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Effect of Ca(OH)2 pretreatment on extruded rice straw anaerobic digestion
Bioresource Technology 196 (2015) 116–122
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Effect of Ca(OH)2 pretreatment on extruded rice straw anaerobic digestion Yu Gu, Yalei Zhang, Xuefei Zhou ⇑ Key Laboratory of Yangtze Water Environment of Ministry of Education, State Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China
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
Enhanced methane production of
>30% by Ca(OH)2 pretreatment of extruded rice straw. Increased cellulose and xylose hydrolysis efficiency after Ca(OH)2 pretreatment. Synergistic effect was observed between extrusion and Ca(OH)2 pretreatment. 8% Ca(OH)2 loading rate led to the highest biogas production of 564.7 mL/g VS.
a r t i c l e
i n f o
Article history: Received 17 May 2015 Received in revised form 30 June 2015 Accepted 1 July 2015 Available online 8 July 2015 Keywords: Biogas production Ca(OH)2 pretreatment Enzyme hydrolysis Extrusion pretreatment Rice straw
a b s t r a c t It has been proven that extrusion can change the structure of rice straw and increase biogas production, but the effect of a single pretreatment is limited. Ca(OH)2 pretreatment was used to enhance the enzyme hydrolysis and biogas production of extruded rice straw. After Ca(OH)2 pretreatment, the glucose and xylose conversion rates in enzymatic hydrolysis increased from 36.0% and 22.4% to 66.8% and 50.2%, respectively. The highest biogas production observed in 8% and 10% Ca(OH)2 pretreated rice straw reached 564.7 mL/g VS and 574.5 mL/g VS, respectively, which are 34.3% and 36.7% higher than the non-Ca(OH)2-loaded sample. The Ca(OH)2 pretreatment can effectively remove the lignin and increase the fermentable sugar content. The structural changes in the extruded rice straw have also been analyzed by XRD, FTIR, and SEM. Considering all of the results, an 8% Ca(OH)2 loading rate is the best option for the pretreatment of extruded rice straw. Ó 2015 Published by Elsevier Ltd.
1. Introduction Lignocellulosic biomass, including agricultural residues, energy crops, forestry wastes and a part of municipal wastes, is a fermentable material for renewable energy production. Because it is widely available and contains large amounts of cellulose and hemicellulose, more and more researchers are focusing on lignocellulose utilization. Anaerobic digestion is a feasible way to ⇑ Corresponding author. E-mail address: [email protected] (X. Zhou). http://dx.doi.org/10.1016/j.biortech.2015.07.004 0960-8524/Ó 2015 Published by Elsevier Ltd.
transform cellulose and hemicellulose, which are the main components of lignocellulose and are composed of potentially fermentable sugars, to biogas. However, the biogas conversion efficiency of lignocellulosic biomass is limited by the biomass recalcitrance, which refers to the natural resistance of plants to enzymatic hydrolysis and microbial deconstruction (Himmel et al., 2007). The biomass recalcitrance is caused by the spatial structure and chemical composition of lignocellulose, including its crystalline cellulose content, degree of polymerization, accessibility and lignin protection, and these properties cause its low hydrolysis efficiency (Zhang and Lynd, 2004). The lignocellulose
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hydrolysis process is therefore considered to be the speed-limiting step. To enhance the lignocellulose hydrolysis process and achieve profitable biogas production, pretreatment is required to change the chemical and physical structure of lignocellulose and overcome the biomass recalcitrance. Many pretreatment methods can be adopted to reduce the biomass recalcitrance of lignocellulose, enhance enzyme accessibility and increase the fermentation efficiency. It is worth noting that mechanical crushing not only is important but is an essential pretreatment in lignocellulosic material anaerobic digestion. Mechanical pretreatment can efficiently reduce the material particle size and increase the accessible surface. The main effect of the increased surface area is to enhance the cellulose accessibility (Kratky and Jirout, 2011). Many types of machines have been applied in lignocellulosic material treatment. Extrusion is one effective mechanical pretreatment method. Compared with traditional physical pretreatment methods such as chipping, grinding, and milling, extrusion can not only reduce particle size but also provides a continuous thermo-mechanical treatment that will cause a further expansion of rice straw fibril structure (Chen et al., 2014b; Kratky and Jirout, 2011). Therefore, extruded lignocellulosic materials achieve a lower bulk density, higher water-holding capacity and better biodegradability. In a previous study, it was reported that the biogas production observed in extrusion-pretreated rice straw was 32.5% and 72.2% higher than milling-pretreated rice straw and paper trimmer-pretreated rice straw, respectively (Chen et al., 2014b). In addition, an extruder is suitable for large-scale production and has many other advantages such as a high shear force and efficient