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Evaluation of soluble fraction and enzymatic residual fraction of dilute dry acid, ethylenediamine, and steam explosion pretreated corn stover on the enzymatic hydrolysis of cellulose
Bioresource Technology 209 (2016) 172–179
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Evaluation of soluble fraction and enzymatic residual fraction of dilute dry acid, ethylenediamine, and steam explosion pretreated corn stover on the enzymatic hydrolysis of cellulose Lei Qin, Li Liu, Wen-Chao Li, Jia-Qing Zhu, Bing-Zhi Li ⇑, Ying-Jin Yuan Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Weijin Road 92, Nankai District, Tianjin 300072, PR China SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Weijin Road 92, Nankai District, Tianjin 300072, PR China
h i g h l i g h t s Enzymatic hydrolysis of different pretreated CS are compared. Soluble and enzymatic residual fraction are prepared to examine the inhibition. Inhibitions are compared between soluble fraction and enzymatic residual fraction.
a r t i c l e
i n f o
Article history: Received 29 December 2015 Received in revised form 25 February 2016 Accepted 26 February 2016 Available online 4 March 2016 Keywords: Inhibition Ethylenediamine Lignocellulose Dilute dry acid Steam explosion
a b s t r a c t This study is aimed to examine the inhibition of soluble fraction (SF) and enzymatic residual fraction (ERF) in dry dilute acid (DDA), ethylenediamine (EDA) and steam explosion (SE) pretreated corn stover (CS) on the enzymatic digestibility of cellulose. SF of DDA, EDA and SE pretreated CS has high xylose, soluble lignin and xylo-oligomer content, respectively. SF of EDA pretreated CS leads to the highest inhibition, followed by SE and DDA pretreated CS. Inhibition of ERF of DDA and SE pretreated CS is higher than that of EDA pretreated CS. The inhibition degree (A0/A) of SF is 1.76 and 1.21 times to that of ERF for EDA and SE pretreated CS, respectively. The inhibition degree of ERF is 1.05 times to that of SF in DDA pretreated CS. The quantitative analysis shows that SF of EDA pretreated CS, SF and ERF of SE pretreated CS cause significant inhibition during enzymatic hydrolysis. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Lignocellulosic biomass is considered as a renewable and sustainable feedstock to produce biofuels or chemicals (Zhong et al., 2010). Enzymatic hydrolysis of heteropolysaccharide to fermentable sugar is a feasible and effective route in biomass biorefinery (Li et al., 2010). Pretreatment of biomass is an essential step to achieve high sugar yields. Pretreatment brings about the compositional or structural changes and makes cellulose more digestible (Liu et al., 2015). Pretreatment affects the operation and yields of downstream processes, which ultimately determines the efficiency of the whole process (Garlock et al., 2011).
⇑ Corresponding author at: Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Weijin Road 92, Nankai District, Tianjin 300072, PR China. E-mail address: [email protected] (B.-Z. Li). http://dx.doi.org/10.1016/j.biortech.2016.02.123 0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.
Pretreatment process generates amounts of soluble or insoluble materials, which inhibit the enzymatic digestibility of cellulose, especially in the enzymatic hydrolysis with high solid loading (Zhong et al., 2009; Qin et al., 2013). Soluble phenolics derived from lignin are generally produced after most pretreatment processes (Du et al., 2010). Phenolics are more detrimental to enzyme than soluble sugars, furan derivatives or organic acids at the comparative concentrations (Kim et al., 2011; Qin et al., 2013). Other work on phenolics inhibition also found that cellulase is more susceptible to be inhibited than b-glucosidase (Ximenes et al., 2011). Oligosaccharides were identified as strong cellulase inhibitors from the liquid fraction of the pretreated biomass (Kont et al., 2013; Xue et al., 2015). Xylo-oligomers were found more inhibitory than monomeric sugars, cellobiose or xylan for equal amounts (Qing et al., 2010). Xylo-oligomers and arabino-oligomers were shown to have high recalcitrance to enzyme activities of cellulase, b-glucosidase, hemicellulase and pectinase (Xue et al., 2015).
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Inhibition degree of soluble materials was found to depend on their concentrations with linear correlation (Holtzapple et al., 1990). The inhibition of insoluble materials is mainly from the enzymatic residual lignin. Lignin inhibition caused by non-productive enzyme adsorption has been deeply studied in past years (Berlin et al., 2006; Nakagame et al., 2010; Rahikainen et al., 2013; Guo et al., 2014). The adsorption of enzymes on lignin differs for enzyme species, lignin properties and pretreatment methods. Lignin inhibits cellulases most, followed by xylanases and b-glucosidase (Berlin et al., 2006; Guo et al., 2014). Dry-to-dry pretreatment process is drawing more and more attention due to its operation convenience and thrift of water usage and energy consumption. Dry dilute acid pretreatment (DDA) is an improved technology of dilute acid pretreatment, which pretreats the biomass at high solid-to-liquid ratio (He et al., 2014a,b). No waste water is generated after DDA, and hence the pretreated materials with high solids content can be utilized directly in subsequent enzymatic hydrolysis and fermentation without washing or solid–liquid separation (Zhang et al., 2011; Zhu et al., 2015). Similar to dilute acid pretreatment (Hsu et al., 2010; Weiss et al., 2010; Noureddini and Byun, 2010), DDA degrades most of hemicellulose to xylose and by-products. Ethylenediamine pretreatment (EDA) is a relatively new alkaline pretreatment method and it can be operated at ambient pressure and high solid-to-liquid ratio without water addition (Qin et al., 2015). EDA pretreatment transforms cellulose crystal form, breaks the ether bonds in lignin and hemicellulose, and re-localizes lignin, thus drastically improves cellulose conversion in enzymatic hydrolysis. Both DDA and EDA are dry-to-dry processes. Steam explosion pretreatment (SE) is an extensively studied process in which the lignocellulosic biomass is heated by high-pressure saturated steam, followed by an explosive decompression (Oliveira et al., 2013; Cotana et al., 2014). SE partially solubilizes hemicellulose, but preserves most of cellulose and lignin in solid (Liu et al., 2014). All these three pretreatment methods can lead to high sugar yield in enzymatic hydrolysis process. However, the chemical properties of biomass pretreated by DDA and EDA have not been fully characterized yet. In addition, the inhibition of soluble and insoluble materials in the pretreated biomass on the cellulose conversion has not been compared. In this study, we prepare the soluble fraction (SF) and the enzymatic residual fraction (ERF) from corn stover (CS) of DDA, EDA and SE, and present the chemical compositions of them. Besides, we investigate the relationship between the amounts of these two parts (SF and ERF) and their inhibitory effects. Previous studies often compared the effect of one component (soluble content or insoluble lignin) on cellulose conversion between different pretreated biomass. Here we compare the effect of SF and ERF to find out the main cause of inhibition in each pretreated corn stover (PCS).
