Bioresource Technology 179 (2015) 407–413
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Fermentative hydrogen and methane cogeneration from cassava residues: Effect of pretreatment on structural characterization and fermentation performance Jun Cheng a,⇑, Richen Lin a, Lingkan Ding a, Wenlu Song a,b, Yuyou Li c, Junhu Zhou a, Kefa Cen a a b c
State Key Laboratory of Clean Energy Utilization, Zhejiang University, Hangzhou 310027, China Department of Life Science and Engineering, Jining University, Jining 273155, China Department of Civil and Environmental Engineering, Tohoku University, Sendai 9808579, Japan
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Cassava residue was pretreated by
microwave (or steam)-heated acid (MHAP or SHAP). MHAP generated many regular micropores and SHAP generated many irregular fragments. SHAP generated wider cracks (0.2 lm) in delaminated cell walls than MHAP (0.1 lm). MHAP resulted in a higher crystallinity index (33.00) than SHAP (25.88). MHAP with enzymolysis led to a higher H2 yield than SHAP, but CH4 yield reversed.
a r t i c l e
i n f o
Article history: Received 14 October 2014 Received in revised form 12 December 2014 Accepted 13 December 2014 Available online 19 December 2014 Keywords: Cassava residue Microwave-heated acid Steam-heated acid Hydrogen Methane
a b s t r a c t The physicochemical properties of cassava residues subjected to microwave (or steam)-heated acid pretreatment (MHAP or SHAP) were comparatively investigated to improve fermentative hydrogen and methane cogeneration. The hydrogen yield from cassava residues with MHAP and enzymolysis was higher (106.2 mL/g TVS) than that with SHAP and enzymolysis (102.1 mL/g TVS), whereas the subsequent methane yields showed opposite results (75.4 and 93.2 mL/g TVS). Total energy conversion efficiency increased to 24.7%. Scanning electron microscopy images revealed MHAP generated numerous regular micropores (6 lm) and SHAP generated irregular fragments (23 lm) in the destroyed lignocellulose matrix. Transmission electron microscopy images showed SHAP generated wider cracks (0.2 lm) in delaminated cell walls than MHAP (0.1 lm). X-ray diffraction patterns indicated MHAP caused a higher crystallinity index (33.00) than SHAP (25.88), due to the deconstruction of amorphous cellulose. Fourier transform infrared spectroscopy indicated MHAP caused a higher crystallinity coefficient (1.20) than SHAP (1.12). Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
⇑ Corresponding author. Tel.: +86 571 87952889; fax: +86 571 87951616. E-mail address: [email protected] (J. Cheng). http://dx.doi.org/10.1016/j.biortech.2014.12.050 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
The search for sustainable and alternative energy is significant considering the increasing energy demands and diminishing fossil fuels. Hydrogen is a promising candidate because it is renewable, carbon neutral, and environment-friendly (Turner, 2004).
408
J. Cheng et al. / Bioresource Technology 179 (2015) 407–413
Currently, more attention has been focused on bio-hydrogen production from lignocellulosic wastes (Cheng et al., 2011; Chu and Majumdar, 2012; Somerville et al., 2010). Cassava is widely grown throughout southern China, and is an important raw material for starch production in industry. The pulverized cassava was screened through a mesh sieve to produce starch (125 lm), which were mainly composed of lignocellulose biomass. According to the statistics from the Food Agricultural Organization of the United Nations, cassava production in China was approximately 4.56 million tons in 2012, which indicates that large amounts of cassava residues was also produced by cassavabased starch and ethanol industries. If not properly managed, these cassava residues can cause severe environmental pollution. However, cassava residue is a typical lignocellulosic material that could potentially provide a sustainable source for biofuel production owing to its abundance, low cost, and availability. Cassava residues, which mainly consist of cellulose, hemicellulose, and lignin, do not biodegrade easily due to the three lignocellulosic components being cross-linked to each other, forming a cellulose–hemicellulose–lignin matrix. Cassava residues consist of both crystalline and amorphous cellulose structures, and the microfibril bundles of cellulose are bound by hydrogen bonding. Lignin consists of a complex array of polymers that are associated with each other, and provides structural support. Hemicellulose connects the cellulose and lignin, which provides more rigidity to the entire matrix. To improve the digestibility of lignocellulosic biomass and further enhance subsequent biofuel yields, various pretreatment technologies have been recently reviewed (Hendriks and Zeeman, 2009; Kallioinen et al., 2013; Monlau et al., 2013; Zheng et al., 2014), which can be classified into biological, physical, chemical, and physicochemical pretreatments. The degradation of cassava residues by complex microbial communities with high cellulose-degradation ability is an effective pretreatment method, which resulted in 96.6% increase in methane yield (Zhang et al., 2011). When subjected to mechanical activation pretreatment, the crystal structure of cassava residues is significantly destroyed, resulting in increased amorphization and decreased crystallinity (Liao et al., 2011). The hydrolysis of sugar beet residues significantly improved under dilute acid pretreatment with 150% increase in ethanol yield (Zheng et al., 2013). Among these pretreatments, dilute sulfuric acid pretreatment is one of the most studied and widely used methods (Chen et al., 2012; Cheng et al., 2014; Guragain et al., 2011; Zheng et al., 2013). However, to our knowledge, few studies discussed the use of microwave-heated acid pretreatment (MHAP) or steam-heated acid pretreatment (SHAP) on cassava residues to enhance its bio-hydrogen and biomethane production. Still, few attempts tried to reveal the physicochemical properties of cassava residues after MHAP and SHAP. Therefore, this study investigated the effects of MHAP and SHAP on the microstructure changes of cassava residues. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR) were used to determine the physicochemical properties of cassava residues after MHAP and SHAP. A two-stage fermentation process, which consisted of dark hydrogen and methane fermentation, was conducted to increase the energy conversion efficiency.