mixing (Kratky and Jirout, 2011). Although extrusion can effectively enhance the anaerobic digestion efficiency, the effect is still limited because it only changes the structural characterization. The chemical characterization such as lignin content is also an important factor of the biomass recalcitrance. Alkaline pretreatment is an efficient pretreatment to change the chemical characterization of lignocellulosic materials, especially through lignin removal (Krishania et al., 2013). Approximately 10–40% of the lignin can be removed when 2–10% NaOH was used to treat lignocellulosic materials, and there is no significant cellulose degradation during the pretreatment (Chen et al., 2014a; Zhu et al., 2010). During the alkali pretreatment, solvation and saponification reactions happen rapidly (Park and Kim, 2012). This will result in a swelling effect on the biomass and decompose the polysaccharides, which increases the enzyme accessibility and decrease the biomass recalcitrance (Hendriks and Zeeman, 2009; Krishania et al., 2013). The alkali pretreatment not only enhances the lignocellulosic biomass digestibility but also provides extra alkalinity, neutralizes acid production and prevents a pH drop during the anaerobic digestion process (Chen et al., 2014a; Pavlostathis and Gossett, 1985). As mentioned above, the individual extrusion pretreatment has inevitable limitations. To further enhance biogas production from extruded lignocellulosic materials, alkali pretreatment is a good application. Extrusion may also have a positive effect on the subsequent alkali pretreatment. NaOH and Ca(OH)2 are two alkalis widely used in lignocellulosic material pretreatment. Compared with NaOH, Ca(OH)2 is a much cheaper reagent, and Ca2+ has no inhibitory effect on anaerobic digestion (Chen et al., 2008). Extrusion and Ca(OH)2 pretreatment were therefore performed. The aims of this study were (1) to analyze the effect of Ca(OH)2 on enhancing the biogas production and enzymatic hydrolysis of extruded rice straw, (2) to analyze the effect of Ca(OH)2 on extruded rice straw’s physical and chemical characterizations, and (3) to determine the best Ca(OH)2 loading rate for extruded rice straw.
2. Methods 2.1. Feedstock and inoculum The rice straw used in the experiment was collected from Chongming Island. The rice straw was dried at room temperature after harvesting and then extruded with a twin-screw extruder (JXM80, Jinwor Machinery Co., Ltd., Nanjing, China), as described previously (Chen et al., 2014b). Then, the rice straw was screened. Particles that passed through a 20-mesh sieve but were retained by a 40-mesh sieve were collected and air-dried at 40 °C. The dried rice straw was then stored in a vacuum plastic bag at room temperature to prevent possible degradation. Digested dairy manure obtained from a livestock and poultry farm in Chongming was used as an inoculum in this study. The characterizations of the extruded rice straw and inoculum are presented in Table 1. 2.2. Ca(OH)2 pretreatment The concentrations of Ca(OH)2 (based on the dry matter of the rice straw) used in the experiment were 5%, 8%, 10%, 12%, and 15%. Although all of the rice straw was extruded, the rice straw that was not pretreated with Ca(OH)2 was referred to as untreated. 500 mL wide mouthed bottles (with a 400 mL working volume) were used as the reactors. Rubber stoppers were used to seal the reactors during the pretreatment process. Distilled water was added to adjust the solid/liquid ratio to 5% (w/v). The mixtures were stirred with a magnetic stirrer and incubated at ambient temperature (25 °C) for 72 h. After the pretreatment, the pretreated rice straw was filtered and washed with distilled water to neutral pH. Then, the pretreated rice straw was dried and stored in a vacuum plastic bag for the subsequent analysis and experiments. 2.3. Anaerobic digestion The anaerobic digestion of Ca(OH)2 pretreated and untreated rice straw was performed in batch reactors. 1 g rice straw (based on TS) was inoculated with 40 mL digested diary manure. The mixed feedstock was then digested in 100 mL serum bottles. The serum bottles were sealed with rubber stoppers, and aluminum caps were used to provide extra reinforcement. All of the reactors were then placed in a thermostatic chamber maintained at 35 °C and digested for 40 days. The blank group, with the same inoculum quantity and no rice straw, was used to exclude the biogas production from the inoculum. 2.4. Enzymatic hydrolysis The commercial cellulase and xylanase used in the experiments was from Sigma–Aldrich. The enzyme activities of the enzyme preparations are shown in Table 2. For these experiments, 50-mL tubes with working volumes of 40 mL were used as the hydrolysis reactors. The pretreated rice straw was hydrolyzed in sodium acetate buffer (50 mM, pH 5.0) with 2% (w/v) solids loading. 0.1 mL of
Table 1 Characteristics of rice straw and inoculum. Parameter
Rice straw
Inoculum
Total solids (%) Volatile solids (% TS) Total carbon (% TS) Total nitrogen (% TS) C/N ratio
97.3 ± 0.3 86.3 ± 0.5 35.2 ± 1.1 0.6 ± 0.0 58.6
2.8 ± 0.0 59.1 ± 0.0 20.5 ± 1.2 3.1 ± 0.1 6.7
Data is expressed as means ± SD (n P 3).