2. Methods
protein/mL, 77 FPU/mL) and hemicellulase Multifect XylanaseTM (42 mg protein/mL) were gifted by Genencor (NY, US). 2.2. Pretreatment All pretreatment processes were carried out according to previous experiences. An appropriate condition of each pretreatment was applied in order to achieve a relative high sugar yield. The pretreatment conditions were summarized in Table 1. Dry dilute acid pretreatment (DDA) was conducted by the collaboration partners from East China University of Science and Technology as described previously (He et al., 2014a,b). In brief, 2100 g of the presoaked CS (1400 g of dry CS plus 700 g of dilute acid solution) was fed into a 20-L stainless cylinder reactor with a helical ribbon stirrer. The desired temperature (175 °C) was reached by replenishing saturated steam and the condition was maintained for 3 min. To stop the pretreatment operation, the steam supply was switched off and the steam inside the reactor was quickly released from the outlet of the reactor. The pretreated CS solid was taken out directly from the bottom of the reactor, airdried and stored in 20 °C. Steam explosion pretreatment (SE) was conducted in a 15-L reactor system as previous description (Liu et al., 2013a,b). During pretreatment, 150 g CS (dry matter) was top-loaded into the reactor. Steam was filled into the reactor until the temperature reached 200 °C (1.8 MPa). After 5 min of exposure to the saturated steam, CS was exploded into the reception chamber by the ball-valve. After pretreatment, the pretreated CS was separated from the liquid fraction by vacuum filtration. The pretreated solid was airdried and stored in 20 °C. Ethylenediamine pretreatment (EDA) was conducted in a vacuum drying oven (Qin et al., 2015). 100 g CS was mixed with 100 g ethylenediamine on a stainless tray. The mixture was then held in the vacuum oven at 130 °C for 10 min. After the holding time, vacuum pump was opened to vent ethylenediamine until the residual ethylenediamine was less than 5% (wt) in pretreated CS. All PCS was used without any washing or detoxification process. The compositions of PCS were listed in Table 2. 2.3. Enzymatic hydrolysis of PCS The enzymatic hydrolysis (EH) was conducted at 1% or 6% glucan loading with a 20-mL reaction volume in a 100-mL Erlenmeyer flask. Cellulase and hemicellulase loading were 15 and 10 mg protein/g glucan, respectively. Sodium azide (0.2%, w/v) was used to inhibit microbial contamination. Citrate buffer (5 mM) was used to keep solution pH = 4.8. Flasks were incubated at 50 °C in a shaker at 150 rpm. Samples were withdrawn and subjected to sugar analysis by HPLC with Aminex HPX-87H column as described previously (Qin et al., 2012). Glucose yield and xylose yield are calculated as following equation:
2.1. Materials CS harvested from Tianjin (China) was air-dried and milled. The moisture content of the milled CS was 5%. The particles between 20 and 80 meshes were collected and stored in air-tight containers prior to pretreatment. Moisture content and composition analysis of CS were determined according to the Laboratory Analysis Protocol (LAP) of the National Renewable Energy Laboratory (NREL). The dry matter of CS composed of 30.5% glucan, 19.8% xylan and 16.5% acid-insoluble lignin (AIL). Pure cellulose, Avicel PH-101, was purchased from Sigma– Aldrich (MO, US). Commercial cellulase Accellerase 1500TM (89 mg
Table 1 Pretreatment conditions. Pretreatment conditions
Chemicals Loadings Liquid to solid ratio Temperature (°C) Time (min)
Pretreatment DDA
EDA
SE
Sulfuric acid 2.5% wt 1:2 175 3
Ethylenediamine 99% wt 1:1 130 10
/ / / 200 5
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Table 2 Compositionsa of PCS.
Water extractives Glucose Xylose Gluco-oligomer Xylo-oligomer Soluble lignin Acetic acid 5-HMF Furfural Ethanol extractives Insoluble contents Glucan Xylan Acid-soluble lignin Acid-insoluble lignin Acetyl
DDAPCS, %
EDAPCS, %
SEPCS, %
28.6 ± 1.1 1.8 ± 0.1 8.9 ± 0.2 0.9 ± 0.1 2.0 ± 0.4 3.2 ± 0.0 0.4 ± 0.1 0.2 ± 0.0 0.1 ± 0.0 11.1 ± 0.8 60.8 ± 2.3 38.6 ± 0.6 2.3 ± 0.0 0.4 ± 0.0 16.6 ± 0.3 1.0 ± 0.3
48.1 ± 0.8 0.4 ± 0.4 0.8 ± 0.1 1.4 ± 0.1 5.9 ± 0.1 14.1 ± 1.6 0.2 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 1.8 ± 0.9 50.6 ± 1.7 27.9 ± 0.1 9.9 ± 0.2 3.0 ± 0.0 5.0 ± 0.3 0.2 ± 0.1
38.9 ± 0.5 1.1 ± 0.1 1.9 ± 0.0 1.8 ± 0.0 6.3 ± 0.1 7.3 ± 0.4 0.9 ± 0.1 0.6 ± 0.0 0.3 ± 0.0 8.2 ± 1.1 52.3 ± 2.1 26.8 ± 0.8 6.0 ± 0.1 0.6 ± 0.0 17.1 ± 0.6 3.1 ± 0.2
a All compositions were based on dry matter. Data were determined in triplicates.
4500 rpm for 10 min and the supernatant was filtrated through Whatman No. 1 filter paper. The washing liquids (namely soluble fraction) were stored at 20 °C until used. The composition of the SF (monomeric and oligomeric sugars, soluble lignin, and byproducts) were determined according to LAP of NREL. The solid fraction was freeze-dried and then subjected to an extensive enzymatic hydrolysis according to previous report (Berlin et al., 2006). Cellulase and hemicellulase were added to the solid residue with 60 and 40 mg protein/g glucan, respectively, with 3% glucan loading at pH 4.8, 50 °C and 200 rpm for 168 h. After hydrolysis, the enzymatic residue was recovered by centrifugation and washed three times in deionized water and sonicated for 60 min. The enzymatic residue was then incubated at 37 °C for 24 h in 50 mM phosphate buffer (pH = 7) containing 1 U/mL Pronase (Sigma, US). The protease was then deactivated by incubation at 90 °C for 2 h. The enzymatic residue was washed extensively with deionized water and freeze-dried. The ERF was stored in sealed vials at room temperature in a desiccator. 2.5. Characteristics of ERF
Glucose ðxyloseÞ yield ¼ ½glucose ðxyloseÞ concentration; g=L 20 mL 0:9ð0:88Þ =½glucan ðxylanÞ weight in substrate; mg 100%
ð1Þ
2.4. Preparation of soluble fraction and enzymatic residual fraction from PCS Soluble fraction (SF) and enzymatic residual fraction (ERF) were prepared by the following procedure (Scheme 1). PCS was milled in a planetary ball mill (DECO, China) with zirconium dioxide balls at 300 rpm for 4 h. The particles passed through 80 meshes were collected. The milled PCS was incubated with 5 mM citrate buffer in solid to water ratio of 1:7 for 1 h. The mixture was centrifuged at
Elemental analysis of ERF was performed using a LECO CN-2000 elemental analyzer (Leco, MI, US). FT-IR spectra were obtained using a Nicolet IZ10 Fourier transform infrared spectroscopy and potassium bromide disc containing about 1% solid. Spectra were obtained by averaging 32 scans from 4000 to 400 cm1. 2.6. Determining the effects of SF and ERF on EH The hydrolysis of pure cellulose (Avicel) was performed for carrying out the comparison of SF and ERF in PCS. Cellulose conversion is defined as:
Cellulose conversion ¼ ½glucose concentration; g=L 20 mL 0:9= ½cellulose weight in Avicel; mg 100% ð2Þ Washing liquid (SF) prepared from PCS was diluted into different ratios (0%, 10%, 25%, 50% and 100%). Avicel was hydrolyzed in the diluted washing liquid with 1% glucan loading at pH 4.8, 50 °C and 150 rpm. Cellulase loading was 15 mg protein/g glucan. To explore the effect of ERF, 10 mg/mL ERF solid was added into the enzymatic hydrolysate of Avicel (pH 4.8, 50 °C and 150 rpm). Glucan loading was 1% and cellulase loading was 15 mg protein/ g glucan. Glucose concentration in hydrolysates was analyzed by HPLC with Aminex HPX-87H column. 3. Results and discussion 3.1. Composition and EH of PCS
Scheme 1. Preparation process of SF and ERF.