2. Methods 2.1. Feedstock and bacteria Cassava residues were obtained from a cassava processing plant in Guangxi Province, China. The cassava residues were oven dried,
powdered to a 0.02 mm mesh size, and stored for use in subsequent experiments. Mixed hydrogen-producing bacteria (HPB) and methane-producing bacteria (MPB) were obtained from a biogas plant in Zhejiang Province, China. Bacterial isolation and enrichment were described in previous studies (Cheng et al., 2010). 2.2. Pretreatment and fermentation methods 2.2.1. MHAP Microwave-heating pretreatment was conducted in a microwave digestion system (Shanghai Yiyao WX-4000, China). Due to the capacity limit of the microwave digestion system, 5 g of cassava residues were divided into four polytetrafluoroethylene reactors. 1.25 g of dried cassava residues were separately added into the four reactors, and then dilute H2SO4 (1.0%, v/v) was added to bring the combined volume to 25 ml in each reactor. The four reactors were then sealed and heated by microwaves to 135 °C for 15 min. 2.2.2. SHAP Steam-heating pretreatment was performed in an autoclave (Sanyo MLS-3780, Japan). 5.0 g of dried cassava residues were placed in a conical flask, and then dilute H2SO4 (1.0%, v/v) was added to bring the combined volume to 100 ml. The conical flasks were then placed in the autoclave and heated by steam at 135 °C for 15 min. 2.2.3. Enzymatic hydrolysis The enzymatic hydrolysis was performed in 250 mL flasks. After MHAP or SHAP, the pH of cassava residues solution was adjusted to 4.5 using NaOH. Trichoderma reesei cellulase (Shanghai Boao Biotechnology Corp., China) was added to the solution at 5 wt.% of the original cassava residues. The flasks were then sealed and placed in a shaker at 120 r/min for 120 h at 45 °C. 2.2.4. Dark hydrogen fermentation Fermentation experiments were conducted in 300 ml glass bottles. Approximately 100 ml of hydrolyzed solution (containing 5.0 g of cassava residues) and 125 ml of deionized water were added to each bottle and then mixed with 0.5 g of yeast extract. The initial pH was adjusted to 6.0 ± 0.1 using 6 M HCl and 6 M NaOH solution. The bottles were then inoculated with 25 ml HPB, sealed with rubber stoppers, purged with nitrogen gas for 10 min, and maintained at 35 ± 1.0 °C for dark hydrogen fermentation. 2.2.5. Dark methane fermentation The residual solutions of dark hydrogen fermentation were autoclaved at 121 °C for 20 min to inactivate the HPB. The pH of autoclaved solution was adjusted to 8.0 ± 0.1 using 6 M HCl and 6 M NaOH solution. The bottles were subsequently inoculated with 15 ml MPB, sealed with rubber stoppers, purged with nitrogen gas for 10 min, and maintained at 35 ± 1.0 °C for dark methane fermentation. 2.3. Analytical methods Electron micrographs of the cassava residues before and after MHAP/SHAP were obtained under a field emission SEM (Hitachi S3700, Japan) after the samples were sputtered with a thick layer of gold. The structural changes of cassava residues were also examined by TEM (Hitachi H-7650, Japan) at 120 keV electron-energy emission after staining the samples with KMnO4 and UO2(CH3 COO)2. XRD analyses were conducted on an X-ray diffractometer (X’Pert PRO, Netherlands) to determine changes in the crystallinity of cellulose from cassava residues. Chemical changes were
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J. Cheng et al. / Bioresource Technology 179 (2015) 407–413
