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Urea for enhanced enzymatic conversion and hydrogen production
Bioresource Technology 287 (2019) 121399
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High-solid pretreatment of rice straw at cold temperature using NaOH/Urea for enhanced enzymatic conversion and hydrogen production
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Lili Dong, Guangli Cao , Jiwen Wu, Bingfeng Liu, Defeng Xing, Lei Zhao, Chunshuang Zhou, Liping Feng, Nanqi Ren State Key Laboratory of Urban Water Resource and Environment, School of Environment, Harbin Institute of Technology, Harbin 150090, China
A R T I C LE I N FO
A B S T R A C T
Keywords: High-solid loading NaOH/Urea pretreatment Low temperature Rice straw Enzymatic Hydrogen
A high-solid loading pretreatment using NaOH/Urea solution at −12 °C was proposed to pretreat rice straw (RS) for enhanced saccharify and hydrogen production. Results shown NaOH/Urea pretreatment exhibited excellent pretreatment performance at solid loading ranged from 10% to 100% (w/v) with an average reducing sugar conversion of 80.22% was obtained which was 31.89% higher than that untreated RS. Upon fermentation of 100% solid loading pretreated hydrolysate, the H2 yield of 72.5 mL/g-pretreated RS was calculated based on substrate consumption, which enabled 49.5% higher reducing sugar transfer to H2 through material balance. FTIR and XRD analysis further demonstrated that the cold NaOH/Urea pretreatment at 100% (w/v) could effectively disrupt the lignin structure and decrease the cellulose crystallinity. The present study suggested a high solid loading pretreatment with NaOH/Urea at cold temperature could be a valuable alternative for better techno-economic of the lignocelluloses – to – sugars – to H2 routes.
1. Introduction During last decades, the industrial revolution has brought unimaginable benefits to human societies by providing optimized material products and human activity, along with higher living standards for humanity, but has also brought climate change and energy crisis. Hence, the development of renewable energies have promoted in the worldwide (Wang et al., 2018; Toquero and Bolado, 2014; Mahmood et al., 2016). Conversion of abundant lignocellulosic biomass to biofuels presents a promising option for improving energy security along with reducing the emission of green-house gases, preventing the consequent increase in the average global temperature, and producing a sustainable energy (Dyk and Pletschke, 2012; Zhao et al., 2012a,b; Sindhu et al., 2016). However, due to the recalcitrant structure of lignocellulose, the low accessibility of enzymes to the carbohydrate polymers is the primary obstacle for the bioconversion of lignocellulosic biomass (Sindhu et al., 2016; Dong et al. 2018). It’s reported that only 20% theoretical maximum sugar yield can be obtained from enzymatic hydrolysis of lignocellulosic biomass without appropriate pretreatment (Zhao et al., 2012a,b; Kim et al., 2006; Mosier et al., 2005). Thus, efficient pretreatment is required to disrupt the heterogeneous matrix, increase surface area and porosity of the cellulosic material, so as to enhance the enzymatic hydrolysis and cellulose conversion.
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Various techniques including physical pretreatments (size reduction), physico-chemical pretreatments (liquid hot water, steam explosion, ammonia fiber explosion), chemical pretreatments (acid, alkaline, alkaline/oxidative, wet oxidation, ozonolysis) and biological pretreatments have been developed to improve the accessibility of enzymes to cellulosic materials (Toquero and Bolado, 2014). Among these methods, alkali is relatively inexpensive and more effective in reducing lignin content and increasing the accessibility of cellulose (Sivers and Zacchi, 1995, Chang et al., 2001). However, alkali pretreatments commonly require over 100 °C pretreatment temperature, long duration time, and with the drawbacks of special equipment requirement (Mosier et al., 2005; Lee et al., 1999; Palmqvist and Hahn – Hagerdal, 2000; Tim and Riehard, 2005; Ren et al., 2009). Recently, researchers found that alkali based solvent with urea at low temperature can effectively enhance the biodegradability of lignocellulosic material. This process has been receiving more and more attention for the enhancement of enzymatic conversion and biofuel production. In the cold climate region, the average daily temperature in winter is between −5 and −20 °C, if NaOH/Urea pretreatment can be taken at a high solid loading, both scientific and economic breakthroughs can be obtained (Zhao et al., 2008; Li et al., 2016). Kuo and Lee (2009) have reported that the enzymatic hydrolysis efficiency of cotton cellulose pretreated with NaOH/Urea solution at −20 °C was improved from 23% to 85%,
Corresponding author. E-mail address: [email protected] (G. Cao).
https://doi.org/10.1016/j.biortech.2019.121399 Received 3 April 2019; Received in revised form 26 April 2019; Accepted 28 April 2019 Available online 30 April 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
Bioresource Technology 287 (2019) 121399
L. Dong, et al.
180%, 200% (w/v) and quickly stirred for 3 min, respectively. In the stirring process, we adopted mechanical stirring, and the speed of the agitator was set at 350 rpm. After that, the pretreated RS samples were filtered and washed thoroughly with distilled water, and neutralize to pH 7.0, and then dried at 60 °C until constant weight was obtained and preserved for further use.
which implied that approximately 3-fold enhancement on cellulose saccharification conversion. Zhao et al. (2008) have demonstrated that the enzymatic efficiency was improved significantly when spruce was pretreated with NaOH/Urea solution at −15 °C. Our previous research also showed that the cold NaOH/Urea based pretreatment could efficiently remove lignin and retain most of cellulose and hemicellulose in rice straw. Upon fermentation of pretreated rice straw, 113.53% higher hydrogen yield was obtained by NaOH/Urea pretreatment at −12 °C compared with untreated sample (Dong et al., 2018). However, lowsolids loadings (≤5% solids, w/v) are usually used in this pretreatment process. Over the last few years, numerous studies utilizing low-solids loadings (≤10% solids, w/v) are efficient and helpful, but the use of high-solids loadings (> 15% solids, w/v) potentially offers many advantages over lower solids loadings such as decreased operation costs and increased industrial application or the economics (Wang et al., 2018; Modenbach and Nokes, 2012; Martínez-Patino et al. 2015; Zhang et al., 2010; Cheng et al., 2010). Although efforts have been made to carry out the effects of high-solids loadings on lignocellulosic materials pretreatment process for improving the saccharification efficiency of enzymatic hydrolysis. However, current technology allows only up to 50% of the solid loading to be used in the pretreatment process, the effect of higher solid loading more than 50% on pretreatment efficiency has not been studied (Modenbach and Nokes, 2012; Luterbacher et al., 2010; Zhu et al., 2009; Roche et al., 2009; Cheng et al., 2010). Meanwhile, the adoption of high-solids loadings with NaOH/Urea pretreatment at low temperature is still relatively unexplored. So breakthroughs in increasing the solid loading in cold NaOH/Urea pretreatment are needed for both economic and practical reasons. At industrial level, even a small improvement in the pretreatment process efficiency may have outstanding economic benefits. The goal of this study is to evaluate the feasibility of using highsolids loadings in NaOH/Urea pretreatment at cold temperature for pretreated rice straw (RS). The changes of chemical properties of NaOH/Urea pretreated RS with solid loadings ranging from 10% up to 200% were firstly described. Then the enzymatic hydrolysis efficiency of pretreated RS was evaluated to determine whether NaOH/Urea solution pretreatment at high-solids loadings could enhance reducing sugar production. After that, the produced reducing sugars were fermented to H2 by T. thermosaccharolyticum W16. At last, fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) were adopted to give a micro perspective on how the pretreatment process at high-solid loading induced the structural changes of RS. The feasibility of lignocellulosic conversion would be greatly improved if high-solids loadings could be used in the unit of pretreatment. Meanwhile, it could be helpful to enlarge the application scale of this solution system.