The compositions of the PCS with different pretreatments are listed in Table 2. In order to determine the soluble sugars, extraction process was carried out prior to two-stage acid hydrolysis. Extraction also dissolves part of lignin, making acid-soluble lignin (ASL) and acid-insoluble lignin (AIL) content lower than other reports. Statistically, ethylenediamine pretreated corn stover (EDACS) has the highest water extractives, followed by steam exploded corn stover (SECS) and dry dilute acid pretreated corn stover (DDACS). The xylose content of DDACS is significantly higher than other groups of PCS. The soluble lignin content is highest in EDACS. The xylo-oligomer content is similar for EDACS and SECS. Ethanol extractives in EDACS is lower than DDACS and SECS. For insoluble contents, DDACS has the highest glucan content and
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the lowest xylan content, while EDACS has the highest xylan and ASL content and the lowest AIL content. The FT-IR spectra of untreated CS (UTCS) and PCS are shown in Fig. S1. The decrease in the peaks around 1730 and 1250 cm1 of all PCS are due to the acetyl groups cleavage of hemicellulose (Hsu et al., 2010). A peak around 1642 cm1 of EDACS indicates the ammonolysis of the acetyl groups in hemicellulose and lignin. The EH of PCS at 1% and 6% glucan loading were implemented (Figs. 1 and 2). Previous studies showed that EH of SECS with enzyme loading of 15 FPU/g glucan achieved glucose yield of 60– 80% at different glucan loadings (Liu et al., 2013a,b). EDACS exhibited a glucose yield of 84% at 1% glucan loading (Qin et al., 2015). Previous report showed that glucose yield of DDACS reached 70% with the same pretreatment and hydrolysis conditions (He et al., 2014b). Our results just agree with these reports. Figs. 1 and 2 show that the glucose yield of EDACS is higher than that of DDACS and SECS. DDACS has higher xylan conversion due to its lower xylan content. The increase of glucan loading decreases glucose yield for all PCS. When glucan loading increases from 1% to 6%, glucose yield at 120 h of DDACS, EDACS and SECS decreases from 80.4%, 92.0% and 69.6% to 56.6%, 68.8% and 49.8% (decreases by 28%, 25% and 29%), respectively. As known, the reduction of sugars yield at high solid loading is due to insufficient mass and heat transfer, increased concentration of inhibitors, lack of available water, and so on (Modenbach and Nokes, 2013). Herein, the inhibitors may exhibit significant impact on EH because the PCS is washing-free. The impact of inhibitors on sugar yield and enzyme activity is dependent on the types and amounts of inhibitors, which needs intensive studies.
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3.2. Effect of SF on the EH of cellulose In order to explore the impact of SF on enzyme digestibility, PCS was washed by 5 mM citrate buffer with a solid to liquid ratio of 1:7, and each washing liquid with different dilution ratios was added into the EH of Avicel. The components of washing liquid are listed in Table 3. The concentration of each component is generally proportional to the corresponding composition in PCS. The glucose concentrations in EH with different species and amounts of SF are detected. Control groups without Avicel addition were carried out to determine the glucose produced from SF (Fig. S3). The resulting cellulose conversions based on glucose concentrations are shown in Fig. 3. All the washing liquids decrease the cellulose conversion. Higher washing liquid concentration leads to lower cellulose conversion. It is observed that washing liquid of SECS results in the lowest cellulose conversion, followed by EDACS, DDACS, and UTCS. Washing liquid (100%) of DDACS, EDACS, SECS and UTCS decrease cellulose conversion from 58.3% (0% washing liquid) to 39.4%, 17.7%, 16.3%, and 42.9% at 72 h, respectively. According to previous study (Holtzapple et al., 1990), the ratio of cellulose conversion without inhibitors to cellulose conversion with inhibitor (A0/A) has a linear relationship with the concentration of inhibitor as following equation:
A0 =A ¼ 1 þ bI
ð3Þ
where I is the concentration of inhibitor (g/L); b is the inhibition constant (L/g). The value of A0/A represents the inhibition degree. In our study, the dilution ratio of washing liquid is proportional to the inhibitor concentration. Thus Eq. (3) can also be expressed as:
A0 =A ¼ 1 þ b0 n
ð4Þ
Fig. 1. EH of PCS by DDA, EDA and SE at 1% glucan loading. (a) Sugar concentrations; (b) sugar yields (based on glucan and xylan content in PCS). The default hydrolysis conditions were 15 mg cellulase per g glucan, 10 mg hemicellulase per g glucan, pH 4.8, 50 °C and 150 rpm. Error bars represented standard deviations, n = 3.
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Fig. 2. EH of PCS by DDA, EDA and SE at 6% glucan loading. (a) Sugar concentrations; (b) sugar yields (based on glucan and xylan content in PCS). The default hydrolysis conditions were 15 mg cellulase per g glucan, 10 mg hemicellulase per g glucan, pH 4.8, 50 °C and 200 rpm. Error bars represented standard deviations, n = 3.
Table 3 Components of SF in concentration (g/L).
Glucose Xylose Gluco-oligomer Xylo-oligomer Soluble lignin Acetic acid 5-HMF Furfural
3.3. Effect of ERF on the EH of cellulose
DDA
EDA
SE
UT
2.6 ± 0.0 13.0 ± 0.2 0.8 ± 0.1 3.7 ± 0.2 2.3 ± 0.1 0.5 ± 0.1 0.3 ± 0.0 0.2 ± 0.0
0.3 ± 0.0 0.2 ± 0.0 0.8 ± 0.1 7.6 ± 0.1 10.5 ± 0.5 0.3 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.7 ± 0.0 1.5 ± 0.1 2.4 ± 0.2 7.9 ± 0.1 4.4 ± 0.2 1.2 ± 0.0 0.8 ± 0.0 0.3 ± 0.0
0.8 ± 0.0 1.2 ± 0.0 0.5 ± 0.1 0.2 ± 0.1 1.3 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
where n is the dilution ratio; b0 is another expression of inhibition constant. Dilution ratio of washing liquid from each PCS shows a significant linear correlation with A0/A (Fig. 4). The slope of the line represents the inhibition constant b0 . Inhibition constants of DDACS, EDACS, SECS and UTCS are 0.648, 2.941, 1.859 and 0.531, respectively. It is obvious that higher inhibition constant leads to greater inhibitory effect. The relationship between the inhibition constant and the concentration of washing liquid showed that xylo-oligomer and soluble lignin concentration may have the greatest impact on inhibition degree (Fig. S4). Previous researchers proposed that xylo-oligomer simulate the structure of cellulose chain and bind to the active site tunnel of cellobiohydrolase (Kont et al., 2013). The oligosaccharides are hardly hydrolyzed by homologous cellulase in the absence of accessory enzymes (Xue et al., 2015). Soluble phenolics predominates over oligosaccharides in decreasing the final cellulose conversion, because phenolics lead to precipitation and irreversible inhibition of cellulase (Kim et al., 2011).
The compositions of ERF for each PCS are listed in Table 4. ERF of DDACS has the highest AIL content (84.0%), followed by EDACS (73.8%), SECS (59.5%) and UTCS (26.5%). The content of unhydrolyzed glucan and xylan in UTCS are significantly higher than that of PCS. FT-IR spectra of ERF show that the peaks related to cellulose (e.g. 1050 and 1161 cm1) decrease and the peaks related to lignin (e.g. 1428, 1513 and 1604 cm1) increase compared to PCS (Fig. S2), which indicates the removal of cellulose content in ERF. Noticeable drops in peak intensity was observed near 1260–1270 cm1 in ERF of DDACS and EDACS compared to UTCS and SECS, corresponding to the guaiacyl (G) ring breathing with CAO stretching. There is no appreciable change in the 1300–1330 cm1 region of all ERF that represents the syringyl (S) ring breathing (Davidson et al., 2005). This shows that the guaiacyl type of lignin is more prone to be hydrolyzed than the syringyl type after DDA and EDA pretreatment. Previous finding show that low S/G ratio leads to high protein adsorption capacity for lignin, which could decrease the adsorption of enzyme to cellulose (Guo et al., 2014). Therefore, the residual lignin of DDACS and EDACS could be less detrimental to enzymatic hydrolysis than UTCS and SECS. Elemental analysis was conducted to determine the residual protein in ERF. Table 5 shows that there is little nitrogen content (less than 1%) in ERF, except for EDACS. The high nitrogen content in ERF of EDACS is due to the ammonolysis of lignin during pretreatment (Qin et al., 2015). Comparative nitrogen contents were observed in the enzymatic residues in previous reports (Berlin et al., 2006; Nakagame et al., 2010).
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Fig. 3. Effect of SF from different PCS on Avicel conversion. (a) DDACS; (b) EDACS; (c) SECS; (d) UTCS. Washing liquids were added into hydrolysates in terms of 0, 0.1, 0.2, 0.5 and 1.0 mL/mL hydrolysate.
Table 4 Compositions of ERF.
Glucan Xylan Acid-insoluble lignin
DDA, %
EDA, %
SE, %
UT, %
6.2 ± 0.9 1.4 ± 0.7 84.0 ± 0.4
2.8 ± 0.7 1.4 ± 0.4 73.8 ± 0.7
20.0 ± 1.1 8.0 ± 0.2 59.5 ± 0.7
33.8 ± 0.9 20.8 ± 0.2 26.5 ± 0.2
Table 5 Elemental analysis of ERF.