3. Results and discussion 3.1. Effects of pretreatments on cassava residues saccharification The lignocellulosic compositions of cassava residues mainly contained 26.3% cellulose, 17.6% hemicellulose, and 7.0% lignin. The TVS content was 81.3%, and the heating value was 17.9 kJ/g TVS. To improve enzymatic digestibility and bio-hydrogen production, an effective pretreatment is necessary for disrupting the cellulose–hemicellulose–lignin matrix in cassava residues. In this study, MHAP and SHAP were conducted to examine their effects on saccharification. According to the calculation method proposed in a previous study (Cheng et al., 2010), the reducing sugar yield of dried cassava residues was theoretically 49.4 g/100 g. The reducing sugars yields of cassava residues measured by DNS method after MHAP and SHAP were 29.6 and 35.0 g/100 g cassava residues, respectively. After further enzymatic hydrolysis, the reducing sugars yields of cassava residues increased to 38.9 and 43.6 g/100 g cassava residues, which corresponded to 78.6% and 88.2% of the theoretical yield, respectively. Thus, using MHAP and SHAP followed by enzymatic hydrolysis, the lignocellulosic structure of cassava residues can be significantly damaged, and most of the cellulose and hemicellulose can be degraded into reducing sugars (e.g., glucose and xylose). The reducing sugars released from pretreated cassava residues were measured on a HPLC system, as shown in Fig. 1. Cellobiose, glucose, xylose, galactose, mannose, and arabinose were the main reducing sugars after enzymatic hydrolysis. Glucose and xylose comprised of over 70% of total sugars. The glucose yields for MHAP and SHAP were 18.9 and 22.7 g/100 g cassava residues, respectively. The xylose yields for MHAP and SHAP were 5.5 and 7.5 g/ 100 g cassava residues, respectively. The total sugars yield after SHAP and enzymatic hydrolysis (41.2 g/100 g cassava residues) was higher than that after MHAP and enzymatic hydrolysis (34.8/100 g cassava residues), which was in accordance with the result by DNS method.
50 Microwave-heated acid pretreatment+enzymatic hydrolysis Steam-heated acid pretreatment+enzymatic hydrolysis
Reducing sugars yields (g/100 g cassava residues)
examined using a FTIR spectrometer (Nicolet 5700, USA) equipped with a universal attenuated total reflectance accessory. The cellulose, hemicellulose, and lignin contents of the cassava residues were determined using a raw-fiber extractor (FIWE, VELP Scientific Corp., Italy) based on the Van Soest detergent method. Moisture content was determined by oven drying at 105 °C until a constant mass was obtained. Total volatile solids (TVS) and ash contents were determined by heating at 600 °C for 2 h. The total reducing sugar contents of the hydrolyzed cassava residues were determined using the 3,5-dinitrosalicylic acid method. The heating values of the cassava residues were determined using a fast calorimeter (Changsha Kaiyuan 5E-KC5410, China). The concentrations of cellobiose, glucose, xylose, galactose, mannose and arabinose were determined on a high performance liquid chromatography (HPLC) system (Agilent 1200, USA) using refractive index detector and Aminex HPX-87P column at 85 °C with H2O as mobile phase at 1 mm/min. The concentrations of hydrogen, methane, and soluble metabolite products (SMPs) were analyzed by gas chromatography (GC) systems. The hydrogen and methane yields were simulated by the modified Gompertz equation, and the dynamic parameters (H, the cumulative hydrogen and methane yields, mL/g TVS; Hm, the maximum hydrogen and methane yields potential, mL/g TVS, Rm, the peak rate of hydrogen and methane production, mL/g TVS/h; k, the lag-phase time of hydrogen and methane production, h; and Tm, the peak time, h) were calculated using Origin 8.5 software. The experiments were conducted in triplicates under all the conditions stated, and the results were expressed as mean (±SD).
40 30 20 10 0 Cellobiose
Glucose
Xylose
Galactose
Mannose
Arabinose Total sugars
Fig. 1. Reducing sugars release from pretreated cassava residues.