2.3. Enzymatic hydrolysis Enzymatic hydrolysis experiment was carried out at a substrate concentration of 2% (w/v) with a mixture cellulolytic hybrid enzyme from Cellulase in sodium citrate buffer solution for pretreated and untreated RS. In the subsequent enzymatic hydrolysis, the effects of different cellulase dosage (1–5 FPU/g RS) were firstly determined for the optimal enzyme units of enzymatic hydrolysis using NaOH /Urea pretreated RS at solid content of 3% (w/v). Secondly, the RS pretreated with different solid loading was hydrolyzed by enzymes at the the optimal amount previously optimized. In all cases, enzymatic hydrolysis was carried out at pH 6.0, 50 °C in a rotary shaker at 135 rpm. Each experiment was performed in triplicate. 2.4. Biohydrogen production in batch culture The RS hydrolysate was used as carbon source to produce H2 by T. thermosaccharolyticum W16 isolated by Ren et al. (2009). Hydrogen production was performed in 100 mL serum vials with working volume of 50 mL. The tested hydrolysate was diluted to reduce the sugar concentration to 10 g/L and supplemented with the following nutrients (L−1): 1.0 g NH4Cl, 3.0 g K2HPO4, 1.5 g KH2PO4, 0.5 g MgCl2·6H2O, 1.0 g NaCl, 0.2 g KCl, 0.5 g cysteine-HCl, 2.0 g yeast extract, and 2.0 g tryptone. The culture temperature and pH were 60 °C and 7.0, respectively. During the course of fermentation, biogas production and compositions of produced biogas were monitored with respect to culture time. All tests mentioned above were performed in triplicate to determine the reproducibility of the experiments. 2.5. Analytical methods The cellulose, hemicellulose and lignin contents of RS were measured using a Fiber analyzer A200i (ANKOM Co., US). Microstructural changes in the RS before and after pretreatment were observed by FTIR and XRD. FTIR spectroscopy analysis was carried out using a Magna-IR 750 (Nicolet Instrument Co., USA) according to the method described by Dong et al. (2018). The crystallinity of the RS was determined by Xray diffraction (XRD) using a D8 X’Pert PRO MPD diffractometer (PANalytical Ltd., Holland). The cellulase activity was measured according to the method reported by Adney and Baker, 2008. The final of reducing sugar content of the hydrolytic samples was analyzed by the dinitrosalicylic acid (DNS) method (Wang et al., 2016). High performance liquid chromatography (HPLC) system (LC-10A, Shimadzu Corporation, Kyoto, Japan) was used to detect the initial sugar compositions of the hydrolysate, and the sugar consumption during fermentation (Qi et al., 2008). The gas products (H2 and CO2) were analyzed by gas chromatography (7890D, Agilent Cooperation, USA) using a thermal conductivity detector.
2. Materials and methods 2.1. Raw material The RS used in this study was collected from Wuchang, Heilongjiang province, China. The raw material was milled to pass through a 40mesh (0.425 mm) screen, and then dried at 60 °C until constant weight was acquired. The main constituents of RS on a dry basis were 35.97% cellulose, 27.45% hemicellulose, 14.08% lignin. Cellulase was kindly provided by Novozymes (China) Investment Co., Ltd. Sodium hydroxide and urea were used as received from Sigma-Aldrich (St. Louis, MO).
2.6. Data analysis
2.2. Pretreatment of RS at different solid loadings
The enzyme saccharification rate was calculated as (Li et al., 2009): enzymatic saccharification (%) = reducing sugars released (g) × 0.9 × 100/polysaccharides in substrate (g) where 0.9 is the correction coefficient for hydrolysis. Acid detergent fiber (ADF), neutral detergent fiber (NDF), residue after 72% H2SO4 treatment, and acid detergent lignin (ADL) were determined to calculate the contents of cellulose, hemicellulose and lignin. Calculation equations as follow:
NaOH /Urea pretreatment solutions were prepared by mixing 7% w/v NaOH and 12% w/v urea in deionized water and pre-cooled and maintained at the temperature of −12 °C according to previous study (Dong et al. 2018). To investigate the effect of different solid loading on pretreatment of RS, the RS was immersed immediately in the solvents with different solid content of 10%, 20%, 50%, 100%, 130%, 160%, 2
Bioresource Technology 287 (2019) 121399
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Hemicellulose (%) = NDF% − ADF%
(1)
Lignin (%) = ADL%
(2)
Cellulose(%) = ADF% − Residue after 72% H2 SO4
(3)
based on dry matter after pretreatment generally decreased at solid loading more than 100%. Although a higher the residue solid yield obtained at solid loading more than 100%, the contents of cellulose and hemicellulose were 37.76% and 30.60% at 200% solid loading, which were similar to that in raw RS. It suggested that pretreatment efficiency appeared a mild agent at solid loading more than 100%. The results demonstrated that a high solid loading pretreatment with NaOH/Urea solution at low temperature were helpful for the saccharification of RS.
The X-ray diffractometer was set at 45 kV and 200 mA. XRD patterns of the samples were recorded over the range of 2θ = 5–90° at a scan speed of 2 °min−1 with a step size of 0.02°. The crystallinity index (CrI) was calculated according to Eq. (1) (Kim and Holtzapple, 2006).
CrI (%) = [(I002 − Iam)/ I002] × 100
3.2. Enzymatic hydrolysis of high solids based pretreated RS
(4)
I002 is the maximum diffraction intensity of the crystalline structure and Iam is the diffraction intensity of the amorphous fraction at 2θ ≈ 18°.
3.2.1. Production of reducing sugars in optimal enzyme units of enzymatic hydrolysis The optimal ratio of enzyme to substrate is crucial for the efficient utilization of enzymes. Thus, the effects of cellulase dosage on the production of reducing sugars were firstly investigated, and the results were presented in Fig. 1a. Various concentrations (1, 3 and 5 FPU g−1 RS) of cellulase was used for hydrolysis of RS which was pretreated at solid loading of 3% (w/v) with NaOH /Urea at −12 °C. The profiles of reducing sugars were examined over a 60 h period. The release of reducing sugars continuously increased for 48 h and then remained stable at tested cellulase dosage. Moreover, with the increase of cellulase dosage, the saccharification efficiency increased. However, cellulase with 5 FPU g−1 RS could achieve a higher enzyme saccharification efficiency, which only increased 2.84% compared with 3 FPU g−1 RS. Considering the comprehensive treatment effect and economy, 3 FPU cellulase g−1 RS were selected as the optimal concentrations for the hydrolysis experiments in further experiments.
