS (16.9 g of XOS from 1 kg poplar) was produced from HPAC-pretreated poplar by XYL, a very low xylose/XOS ratio of 0.14 was afforded (Fig. 1C), indicating that the XOS produced was of relatively high purity and that HPAC-pretreated poplar was suitable for XOS production by XYL. 3.3 Glucose production from poplar after HPAC-pretreatment and XYL hydrolysis After XYL hydrolysis of HPAC-pretreated poplar, xylan in poplar was hydrolyzed to XOS and xylose (Fig. 1), and the solid residues were further hydrolyzed by CEL to produce monosaccharides (Fig. 2). Higher glucose (75.2%) and xylose (61.4%) yields were obtained from poplar pretreated using 60% HPAC with 50 mM H2SO4 as catalyst than that (33.3% glucose yield and 43.4% xylose yield) with 200 mM H2SO4 as catalyst using 10 mg CEL/g DM. Furthermore, 1.17 mg/mL and 1.02 mg/mL cellobiose were produced by 60% HPAC with 50 and 200 mM H2SO4 as catalysts, respectively. This cellobiose accumulation indicated that the enzymatic activity of -glucosidase in the CEL preparation was relatively low. As cellobiose is a strong inhibitor of cellobiohydrolase (Haven and Jørgensen, 2013), cellobiose accumulation decreased the cellulolytic hydrolysis capacity. To enhance the glucose yield, it was necessary to add more -glucosidase to convert cellobiose into 8
glucose. Surprisingly, compared with 200 mM H2SO4-catalyzed poplar with a lower lignin content (5.2%), 50 mM H2SO4-catalyzed poplar with a higher lignin content (11.8%) afforded a higher hydrolysis yield (Fig. 2 and Table 1). This phenomenon might be attributed to the more severe acid pretreatment resulting in higher inhibitory effect of lignin in poplar and decreased the accessibility of cellulase to cellulose in poplar, which has been reported previously (Nakagame et al. 2010; Rahikainen et al., 2011). These data suggested that H2SO4 concentrations above 200 mM might not be suitable for monosaccharide production from poplar, despite exhibiting stronger delignification ability (Fig. 2 and Table 1). 3.4 Mass balance Under the tested conditions, the highest XOS yield was 19.8% from xylan in the HPAC-pretreated poplar (60%, 200 mM H2SO4, 10,000 nkat XYL/g DM, Fig. 1), with 14.2 g of XOS produced from 1 kg of poplar by XYL (Fig. 3). For poplar pretreated using 60% HPAC with 50 mM H2SO4 as catalyst, 16.9 g of XOS was produced from 1 kg of poplar by XYL. After enzymatic hydrolysis of poplar by CEL, less monosaccharides (137.5 g of glucose; 31.2 g of xylose) were produced from poplar pretreated using 200 mM H2SO4 as catalyst compared with that pretreated using 50 mM H2SO4 as catalyst (296.4 g of glucose; 78.6 g of xylose). These results indicated that the poplar pretreated using 60% HPAC with 50 mM H2SO4 was more suitable for XOS and monosaccharide production (Fig, 3). Lai et al. (2019) reported that 73 g XOS and 356 g glucose was obtained from 1 kg poplar. Wen et al. (2019) reported that 69 g XOS and 403 g glucose was obtained from 1 kg poplar. In their work, acetic acid pretreatment was used to produce XOS from poplar and high XOS yields with relatively low purity of XOS were obtained (Lai et al., 2019; Wen et al., 2019). Lee et al. (2017) reported that 312 g glucose was produced from 1 kg poplar pretreated by hot compressed water and peracetic acid. In this study, a two-step enzymatic hydrolysis was proposed for the coproduction of XOS and monosaccharides from HPAC-pretreated poplar. Compared with the conventional XOS production process, consisting of xylan extraction, xylan hydrolysis, and cellulose-rich solid hydrolysis, this method showed many advantages, including the high purity of XOS produced, a simple process with 9
easy handling, and reduced pollution. Notably, the XOS yield in the hydrolysis of HPAC-pretreated poplar by 10,000 nkat XYL/g DM was limited (13.6%–19.8%). The data in this study (Fig. 1) indicated that XOS production from poplar could be improved by optimizing the pretreatment, increasing the XYL dosage, extending the hydrolysis time, or selecting an efficient xylanase. Furthermore, the effects of HPAC pretreatment and the role of poplar cellulose and lignin in XOS production need to be further investigated in future. 4. Conclusions HPAC-pretreated poplar showed efficient XOS and glucose production via a two-step enzymatic hydrolysis. Poplar pretreated using 60% HPAC with 50 mM H2SO4 as catalyst was more suitable for this process, producing 16.9 g of XOS and 296.4 g of glucose from 1 kg of poplar using XYL and CEL. Pretreatment with 200 mM H2SO4 resulted in lower amounts of XOS and glucose owing to a low solid recovery (55.7%). XOS produced from HPAC-pretreated poplar by XYL showed a very high purity. This study provides an alternative approach to the production of functional oligosaccharides and fermentable sugars from poplar using a simple and less polluting strategy. Acknowledgements This work was supported by the National Key R&D Program of China (No. 2017YFD0601001) and the National Natural Science Foundation of China (No. 31670598, 31270622). Appendix A. Supplementary data E-supplementary data to this article can be found online. References 1.