3.2. Physicochemical characterization of cassava residues before and after pretreatments 3.2.1. Scanning electron microscopy The physical structure changes of cassava residues after MHAP and SHAP were determined by SEM, as shown in Fig. S1. Raw cassava residues consisted of compact fiber bundles with different particle sizes (1–14 lm) on smooth flat surface, and the cellulose–hemicellulose–lignin matrix was also clearly visible, as shown in Fig. S1a. The surface morphologies of cassava residues were significantly damaged after MHAP and SHAP, which resulted in increased surface areas that evidently enhanced the digestibility. In Fig. S1b, the surface of cassava residues were obviously modified by MHAP. The compact fiber bundles were split and fractured, which contributed to an increase in surface roughness and the generation of irregular fragments (18 lm). In Fig. S1c, the lignocellulosic structure was split and fractured with larger fragments (23 lm) and fewer micropores after SHAP. The appearance of micropores (6 lm) at regular intervals was very prominent after MHAP, which could be attributed to the hydrolysis of hemicellulose and lignin during MHAP. Similar structural changes were reported in hemp hurd biomass that were pretreated by electron-beam irradiation (Abraham et al., 2013), in poplar solids that were pretreated by flow-through (Kumar et al., 2009), and in sugarcane bagasse that were pretreated by microwave-alkali or acid (Binod et al., 2012; Chen et al., 2011). When cassava residues were pretreated by microwave heating, the polar component was heated rapidly, whereas the nonpolar components did not absorb the microwaves and were not directly heated. This difference in behavior caused the inhomogeneity of surface morphologies. The rapid oscillation of the polar substances and along with the temperature inhomogeneity facilitated the disruption of the physical structure of cassava residues. In contrast, steam heating was slowly introduced to the sample surface, resulting in fewer structural changes (Hu and Wen, 2008). 3.2.2. Transmission electron microscopy To further characterize the ultrastructural change of cassava residues that were pretreated by MHAP and SHAP, TEM images were illustrated in Fig. S2. The cell wall layers were clearly observed in raw cassava residues, as shown in Fig. S2a. The cell walls of raw cassava residues composed of intercellular layers, namely, the compound middle lamella (CML), outer secondary wall (S1), middle secondary wall (S2), and inner secondary wall (S3). The CML (170 nm width) that fixed the cells together was clearly shown against the adjacent cell wall layers. The secondary wall was a thick layer inside the CML in inner parts of the cell. The
J. Cheng et al. / Bioresource Technology 179 (2015) 407–413
3.2.3. X-ray diffraction analysis The XRD patterns of the pretreated cassava residues were shown in Fig. S3a to determine the change in crystallinity after MHAP and SHAP. A major diffraction peak on cellulose crystallographic plane has been identified at 2h = 22–23°, which indicated the presence of a highly organized crystalline region, whereas the peak at 2h = 18° indicated a less organized region (Chen et al., 2012). The crystallinity index (CrI), which represented the percentage of crystalline materials, was calculated by an empirical method. Compared with raw cassava residues, the peak intensity representing the amorphous cellulose in the MHAP and SHAP samples decreased significantly, whereas the peak intensity representing the crystalline cellulose decreased slightly. The CrI of raw, MHAP, and SHAP samples were 23.67, 33.00 and 25.88, respectively. The increase in CrI of the pretreated samples was attributed to the dissolution of amorphous cellulose, hemicellulose, and lignin during pretreatment. Compared with SHAP sample, the CrI of MHAP sample increased significantly, which indicated that the amorphous cellulose of cassava residues experienced more damages. 3.2.4. FTIR analysis The FTIR spectra of the raw and pretreated cassava residues were used to obtain information on their structural and chemical changes, as illustrated in Fig. S3b. The adsorption band at 1430 cm1 was assigned to CH2 scissoring vibrations, which was strong in type I crystalline (cellulose I) and very weak in type II crystalline (cellulose II) and amorphous celluloses. The adsorption band at 894 cm1 was assigned to the C–O–C stretching vibration at the b-(1,4)-glycosidic linkage, which was weak and broad in cellulose I, but strong and sharp in cellulose II and amorphous cellulose. The intensity of the carbonyl bond vibration at 1740 cm1, which has been ascribed to the C@O vibration of the acetyl groups in hemicellulose became weaker after MHAP and SHAP. This phenomenon was due to a proportion of the hemicellulose was removed during pretreatment (Moretti et al., 2014). An additional change was the increased intensity of the band at 894 cm1, which was due to the increased relative cellulose content. Compared to raw material, the lignin bond vibration at 1510 cm1 (aromatic ring of lignin) in MHAP and SHAP samples was significantly enhanced. This phenomenon can be attributed to the removal of most of the hemicellulose and a portion of cellulose during pretreatment. The absorption ratio A1430 cm1/A894 cm1, which was known as the lateral order index (LOI) was used to reflect the coefficient of crystalline cellulose in the cellulose structure (Oh et al., 2005). The
LOI of raw, MHAP, and SHAP samples were 0.77, 1.18, and 1.12, respectively. The increase in LOI of the pretreated samples was attributed to the hydrolysis of amorphous cellulose, hemicellulose, and lignin during pretreatment, which was consistent with XRD results.