C N
Fig. 4. Effect of SF on inhibition degree (A0/A). (a) DDACS; (b) EDACS; (c) SECS; (d) UTCS. A0/A were calculated based on the data at 24 h hydrolysis.
The Avicel was enzymatic hydrolyzed with the addition of 10 mg/mL ERF. Control groups without Avicel addition were also conducted (Fig. S5). Avicel cellulose conversions with different ERF addition are shown in Fig. 5. All enzymatic residual solids lead
DA, %
EDA, %
SE, %
UT, %
53.66 ± 0.03 0.82 ± 0.01
48.57 ± 0.64 2.83 ± 0.07
55.65 ± 0.39 0.90 ± 0.04
43.42 ± 0.19 0.50 ± 0.01
to decrease of cellulose conversion. The conversion decreases from control (46.9%) > UT (42.4%) > EDA (41.8%) > SE (37.2%) > DDA (34.4%) at 24 h. The values of A0/A at 24 h were 1.105, 1.361, 1.122 and 1.260 for the ERF of UT, DDA, EDA and SE, respectively. The inhibition degree almost keeps stable along with the hydrolysis time. It is believed that the inhibition of lignin on hydrolysis is mainly due to the unproductive binding of the enzymes. In general, higher lignin content in ERF should induce to higher inhibition. However, the lower lignin content in SECS–ERF results in more inhibition than EDA–ERF (SECS–ERF and EDA–ERF contain 59.5% and 73.8%
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Fig. 5. Effect of ERF from different PCS on Avicel conversion. Cellulase loading is 15 mg protein/g glucan. ERF was added at 10 lg/mL. Control means no ERF was added in Avicel’s hydrolysis.
lignin, achieve 37.2% and 41.8% glucose yield, respectively). This result may be due to the different enzyme adsorption to lignin, due to lignin sources and structural features, such as S/G ratio (Guo et al., 2014). The inhibition of lignin is an important issue in SE pretreated biomass. The lignin isolated from steam pretreated poplar decreased the hydrolysis yields of cellulose, whereas the other isolated lignins did not appear to decrease the hydrolysis yields significantly (Nakagame et al., 2010). Rahikainen et al. (2013) showed that SE alters the lignin structure, leading to increased enzyme adsorption. Adsorption and enzyme specific activity were shown to be inversely proportional to lignin content and hydrophobicity of SE pretreated biomass (Heiss-Blanquet et al., 2011). It is also suggested that the deposition of small spherical lignin droplets on the fiber surface during acidic pretreatment likely limits the access of enzymes to cellulose. In contrast, the ammoniation of lignin was reported to decrease the lignin–enzyme interaction (Sewalt et al., 1997), which results in lower inhibition of enzymatic residue of EDACS. These could explain the higher inhibition of enzymatic residue of SECS.
3.4. Comparison of SF and ERF in PCS The effects of SF and ERF have been separately analyzed and quantified by inhibition constant, which provides an approach to figure out the dominant inhibitory factor from SF and ERF. Here we stimulate the effect of SF and ERF on cellulose conversion in PCS by adding the SF and ERF preparation into the enzymatic hydrolysate of Avicel. The addition amount of SF and ERF was calculated and equal to that in the real enzymatic hydrolysate of untreated/pretreated CS with 1% glucan loading. The resulting inhibition A0/A from SF and ERF can be calculated through dilution ratio n and ERF concentration I in Eqs. (4) and (3), respectively. As untreated/pretreated CS was washed with solid-to-liquid ratio of 1:7 (12.5% solid loading), the dilution ratio of hydrolysate in 1% glucan loading equals to the corresponding solid loading divided by 12.5%. The actual loading of AIL in the hydrolysate of untreated/pretreated CS with 1% glucan loading determined the calculated ERF concentration (that is, the calculated ERF concentration contain the same AIL concentration with real enzymatic hydrolysate of untreated/pretreated CS, as shown in Table S2). It is noticeable that b and b0 refer to the inhibition per unit inhibitor,
Fig. 6. Calculated inhibition degree (A0/A) of SF and ERF in the EH of untreated/ pretreated CS with 1% glucan loading.
and A0/A stands for the inhibition from actual quantity of inhibitors in hydrolysate. From Fig. 6, we can see that, for EDACS, A0/A of SF is 1.76 times to A0/A of ERF. For SECS, A0/A of SF is 1.21 times to A0/A of ERF. The significant higher A0/A of SF than ERF in EDACS and SECS indicates that SF of them are the dominant factor inhibiting the cellulose conversion. The inhibition of ERF in UTCS and DDACS is slightly higher than that of SF (for UTCS and DDACS, A0/A of ERF is 1.07 and 1.05 times to A0/A of SF, respectively). Through this quantitative analysis, not only the inhibitory effect between the pretreated biomass, but also the inhibitory effect between SF and ERF can be revealed clearly. It shows that SF of EDACS, SF and ERF of SECS cause significant inhibition during enzymatic hydrolysis.
4. Conclusions The SF and ERF were separated from different PCS to examine the inhibition degree on enzymatic digestibility of cellulose. The SF of EDACS and SECS exhibited higher inhibitory effect compared to that of DDACS, due to the higher concentration of soluble lignin and xylo-oligomer. The ERF of DDACS and SECS exhibited higher inhibition than that of EDACS. The inhibition of SF was slightly lower than ERF in DDACS, while the inhibition of SF was significantly higher than ERF in EDACS and SECS. Thus, in order to reduce the inhibition on enzymatic hydrolysis from feedstock, different detoxification strategies should be developed. Acknowledgements This study was financially supported by the Ministry of Science and Technology of China (‘‘973” Program: 2013CB733601), the National Natural Science Foundation of China (Major program: 21390203), Tianjin Municipal Science and Technology Commission (13RCGFSY19800).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2016.02. 123.