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Table 2 Activities of the commercial enzyme preparations. Enzyme preparation
CMCase (U/mL)
FPA (FPU/mL)
Xylanase (U/mL)
Cellulase Xylanase
263.7 ± 15.8
9.9 ± 3.7
79.7 ± 10.6 82.4 ± 29.6
Data is expressed as means ± SD (n P 3).
cellulase and 0.2 mL of xylanase were added into the tubes, which were incubated at 50 °C with shaking at 190 rpm for 3–72 h. At the end of the hydrolysis, the tubes were incubated in a water bath at 100 °C for 10 min to inactivate the enzymes. When the hydrolysis process was finished, the samples were centrifuged at 12,000 rpm for 15 min, and the supernatants were collected for the reducing sugar, glucose, and cellulose content determinations.
A Fourier transform infrared spectrophotometer (Thermo Fisher Scientific, Nicolet 5700) was used to examine the changes in the functional groups of Ca(OH)2-pretreated and untreated rice straw. The samples were dried and dispersed in KBr, and then approximately 1 MPa pressure was added to the mixture to form sample discs for analysis. Spectra were obtained from 32 scans using a resolution from 4000 to 400 cm 1. The spectral analysis was performed using OMNIC software. Pure KBr was used to calibrate the background. A scanning electron microscope (Phenom, Pro) was used to examine the structural changes in the Ca(OH)2-pretreated and untreated rice straw. The samples were coated with gold and then analyzed with an accelerating voltage of 10 kV. 3. Results and discussion 3.1. Compositional change of pretreated rice straw
2.5. Analytical methods The total solids (TS) and volatile solids (VS) of the extruded rice straw and inoculum were measured according to the standard methods (APHA, 2012). The total carbon and nitrogen content were analyzed using an elemental analyzer (Elementar, Vario EL III). The cellulase and xylanase activities were measured according to the NREL LAP protocol, and the reducing sugar yields were determined by the DNS method (Adney and Baker, 1996). The cellulose and hemicellulose contents in the Ca(OH)2-pretreated and untreated rice straw were analyzed according to the standard NREL LAP protocol (Sluiter et al., 2008). The biogas production was analyzed by calculating the volume and pressure in the headspace of the serum bottle. In the first 10 days, the pressure was tested every day, and from the 11th to the 40th day the pressure was tested every two days. The pressure was determined by a pressure meter (VWR, PS 100-2BAR), and the biogas production was transferred to the normal pressure and temperature. Disposable syringes were used to collect a biogas sample, and then the methane content was analyzed by a gas chromatograph (Agilent, 6890) (Gu et al., 2014). The methane production was calculated by multiplying the methane content by the biogas production. The glucose and xylose yields in the enzyme hydrolysis process were analyzed by an Agilent 1200 high performance liquid chromatography (HPLC) system equipped with a refractive index detector using a SP0810 sugar column (Shodex). The column temperature and the detector temperature were set at 80 and 55 °C, respectively. Degassed HPLC-grade water was used as the mobile phase at a flow rate of 0.6 mL/min. X-ray diffraction was performed on an X-ray diffractometer (Bruker, D8 Advance) with Cu Ka radiation at a wavelength of 0.1542 nm (50 kV, 300 mA). The rice straw was scanned at angles varying from 10° to 50° with an increment of 2°/min. The cellulose crystallinity was calculated as described by Segal et al. (1959).
The compositions of the rice straw before and after Ca(OH)2 pretreatment are presented in Table 3. A significant degradation in hot water extraction (HWE) and ethanol extraction (EE) was observed in all Ca(OH)2-pretreated samples and this led to the incensement of total lignocellulose. The structural carbohydrates contained in the different samples were analyzed to study the impact of Ca(OH)2 pretreatment. The glucose content, which refers to the remaining cellulose, was the highest and most important component in the substrate. In the 5% and 8% samples, the glucose content was in a relatively high level and reached 40.6% and 38.1%, respectively. This indicates superior biodegradability and higher fermentable sugar content in these two samples. Compared with glucose, hemicellulose, mainly composed of xylan, is more easily solubilized in Ca(OH)2. This partially contributes to the spatial structure of lignocellulose, in which cellulose is covered by hemicellulose and lignin (Chen et al., 2014a). The xylose content, which refers to the xylan content, decreased sharply when the Ca(OH)2 loading was higher than 8%. Although lower xylan content will accelerate the cellulose hydrolysis, xylose loss will also decrease the potential biogas production. Galactose, arabinose and mannose, as a part of hemicellulose, were also significantly removed in the high Ca(OH)2 loading pretreatment, but their affections to biogas production were limited. Lignin has been regarded as a cellulase adsorbent, which reduces the cellulase accessibility in lignocellulosic material hydrolysis (Zhang and Lynd, 2004). After the Ca(OH)2 pretreatment, the acid insoluble lignin (AIL) was decreased in all pretreated samples, and its removal rate increased with the Ca(OH)2 loading rate. The original lignin content was 13.7%, and the lowest content, observed in the 15% Ca(OH)2 treated sample, was 10.6%. Considering the compositional change caused by the decreases in HWE and EE, the AIL removal efficiency increased slightly in the high Ca(OH)2 loading rate. Although less lignin was contained in the high Ca(OH)2 loading samples, the potentially fermentable sugar content (mainly glucose and xylose)
Table 3 Chemical composition of different Ca(OH)2 pretreated extruded rice straw. Ca(OH)2 addition
Untreated 5% 8% 10% 12% 15%
HWE (%)
6.7 ± 0.6 0.7 ± 0.1 0.3 ± 0.0 0.2 ± 0.0
Data is expressed as means ± SD (n P 3).