3. Results and discussion 3.1. Effect of solid loadings on the composition of RS Solid loading is an important factor that not only affects the improvement of cellulose digestibility by the removal of lignin component or partial depolymerization but also influences the reduction of the volume of pretreatment equipment and the consumption of water (Modenbach and Nokes, 2012). The effect of solid loading increasing from 10% to 200% (w/v) on NaOH/Urea pretreatment was investigated using a low temperature (−12 °C) and short reaction time (3 min). The chemical compositions of RS and the percentage of weight loss of each different solid loading through NaOH/Urea solution pretreatment was shown in Table 1. The solid loading ranged from 10% to 50% had a significant influence on the delignification of RS, the amount of lignin removal ranged from 59.91–62.35%. When the solid loading continued improving to 100%, only 4.39% decreased on the content of lignin of RS. It suggested that NaOH/Urea solution pretreatment at high solid loading 100% obtained a satisfactory delignification result. However, when the solid loading future increased until to 200%, the reduction of lignin decreased sharply, only 38.76% removal rate at 200% solid loading pretreatment process. This implied that the effect of NaOH/ Urea on RS was not as good as previous at solid content exceeds 100%. In term of lagre-scale and industrial application, an ideal pretreatment is not only to remove as much unusable lignin as possible, but also to maximally retain all available carbohydrates for further bioenergy production (Lou et al., 2016). In contrast to large amount of lignin removal at solids loadings ranged from 10% to 100%, more than 89% cellulose and 81% hemicellulose were retained after pretreatment. It can be obtained from Table 1 that the content of cellulose and hemicellulose slightly decreased 1.28% and 8.75% at solid loading was 100% compared to solid loading 10–50%, indicated that most of the cellulose and hemicellulose remained in RS at high solid loading. Meanwhile, it was observed that cellulose and hemicellulose contents
3.2.2. Sugar yields from high solids based pretreated RS To evaluate the efficiency of high-solids loadings pretreatment with NaOH/Urea at cold temperature, the RS residues after pretreatment were subsequently hydrolyzed with 3 FPU g−1 RS commercially available cellulase for 60 h. As shown in Fig. 1b, the solid loading could greatly affect enzymatic hydrolysis. The reducing sugar conversion ratio of RS decreased from 80.22% to 50.67% as solid loading increased. It should be noted that the reducing sugar conversion yields ranged from 77.59 to 80.22% at 10–100% solid loading. Although, there was no significant difference (P > 0.05) was found among these solid loadings indicating that NaOH/Urea pretreatment had a considerable effective effect on enzymatic hydrolysis when solid loading was less than 100%, nevertheless, the enzymatic hydrolysis efficiency decreased significantly when the solid loading exceeded 100%. It can be found that the reducing sugar conversion was decreased from 77.59% at 100% solid loading to 56.46% at 130% solid loading. What’s more, the reducing sugar conversion was nearly the same to that in the raw RS without pretreatment when the solid loading increased to 200% during pretreatment. This result was consistent with the result of
Table 1 Compositions of raw at different solid-loading NaOH/Urea solution pretreated RS at −12 °C. Solid-loading (w/v)
Composition (%)a Cellulose
10% 20% 50% 100% 130% 160% 180% 200% Untreated a b c
45.59 45.00 44.12 41.02 38.57 38.03 38.00 37.76 35.97
± ± ± ± ± ± ± ± ±
0.05 0.98 0.05 0.00 0.38 0.06 0.66 0.50 0.14
Residue solid (%)b Hemicellulose
Lignin
32.00 31.57 31.09 31.37 30.67 31.43 31.17 30.60 27.45
7.84 ± 1.30 7.88 ± 1.09 7.88 ± 0.08 7.84 ± 1.02 8.45 ± 0.92 9.04 ± 0.00 9.02 ± 0.05 9.84 ± 1.90 14.08 ± 3.20
± ± ± ± ± ± ± ± ±
1.00 1.67 3.17 4.16 2.72 1.51 1.23 1.00 1.00
70.40 71.50 74.30 80.00 85.00 86.50 87.80 89.00 –
± ± ± ± ± ± ± ±
Composition is shown as percentage of the solid fraction before and after pretreatment. Solid yield is shown as percentage of the initial amount of dry matter. Removal yield is shown as percentage of the amount in the initial material. 3
2.84 1.61 1.50 1.80 1.00 2.74 6.10 1.81
Removal yield (%)c Cellulose
Hemicellulose
Lignin
10.72 ± 0.01 10.50 ± 0.02 8.82 ± 0.00 8.73 ± 0.04 8.81 ± 0.01 8.51 ± 0.02 7.21 ± 0.03 6.54 ± 0.06 –
18.23 ± 0.03 18.06 ± 0.05 16.11 ± 0.07 8.72 ± 0.05 5.11 ± 0.03 0.97 ± 0.01 0.31 ± 0.03 0.80 ± 0.03 –
62.35 61.51 59.91 56.87 50.24 45.60 44.87 38.76 –
± ± ± ± ± ± ± ±
0.05 0.00 0.06 0.04 0.04 0.00 0.04 0.12
Bioresource Technology 287 (2019) 121399
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Fig. 1. a. Reducing sugar released from hydrolysis of RS by various concentrations (1 FPU, 3 FPU, 5 FPU g−1 RS) of cellulase from Novozym at pH 6.0, 50 °C and 135 rpm. b. Reducing sugar conversion rate from hydrolysis of untreated and pretreated RS using NaOH/Urea aqueous solution at 10%, 20%, 50%, 100%, 130%, 160%, 180% and 200% solid loading pretreatment at 3 FPU g−1 RS, pH 6.0, 50 °C and 135 rpm. c. Composition of hydrolysate of untreated and pretreated RS using NaOH/Urea aqueous solution at 10%, 20%, 50%, 100%, 130%, 160%, 180% and 200% solid loading pretreatment of cellulase from Novozym at 3 FPU g−1 RS, pH 6.0, 50 °C and 135 rpm.
chemical analysis. Apparently, further increases in solid loading beyond 100% have little or no beneficial effect. HPLC analysis showed that the sugar composition of enzymatic hydrolysate were mainly composed of glucose and xylose, and a small amount of arabinose, galactose and rhamnose for untreated and pretreated RS (Fig. 1c). And, on average over 51% and 39% of the hydrolysate is glucose and xylose. So far, various pretreatment methods such as stream-explosion, diluted acid hydrolysis, ammonia pretreatment have been employed for enhancing enzymatic hydrolysis from lignocellulosic biomass, and solid loadings used in pretreatment procedure between 5% and 50% with a saccharification ratio of about 80% was obtained (Wang et al., 2018; Zhao et al., 2008). In this work, a comparable reducing sugar conversion was acquired using NaOH /Urea pretreatment at cold temperature with solid loading as high as 100% (w/v), indicating that cold NaOH /Urea pretreatment is effective at high solid loading. In other words, it can acquire reasonable pretreatment effect by increasing of solid loading more than 2 times, which means that the amount of water and chemical reagent used in the pretreatment process were reduced by more than 2 times. Usually, solid-loading in studies pretreated with alkali/urea was less than 5%, according to Zhao et al. (2008), 7% NaOH and 12% urea were used to pretreatment spruce at −15 °C with solid loading of 5%, results showed that a 70% sugar conversion was obtained after pretreatment. Recently, some pretreatment technologies were also explored at high solid loadings such as ammonia fiber expansion (AFEX), which have been reported to effectively improve biomass biodegradability when solid loading was as high as 90%. However, high temperature and pressure are needed in the AFEX pretreatment process (Wyman et al., 2005). Accordingly, it can be inferred that low temperature NaOH/Urea solvent at the high solid loading could still achieve an attractive treatment effect.
Fig. 2. H2 production by Thermoanaerobacterium thermosaccharolyticum W16 using enzymatic hydrolysate from 100% solid loading NaOH/Urea pretreated RS.
work is comparable to that of pretreated lignocellulose with bio-or physicochemical pretreatment at relative low solid loading reported in related studies (Li and Chen, 2007; Zhao et al., 2012a,b; Varrone et al., 2013; Cao et al., 2009). These results further indicate that bio-H2 production from RS was successful when using NaOH/Urea at 100% solid loading under low temperature pretreatment method.
3.4. The exploration of the high solid loading NaOH/Urea pretreatment on morphological and structural changes of RS
3.3. Hydrogen fermentation of RS based – hydrolysate FTIR was applied to further determine the chemical structure changes of RS at 100% solid loading NaOH/Urea pretreatment process. FTIR data showed band at approximately1250 cm−1 corresponding to guaiacyl ring breathing, CeO stretch in lignin and for CeO linkage in guaiacyl aromatic methoxyl groups, which are the main constituting units of lignin and xylan (Supplementary data). The absorption at 1733 cm−1 corresponding to either the acetyl and uranic ester groups of the hemicelluloses or the ester linkage of carboxylic group of the ferulic and p-coumaric acids of lignin and/or hemicelluloses. The bands near 1530 and 1059 cm−1 represent aromatic skeletal vibration CeH and CeO of lignin (supplementary material) (Dong et al., 2018; Kaplan and David, 1998; Zhao et al., 2012a,b). Compared with untreated RS, all of the functional group mentioned above decreased after pretreatment. However, the broad absorption band at 3407 cm−1 corresponds to the hydrogen-bond OeH stretching vibrations, which belong to the
Fermentative conversion of enzymatic hydrolysate of 100% solid loading pretreated RS into H2 was evaluated with T. thermosaccharolyticum W16. H2 was immediately produced after 5 h incubation with no lag phase, and maintained a high level of production rate before the incubation of 24 h (Fig. 2). After 36 h of fermentation, the cumulative hydrogen production reached 2540 mL/L. Coincide with the production of hydrogen, the sugars in hydrolysate were consumed quickly before 24 h of fermentation. At the end of fermentation, arabinose was completely consumed, 79.1% of glucose, 50% xylose and small amount of rhamnose were consumed, and the hydrogen yield based on substrate consumption was 1.9 mol H2 mol−1 sugar. On the basis of RS weight, H2 yield was 72.5 mL/g-pretreated RS. Compared H2 yields with reported in lignocellulosic biomass fermentation studies, it can be inferred that the performance of hydrogen production in this 4
Bioresource Technology 287 (2019) 121399
L. Dong, et al.
Fig. 3. Material balances of biohydrogen production from RS with and without NaOH/Urea pretreatment at 100% solid loading.