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Liu, Z.H., Qin, L., Pang, F., Jin, M.J., Li, B.Z., Kang, Y., Dale, B.E., Yuan, Y.J., 2013b. Effects of biomass particle size on steam explosion pretreatment performance for improving the enzyme digestibility of corn stover. Ind. Crops Prod. 44, 176–184. Liu, Z.H., Qin, L., Zhu, J.Q., Li, B.Z., Yuan, Y.J., 2014. Simultaneous saccharification and fermentation of steam-exploded corn stover at high glucan loading and high temperature. Biotechnol. Biofuels 7, 167. Modenbach, A.A., Nokes, S.E., 2013. Enzymatic hydrolysis of biomass at high-solids loadings – a review. Biomass Bioenergy 56, 526–544. Nakagame, S., Chandra, R.P., Saddler, J.N., 2010. The effect of isolated lignins, obtained from arrange of pretreated lignocellulosic substrates, on enzymatic hydrolysis. Biotechnol. Bioeng. 105 (5), 871–879. Noureddini, H., Byun, J., 2010. Dilute-acid pretreatment of distillers’ grains and corn fiber. Bioresour. Technol. 101, 1060–1067. Oliveira, F.M.V., Pinheiro, I.O., Souto-Maior, A.M., Martin, C., Gonçalves, A.R., Rocha, G.J.M., 2013. Industrial-scale steam explosion pretreatment of sugarcane straw for enzymatic hydrolysis of cellulose for production of second generation ethanol and value-added products. Bioresour. Technol. 130 (1), 168–173. Qin, L., Li, W.C., Zhu, J.Q., Liang, J.N., Li, B.Z., Yuan, Y.J., 2015. Ethylenediamine pretreatment changes cellulose allomorph and lignin structure of lignocellulose at ambient pressure. Biotechnol. Biofuels 8, 174. Qin, L., Liu, Z.H., Jin, M.J., Li, B.Z., Yuan, Y.J., 2013. High temperature aqueous ammonia pretreatment and post-washing enhance the high solids enzymatic hydrolysis of corn stover. Bioresour. Technol. 146, 504–511. Qin, L., Liu, Z.H., Li, B.Z., Dale, B.E., Yuan, Y.J., 2012. Mass balance and transformation of corn stover by pretreatment with different dilute organic acids. Bioresour. Technol. 112, 319–326. Qing, Q., Yang, B., Wyman, C.E., 2010. Xylooligomers are strong inhibitors of cellulose hydrolysis by enzymes. Bioresour. Technol. 101, 9624–9630. Rahikainen, J.L., Martin-Sampedro, R., Heikkinen, H., Rovio, S., Marjamaa, K., Tamminen, T., Rojas, O.J., Kruus, K., 2013. Inhibitory effect of lignin during cellulose bioconversion: the effect of lignin chemistry on non-productive enzyme adsorption. Bioresour. Technol. 133, 270–278. Sewalt, V.J.H., Glasser, W.G., Beauchemin, K.A., 1997. Lignin impact on fiber degradation. 3. Reversal of inhibition of enzymatic hydrolysis by chemical modification of lignin and by additives. J. Agric. Food Chem. 45, 1823–1828. Weiss, N.D., Farmer, J.D., Schell, D.J., 2010. Impact of corn stover composition on hemicellulose conversion during dilute acid pretreatment and enzymatic cellulose digestibility of the pretreated solids. Bioresour. Technol. 101, 674–678. Ximenes, E., Kim, Y., Mosier, N., Dien, B., Ladisch, M., 2011. Deactivation of cellulases by phenols. Enzyme Microb. Technol. 48, 54–60. Xue, S., Uppugundla, N., Bowman, M.J., Cavalier, D., Da Costa Sousa, L., Dale, B.E., Balan, V., 2015. Sugar loss and enzyme inhibition due to oligosaccharide accumulation during high solids-loading enzymatic hydrolysis. Biotechnol. Biofuels 8, 195. Zhang, J., Wang, X., Chu, D., He, Y., Bao, J., 2011. Dry pretreatment of lignocellulose with extremely low steam and water usage for bioethanol production. Bioresour. Technol. 102, 4480–4488. Zhong, C., Cao, Y.X., Li, B.Z., Yuan, Y.J., 2010. Biofuels in China: past, present and future. Biofuels Bioprod. Biorefin. 4, 326–342. Zhong, C., Lau, M.W., Balan, V., Dale, B.E., Yuan, Y.J., 2009. Optimization of enzymatic hydrolysis and ethanol fermentation from AFEX-treated rice straw. Appl. Microbiol. Biotechnol. 84, 667–676. Zhu, J.Q., Qin, L., Li, W.C., Li, B.Z., Zhang, J., Bao, J., Huang, Y.D., Yuan, Y.J., 2015. Simultaneous saccharification and co-fermentation of dry diluted acid pretreated corn stover at high dry matter loading: overcoming the inhibitors by non-tolerant yeast. Bioresour. Technol. 198, 39–46.
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Evaluation of soluble fraction and enzymatic residual fraction of dilute dry acid, ethylenediamine, and steam explosion pretreated corn stover on the enzymatic hydrolysis of cellulose Lei Qin, Li Liu, Wen-Chao Li, Jia-Qing Zhu, Bing-Zhi Li ⇑, Ying-Jin Yuan Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Weijin Road 92, Nankai District, Tianjin 300072, PR China SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Weijin Road 92, Nankai District, Tianjin 300072, PR China
h i g h l i g h t s Enzymatic hydrolysis of different pretreated CS are compared. Soluble and enzymatic residual fraction are prepared to examine the inhibition. Inhibitions are compared between soluble fraction and enzymatic residual fraction.
a r t i c l e
i n f o
Article history: Received 29 December 2015 Received in revised form 25 February 2016 Accepted 26 February 2016 Available online 4 March 2016 Keywords: Inhibition Ethylenediamine Lignocellulose Dilute dry acid Steam explosion
a b s t r a c t This study is aimed to examine the inhibition of soluble fraction (SF) and enzymatic residual fraction (ERF) in dry dilute acid (DDA), ethylenediamine (EDA) and steam explosion (SE) pretreated corn stover (CS) on the enzymatic digestibility of cellulose. SF of DDA, EDA and SE pretreated CS has high xylose, soluble lignin and xylo-oligomer content, respectively. SF of EDA pretreated CS leads to the highest inhibition, followed by SE and DDA pretreated CS. Inhibition of ERF of DDA and SE pretreated CS is higher than that of EDA pretreated CS. The inhibition degree (A0/A) of SF is 1.76 and 1.21 times to that of ERF for EDA and SE pretreated CS, respectively. The inhibition degree of ERF is 1.05 times to that of SF in DDA pretreated CS. The quantitative analysis shows that SF of EDA pretreated CS, SF and ERF of SE pretreated CS cause significant inhibition during enzymatic hydrolysis. Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction Lignocellulosic biomass is considered as a renewable and sustainable feedstock to produce biofuels or chemicals (Zhong et al., 2010). Enzymatic hydrolysis of heteropolysaccharide to fermentable sugar is a feasible and effective route in biomass biorefinery (Li et al., 2010). Pretreatment of biomass is an essential step to achieve high sugar yields. Pretreatment brings about the compositional or structural changes and makes cellulose more digestible (Liu et al., 2015). Pretreatment affects the operation and yields of downstream processes, which ultimately determines the efficiency of the whole process (Garlock et al., 2011).
⇑ Corresponding author at: Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Weijin Road 92, Nankai District, Tianjin 300072, PR China. E-mail address: [email protected] (B.-Z. Li). http://dx.doi.org/10.1016/j.biortech.2016.02.123 0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.
Pretreatment process generates amounts of soluble or insoluble materials, which inhibit the enzymatic digestibility of cellulose, especially in the enzymatic hydrolysis with high solid loading (Zhong et al., 2009; Qin et al., 2013). Soluble phenolics derived from lignin are generally produced after most pretreatment processes (Du et al., 2010). Phenolics are more detrimental to enzyme than soluble sugars, furan derivatives or organic acids at the comparative concentrations (Kim et al., 2011; Qin et al., 2013). Other work on phenolics inhibition also found that cellulase is more susceptible to be inhibited than b-glucosidase (Ximenes et al., 2011). Oligosaccharides were identified as strong cellulase inhibitors from the liquid fraction of the pretreated biomass (Kont et al., 2013; Xue et al., 2015). Xylo-oligomers were found more inhibitory than monomeric sugars, cellobiose or xylan for equal amounts (Qing et al., 2010). Xylo-oligomers and arabino-oligomers were shown to have high recalcitrance to enzyme activities of cellulase, b-glucosidase, hemicellulase and pectinase (Xue et al., 2015).
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Inhibition degree of soluble materials was found to depend on their concentrations with linear correlation (Holtzapple et al., 1990). The inhibition of insoluble materials is mainly from the enzymatic residual lignin. Lignin inhibition caused by non-productive enzyme adsorption has been deeply studied in past years (Berlin et al., 2006; Nakagame et al., 2010; Rahikainen et al., 2013; Guo et al., 2014). The adsorption of enzymes on lignin differs for enzyme species, lignin properties and pretreatment methods. Lignin inhibits cellulases most, followed by xylanases and b-glucosidase (Berlin et al., 2006; Guo et al., 2014). Dry-to-dry pretreatment process is drawing more and more attention due to its operation convenience and thrift of water usage and energy consumption. Dry dilute acid pretreatment (DDA) is an improved technology of dilute acid pretreatment, which pretreats the biomass at high solid-to-liquid ratio (He et al., 2014a,b). No waste water is generated after DDA, and hence the pretreated materials with high solids content can be utilized directly in subsequent enzymatic hydrolysis and fermentation without washing or solid–liquid separation (Zhang et al., 2011; Zhu et al., 2015). Similar to dilute acid pretreatment (Hsu et al., 2010; Weiss et al., 2010; Noureddini and Byun, 2010), DDA degrades most of hemicellulose to xylose and by-products. Ethylenediamine pretreatment (EDA) is a relatively new alkaline pretreatment method and it can be operated at ambient pressure and high solid-to-liquid ratio without water addition (Qin et al., 2015). EDA pretreatment transforms cellulose crystal form, breaks the ether bonds in lignin and hemicellulose, and re-localizes lignin, thus drastically improves cellulose conversion in enzymatic hydrolysis. Both DDA and EDA are dry-to-dry processes. Steam explosion pretreatment (SE) is an extensively studied process in which the lignocellulosic biomass is heated by high-pressure saturated steam, followed by an explosive decompression (Oliveira et al., 2013; Cotana et al., 2014). SE partially solubilizes hemicellulose, but preserves most of cellulose and lignin in solid (Liu et al., 2014). All these three pretreatment methods can lead to high sugar yield in enzymatic hydrolysis process. However, the chemical properties of biomass pretreated by DDA and EDA have not been fully characterized yet. In addition, the inhibition of soluble and insoluble materials in the pretreated biomass on the cellulose conversion has not been compared. In this study, we prepare the soluble fraction (SF) and the enzymatic residual fraction (ERF) from corn stover (CS) of DDA, EDA and SE, and present the chemical compositions of them. Besides, we investigate the relationship between the amounts of these two parts (SF and ERF) and their inhibitory effects. Previous studies often compared the effect of one component (soluble content or insoluble lignin) on cellulose conversion between different pretreated biomass. Here we compare the effect of SF and ERF to find out the main cause of inhibition in each pretreated corn stover (PCS).