EE (%)
5.3 ± 0.8 3.1 ± 0.3 0.8 ± 0.1 0.3 ± 0.0 0.1 ± 0.0
Structural carbohydrates content (%)
AIL (%)
Glu
Xyl
Gal
Ara
Man
36.8 ± 0.4 40.6 ± 1.2 38.1 ± 0.6 34.6 ± 1.1 34.1 ± 0.9 31.0 ± 0.4
17.8 ± 0.2 18.0 ± 0.6 13.0 ± 0.3 10.8 ± 0.2 10.5 ± 0.0 9.3 ± 0.3
0.8 ± 0.1 0.7 ± 0.0 0.3 ± 0.0
6.2 ± 0.1 6.5 ± 0.2 6.9 ± 0.1 4.4 ± 0.0 3.6 ± 0.4 4.0 ± 0.1
0.3 ± 0.0
13.7 ± 0.1 12.8 ± 0.1 11.7 ± 0.2 11.3 ± 0.0 10.9 ± 0.0 10.6 ± 0.2
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also decreased with the Ca(OH)2 loading rate increase. Considering the combined effects of these two factors, an 8% Ca(OH)2 loading would be the best choice. 3.2. Biogas production Inoculated rice straw was digested at 35°C for 40 days to investigate the effect of Ca(OH)2 pretreatment on biogas production. The daily biogas production, cumulative biogas production and methane production are depicted in Fig. 1. Compared with untreated rice straw, the biogas production of the Ca(OH)2 pretreated rice straw was much more severe. It is obvious that the Ca(OH)2-pretreated rice straw had a higher daily biogas production, and peaks appeared much earlier. There is therefore no doubt that the Ca(OH)2-pretreated rice straw is more suitable for biogas production. Similar trends of biogas production were found in different Ca(OH)2-pretreated samples. The biogas was generated immediately after the rice straw was seeded, and it continued to increase until reaching the first peak. Before the fermentation ceased, there were several peaks during the process. The peak value is the main difference between the different pretreated substrates. The daily biogas production of the 5–15% Ca(OH)2 pretreated rice straws reached their first peaks between day 4 and day 6 with 135.8, 156.4, 159.9, 188.6 and 174.6 mL/g VS biogas productions, respectively. The second peaks appeared between day 12 and day 20, with 136.4, 178.7, 160.7, 152.0 and 141 mL/g VS biogas productions, respectively. It can be concluded that the higher Ca(OH)2 loading pretreated rice straw produced more biogas in the initial period but had a lower subsequent potential. Fig. 1(b) shows the cumulative biogas production, which was calculated based on the daily biogas production. The total methane production is shown in Fig. 1(c). The total biogas and methane in the Ca(OH)2-pretreated rice straws were significantly higher than those in the untreated straw. There was no significant difference between the 8%, 10%, and 12% Ca(OH)2 pretreated rice straws in the final results, with their total methane productions reaching 330.9, 337.8, and 322.6 mL/g VS, respectively. The biogas productions in the 5% and 15% Ca(OH)2 pretreated samples were lower. Although the 5% Ca(OH)2 pretreated rice straw had the highest cellulose content, its methane production was still 10% lower than that of the 8% Ca(OH)2 pretreated straw. This might be caused by its high lignin content, which was the main reason for the biomass recalcitrance. When the Ca(OH)2 loading rate was 15%, the total biogas production decreased, and the methane production was less than 300 mL/g VS. This phenomenon might be caused by the over dissolution of cellulose and hemicellulose in the high Ca(OH)2 process. These results indicate that 8% is the best Ca(OH)2 loading rate for extruded rice straw. The Ca(OH)2 was insufficient in lower loading rates, and overloading was also inappropriate. Compared with the combined mill-alkali pretreatment process (Cheng et al., 2010; He et al., 2008), the extruded rice straw in this study achieved higher biogas productions in both the Ca(OH)2-pretreated and untreated samples. The optimal alkali addition was also lower than that of milled rice straw (Song et al., 2013). This may be attributed to its larger specific surface area and looser spatial structure (Chen et al., 2014b; Chen et al., 2008).