3.3. Dark hydrogen production from pretreated cassava residues Untreated cassava residues can hardly produce biohydrogen (
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Fermentative hydrogen and methane cogeneration from cassava residues: Effect of pretreatment on structural characterization and fermentation performance Jun Cheng a,⇑, Richen Lin a, Lingkan Ding a, Wenlu Song a,b, Yuyou Li c, Junhu Zhou a, Kefa Cen a a b c
State Key Laboratory of Clean Energy Utilization, Zhejiang University, Hangzhou 310027, China Department of Life Science and Engineering, Jining University, Jining 273155, China Department of Civil and Environmental Engineering, Tohoku University, Sendai 9808579, Japan
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Cassava residue was pretreated by
microwave (or steam)-heated acid (MHAP or SHAP). MHAP generated many regular micropores and SHAP generated many irregular fragments. SHAP generated wider cracks (0.2 lm) in delaminated cell walls than MHAP (0.1 lm). MHAP resulted in a higher crystallinity index (33.00) than SHAP (25.88). MHAP with enzymolysis led to a higher H2 yield than SHAP, but CH4 yield reversed.
a r t i c l e
i n f o
Article history: Received 14 October 2014 Received in revised form 12 December 2014 Accepted 13 December 2014 Available online 19 December 2014 Keywords: Cassava residue Microwave-heated acid Steam-heated acid Hydrogen Methane
a b s t r a c t The physicochemical properties of cassava residues subjected to microwave (or steam)-heated acid pretreatment (MHAP or SHAP) were comparatively investigated to improve fermentative hydrogen and methane cogeneration. The hydrogen yield from cassava residues with MHAP and enzymolysis was higher (106.2 mL/g TVS) than that with SHAP and enzymolysis (102.1 mL/g TVS), whereas the subsequent methane yields showed opposite results (75.4 and 93.2 mL/g TVS). Total energy conversion efficiency increased to 24.7%. Scanning electron microscopy images revealed MHAP generated numerous regular micropores (6 lm) and SHAP generated irregular fragments (23 lm) in the destroyed lignocellulose matrix. Transmission electron microscopy images showed SHAP generated wider cracks (0.2 lm) in delaminated cell walls than MHAP (0.1 lm). X-ray diffraction patterns indicated MHAP caused a higher crystallinity index (33.00) than SHAP (25.88), due to the deconstruction of amorphous cellulose. Fourier transform infrared spectroscopy indicated MHAP caused a higher crystallinity coefficient (1.20) than SHAP (1.12). Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
⇑ Corresponding author. Tel.: +86 571 87952889; fax: +86 571 87951616. E-mail address: [email protected] (J. Cheng). http://dx.doi.org/10.1016/j.biortech.2014.12.050 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
The search for sustainable and alternative energy is significant considering the increasing energy demands and diminishing fossil fuels. Hydrogen is a promising candidate because it is renewable, carbon neutral, and environment-friendly (Turner, 2004).
408
J. Cheng et al. / Bioresource Technology 179 (2015) 407–413
Currently, more attention has been focused on bio-hydrogen production from lignocellulosic wastes (Cheng et al., 2011; Chu and Majumdar, 2012; Somerville et al., 2010). Cassava is widely grown throughout southern China, and is an important raw material for starch production in industry. The pulverized cassava was screened through a mesh sieve to produce starch (125 lm), which were mainly composed of lignocellulose biomass. According to the statistics from the Food Agricultural Organization of the United Nations, cassava production in China was approximately 4.56 million tons in 2012, which indicates that large amounts of cassava residues was also produced by cassavabased starch and ethanol industries. If not properly managed, these cassava residues can cause severe environmental pollution. However, cassava residue is a typical lignocellulosic material that could potentially provide a sustainable source for biofuel production owing to its abundance, low cost, and availability. Cassava residues, which mainly consist of cellulose, hemicellulose, and lignin, do not biodegrade easily due to the three lignocellulosic components being cross-linked to each other, forming a cellulose–hemicellulose–lignin matrix. Cassava residues consist of both crystalline and amorphous cellulose structures, and the microfibril bundles of cellulose are bound by hydrogen bonding. Lignin consists of a complex array of polymers that are associated with each other, and provides structural support. Hemicellulose connects the cellulose and lignin, which provides more rigidity to the entire matrix. To improve the digestibility of lignocellulosic biomass and further enhance subsequent biofuel yields, various pretreatment technologies have been recently reviewed (Hendriks and Zeeman, 2009; Kallioinen et al., 2013; Monlau et al., 2013; Zheng et al., 2014), which can be classified into biological, physical, chemical, and physicochemical pretreatments. The degradation of cassava residues by complex microbial communities with high cellulose-degradation ability is an effective pretreatment method, which resulted in 96.6% increase in methane yield (Zhang et al., 2011). When subjected to mechanical activation pretreatment, the crystal structure of cassava residues is significantly destroyed, resulting in increased amorphization and decreased crystallinity (Liao et al., 2011). The hydrolysis of sugar beet residues significantly improved under dilute acid pretreatment with 150% increase in ethanol yield (Zheng et al., 2013). Among these pretreatments, dilute sulfuric acid pretreatment is one of the most studied and widely used methods (Chen et al., 2012; Cheng et al., 2014; Guragain et al., 2011; Zheng et al., 2013). However, to our knowledge, few studies discussed the use of microwave-heated acid pretreatment (MHAP) or steam-heated acid pretreatment (SHAP) on cassava residues to enhance its bio-hydrogen and biomethane production. Still, few attempts tried to reveal the physicochemical properties of cassava residues after MHAP and SHAP. Therefore, this study investigated the effects of MHAP and SHAP on the microstructure changes of cassava residues. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR) were used to determine the physicochemical properties of cassava residues after MHAP and SHAP. A two-stage fermentation process, which consisted of dark hydrogen and methane fermentation, was conducted to increase the energy conversion efficiency.