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Figure legends: Fig. 1 Production of XOS from HPAC-pretreated poplar (5%, w/v) by XYL (1,000-10,000 nkat/g DM) at pH 5.0 and 50 °C for 24 h. HPAC pretreatment was performed by 60% HPAC with 50 and 200 mM H2SO4 as catalyst, respectively. Error bars represent standard errors. Fig. 2 Enzymatic hydrolysis of HPAC-pretreated poplar (5%, w/v) by 10 mg CTec2/g DM at 50 °C for 48 h. Error bars represent standard errors. Fig. 3 Mass balance of XOS and monosaccharide production from HPAC-pretreated poplar by XYL and CEL. Error bars represent the standard errors. Highlights:
Two-step enzymatic hydrolysis was proposed for XOS and monosaccharide production.
H2SO4 concentration in HPAC pretreatment played an important role in lignin removal.
Poplar pretreated with 50 mM H2SO4 improved XOS and monosaccharide production.
1 kg poplar could produce 16.9 g XOS, 296.4 g glucose and 78.6 g xylose.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
13
Fig. 1
5
12 Time (h)
18
0.6 10 0.4 5
Yield (%)
Xylose/XOS
1.0 6 12 Xylose Time (h） XOS Xylose/XOS
24 0.8 0.6
10 0.4
12 Time (h）
(E) 200-60% HPAC;
18
24
1.00
5,000 nkat/g
Xylose XOS Xylose/XOS
0.80 0.60
10 0.40 0.20 1.0
(F) 200-60% HPAC; 10,000 nkat/g
20 0
0.0 18
6
5
0.2 (C) 50-60% HPAC; 10,000 nkat/g
0.0
15
0 Xylose / XOS Yield (%)
Yield (%)
0.8
15
Xylose
6
XOS
12 Time （h)
0.00 18
24
0.8
Xylose/XOS
0.6 10 0.4
14
5
0.2
0
0.0 0
6
12 Time (h)
18
24
Fig. 2
0.2
20
1.0
XOS
Yield (%）
5
24
Xylose
15
0.4
0
Xylose / XOS
6
(B) 50-60% HPAC; 5,000 nkat/g
0
10
0
0.0 0
20 0
0.6
0.2
0
15
0.8 Xylose / XOS
0.4
Xylose XOS Xylose/XOS
15 Yield (%)
10
1.0
(D) 200-60% HPAC; 1,000 nkat/g
Xylose / XOS
0.8 0.6
20
20
5
0.2
0
0.0 0
6
12 Time (h)
18
24
Xylose / XOS
Xylose XOS Xylose/XOS
15 Yield (%)
1.0
(A) 50-60% HPAC; 1,000 nkat/g
Xylose / XOS
20
Fig.
3
1 kg Poplar Glucan 421.4 g
HPAC
H2SO4
Glucan (%)
Xylan 157.9 g Xylan (%)
Lignin (%)
Solid
HPAC pretreatment
HPAC pretreatment
50 mM H2SO4, 60% HPAC, 60 ˚C
200 mM H2SO4, 60% HPAC, 60 ˚C
752.8 g HPAC-pretreated poplar
557.2 g HPAC-pretreated poplar
Glucan 394.2 g
Glucan 413.3 g
Xylan 124 g
Xylan 71.9 g
Lignin 88.9 g
Lignin 28.9 g
Others 145.7 g
Others 43.1 g
Xylanase hydrolysis
Xylanase hydrolysis
10,000 nkat /g DM, 24 h
10,000 nkat /g DM, 24 h
Hydrolysate liquid
Hydrolysate liquid
XOS 16.9 g
XOS 14.2 g
Xylose 2.4 g
Xylose 1.8 g
Xylan
Lignin
Cellulase hydrolysis
Cellulase hydrolysis 10 mg/g DM, 48 h
10 mg/g DM, 48 h
Hydrolysate liquid
Hydrolysate liquid
Glucose 296.4 g
Glucose 137.5 g
Xylose 76.2 g
Xylose 31.2 g
Cellobiose 18.1 g
Cellobiose 14.3 g
Xylose Table 1
Glucan
Lignin 256.6 g
Xylose
Chemical compositions of poplar pretreated by 60%-100% HPAC with 50200 mM H2SO4 as catalyst. The derivations are standard errors of three independent experiment.
15
(mM)
recovery
removal
removal
removal
(%)
(%)
(%)
(%)
None
42.1±0.6
15.8±0.2
25.7±0.4
－
－
－
60%
50
52.4±1.6
16.5±0.9
11.8±0.7
75.3
6.5
21.5
65.4
60%
200
74.2±1.4
12.9±0.1
5.2±0.3
55.7
1.9
54.4
88.8
16
－