2. Methods
protein/mL, 77 FPU/mL) and hemicellulase Multifect XylanaseTM (42 mg protein/mL) were gifted by Genencor (NY, US). 2.2. Pretreatment All pretreatment processes were carried out according to previous experiences. An appropriate condition of each pretreatment was applied in order to achieve a relative high sugar yield. The pretreatment conditions were summarized in Table 1. Dry dilute acid pretreatment (DDA) was conducted by the collaboration partners from East China University of Science and Technology as described previously (He et al., 2014a,b). In brief, 2100 g of the presoaked CS (1400 g of dry CS plus 700 g of dilute acid solution) was fed into a 20-L stainless cylinder reactor with a helical ribbon stirrer. The desired temperature (175 °C) was reached by replenishing saturated steam and the condition was maintained for 3 min. To stop the pretreatment operation, the steam supply was switched off and the steam inside the reactor was quickly released from the outlet of the reactor. The pretreated CS solid was taken out directly from the bottom of the reactor, airdried and stored in 20 °C. Steam explosion pretreatment (SE) was conducted in a 15-L reactor system as previous description (Liu et al., 2013a,b). During pretreatment, 150 g CS (dry matter) was top-loaded into the reactor. Steam was filled into the reactor until the temperature reached 200 °C (1.8 MPa). After 5 min of exposure to the saturated steam, CS was exploded into the reception chamber by the ball-valve. After pretreatment, the pretreated CS was separated from the liquid fraction by vacuum filtration. The pretreated solid was airdried and stored in 20 °C. Ethylenediamine pretreatment (EDA) was conducted in a vacuum drying oven (Qin et al., 2015). 100 g CS was mixed with 100 g ethylenediamine on a stainless tray. The mixture was then held in the vacuum oven at 130 °C for 10 min. After the holding time, vacuum pump was opened to vent ethylenediamine until the residual ethylenediamine was less than 5% (wt) in pretreated CS. All PCS was used without any washing or detoxification process. The compositions of PCS were listed in Table 2. 2.3. Enzymatic hydrolysis of PCS The enzymatic hydrolysis (EH) was conducted at 1% or 6% glucan loading with a 20-mL reaction volume in a 100-mL Erlenmeyer flask. Cellulase and hemicellulase loading were 15 and 10 mg protein/g glucan, respectively. Sodium azide (0.2%, w/v) was used to inhibit microbial contamination. Citrate buffer (5 mM) was used to keep solution pH = 4.8. Flasks were incubated at 50 °C in a shaker at 150 rpm. Samples were withdrawn and subjected to sugar analysis by HPLC with Aminex HPX-87H column as described previously (Qin et al., 2012). Glucose yield and xylose yield are calculated as following equation:
2.1. Materials CS harvested from Tianjin (China) was air-dried and milled. The moisture content of the milled CS was 5%. The particles between 20 and 80 meshes were collected and stored in air-tight containers prior to pretreatment. Moisture content and composition analysis of CS were determined according to the Laboratory Analysis Protocol (LAP) of the National Renewable Energy Laboratory (NREL). The dry matter of CS composed of 30.5% glucan, 19.8% xylan and 16.5% acid-insoluble lignin (AIL). Pure cellulose, Avicel PH-101, was purchased from Sigma– Aldrich (MO, US). Commercial cellulase Accellerase 1500TM (89 mg
Table 1 Pretreatment conditions. Pretreatment conditions
Chemicals Loadings Liquid to solid ratio Temperature (°C) Time (min)
Pretreatment DDA
EDA
SE
Sulfuric acid 2.5% wt 1:2 175 3
Ethylenediamine 99% wt 1:1 130 10
/ / / 200 5
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Table 2 Compositionsa of PCS.
Water extractives Glucose Xylose Gluco-oligomer Xylo-oligomer Soluble lignin Acetic acid 5-HMF Furfural Ethanol extractives Insoluble contents Glucan Xylan Acid-soluble lignin Acid-insoluble lignin Acetyl
DDAPCS, %
EDAPCS, %
SEPCS, %
28.6 ± 1.1 1.8 ± 0.1 8.9 ± 0.2 0.9 ± 0.1 2.0 ± 0.4 3.2 ± 0.0 0.4 ± 0.1 0.2 ± 0.0 0.1 ± 0.0 11.1 ± 0.8 60.8 ± 2.3 38.6 ± 0.6 2.3 ± 0.0 0.4 ± 0.0 16.6 ± 0.3 1.0 ± 0.3
48.1 ± 0.8 0.4 ± 0.4 0.8 ± 0.1 1.4 ± 0.1 5.9 ± 0.1 14.1 ± 1.6 0.2 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 1.8 ± 0.9 50.6 ± 1.7 27.9 ± 0.1 9.9 ± 0.2 3.0 ± 0.0 5.0 ± 0.3 0.2 ± 0.1
38.9 ± 0.5 1.1 ± 0.1 1.9 ± 0.0 1.8 ± 0.0 6.3 ± 0.1 7.3 ± 0.4 0.9 ± 0.1 0.6 ± 0.0 0.3 ± 0.0 8.2 ± 1.1 52.3 ± 2.1 26.8 ± 0.8 6.0 ± 0.1 0.6 ± 0.0 17.1 ± 0.6 3.1 ± 0.2
a All compositions were based on dry matter. Data were determined in triplicates.
4500 rpm for 10 min and the supernatant was filtrated through Whatman No. 1 filter paper. The washing liquids (namely soluble fraction) were stored at 20 °C until used. The composition of the SF (monomeric and oligomeric sugars, soluble lignin, and byproducts) were determined according to LAP of NREL. The solid fraction was freeze-dried and then subjected to an extensive enzymatic hydrolysis according to previous report (Berlin et al., 2006). Cellulase and hemicellulase were added to the solid residue with 60 and 40 mg protein/g glucan, respectively, with 3% glucan loading at pH 4.8, 50 °C and 200 rpm for 168 h. After hydrolysis, the enzymatic residue was recovered by centrifugation and washed three times in deionized water and sonicated for 60 min. The enzymatic residue was then incubated at 37 °C for 24 h in 50 mM phosphate buffer (pH = 7) containing 1 U/mL Pronase (Sigma, US). The protease was then deactivated by incubation at 90 °C for 2 h. The enzymatic residue was washed extensively with deionized water and freeze-dried. The ERF was stored in sealed vials at room temperature in a desiccator. 2.5. Characteristics of ERF
Glucose ðxyloseÞ yield ¼ ½glucose ðxyloseÞ concentration; g=L 20 mL 0:9ð0:88Þ =½glucan ðxylanÞ weight in substrate; mg 100%
ð1Þ
2.4. Preparation of soluble fraction and enzymatic residual fraction from PCS Soluble fraction (SF) and enzymatic residual fraction (ERF) were prepared by the following procedure (Scheme 1). PCS was milled in a planetary ball mill (DECO, China) with zirconium dioxide balls at 300 rpm for 4 h. The particles passed through 80 meshes were collected. The milled PCS was incubated with 5 mM citrate buffer in solid to water ratio of 1:7 for 1 h. The mixture was centrifuged at
Elemental analysis of ERF was performed using a LECO CN-2000 elemental analyzer (Leco, MI, US). FT-IR spectra were obtained using a Nicolet IZ10 Fourier transform infrared spectroscopy and potassium bromide disc containing about 1% solid. Spectra were obtained by averaging 32 scans from 4000 to 400 cm1. 2.6. Determining the effects of SF and ERF on EH The hydrolysis of pure cellulose (Avicel) was performed for carrying out the comparison of SF and ERF in PCS. Cellulose conversion is defined as:
Cellulose conversion ¼ ½glucose concentration; g=L 20 mL 0:9= ½cellulose weight in Avicel; mg 100% ð2Þ Washing liquid (SF) prepared from PCS was diluted into different ratios (0%, 10%, 25%, 50% and 100%). Avicel was hydrolyzed in the diluted washing liquid with 1% glucan loading at pH 4.8, 50 °C and 150 rpm. Cellulase loading was 15 mg protein/g glucan. To explore the effect of ERF, 10 mg/mL ERF solid was added into the enzymatic hydrolysate of Avicel (pH 4.8, 50 °C and 150 rpm). Glucan loading was 1% and cellulase loading was 15 mg protein/ g glucan. Glucose concentration in hydrolysates was analyzed by HPLC with Aminex HPX-87H column. 3. Results and discussion 3.1. Composition and EH of PCS
Scheme 1. Preparation process of SF and ERF.