be the main reason for reducing crystallinity. The observations mentioned above demonstrated that the high solid loading pretreatment with NaOH/Urea at cold temperature of RS could effectively reduce lignin content and crystalline, which could lead to an increase in susceptibility of RS for enzymatic saccharification, making the production of renewable energy from biomass more efficient.
characteristic absorption peaks of the cellulose (Kaplan and David, 1998). This implied that the hydrogen bonds of RS were broken and their association degree decreased after the pretreatment. Additionally, the peak at the wavelength of 893 cm−1 was stronger after pretreatment than without pretreatment, indicating that the content of cellulose after pretreated RS was higher than untreated RS. Whereas, the bands near 1124 cm−1 and 1078 cm−1 represent aromatic skeletal and C-O stretching in cellulose and hemicelluloses. The changes in intensity ratio R = I (1121 cm−1)/I (1078 cm−1) illustrate the cellulose crystallinity (Zhao et al., 2012a,b), the crystallinity decreases as the ratio increases (supplementary material). Compared with untreated RS (R = 1.16459), the ratio of 100% solid loading NaOH/Urea pretreated RS increased to 1.26666 demonstrated crystallinity decreases after pretreatment. This result is in good agreement with the chemical components analysis To further understand the changes of the crystallinity of cellulose, XRD was employed to investigate the crystallinity of untreated and pretreated RS sample. XRD date showed the crystal decreased after treated compared with untreated RS (Supplementary data). The CrI of the untreated RS was 66%. A lower crystalline was achieved 55% at 100% solid loading pretreatment process. As known, nature cellulose usually show cellulose I crystal (Li et al., 2009). After pretreatment, the part of cellulose I structure was destructed and transformed cellulose II in this study. We suspected the main reason led to this result was the aqueous NaOH/urea solution could destroy the intra- and intermolecular hydrogen bonds and prevent the dissolving cellulose chains from recrystallization more easily. Meanwhile, the solvent in the process of NaOH/Urea treated RS, the effects of the swelling and destruction of the fibers on hydrogen bond lead to reduce crystallinity. Because there was not enough NaOH – water hydrates and Urea – water hydrates in the system of 100% solid loading pretreatment process, so we proposed that swelling and destruction of the hydrogen bond should
3.5. Overall process material balance To provide an overview of the overall process, a mass balance diagram based on 100 g of RS was given with and without NaOH/Urea pretreatment to enzymatic hydrolysis and hydrogen production (Fig. 3). According to the theoretical sugar content in the RS, 100 g raw RS contained 39.7 g glucose and 24.3 g xylose (Zhao et al., 2014). Although 100% solid loading NaOH/Urea pretreatment led to 0.19 g/g solid substrate loss, 4.43 g available sugar run off. What’s more, the corresponding hydrogen yield increased to 1.176 × 104 mL/100 g with pretreated RS, 49.5% higher than the counterpart without pretreatment. The material balance indicates that the high removal efficiency of lignin, and preservation of cellulose and hemicellulose by NaOH/Urea pretreatment at 100% solid loading under low temperature enable 49.5% higher reduce sugar transfer to H2. Besides that, NaOH/Urea solution at high solid loading pretreated RS has been demonstrated favorable to promote the efficiency of enzymatic hydrolysis and improve the overall sugar recovery of the process. Overall, this work has proven that the high solid pretreatment of the lignocellulose was a beneficial way for improving substrate conversion to clean energy. 4. Conclusions An obvious enhancement on enzymatic hydrolysis was achieved which was 77.59–80.22% at 10–100% solid loading NaOH/Urea 5
Bioresource Technology 287 (2019) 121399
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pretreatment at −12 °C with an average reducing sugar conversion of 80.22% was obtained which was 31.89% higher than that untreated RS. Further upon fermentation of the sugars by T. thermosaccharolyticum W16 gives high H2 yield which was 45.98% increased compared with untreated sample. FTIR and XRD analysis further demonstrated that rigid structure of RS was disrupted in pretreatment process and generated more accessible cellulose and hemicellulose. Overall, this high solid loading pretreatment technology could be potentially applied to save energy and chemicals for actual industrial application of the lignocelluloses-to-sugars and hydrogen routes.
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Acknowledgements This research was supported by the National Natural Science Foundation of China (Grant No. 31870110), National key research and development program (No. 2016YFC0401105-06), Heilongjiang Province Foundation for Returness (No. JJ20170414), Special Fund for Scientific and Technological Innovation of Harbin (No. JJ20170385), Postdoctoral Scientific Research Developmental Fund of Heilongjiang Province (No. LBH-Q15054), and State Key Laboratory of Urban Water Resource and Environment (Harbin Institute of Technology) (No. 2018TS02). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.121399. References Adney, B. and Baker, J., 2008. Measurement of cellulase activities. National, Renewable Energy Laboratory (NREL). Cao, G.l., Ren, N.Q., Wang, A.J., Lee, D.J., Guo, W.Q., Liu, B.F., Feng, Y.J., Zhao, Q.L., 2009. Acid hydrolysis of corn stover for biohydrogen production using Thermoanaerobacterium thermosaccharolyticum W16. Int. J. Hydrogen Energy 34 (17), 7182–7188. Chang, V.S., Nagwani, M., Kim, C.H., Holtzapple, M.T., 2001. Oxidative lime pretreatment of high-lignin biomass: poplar wood and newspaper. Appl. Biochem. Biotechnol. 94, 1–28. Cheng, Y.S., Zheng, Y., Yu, C.W., Dooley, M.T., Jenkins, M.B., VanderGheynst, S.J., 2010. Evaluation of High Solids Alkaline Pretreatment of Rice Straw. Appl. Biochem. Biotech. 162 (6), 1768–1784. Dong, L.L., Cao, G.L., Zhao, L., Liu, B.F., Ren, N.Q., 2018. Alkali/urea pretreatment of rice straw at low temperature for enhanced biological hydrogen production. Bioresour. Technol. 267, 71–76. Dyk, J.S.V., Pletschke, B.I., 2012. A review of lignocellulose bioconversion using enzymatic hydrolysis and synergistic cooperation between enzymes-factors affecting enzymes, conversion and synergy. Biotechnol. Adv. 30, 1458–1480. Kaplan and David, 1998. Biopolymers from Renewable Resources. Springer, Berlin Heidelberg, pp. p55. Kim, S., Holtzapple, M.T., 2006. Effect of structural features on enzyme digestibility of corn stover. Bioresour. Technol. 97, 583–591. Kim, T.H., Lee, Y.Y., Sunwoo, C., Kim, J.S., 2006. Pretreatment of corn stover by low – liquid ammonia recycle percolation process. Appl. Biochem. Biotechnol. 133, 41–57. Kuo, C.H., Lee, C.K., 2009. Enhancement of enzymatic saccharification of cellulose by cellulose dissolution pretreatments. Carbohydr. Polym. 77 (1), 41–46. Lee, Y.Y., Lyer, P., Torget, R.W., 1999. Dilute – acid hydrolysis of lignocellulosic biomass. Adv. Biochem. Eng. Biotechnol. 1999 (65), 93. Li, D., Chen, H., 2007. Biological hydrogen production from steam exploded straw by simultaneous saccharification and fermentation. Int. J. Hydrogen Energy 32, 1742–1748. Li, H., Kim, N.J., Jiang, M., Kang, J.W., Chang, H.N., 2009. Simultaneous saccharification and fermentation of lignocellulosic residues pretreated with phosphoric acid –
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High-solid pretreatment of rice straw at cold temperature using NaOH/Urea for enhanced enzymatic conversion and hydrogen production
T
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Lili Dong, Guangli Cao , Jiwen Wu, Bingfeng Liu, Defeng Xing, Lei Zhao, Chunshuang Zhou, Liping Feng, Nanqi Ren State Key Laboratory of Urban Water Resource and Environment, School of Environment, Harbin Institute of Technology, Harbin 150090, China
A R T I C LE I N FO
A B S T R A C T
Keywords: High-solid loading NaOH/Urea pretreatment Low temperature Rice straw Enzymatic Hydrogen
A high-solid loading pretreatment using NaOH/Urea solution at −12 °C was proposed to pretreat rice straw (RS) for enhanced saccharify and hydrogen production. Results shown NaOH/Urea pretreatment exhibited excellent pretreatment performance at solid loading ranged from 10% to 100% (w/v) with an average reducing sugar conversion of 80.22% was obtained which was 31.89% higher than that untreated RS. Upon fermentation of 100% solid loading pretreated hydrolysate, the H2 yield of 72.5 mL/g-pretreated RS was calculated based on substrate consumption, which enabled 49.5% higher reducing sugar transfer to H2 through material balance. FTIR and XRD analysis further demonstrated that the cold NaOH/Urea pretreatment at 100% (w/v) could effectively disrupt the lignin structure and decrease the cellulose crystallinity. The present study suggested a high solid loading pretreatment with NaOH/Urea at cold temperature could be a valuable alternative for better techno-economic of the lignocelluloses – to – sugars – to H2 routes.