Fig. 1. Biogas production for Ca(OH)2 pretreated rice straw: (a) daily biogas production, (b) cumulative biogas production, (c) methane production.
3.3. Enzymatic hydrolysis The enzymatic digestibility of untreated and Ca(OH)2-pretreated rice straws was tested by cellulase and xylanase hydrolysis for 72 h. Fig. 2(a) and (b) plots the glucose and xylose conversion rates in enzymatic hydrolysis as functions of the incubation time for different samples. The glucose and xylose conversion rates were defined as the percentages of glucose and xylose in the pretreated rice straw that were solubilized during
the hydrolysis process. The glucose and xylose produced during the pretreatment were not included. The untreated rice straw was resistant to enzymatic hydrolysis, so the conversion rates of untreated rice straw were low for both cellulase and xylanase hydrolysis. The glucose and xylose conversion rates increased dramatically in the Ca(OH)2-pretreated samples. All of the curves had the same trend, with most of the
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experiments. Although the alkali pretreatment caused the lignocellulose to dissolve, its effect on the fiber size was limited. Samples with lower lignin contents achieved higher reducing sugar production. Delignification was considered as a more important reason for the enhanced glucose and xylose conversion rates. The hydrolysis is the rate-limiting step in the anaerobic digestion. Cellulose and hemicellulose (mainly xylan) are converted to glucose and xylose by enzymes and their conversion efficiencies greatly affects the biogas production. The enhanced conversion efficiency usually led to more efficient biogas production, but the 15% Ca(OH)2 pretreated rice straw that achieved the highest conversion rates produced less biogas than the 8% Ca(OH)2 pretreated sample. This was due to the different structural carbohydrates contained in the different samples. Because there was more glucose and xylose, the 8% Ca(OH)2 pretreated rice straw could release more fermentable sugars for biogas production than the 15% Ca(OH)2 pretreated rice straw. Other studies also found that a high alkali loading rate would decrease the total sugar release in enzyme hydrolysis (Saha and Cotta, 2007; Sills and Gossett, 2011). When using both the structural carbohydrate content and conversion rate to estimate the effect of Ca(OH)2 pretreatment on the extruded rice straw, 8% Ca(OH)2 should be the best option. 3.4. Structural change analysis
Fig. 2. The effect of different Ca(OH)2 pretreatment on glucose and xylose conversion rate during 72 h hydrolysis.
glucan converted in 24 h. For instance, the glucose yields after 72 h hydrolysis, from the untreated rice straw to the 15% Ca(OH)2 treated rice straw, were 24.7%, 35.7%, 45.2%, 50.2%, 53.7% and 58.4%, which represent 68.6%, 76.4%, 75.8%, 83.1%, 85.2% and 87.4% of the total converted glucan, respectively. This indicates that higher Ca(OH)2 pretreated rice straw had a higher digestibility and also digested more quickly. The same phenomenon could also be observed in xylan hydrolysis. The xylan conversion rate represents the hemicellulose digestibility because xylan is the main component of the hemicellulose in rice straw (especially in the Ca(OH)2-pretreated samples). Although hemicellulose can be transformed to a fermentable sugar and used to produce biogas, it exists as a type of physical barrier for cellulose (Hu et al., 2011). Therefore, the better hemicellulose digestibility would not only affect the xylose conversion but also had a positive effect on the glucan conversion. The results also prove that a higher glucose conversion always accompanies a high xylose conversion. The high hydrolysis rate observed in Ca(OH)2-pretreated rice straws indicated better enzyme accessibility and lower biomass recalcitrance. The enzyme accessibility was affected by many facts, such as fiber size and lignin content. As previously described, the extruded rice straw was sieved to a uniform size before the
3.4.1. Changes in cellulose crystallinity The cellulose structure and cellulose crystallinity was measured by X-ray diffraction. The X-ray diffraction pattern did not change between the different Ca(OH)2 pretreated samples, indicating that the cellulose style was not obviously changed during the Ca(OH)2 pretreatment. This might indicate that the Ca(OH)2 could only reach the surface of the cellulose, and the same phenomenon was also observed in the NaOH-pretreated rice straw (He et al., 2008). According to previous study, the peak observed at 22.0° represents the presence of crystalline cellulose and the peak at 18.0 represents amorphous cellulose (Segal et al., 1959). The crystallinity, which is defined as the ratio of the crystalline regions to the total cellulose (including crystalline and amorphous cellulose), have been calculated. The crystallinity in the untreated rice straw was 33.2%, and it increased to 36.2%, 38.8%, 40.4%, 39.4% and 34.9% in the 5–15% Ca(OH)2 pretreated samples, respectively. The slightly increase in crystallinity after pretreatment had also been reported (Liang et al., 2014). The possible reasons for the increased crystallinity are (1) the crystalline cellulose being more resistant to the alkali solution than the amorphous cellulose (Himmel et al., 2007); (2) the decreased contents of lignin and hemicellulose, both of which are amorphous (Zhao et al., 2012). Wu et al. reported that the crystalline cellulose increased after the lignin removal (Wu et al., 2014). Although the crystallinity increased slightly after the alkali pretreatment, the final value was still much lower than that of the combined milled-alkali pretreated samples reported elsewhere (He et al., 2008; Liang et al., 2014; Yang et al., 2009). The crystallinity affects the cellulose digestion speed but not the limitation. The low crystallinity in the extruded rice straw promises an efficient digestion, and the increased crystallinity after the Ca(OH)2 pretreatment had no obvious negative effect on the biogas production and enzymatic hydrolysis. 