2. Methods 2.1. Feedstock and bacteria Cassava residues were obtained from a cassava processing plant in Guangxi Province, China. The cassava residues were oven dried,
powdered to a 0.02 mm mesh size, and stored for use in subsequent experiments. Mixed hydrogen-producing bacteria (HPB) and methane-producing bacteria (MPB) were obtained from a biogas plant in Zhejiang Province, China. Bacterial isolation and enrichment were described in previous studies (Cheng et al., 2010). 2.2. Pretreatment and fermentation methods 2.2.1. MHAP Microwave-heating pretreatment was conducted in a microwave digestion system (Shanghai Yiyao WX-4000, China). Due to the capacity limit of the microwave digestion system, 5 g of cassava residues were divided into four polytetrafluoroethylene reactors. 1.25 g of dried cassava residues were separately added into the four reactors, and then dilute H2SO4 (1.0%, v/v) was added to bring the combined volume to 25 ml in each reactor. The four reactors were then sealed and heated by microwaves to 135 °C for 15 min. 2.2.2. SHAP Steam-heating pretreatment was performed in an autoclave (Sanyo MLS-3780, Japan). 5.0 g of dried cassava residues were placed in a conical flask, and then dilute H2SO4 (1.0%, v/v) was added to bring the combined volume to 100 ml. The conical flasks were then placed in the autoclave and heated by steam at 135 °C for 15 min. 2.2.3. Enzymatic hydrolysis The enzymatic hydrolysis was performed in 250 mL flasks. After MHAP or SHAP, the pH of cassava residues solution was adjusted to 4.5 using NaOH. Trichoderma reesei cellulase (Shanghai Boao Biotechnology Corp., China) was added to the solution at 5 wt.% of the original cassava residues. The flasks were then sealed and placed in a shaker at 120 r/min for 120 h at 45 °C. 2.2.4. Dark hydrogen fermentation Fermentation experiments were conducted in 300 ml glass bottles. Approximately 100 ml of hydrolyzed solution (containing 5.0 g of cassava residues) and 125 ml of deionized water were added to each bottle and then mixed with 0.5 g of yeast extract. The initial pH was adjusted to 6.0 ± 0.1 using 6 M HCl and 6 M NaOH solution. The bottles were then inoculated with 25 ml HPB, sealed with rubber stoppers, purged with nitrogen gas for 10 min, and maintained at 35 ± 1.0 °C for dark hydrogen fermentation. 2.2.5. Dark methane fermentation The residual solutions of dark hydrogen fermentation were autoclaved at 121 °C for 20 min to inactivate the HPB. The pH of autoclaved solution was adjusted to 8.0 ± 0.1 using 6 M HCl and 6 M NaOH solution. The bottles were subsequently inoculated with 15 ml MPB, sealed with rubber stoppers, purged with nitrogen gas for 10 min, and maintained at 35 ± 1.0 °C for dark methane fermentation. 2.3. Analytical methods Electron micrographs of the cassava residues before and after MHAP/SHAP were obtained under a field emission SEM (Hitachi S3700, Japan) after the samples were sputtered with a thick layer of gold. The structural changes of cassava residues were also examined by TEM (Hitachi H-7650, Japan) at 120 keV electron-energy emission after staining the samples with KMnO4 and UO2(CH3 COO)2. XRD analyses were conducted on an X-ray diffractometer (X’Pert PRO, Netherlands) to determine changes in the crystallinity of cellulose from cassava residues. Chemical changes were
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J. Cheng et al. / Bioresource Technology 179 (2015) 407–413