1. Introduction During last decades, the industrial revolution has brought unimaginable benefits to human societies by providing optimized material products and human activity, along with higher living standards for humanity, but has also brought climate change and energy crisis. Hence, the development of renewable energies have promoted in the worldwide (Wang et al., 2018; Toquero and Bolado, 2014; Mahmood et al., 2016). Conversion of abundant lignocellulosic biomass to biofuels presents a promising option for improving energy security along with reducing the emission of green-house gases, preventing the consequent increase in the average global temperature, and producing a sustainable energy (Dyk and Pletschke, 2012; Zhao et al., 2012a,b; Sindhu et al., 2016). However, due to the recalcitrant structure of lignocellulose, the low accessibility of enzymes to the carbohydrate polymers is the primary obstacle for the bioconversion of lignocellulosic biomass (Sindhu et al., 2016; Dong et al. 2018). It’s reported that only 20% theoretical maximum sugar yield can be obtained from enzymatic hydrolysis of lignocellulosic biomass without appropriate pretreatment (Zhao et al., 2012a,b; Kim et al., 2006; Mosier et al., 2005). Thus, efficient pretreatment is required to disrupt the heterogeneous matrix, increase surface area and porosity of the cellulosic material, so as to enhance the enzymatic hydrolysis and cellulose conversion.
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Various techniques including physical pretreatments (size reduction), physico-chemical pretreatments (liquid hot water, steam explosion, ammonia fiber explosion), chemical pretreatments (acid, alkaline, alkaline/oxidative, wet oxidation, ozonolysis) and biological pretreatments have been developed to improve the accessibility of enzymes to cellulosic materials (Toquero and Bolado, 2014). Among these methods, alkali is relatively inexpensive and more effective in reducing lignin content and increasing the accessibility of cellulose (Sivers and Zacchi, 1995, Chang et al., 2001). However, alkali pretreatments commonly require over 100 °C pretreatment temperature, long duration time, and with the drawbacks of special equipment requirement (Mosier et al., 2005; Lee et al., 1999; Palmqvist and Hahn – Hagerdal, 2000; Tim and Riehard, 2005; Ren et al., 2009). Recently, researchers found that alkali based solvent with urea at low temperature can effectively enhance the biodegradability of lignocellulosic material. This process has been receiving more and more attention for the enhancement of enzymatic conversion and biofuel production. In the cold climate region, the average daily temperature in winter is between −5 and −20 °C, if NaOH/Urea pretreatment can be taken at a high solid loading, both scientific and economic breakthroughs can be obtained (Zhao et al., 2008; Li et al., 2016). Kuo and Lee (2009) have reported that the enzymatic hydrolysis efficiency of cotton cellulose pretreated with NaOH/Urea solution at −20 °C was improved from 23% to 85%,
Corresponding author. E-mail address: [email protected] (G. Cao).
https://doi.org/10.1016/j.biortech.2019.121399 Received 3 April 2019; Received in revised form 26 April 2019; Accepted 28 April 2019 Available online 30 April 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
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180%, 200% (w/v) and quickly stirred for 3 min, respectively. In the stirring process, we adopted mechanical stirring, and the speed of the agitator was set at 350 rpm. After that, the pretreated RS samples were filtered and washed thoroughly with distilled water, and neutralize to pH 7.0, and then dried at 60 °C until constant weight was obtained and preserved for further use.
which implied that approximately 3-fold enhancement on cellulose saccharification conversion. Zhao et al. (2008) have demonstrated that the enzymatic efficiency was improved significantly when spruce was pretreated with NaOH/Urea solution at −15 °C. Our previous research also showed that the cold NaOH/Urea based pretreatment could efficiently remove lignin and retain most of cellulose and hemicellulose in rice straw. Upon fermentation of pretreated rice straw, 113.53% higher hydrogen yield was obtained by NaOH/Urea pretreatment at −12 °C compared with untreated sample (Dong et al., 2018). However, lowsolids loadings (≤5% solids, w/v) are usually used in this pretreatment process. Over the last few years, numerous studies utilizing low-solids loadings (≤10% solids, w/v) are efficient and helpful, but the use of high-solids loadings (> 15% solids, w/v) potentially offers many advantages over lower solids loadings such as decreased operation costs and increased industrial application or the economics (Wang et al., 2018; Modenbach and Nokes, 2012; Martínez-Patino et al. 2015; Zhang et al., 2010; Cheng et al., 2010). Although efforts have been made to carry out the effects of high-solids loadings on lignocellulosic materials pretreatment process for improving the saccharification efficiency of enzymatic hydrolysis. However, current technology allows only up to 50% of the solid loading to be used in the pretreatment process, the effect of higher solid loading more than 50% on pretreatment efficiency has not been studied (Modenbach and Nokes, 2012; Luterbacher et al., 2010; Zhu et al., 2009; Roche et al., 2009; Cheng et al., 2010). Meanwhile, the adoption of high-solids loadings with NaOH/Urea pretreatment at low temperature is still relatively unexplored. So breakthroughs in increasing the solid loading in cold NaOH/Urea pretreatment are needed for both economic and practical reasons. At industrial level, even a small improvement in the pretreatment process efficiency may have outstanding economic benefits. The goal of this study is to evaluate the feasibility of using highsolids loadings in NaOH/Urea pretreatment at cold temperature for pretreated rice straw (RS). The changes of chemical properties of NaOH/Urea pretreated RS with solid loadings ranging from 10% up to 200% were firstly described. Then the enzymatic hydrolysis efficiency of pretreated RS was evaluated to determine whether NaOH/Urea solution pretreatment at high-solids loadings could enhance reducing sugar production. After that, the produced reducing sugars were fermented to H2 by T. thermosaccharolyticum W16. At last, fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) were adopted to give a micro perspective on how the pretreatment process at high-solid loading induced the structural changes of RS. The feasibility of lignocellulosic conversion would be greatly improved if high-solids loadings could be used in the unit of pretreatment. Meanwhile, it could be helpful to enlarge the application scale of this solution system.