3.4.2. Changes in functional groups The changes in functional groups of Ca(OH)2 pretreated rice straw were analyzed by Fourier transform infrared (FTIR) spectroscopy. The untreated rice straw was compared with the 8% Ca(OH)2 pretreated rice straw, which was believed to have
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achieved the best performance. The spectra were analyzed according to previous studies (Liang et al., 2014; Popescu et al., 2010; Yang et al., 2009). Although the results showed same trend and had almost the same peaks, it is obvious that all of the peaks in the Ca(OH)2-pretreated rice straw shifted relative to the untreated straw. This implies that the inter- and intramolecular bonds had changed during the Ca(OH)2 pretreatment process. Compared with untreated rice straw, the O–H stretching absorbance and C–H stretching absorbance in Ca(OH)2-pretreated rice straw decreased significantly, indicating an influence of pretreatment on the hydroxyl-stretching region (Popescu et al., 2010). The lower absorbance of the hydroxyl-stretching region observed in Ca(OH)2-pretreated rice straw indicates a removal of hydrogen bonds during the pretreatment process. In the fingerprint region, there were remarkable differences in the observed O–H and conjugated C–O, C–C of aromatic ring, and C–O stretching, which reflect modifications of lignin in the pretreated rice straw. The C–O stretching in different samples did not change much, indicating the low effect of Ca(OH)2 on the crystalline structure, which agrees with the XRD results. These changes in the functional groups demonstrate the lignin removal and cellulose depolymerization after the Ca(OH)2 pretreatment. The results indicate a better biological and enzymatic digestibility of 8% Ca(OH)2 pretreated rice straw, which explains the increased biogas production efficiency and enzyme hydrolysis in the pretreated samples. 3.4.3. Physical surface structural changes The physical surface structural changes also had been analyzed in this study. The 8% Ca(OH)2 pretreated rice straw was chosen for comparison with the untreated rice straw for its superior digestibility and higher biogas production. Compared with other physically pretreated lignocellulosic materials (Hu and Wen, 2008; Zeng et al., 2007), the extruded rice straw seemed to have a gullied surface rather than a smooth surface. The water holding capacity also increased according to previous study (Chen et al., 2014b). These changes create a larger accessible surface for the alkali to react. Although the extrusion could partially tear the rice straw surface and expose the cellulose fiber in part (Chen et al., 2014b), the surface was still covered with the wax layer and lignin. After the Ca(OH)2 pretreatment, the rice straw was further damaged, and the compact surface was destroyed. The wax layer was also destroyed and removed during the pretreatment process. Moreover, inner voids, which are important for the enzymes and microbes to approach the internal cellulose fibrils, were exposed after the pretreatment, as the outer structure had been broken. It is obvious that more cellulose fibrils were exposed after the alkali pretreatment, which increased the cellulose accessibility. The changes observed in the surface structure could well explain the enhanced biogas production and enzymatic hydrolysis in Ca(OH)2-pretreated rice straw. Compared with other physical pretreatments such as the mill pretreatment, extrusion seemed to cause a lower consumption in the following alkali pretreatment. This might suggest a synergistic effect of extrusion in enhancing the effect of the alkali pretreatment.
4. Conclusions The results of this study show that using a Ca(OH)2 pretreatment could significantly enhance the extruded rice straw biogas production rate and enzymatic hydrolysis efficiency. Lignin and fermentable sugar contents changed during the Ca(OH)2 pretreatment process. The physical and chemical structures of the extruded rice straw also have changed after the pretreatment, which lead to better biodegradability. Positive effects were observed in all Ca(OH)2 loading rates, but overloading Ca(OH)2
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would cause a greater fermentable carbohydrate loss, which decreased the total biogas production. The optimal Ca(OH)2 loading rate was 8%, which achieved both efficient hydrolysis and high biogas production. Acknowledgement This research was funded by the National key technologies R&D program of China (No. 2012BAJ25B04).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2015.07. 004.