3. Results and discussion 3.1. Effects of pretreatments on cassava residues saccharification The lignocellulosic compositions of cassava residues mainly contained 26.3% cellulose, 17.6% hemicellulose, and 7.0% lignin. The TVS content was 81.3%, and the heating value was 17.9 kJ/g TVS. To improve enzymatic digestibility and bio-hydrogen production, an effective pretreatment is necessary for disrupting the cellulose–hemicellulose–lignin matrix in cassava residues. In this study, MHAP and SHAP were conducted to examine their effects on saccharification. According to the calculation method proposed in a previous study (Cheng et al., 2010), the reducing sugar yield of dried cassava residues was theoretically 49.4 g/100 g. The reducing sugars yields of cassava residues measured by DNS method after MHAP and SHAP were 29.6 and 35.0 g/100 g cassava residues, respectively. After further enzymatic hydrolysis, the reducing sugars yields of cassava residues increased to 38.9 and 43.6 g/100 g cassava residues, which corresponded to 78.6% and 88.2% of the theoretical yield, respectively. Thus, using MHAP and SHAP followed by enzymatic hydrolysis, the lignocellulosic structure of cassava residues can be significantly damaged, and most of the cellulose and hemicellulose can be degraded into reducing sugars (e.g., glucose and xylose). The reducing sugars released from pretreated cassava residues were measured on a HPLC system, as shown in Fig. 1. Cellobiose, glucose, xylose, galactose, mannose, and arabinose were the main reducing sugars after enzymatic hydrolysis. Glucose and xylose comprised of over 70% of total sugars. The glucose yields for MHAP and SHAP were 18.9 and 22.7 g/100 g cassava residues, respectively. The xylose yields for MHAP and SHAP were 5.5 and 7.5 g/ 100 g cassava residues, respectively. The total sugars yield after SHAP and enzymatic hydrolysis (41.2 g/100 g cassava residues) was higher than that after MHAP and enzymatic hydrolysis (34.8/100 g cassava residues), which was in accordance with the result by DNS method.
50 Microwave-heated acid pretreatment+enzymatic hydrolysis Steam-heated acid pretreatment+enzymatic hydrolysis
Reducing sugars yields (g/100 g cassava residues)
examined using a FTIR spectrometer (Nicolet 5700, USA) equipped with a universal attenuated total reflectance accessory. The cellulose, hemicellulose, and lignin contents of the cassava residues were determined using a raw-fiber extractor (FIWE, VELP Scientific Corp., Italy) based on the Van Soest detergent method. Moisture content was determined by oven drying at 105 °C until a constant mass was obtained. Total volatile solids (TVS) and ash contents were determined by heating at 600 °C for 2 h. The total reducing sugar contents of the hydrolyzed cassava residues were determined using the 3,5-dinitrosalicylic acid method. The heating values of the cassava residues were determined using a fast calorimeter (Changsha Kaiyuan 5E-KC5410, China). The concentrations of cellobiose, glucose, xylose, galactose, mannose and arabinose were determined on a high performance liquid chromatography (HPLC) system (Agilent 1200, USA) using refractive index detector and Aminex HPX-87P column at 85 °C with H2O as mobile phase at 1 mm/min. The concentrations of hydrogen, methane, and soluble metabolite products (SMPs) were analyzed by gas chromatography (GC) systems. The hydrogen and methane yields were simulated by the modified Gompertz equation, and the dynamic parameters (H, the cumulative hydrogen and methane yields, mL/g TVS; Hm, the maximum hydrogen and methane yields potential, mL/g TVS, Rm, the peak rate of hydrogen and methane production, mL/g TVS/h; k, the lag-phase time of hydrogen and methane production, h; and Tm, the peak time, h) were calculated using Origin 8.5 software. The experiments were conducted in triplicates under all the conditions stated, and the results were expressed as mean (±SD).
40 30 20 10 0 Cellobiose
Glucose
Xylose
Galactose
Mannose
Arabinose Total sugars
Fig. 1. Reducing sugars release from pretreated cassava residues.