2.3. Enzymatic hydrolysis Enzymatic hydrolysis experiment was carried out at a substrate concentration of 2% (w/v) with a mixture cellulolytic hybrid enzyme from Cellulase in sodium citrate buffer solution for pretreated and untreated RS. In the subsequent enzymatic hydrolysis, the effects of different cellulase dosage (1–5 FPU/g RS) were firstly determined for the optimal enzyme units of enzymatic hydrolysis using NaOH /Urea pretreated RS at solid content of 3% (w/v). Secondly, the RS pretreated with different solid loading was hydrolyzed by enzymes at the the optimal amount previously optimized. In all cases, enzymatic hydrolysis was carried out at pH 6.0, 50 °C in a rotary shaker at 135 rpm. Each experiment was performed in triplicate. 2.4. Biohydrogen production in batch culture The RS hydrolysate was used as carbon source to produce H2 by T. thermosaccharolyticum W16 isolated by Ren et al. (2009). Hydrogen production was performed in 100 mL serum vials with working volume of 50 mL. The tested hydrolysate was diluted to reduce the sugar concentration to 10 g/L and supplemented with the following nutrients (L−1): 1.0 g NH4Cl, 3.0 g K2HPO4, 1.5 g KH2PO4, 0.5 g MgCl2·6H2O, 1.0 g NaCl, 0.2 g KCl, 0.5 g cysteine-HCl, 2.0 g yeast extract, and 2.0 g tryptone. The culture temperature and pH were 60 °C and 7.0, respectively. During the course of fermentation, biogas production and compositions of produced biogas were monitored with respect to culture time. All tests mentioned above were performed in triplicate to determine the reproducibility of the experiments. 2.5. Analytical methods The cellulose, hemicellulose and lignin contents of RS were measured using a Fiber analyzer A200i (ANKOM Co., US). Microstructural changes in the RS before and after pretreatment were observed by FTIR and XRD. FTIR spectroscopy analysis was carried out using a Magna-IR 750 (Nicolet Instrument Co., USA) according to the method described by Dong et al. (2018). The crystallinity of the RS was determined by Xray diffraction (XRD) using a D8 X’Pert PRO MPD diffractometer (PANalytical Ltd., Holland). The cellulase activity was measured according to the method reported by Adney and Baker, 2008. The final of reducing sugar content of the hydrolytic samples was analyzed by the dinitrosalicylic acid (DNS) method (Wang et al., 2016). High performance liquid chromatography (HPLC) system (LC-10A, Shimadzu Corporation, Kyoto, Japan) was used to detect the initial sugar compositions of the hydrolysate, and the sugar consumption during fermentation (Qi et al., 2008). The gas products (H2 and CO2) were analyzed by gas chromatography (7890D, Agilent Cooperation, USA) using a thermal conductivity detector.
2. Materials and methods 2.1. Raw material The RS used in this study was collected from Wuchang, Heilongjiang province, China. The raw material was milled to pass through a 40mesh (0.425 mm) screen, and then dried at 60 °C until constant weight was acquired. The main constituents of RS on a dry basis were 35.97% cellulose, 27.45% hemicellulose, 14.08% lignin. Cellulase was kindly provided by Novozymes (China) Investment Co., Ltd. Sodium hydroxide and urea were used as received from Sigma-Aldrich (St. Louis, MO).
2.6. Data analysis
2.2. Pretreatment of RS at different solid loadings
The enzyme saccharification rate was calculated as (Li et al., 2009): enzymatic saccharification (%) = reducing sugars released (g) × 0.9 × 100/polysaccharides in substrate (g) where 0.9 is the correction coefficient for hydrolysis. Acid detergent fiber (ADF), neutral detergent fiber (NDF), residue after 72% H2SO4 treatment, and acid detergent lignin (ADL) were determined to calculate the contents of cellulose, hemicellulose and lignin. Calculation equations as follow:
NaOH /Urea pretreatment solutions were prepared by mixing 7% w/v NaOH and 12% w/v urea in deionized water and pre-cooled and maintained at the temperature of −12 °C according to previous study (Dong et al. 2018). To investigate the effect of different solid loading on pretreatment of RS, the RS was immersed immediately in the solvents with different solid content of 10%, 20%, 50%, 100%, 130%, 160%, 2
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Hemicellulose (%) = NDF% − ADF%
(1)
Lignin (%) = ADL%
(2)
Cellulose(%) = ADF% − Residue after 72% H2 SO4
(3)
based on dry matter after pretreatment generally decreased at solid loading more than 100%. Although a higher the residue solid yield obtained at solid loading more than 100%, the contents of cellulose and hemicellulose were 37.76% and 30.60% at 200% solid loading, which were similar to that in raw RS. It suggested that pretreatment efficiency appeared a mild agent at solid loading more than 100%. The results demonstrated that a high solid loading pretreatment with NaOH/Urea solution at low temperature were helpful for the saccharification of RS.
The X-ray diffractometer was set at 45 kV and 200 mA. XRD patterns of the samples were recorded over the range of 2θ = 5–90° at a scan speed of 2 °min−1 with a step size of 0.02°. The crystallinity index (CrI) was calculated according to Eq. (1) (Kim and Holtzapple, 2006).
CrI (%) = [(I002 − Iam)/ I002] × 100
3.2. Enzymatic hydrolysis of high solids based pretreated RS
(4)
I002 is the maximum diffraction intensity of the crystalline structure and Iam is the diffraction intensity of the amorphous fraction at 2θ ≈ 18°.
3.2.1. Production of reducing sugars in optimal enzyme units of enzymatic hydrolysis The optimal ratio of enzyme to substrate is crucial for the efficient utilization of enzymes. Thus, the effects of cellulase dosage on the production of reducing sugars were firstly investigated, and the results were presented in Fig. 1a. Various concentrations (1, 3 and 5 FPU g−1 RS) of cellulase was used for hydrolysis of RS which was pretreated at solid loading of 3% (w/v) with NaOH /Urea at −12 °C. The profiles of reducing sugars were examined over a 60 h period. The release of reducing sugars continuously increased for 48 h and then remained stable at tested cellulase dosage. Moreover, with the increase of cellulase dosage, the saccharification efficiency increased. However, cellulase with 5 FPU g−1 RS could achieve a higher enzyme saccharification efficiency, which only increased 2.84% compared with 3 FPU g−1 RS. Considering the comprehensive treatment effect and economy, 3 FPU cellulase g−1 RS were selected as the optimal concentrations for the hydrolysis experiments in further experiments.