The compositions of the PCS with different pretreatments are listed in Table 2. In order to determine the soluble sugars, extraction process was carried out prior to two-stage acid hydrolysis. Extraction also dissolves part of lignin, making acid-soluble lignin (ASL) and acid-insoluble lignin (AIL) content lower than other reports. Statistically, ethylenediamine pretreated corn stover (EDACS) has the highest water extractives, followed by steam exploded corn stover (SECS) and dry dilute acid pretreated corn stover (DDACS). The xylose content of DDACS is significantly higher than other groups of PCS. The soluble lignin content is highest in EDACS. The xylo-oligomer content is similar for EDACS and SECS. Ethanol extractives in EDACS is lower than DDACS and SECS. For insoluble contents, DDACS has the highest glucan content and
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the lowest xylan content, while EDACS has the highest xylan and ASL content and the lowest AIL content. The FT-IR spectra of untreated CS (UTCS) and PCS are shown in Fig. S1. The decrease in the peaks around 1730 and 1250 cm1 of all PCS are due to the acetyl groups cleavage of hemicellulose (Hsu et al., 2010). A peak around 1642 cm1 of EDACS indicates the ammonolysis of the acetyl groups in hemicellulose and lignin. The EH of PCS at 1% and 6% glucan loading were implemented (Figs. 1 and 2). Previous studies showed that EH of SECS with enzyme loading of 15 FPU/g glucan achieved glucose yield of 60– 80% at different glucan loadings (Liu et al., 2013a,b). EDACS exhibited a glucose yield of 84% at 1% glucan loading (Qin et al., 2015). Previous report showed that glucose yield of DDACS reached 70% with the same pretreatment and hydrolysis conditions (He et al., 2014b). Our results just agree with these reports. Figs. 1 and 2 show that the glucose yield of EDACS is higher than that of DDACS and SECS. DDACS has higher xylan conversion due to its lower xylan content. The increase of glucan loading decreases glucose yield for all PCS. When glucan loading increases from 1% to 6%, glucose yield at 120 h of DDACS, EDACS and SECS decreases from 80.4%, 92.0% and 69.6% to 56.6%, 68.8% and 49.8% (decreases by 28%, 25% and 29%), respectively. As known, the reduction of sugars yield at high solid loading is due to insufficient mass and heat transfer, increased concentration of inhibitors, lack of available water, and so on (Modenbach and Nokes, 2013). Herein, the inhibitors may exhibit significant impact on EH because the PCS is washing-free. The impact of inhibitors on sugar yield and enzyme activity is dependent on the types and amounts of inhibitors, which needs intensive studies.
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3.2. Effect of SF on the EH of cellulose In order to explore the impact of SF on enzyme digestibility, PCS was washed by 5 mM citrate buffer with a solid to liquid ratio of 1:7, and each washing liquid with different dilution ratios was added into the EH of Avicel. The components of washing liquid are listed in Table 3. The concentration of each component is generally proportional to the corresponding composition in PCS. The glucose concentrations in EH with different species and amounts of SF are detected. Control groups without Avicel addition were carried out to determine the glucose produced from SF (Fig. S3). The resulting cellulose conversions based on glucose concentrations are shown in Fig. 3. All the washing liquids decrease the cellulose conversion. Higher washing liquid concentration leads to lower cellulose conversion. It is observed that washing liquid of SECS results in the lowest cellulose conversion, followed by EDACS, DDACS, and UTCS. Washing liquid (100%) of DDACS, EDACS, SECS and UTCS decrease cellulose conversion from 58.3% (0% washing liquid) to 39.4%, 17.7%, 16.3%, and 42.9% at 72 h, respectively. According to previous study (Holtzapple et al., 1990), the ratio of cellulose conversion without inhibitors to cellulose conversion with inhibitor (A0/A) has a linear relationship with the concentration of inhibitor as following equation:
A0 =A ¼ 1 þ bI
ð3Þ
where I is the concentration of inhibitor (g/L); b is the inhibition constant (L/g). The value of A0/A represents the inhibition degree. In our study, the dilution ratio of washing liquid is proportional to the inhibitor concentration. Thus Eq. (3) can also be expressed as:
A0 =A ¼ 1 þ b0 n
ð4Þ
Fig. 1. EH of PCS by DDA, EDA and SE at 1% glucan loading. (a) Sugar concentrations; (b) sugar yields (based on glucan and xylan content in PCS). The default hydrolysis conditions were 15 mg cellulase per g glucan, 10 mg hemicellulase per g glucan, pH 4.8, 50 °C and 150 rpm. Error bars represented standard deviations, n = 3.
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Fig. 2. EH of PCS by DDA, EDA and SE at 6% glucan loading. (a) Sugar concentrations; (b) sugar yields (based on glucan and xylan content in PCS). The default hydrolysis conditions were 15 mg cellulase per g glucan, 10 mg hemicellulase per g glucan, pH 4.8, 50 °C and 200 rpm. Error bars represented standard deviations, n = 3.
Table 3 Components of SF in concentration (g/L).
Glucose Xylose Gluco-oligomer Xylo-oligomer Soluble lignin Acetic acid 5-HMF Furfural
3.3. Effect of ERF on the EH of cellulose
DDA
EDA
SE
UT
2.6 ± 0.0 13.0 ± 0.2 0.8 ± 0.1 3.7 ± 0.2 2.3 ± 0.1 0.5 ± 0.1 0.3 ± 0.0 0.2 ± 0.0
0.3 ± 0.0 0.2 ± 0.0 0.8 ± 0.1 7.6 ± 0.1 10.5 ± 0.5 0.3 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
0.7 ± 0.0 1.5 ± 0.1 2.4 ± 0.2 7.9 ± 0.1 4.4 ± 0.2 1.2 ± 0.0 0.8 ± 0.0 0.3 ± 0.0
0.8 ± 0.0 1.2 ± 0.0 0.5 ± 0.1 0.2 ± 0.1 1.3 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
where n is the dilution ratio; b0 is another expression of inhibition constant. Dilution ratio of washing liquid from each PCS shows a significant linear correlation with A0/A (Fig. 4). The slope of the line represents the inhibition constant b0 . Inhibition constants of DDACS, EDACS, SECS and UTCS are 0.648, 2.941, 1.859 and 0.531, respectively. It is obvious that higher inhibition constant leads to greater inhibitory effect. The relationship between the inhibition constant and the concentration of washing liquid showed that xylo-oligomer and soluble lignin concentration may have the greatest impact on inhibition degree (Fig. S4). Previous researchers proposed that xylo-oligomer simulate the structure of cellulose chain and bind to the active site tunnel of cellobiohydrolase (Kont et al., 2013). The oligosaccharides are hardly hydrolyzed by homologous cellulase in the absence of accessory enzymes (Xue et al., 2015). Soluble phenolics predominates over oligosaccharides in decreasing the final cellulose conversion, because phenolics lead to precipitation and irreversible inhibition of cellulase (Kim et al., 2011).