3.2. Physicochemical characterization of cassava residues before and after pretreatments 3.2.1. Scanning electron microscopy The physical structure changes of cassava residues after MHAP and SHAP were determined by SEM, as shown in Fig. S1. Raw cassava residues consisted of compact fiber bundles with different particle sizes (1–14 lm) on smooth flat surface, and the cellulose–hemicellulose–lignin matrix was also clearly visible, as shown in Fig. S1a. The surface morphologies of cassava residues were significantly damaged after MHAP and SHAP, which resulted in increased surface areas that evidently enhanced the digestibility. In Fig. S1b, the surface of cassava residues were obviously modified by MHAP. The compact fiber bundles were split and fractured, which contributed to an increase in surface roughness and the generation of irregular fragments (18 lm). In Fig. S1c, the lignocellulosic structure was split and fractured with larger fragments (23 lm) and fewer micropores after SHAP. The appearance of micropores (6 lm) at regular intervals was very prominent after MHAP, which could be attributed to the hydrolysis of hemicellulose and lignin during MHAP. Similar structural changes were reported in hemp hurd biomass that were pretreated by electron-beam irradiation (Abraham et al., 2013), in poplar solids that were pretreated by flow-through (Kumar et al., 2009), and in sugarcane bagasse that were pretreated by microwave-alkali or acid (Binod et al., 2012; Chen et al., 2011). When cassava residues were pretreated by microwave heating, the polar component was heated rapidly, whereas the nonpolar components did not absorb the microwaves and were not directly heated. This difference in behavior caused the inhomogeneity of surface morphologies. The rapid oscillation of the polar substances and along with the temperature inhomogeneity facilitated the disruption of the physical structure of cassava residues. In contrast, steam heating was slowly introduced to the sample surface, resulting in fewer structural changes (Hu and Wen, 2008). 3.2.2. Transmission electron microscopy To further characterize the ultrastructural change of cassava residues that were pretreated by MHAP and SHAP, TEM images were illustrated in Fig. S2. The cell wall layers were clearly observed in raw cassava residues, as shown in Fig. S2a. The cell walls of raw cassava residues composed of intercellular layers, namely, the compound middle lamella (CML), outer secondary wall (S1), middle secondary wall (S2), and inner secondary wall (S3). The CML (170 nm width) that fixed the cells together was clearly shown against the adjacent cell wall layers. The secondary wall was a thick layer inside the CML in inner parts of the cell. The
J. Cheng et al. / Bioresource Technology 179 (2015) 407–413
3.2.3. X-ray diffraction analysis The XRD patterns of the pretreated cassava residues were shown in Fig. S3a to determine the change in crystallinity after MHAP and SHAP. A major diffraction peak on cellulose crystallographic plane has been identified at 2h = 22–23°, which indicated the presence of a highly organized crystalline region, whereas the peak at 2h = 18° indicated a less organized region (Chen et al., 2012). The crystallinity index (CrI), which represented the percentage of crystalline materials, was calculated by an empirical method. Compared with raw cassava residues, the peak intensity representing the amorphous cellulose in the MHAP and SHAP samples decreased significantly, whereas the peak intensity representing the crystalline cellulose decreased slightly. The CrI of raw, MHAP, and SHAP samples were 23.67, 33.00 and 25.88, respectively. The increase in CrI of the pretreated samples was attributed to the dissolution of amorphous cellulose, hemicellulose, and lignin during pretreatment. Compared with SHAP sample, the CrI of MHAP sample increased significantly, which indicated that the amorphous cellulose of cassava residues experienced more damages. 3.2.4. FTIR analysis The FTIR spectra of the raw and pretreated cassava residues were used to obtain information on their structural and chemical changes, as illustrated in Fig. S3b. The adsorption band at 1430 cm1 was assigned to CH2 scissoring vibrations, which was strong in type I crystalline (cellulose I) and very weak in type II crystalline (cellulose II) and amorphous celluloses. The adsorption band at 894 cm1 was assigned to the C–O–C stretching vibration at the b-(1,4)-glycosidic linkage, which was weak and broad in cellulose I, but strong and sharp in cellulose II and amorphous cellulose. The intensity of the carbonyl bond vibration at 1740 cm1, which has been ascribed to the C@O vibration of the acetyl groups in hemicellulose became weaker after MHAP and SHAP. This phenomenon was due to a proportion of the hemicellulose was removed during pretreatment (Moretti et al., 2014). An additional change was the increased intensity of the band at 894 cm1, which was due to the increased relative cellulose content. Compared to raw material, the lignin bond vibration at 1510 cm1 (aromatic ring of lignin) in MHAP and SHAP samples was significantly enhanced. This phenomenon can be attributed to the removal of most of the hemicellulose and a portion of cellulose during pretreatment. The absorption ratio A1430 cm1/A894 cm1, which was known as the lateral order index (LOI) was used to reflect the coefficient of crystalline cellulose in the cellulose structure (Oh et al., 2005). The
LOI of raw, MHAP, and SHAP samples were 0.77, 1.18, and 1.12, respectively. The increase in LOI of the pretreated samples was attributed to the hydrolysis of amorphous cellulose, hemicellulose, and lignin during pretreatment, which was consistent with XRD results.
3.3. Dark hydrogen production from pretreated cassava residues Untreated cassava residues can hardly produce biohydrogen (