3. Results and discussion 3.1. Effect of solid loadings on the composition of RS Solid loading is an important factor that not only affects the improvement of cellulose digestibility by the removal of lignin component or partial depolymerization but also influences the reduction of the volume of pretreatment equipment and the consumption of water (Modenbach and Nokes, 2012). The effect of solid loading increasing from 10% to 200% (w/v) on NaOH/Urea pretreatment was investigated using a low temperature (−12 °C) and short reaction time (3 min). The chemical compositions of RS and the percentage of weight loss of each different solid loading through NaOH/Urea solution pretreatment was shown in Table 1. The solid loading ranged from 10% to 50% had a significant influence on the delignification of RS, the amount of lignin removal ranged from 59.91–62.35%. When the solid loading continued improving to 100%, only 4.39% decreased on the content of lignin of RS. It suggested that NaOH/Urea solution pretreatment at high solid loading 100% obtained a satisfactory delignification result. However, when the solid loading future increased until to 200%, the reduction of lignin decreased sharply, only 38.76% removal rate at 200% solid loading pretreatment process. This implied that the effect of NaOH/ Urea on RS was not as good as previous at solid content exceeds 100%. In term of lagre-scale and industrial application, an ideal pretreatment is not only to remove as much unusable lignin as possible, but also to maximally retain all available carbohydrates for further bioenergy production (Lou et al., 2016). In contrast to large amount of lignin removal at solids loadings ranged from 10% to 100%, more than 89% cellulose and 81% hemicellulose were retained after pretreatment. It can be obtained from Table 1 that the content of cellulose and hemicellulose slightly decreased 1.28% and 8.75% at solid loading was 100% compared to solid loading 10–50%, indicated that most of the cellulose and hemicellulose remained in RS at high solid loading. Meanwhile, it was observed that cellulose and hemicellulose contents
3.2.2. Sugar yields from high solids based pretreated RS To evaluate the efficiency of high-solids loadings pretreatment with NaOH/Urea at cold temperature, the RS residues after pretreatment were subsequently hydrolyzed with 3 FPU g−1 RS commercially available cellulase for 60 h. As shown in Fig. 1b, the solid loading could greatly affect enzymatic hydrolysis. The reducing sugar conversion ratio of RS decreased from 80.22% to 50.67% as solid loading increased. It should be noted that the reducing sugar conversion yields ranged from 77.59 to 80.22% at 10–100% solid loading. Although, there was no significant difference (P > 0.05) was found among these solid loadings indicating that NaOH/Urea pretreatment had a considerable effective effect on enzymatic hydrolysis when solid loading was less than 100%, nevertheless, the enzymatic hydrolysis efficiency decreased significantly when the solid loading exceeded 100%. It can be found that the reducing sugar conversion was decreased from 77.59% at 100% solid loading to 56.46% at 130% solid loading. What’s more, the reducing sugar conversion was nearly the same to that in the raw RS without pretreatment when the solid loading increased to 200% during pretreatment. This result was consistent with the result of
Table 1 Compositions of raw at different solid-loading NaOH/Urea solution pretreated RS at −12 °C. Solid-loading (w/v)
Composition (%)a Cellulose
10% 20% 50% 100% 130% 160% 180% 200% Untreated a b c
45.59 45.00 44.12 41.02 38.57 38.03 38.00 37.76 35.97
± ± ± ± ± ± ± ± ±
0.05 0.98 0.05 0.00 0.38 0.06 0.66 0.50 0.14
Residue solid (%)b Hemicellulose
Lignin
32.00 31.57 31.09 31.37 30.67 31.43 31.17 30.60 27.45
7.84 ± 1.30 7.88 ± 1.09 7.88 ± 0.08 7.84 ± 1.02 8.45 ± 0.92 9.04 ± 0.00 9.02 ± 0.05 9.84 ± 1.90 14.08 ± 3.20
± ± ± ± ± ± ± ± ±
1.00 1.67 3.17 4.16 2.72 1.51 1.23 1.00 1.00
70.40 71.50 74.30 80.00 85.00 86.50 87.80 89.00 –
± ± ± ± ± ± ± ±
Composition is shown as percentage of the solid fraction before and after pretreatment. Solid yield is shown as percentage of the initial amount of dry matter. Removal yield is shown as percentage of the amount in the initial material. 3
2.84 1.61 1.50 1.80 1.00 2.74 6.10 1.81
Removal yield (%)c Cellulose
Hemicellulose
Lignin
10.72 ± 0.01 10.50 ± 0.02 8.82 ± 0.00 8.73 ± 0.04 8.81 ± 0.01 8.51 ± 0.02 7.21 ± 0.03 6.54 ± 0.06 –
18.23 ± 0.03 18.06 ± 0.05 16.11 ± 0.07 8.72 ± 0.05 5.11 ± 0.03 0.97 ± 0.01 0.31 ± 0.03 0.80 ± 0.03 –
62.35 61.51 59.91 56.87 50.24 45.60 44.87 38.76 –
± ± ± ± ± ± ± ±
0.05 0.00 0.06 0.04 0.04 0.00 0.04 0.12
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Fig. 1. a. Reducing sugar released from hydrolysis of RS by various concentrations (1 FPU, 3 FPU, 5 FPU g−1 RS) of cellulase from Novozym at pH 6.0, 50 °C and 135 rpm. b. Reducing sugar conversion rate from hydrolysis of untreated and pretreated RS using NaOH/Urea aqueous solution at 10%, 20%, 50%, 100%, 130%, 160%, 180% and 200% solid loading pretreatment at 3 FPU g−1 RS, pH 6.0, 50 °C and 135 rpm. c. Composition of hydrolysate of untreated and pretreated RS using NaOH/Urea aqueous solution at 10%, 20%, 50%, 100%, 130%, 160%, 180% and 200% solid loading pretreatment of cellulase from Novozym at 3 FPU g−1 RS, pH 6.0, 50 °C and 135 rpm.
chemical analysis. Apparently, further increases in solid loading beyond 100% have little or no beneficial effect. HPLC analysis showed that the sugar composition of enzymatic hydrolysate were mainly composed of glucose and xylose, and a small amount of arabinose, galactose and rhamnose for untreated and pretreated RS (Fig. 1c). And, on average over 51% and 39% of the hydrolysate is glucose and xylose. So far, various pretreatment methods such as stream-explosion, diluted acid hydrolysis, ammonia pretreatment have been employed for enhancing enzymatic hydrolysis from lignocellulosic biomass, and solid loadings used in pretreatment procedure between 5% and 50% with a saccharification ratio of about 80% was obtained (Wang et al., 2018; Zhao et al., 2008). In this work, a comparable reducing sugar conversion was acquired using NaOH /Urea pretreatment at cold temperature with solid loading as high as 100% (w/v), indicating that cold NaOH /Urea pretreatment is effective at high solid loading. In other words, it can acquire reasonable pretreatment effect by increasing of solid loading more than 2 times, which means that the amount of water and chemical reagent used in the pretreatment process were reduced by more than 2 times. Usually, solid-loading in studies pretreated with alkali/urea was less than 5%, according to Zhao et al. (2008), 7% NaOH and 12% urea were used to pretreatment spruce at −15 °C with solid loading of 5%, results showed that a 70% sugar conversion was obtained after pretreatment. Recently, some pretreatment technologies were also explored at high solid loadings such as ammonia fiber expansion (AFEX), which have been reported to effectively improve biomass biodegradability when solid loading was as high as 90%. However, high temperature and pressure are needed in the AFEX pretreatment process (Wyman et al., 2005). Accordingly, it can be inferred that low temperature NaOH/Urea solvent at the high solid loading could still achieve an attractive treatment effect.
Fig. 2. H2 production by Thermoanaerobacterium thermosaccharolyticum W16 using enzymatic hydrolysate from 100% solid loading NaOH/Urea pretreated RS.
work is comparable to that of pretreated lignocellulose with bio-or physicochemical pretreatment at relative low solid loading reported in related studies (Li and Chen, 2007; Zhao et al., 2012a,b; Varrone et al., 2013; Cao et al., 2009). These results further indicate that bio-H2 production from RS was successful when using NaOH/Urea at 100% solid loading under low temperature pretreatment method.
3.4. The exploration of the high solid loading NaOH/Urea pretreatment on morphological and structural changes of RS
3.3. Hydrogen fermentation of RS based – hydrolysate FTIR was applied to further determine the chemical structure changes of RS at 100% solid loading NaOH/Urea pretreatment process. FTIR data showed band at approximately1250 cm−1 corresponding to guaiacyl ring breathing, CeO stretch in lignin and for CeO linkage in guaiacyl aromatic methoxyl groups, which are the main constituting units of lignin and xylan (Supplementary data). The absorption at 1733 cm−1 corresponding to either the acetyl and uranic ester groups of the hemicelluloses or the ester linkage of carboxylic group of the ferulic and p-coumaric acids of lignin and/or hemicelluloses. The bands near 1530 and 1059 cm−1 represent aromatic skeletal vibration CeH and CeO of lignin (supplementary material) (Dong et al., 2018; Kaplan and David, 1998; Zhao et al., 2012a,b). Compared with untreated RS, all of the functional group mentioned above decreased after pretreatment. However, the broad absorption band at 3407 cm−1 corresponds to the hydrogen-bond OeH stretching vibrations, which belong to the
Fermentative conversion of enzymatic hydrolysate of 100% solid loading pretreated RS into H2 was evaluated with T. thermosaccharolyticum W16. H2 was immediately produced after 5 h incubation with no lag phase, and maintained a high level of production rate before the incubation of 24 h (Fig. 2). After 36 h of fermentation, the cumulative hydrogen production reached 2540 mL/L. Coincide with the production of hydrogen, the sugars in hydrolysate were consumed quickly before 24 h of fermentation. At the end of fermentation, arabinose was completely consumed, 79.1% of glucose, 50% xylose and small amount of rhamnose were consumed, and the hydrogen yield based on substrate consumption was 1.9 mol H2 mol−1 sugar. On the basis of RS weight, H2 yield was 72.5 mL/g-pretreated RS. Compared H2 yields with reported in lignocellulosic biomass fermentation studies, it can be inferred that the performance of hydrogen production in this 4
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Fig. 3. Material balances of biohydrogen production from RS with and without NaOH/Urea pretreatment at 100% solid loading.