The compositions of ERF for each PCS are listed in Table 4. ERF of DDACS has the highest AIL content (84.0%), followed by EDACS (73.8%), SECS (59.5%) and UTCS (26.5%). The content of unhydrolyzed glucan and xylan in UTCS are significantly higher than that of PCS. FT-IR spectra of ERF show that the peaks related to cellulose (e.g. 1050 and 1161 cm1) decrease and the peaks related to lignin (e.g. 1428, 1513 and 1604 cm1) increase compared to PCS (Fig. S2), which indicates the removal of cellulose content in ERF. Noticeable drops in peak intensity was observed near 1260–1270 cm1 in ERF of DDACS and EDACS compared to UTCS and SECS, corresponding to the guaiacyl (G) ring breathing with CAO stretching. There is no appreciable change in the 1300–1330 cm1 region of all ERF that represents the syringyl (S) ring breathing (Davidson et al., 2005). This shows that the guaiacyl type of lignin is more prone to be hydrolyzed than the syringyl type after DDA and EDA pretreatment. Previous finding show that low S/G ratio leads to high protein adsorption capacity for lignin, which could decrease the adsorption of enzyme to cellulose (Guo et al., 2014). Therefore, the residual lignin of DDACS and EDACS could be less detrimental to enzymatic hydrolysis than UTCS and SECS. Elemental analysis was conducted to determine the residual protein in ERF. Table 5 shows that there is little nitrogen content (less than 1%) in ERF, except for EDACS. The high nitrogen content in ERF of EDACS is due to the ammonolysis of lignin during pretreatment (Qin et al., 2015). Comparative nitrogen contents were observed in the enzymatic residues in previous reports (Berlin et al., 2006; Nakagame et al., 2010).
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Fig. 3. Effect of SF from different PCS on Avicel conversion. (a) DDACS; (b) EDACS; (c) SECS; (d) UTCS. Washing liquids were added into hydrolysates in terms of 0, 0.1, 0.2, 0.5 and 1.0 mL/mL hydrolysate.
Table 4 Compositions of ERF.
Glucan Xylan Acid-insoluble lignin
DDA, %
EDA, %
SE, %
UT, %
6.2 ± 0.9 1.4 ± 0.7 84.0 ± 0.4
2.8 ± 0.7 1.4 ± 0.4 73.8 ± 0.7
20.0 ± 1.1 8.0 ± 0.2 59.5 ± 0.7
33.8 ± 0.9 20.8 ± 0.2 26.5 ± 0.2
Table 5 Elemental analysis of ERF.
C N
Fig. 4. Effect of SF on inhibition degree (A0/A). (a) DDACS; (b) EDACS; (c) SECS; (d) UTCS. A0/A were calculated based on the data at 24 h hydrolysis.
The Avicel was enzymatic hydrolyzed with the addition of 10 mg/mL ERF. Control groups without Avicel addition were also conducted (Fig. S5). Avicel cellulose conversions with different ERF addition are shown in Fig. 5. All enzymatic residual solids lead
DA, %
EDA, %
SE, %
UT, %
53.66 ± 0.03 0.82 ± 0.01
48.57 ± 0.64 2.83 ± 0.07
55.65 ± 0.39 0.90 ± 0.04
43.42 ± 0.19 0.50 ± 0.01
to decrease of cellulose conversion. The conversion decreases from control (46.9%) > UT (42.4%) > EDA (41.8%) > SE (37.2%) > DDA (34.4%) at 24 h. The values of A0/A at 24 h were 1.105, 1.361, 1.122 and 1.260 for the ERF of UT, DDA, EDA and SE, respectively. The inhibition degree almost keeps stable along with the hydrolysis time. It is believed that the inhibition of lignin on hydrolysis is mainly due to the unproductive binding of the enzymes. In general, higher lignin content in ERF should induce to higher inhibition. However, the lower lignin content in SECS–ERF results in more inhibition than EDA–ERF (SECS–ERF and EDA–ERF contain 59.5% and 73.8%
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Fig. 5. Effect of ERF from different PCS on Avicel conversion. Cellulase loading is 15 mg protein/g glucan. ERF was added at 10 lg/mL. Control means no ERF was added in Avicel’s hydrolysis.
lignin, achieve 37.2% and 41.8% glucose yield, respectively). This result may be due to the different enzyme adsorption to lignin, due to lignin sources and structural features, such as S/G ratio (Guo et al., 2014). The inhibition of lignin is an important issue in SE pretreated biomass. The lignin isolated from steam pretreated poplar decreased the hydrolysis yields of cellulose, whereas the other isolated lignins did not appear to decrease the hydrolysis yields significantly (Nakagame et al., 2010). Rahikainen et al. (2013) showed that SE alters the lignin structure, leading to increased enzyme adsorption. Adsorption and enzyme specific activity were shown to be inversely proportional to lignin content and hydrophobicity of SE pretreated biomass (Heiss-Blanquet et al., 2011). It is also suggested that the deposition of small spherical lignin droplets on the fiber surface during acidic pretreatment likely limits the access of enzymes to cellulose. In contrast, the ammoniation of lignin was reported to decrease the lignin–enzyme interaction (Sewalt et al., 1997), which results in lower inhibition of enzymatic residue of EDACS. These could explain the higher inhibition of enzymatic residue of SECS.
3.4. Comparison of SF and ERF in PCS The effects of SF and ERF have been separately analyzed and quantified by inhibition constant, which provides an approach to figure out the dominant inhibitory factor from SF and ERF. Here we stimulate the effect of SF and ERF on cellulose conversion in PCS by adding the SF and ERF preparation into the enzymatic hydrolysate of Avicel. The addition amount of SF and ERF was calculated and equal to that in the real enzymatic hydrolysate of untreated/pretreated CS with 1% glucan loading. The resulting inhibition A0/A from SF and ERF can be calculated through dilution ratio n and ERF concentration I in Eqs. (4) and (3), respectively. As untreated/pretreated CS was washed with solid-to-liquid ratio of 1:7 (12.5% solid loading), the dilution ratio of hydrolysate in 1% glucan loading equals to the corresponding solid loading divided by 12.5%. The actual loading of AIL in the hydrolysate of untreated/pretreated CS with 1% glucan loading determined the calculated ERF concentration (that is, the calculated ERF concentration contain the same AIL concentration with real enzymatic hydrolysate of untreated/pretreated CS, as shown in Table S2). It is noticeable that b and b0 refer to the inhibition per unit inhibitor,
Fig. 6. Calculated inhibition degree (A0/A) of SF and ERF in the EH of untreated/ pretreated CS with 1% glucan loading.
and A0/A stands for the inhibition from actual quantity of inhibitors in hydrolysate. From Fig. 6, we can see that, for EDACS, A0/A of SF is 1.76 times to A0/A of ERF. For SECS, A0/A of SF is 1.21 times to A0/A of ERF. The significant higher A0/A of SF than ERF in EDACS and SECS indicates that SF of them are the dominant factor inhibiting the cellulose conversion. The inhibition of ERF in UTCS and DDACS is slightly higher than that of SF (for UTCS and DDACS, A0/A of ERF is 1.07 and 1.05 times to A0/A of SF, respectively). Through this quantitative analysis, not only the inhibitory effect between the pretreated biomass, but also the inhibitory effect between SF and ERF can be revealed clearly. It shows that SF of EDACS, SF and ERF of SECS cause significant inhibition during enzymatic hydrolysis.
4. Conclusions The SF and ERF were separated from different PCS to examine the inhibition degree on enzymatic digestibility of cellulose. The SF of EDACS and SECS exhibited higher inhibitory effect compared to that of DDACS, due to the higher concentration of soluble lignin and xylo-oligomer. The ERF of DDACS and SECS exhibited higher inhibition than that of EDACS. The inhibition of SF was slightly lower than ERF in DDACS, while the inhibition of SF was significantly higher than ERF in EDACS and SECS. Thus, in order to reduce the inhibition on enzymatic hydrolysis from feedstock, different detoxification strategies should be developed. Acknowledgements This study was financially supported by the Ministry of Science and Technology of China (‘‘973” Program: 2013CB733601), the National Natural Science Foundation of China (Major program: 21390203), Tianjin Municipal Science and Technology Commission (13RCGFSY19800).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2016.02. 123.
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