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Effect of Ca(OH)2 pretreatment on extruded rice straw anaerobic digestion Yu Gu, Yalei Zhang, Xuefei Zhou ⇑ Key Laboratory of Yangtze Water Environment of Ministry of Education, State Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China
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
Enhanced methane production of
>30% by Ca(OH)2 pretreatment of extruded rice straw. Increased cellulose and xylose hydrolysis efficiency after Ca(OH)2 pretreatment. Synergistic effect was observed between extrusion and Ca(OH)2 pretreatment. 8% Ca(OH)2 loading rate led to the highest biogas production of 564.7 mL/g VS.
a r t i c l e
i n f o
Article history: Received 17 May 2015 Received in revised form 30 June 2015 Accepted 1 July 2015 Available online 8 July 2015 Keywords: Biogas production Ca(OH)2 pretreatment Enzyme hydrolysis Extrusion pretreatment Rice straw
a b s t r a c t It has been proven that extrusion can change the structure of rice straw and increase biogas production, but the effect of a single pretreatment is limited. Ca(OH)2 pretreatment was used to enhance the enzyme hydrolysis and biogas production of extruded rice straw. After Ca(OH)2 pretreatment, the glucose and xylose conversion rates in enzymatic hydrolysis increased from 36.0% and 22.4% to 66.8% and 50.2%, respectively. The highest biogas production observed in 8% and 10% Ca(OH)2 pretreated rice straw reached 564.7 mL/g VS and 574.5 mL/g VS, respectively, which are 34.3% and 36.7% higher than the non-Ca(OH)2-loaded sample. The Ca(OH)2 pretreatment can effectively remove the lignin and increase the fermentable sugar content. The structural changes in the extruded rice straw have also been analyzed by XRD, FTIR, and SEM. Considering all of the results, an 8% Ca(OH)2 loading rate is the best option for the pretreatment of extruded rice straw. Ó 2015 Published by Elsevier Ltd.
1. Introduction Lignocellulosic biomass, including agricultural residues, energy crops, forestry wastes and a part of municipal wastes, is a fermentable material for renewable energy production. Because it is widely available and contains large amounts of cellulose and hemicellulose, more and more researchers are focusing on lignocellulose utilization. Anaerobic digestion is a feasible way to ⇑ Corresponding author. E-mail address: [email protected] (X. Zhou). http://dx.doi.org/10.1016/j.biortech.2015.07.004 0960-8524/Ó 2015 Published by Elsevier Ltd.
transform cellulose and hemicellulose, which are the main components of lignocellulose and are composed of potentially fermentable sugars, to biogas. However, the biogas conversion efficiency of lignocellulosic biomass is limited by the biomass recalcitrance, which refers to the natural resistance of plants to enzymatic hydrolysis and microbial deconstruction (Himmel et al., 2007). The biomass recalcitrance is caused by the spatial structure and chemical composition of lignocellulose, including its crystalline cellulose content, degree of polymerization, accessibility and lignin protection, and these properties cause its low hydrolysis efficiency (Zhang and Lynd, 2004). The lignocellulose
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hydrolysis process is therefore considered to be the speed-limiting step. To enhance the lignocellulose hydrolysis process and achieve profitable biogas production, pretreatment is required to change the chemical and physical structure of lignocellulose and overcome the biomass recalcitrance. Many pretreatment methods can be adopted to reduce the biomass recalcitrance of lignocellulose, enhance enzyme accessibility and increase the fermentation efficiency. It is worth noting that mechanical crushing not only is important but is an essential pretreatment in lignocellulosic material anaerobic digestion. Mechanical pretreatment can efficiently reduce the material particle size and increase the accessible surface. The main effect of the increased surface area is to enhance the cellulose accessibility (Kratky and Jirout, 2011). Many types of machines have been applied in lignocellulosic material treatment. Extrusion is one effective mechanical pretreatment method. Compared with traditional physical pretreatment methods such as chipping, grinding, and milling, extrusion can not only reduce particle size but also provides a continuous thermo-mechanical treatment that will cause a further expansion of rice straw fibril structure (Chen et al., 2014b; Kratky and Jirout, 2011). Therefore, extruded lignocellulosic materials achieve a lower bulk density, higher water-holding capacity and better biodegradability. In a previous study, it was reported that the biogas production observed in extrusion-pretreated rice straw was 32.5% and 72.2% higher than milling-pretreated rice straw and paper trimmer-pretreated rice straw, respectively (Chen et al., 2014b). In addition, an extruder is suitable for large-scale production and has many other