be the main reason for reducing crystallinity. The observations mentioned above demonstrated that the high solid loading pretreatment with NaOH/Urea at cold temperature of RS could effectively reduce lignin content and crystalline, which could lead to an increase in susceptibility of RS for enzymatic saccharification, making the production of renewable energy from biomass more efficient.
characteristic absorption peaks of the cellulose (Kaplan and David, 1998). This implied that the hydrogen bonds of RS were broken and their association degree decreased after the pretreatment. Additionally, the peak at the wavelength of 893 cm−1 was stronger after pretreatment than without pretreatment, indicating that the content of cellulose after pretreated RS was higher than untreated RS. Whereas, the bands near 1124 cm−1 and 1078 cm−1 represent aromatic skeletal and C-O stretching in cellulose and hemicelluloses. The changes in intensity ratio R = I (1121 cm−1)/I (1078 cm−1) illustrate the cellulose crystallinity (Zhao et al., 2012a,b), the crystallinity decreases as the ratio increases (supplementary material). Compared with untreated RS (R = 1.16459), the ratio of 100% solid loading NaOH/Urea pretreated RS increased to 1.26666 demonstrated crystallinity decreases after pretreatment. This result is in good agreement with the chemical components analysis To further understand the changes of the crystallinity of cellulose, XRD was employed to investigate the crystallinity of untreated and pretreated RS sample. XRD date showed the crystal decreased after treated compared with untreated RS (Supplementary data). The CrI of the untreated RS was 66%. A lower crystalline was achieved 55% at 100% solid loading pretreatment process. As known, nature cellulose usually show cellulose I crystal (Li et al., 2009). After pretreatment, the part of cellulose I structure was destructed and transformed cellulose II in this study. We suspected the main reason led to this result was the aqueous NaOH/urea solution could destroy the intra- and intermolecular hydrogen bonds and prevent the dissolving cellulose chains from recrystallization more easily. Meanwhile, the solvent in the process of NaOH/Urea treated RS, the effects of the swelling and destruction of the fibers on hydrogen bond lead to reduce crystallinity. Because there was not enough NaOH – water hydrates and Urea – water hydrates in the system of 100% solid loading pretreatment process, so we proposed that swelling and destruction of the hydrogen bond should
3.5. Overall process material balance To provide an overview of the overall process, a mass balance diagram based on 100 g of RS was given with and without NaOH/Urea pretreatment to enzymatic hydrolysis and hydrogen production (Fig. 3). According to the theoretical sugar content in the RS, 100 g raw RS contained 39.7 g glucose and 24.3 g xylose (Zhao et al., 2014). Although 100% solid loading NaOH/Urea pretreatment led to 0.19 g/g solid substrate loss, 4.43 g available sugar run off. What’s more, the corresponding hydrogen yield increased to 1.176 × 104 mL/100 g with pretreated RS, 49.5% higher than the counterpart without pretreatment. The material balance indicates that the high removal efficiency of lignin, and preservation of cellulose and hemicellulose by NaOH/Urea pretreatment at 100% solid loading under low temperature enable 49.5% higher reduce sugar transfer to H2. Besides that, NaOH/Urea solution at high solid loading pretreated RS has been demonstrated favorable to promote the efficiency of enzymatic hydrolysis and improve the overall sugar recovery of the process. Overall, this work has proven that the high solid pretreatment of the lignocellulose was a beneficial way for improving substrate conversion to clean energy. 4. Conclusions An obvious enhancement on enzymatic hydrolysis was achieved which was 77.59–80.22% at 10–100% solid loading NaOH/Urea 5
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pretreatment at −12 °C with an average reducing sugar conversion of 80.22% was obtained which was 31.89% higher than that untreated RS. Further upon fermentation of the sugars by T. thermosaccharolyticum W16 gives high H2 yield which was 45.98% increased compared with untreated sample. FTIR and XRD analysis further demonstrated that rigid structure of RS was disrupted in pretreatment process and generated more accessible cellulose and hemicellulose. Overall, this high solid loading pretreatment technology could be potentially applied to save energy and chemicals for actual industrial application of the lignocelluloses-to-sugars and hydrogen routes.
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Acknowledgements This research was supported by the National Natural Science Foundation of China (Grant No. 31870110), National key research and development program (No. 2016YFC0401105-06), Heilongjiang Province Foundation for Returness (No. JJ20170414), Special Fund for Scientific and Technological Innovation of Harbin (No. JJ20170385), Postdoctoral Scientific Research Developmental Fund of Heilongjiang Province (No. LBH-Q15054), and State Key Laboratory of Urban Water Resource and Environment (Harbin Institute of Technology) (No. 2018TS02). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.121399. References Adney, B. and Baker, J., 2008. Measurement of cellulase activities. National, Renewable Energy Laboratory (NREL). Cao, G.l., Ren, N.Q., Wang, A.J., Lee, D.J., Guo, W.Q., Liu, B.F., Feng, Y.J., Zhao, Q.L., 2009. Acid hydrolysis of corn stover for biohydrogen production using Thermoanaerobacterium thermosaccharolyticum W16. Int. J. Hydrogen Energy 34 (17), 7182–7188. Chang, V.S., Nagwani, M., Kim, C.H., Holtzapple, M.T., 2001. Oxidative lime pretreatment of high-lignin biomass: poplar wood and newspaper. Appl. Biochem. Biotechnol. 94, 1–28. Cheng, Y.S., Zheng, Y., Yu, C.W., Dooley, M.T., Jenkins, M.B., VanderGheynst, S.J., 2010. Evaluation of High Solids Alkaline Pretreatment of Rice Straw. Appl. Biochem. Biotech. 162 (6), 1768–1784. Dong, L.L., Cao, G.L., Zhao, L., Liu, B.F., Ren, N.Q., 2018. Alkali/urea pretreatment of rice straw at low temperature for enhanced biological hydrogen production. Bioresour. Technol. 267, 71–76. Dyk, J.S.V., Pletschke, B.I., 2012. A review of lignocellulose bioconversion using enzymatic hydrolysis and synergistic cooperation between enzymes-factors affecting enzymes, conversion and synergy. Biotechnol. Adv. 30, 1458–1480. Kaplan and David, 1998. Biopolymers from Renewable Resources. Springer, Berlin Heidelberg, pp. p55. Kim, S., Holtzapple, M.T., 2006. Effect of structural features on enzyme digestibility of corn stover. Bioresour. Technol. 97, 583–591. Kim, T.H., Lee, Y.Y., Sunwoo, C., Kim, J.S., 2006. Pretreatment of corn stover by low – liquid ammonia recycle percolation process. Appl. Biochem. Biotechnol. 133, 41–57. Kuo, C.H., Lee, C.K., 2009. Enhancement of enzymatic saccharification of cellulose by cellulose dissolution pretreatments. Carbohydr. Polym. 77 (1), 41–46. Lee, Y.Y., Lyer, P., Torget, R.W., 1999. Dilute – acid hydrolysis of lignocellulosic biomass. Adv. Biochem. Eng. Biotechnol. 1999 (65), 93. Li, D., Chen, H., 2007. Biological hydrogen production from steam exploded straw by simultaneous saccharification and fermentation. Int. J. Hydrogen Energy 32, 1742–1748. Li, H., Kim, N.J., Jiang, M., Kang, J.W., Chang, H.N., 2009. Simultaneous saccharification and fermentation of lignocellulosic residues pretreated with phosphoric acid –
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