advantages such as a high shear force and efficient mixing (Kratky and Jirout, 2011). Although extrusion can effectively enhance the anaerobic digestion efficiency, the effect is still limited because it only changes the structural characterization. The chemical characterization such as lignin content is also an important factor of the biomass recalcitrance. Alkaline pretreatment is an efficient pretreatment to change the chemical characterization of lignocellulosic materials, especially through lignin removal (Krishania et al., 2013). Approximately 10–40% of the lignin can be removed when 2–10% NaOH was used to treat lignocellulosic materials, and there is no significant cellulose degradation during the pretreatment (Chen et al., 2014a; Zhu et al., 2010). During the alkali pretreatment, solvation and saponification reactions happen rapidly (Park and Kim, 2012). This will result in a swelling effect on the biomass and decompose the polysaccharides, which increases the enzyme accessibility and decrease the biomass recalcitrance (Hendriks and Zeeman, 2009; Krishania et al., 2013). The alkali pretreatment not only enhances the lignocellulosic biomass digestibility but also provides extra alkalinity, neutralizes acid production and prevents a pH drop during the anaerobic digestion process (Chen et al., 2014a; Pavlostathis and Gossett, 1985). As mentioned above, the individual extrusion pretreatment has inevitable limitations. To further enhance biogas production from extruded lignocellulosic materials, alkali pretreatment is a good application. Extrusion may also have a positive effect on the subsequent alkali pretreatment. NaOH and Ca(OH)2 are two alkalis widely used in lignocellulosic material pretreatment. Compared with NaOH, Ca(OH)2 is a much cheaper reagent, and Ca2+ has no inhibitory effect on anaerobic digestion (Chen et al., 2008). Extrusion and Ca(OH)2 pretreatment were therefore performed. The aims of this study were (1) to analyze the effect of Ca(OH)2 on enhancing the biogas production and enzymatic hydrolysis of extruded rice straw, (2) to analyze the effect of Ca(OH)2 on extruded rice straw’s physical and chemical characterizations, and (3) to determine the best Ca(OH)2 loading rate for extruded rice straw.
2. Methods 2.1. Feedstock and inoculum The rice straw used in the experiment was collected from Chongming Island. The rice straw was dried at room temperature after harvesting and then extruded with a twin-screw extruder (JXM80, Jinwor Machinery Co., Ltd., Nanjing, China), as described previously (Chen et al., 2014b). Then, the rice straw was screened. Particles that passed through a 20-mesh sieve but were retained by a 40-mesh sieve were collected and air-dried at 40 °C. The dried rice straw was then stored in a vacuum plastic bag at room temperature to prevent possible degradation. Digested dairy manure obtained from a livestock and poultry farm in Chongming was used as an inoculum in this study. The characterizations of the extruded rice straw and inoculum are presented in Table 1. 2.2. Ca(OH)2 pretreatment The concentrations of Ca(OH)2 (based on the dry matter of the rice straw) used in the experiment were 5%, 8%, 10%, 12%, and 15%. Although all of the rice straw was extruded, the rice straw that was not pretreated with Ca(OH)2 was referred to as untreated. 500 mL wide mouthed bottles (with a 400 mL working volume) were used as the reactors. Rubber stoppers were used to seal the reactors during the pretreatment process. Distilled water was added to adjust the solid/liquid ratio to 5% (w/v). The mixtures were stirred with a magnetic stirrer and incubated at ambient temperature (25 °C) for 72 h. After the pretreatment, the pretreated rice straw was filtered and washed with distilled water to neutral pH. Then, the pretreated rice straw was dried and stored in a vacuum plastic bag for the subsequent analysis and experiments. 2.3. Anaerobic digestion The anaerobic digestion of Ca(OH)2 pretreated and untreated rice straw was performed in batch reactors. 1 g rice straw (based on TS) was inoculated with 40 mL digested diary manure. The mixed feedstock was then digested in 100 mL serum bottles. The serum bottles were sealed with rubber stoppers, and aluminum caps were used to provide extra reinforcement. All of the reactors were then placed in a thermostatic chamber maintained at 35 °C and digested for 40 days. The blank group, with the same inoculum quantity and no rice straw, was used to exclude the biogas production from the inoculum. 2.4. Enzymatic hydrolysis The commercial cellulase and xylanase used in the experiments was from Sigma–Aldrich. The enzyme activities of the enzyme preparations are shown in Table 2. For these experiments, 50-mL tubes with working volumes of 40 mL were used as the hydrolysis reactors. The pretreated rice straw was hydrolyzed in sodium acetate buffer (50 mM, pH 5.0) with 2% (w/v) solids loading. 0.1 mL of
Table 1 Characteristics of rice straw and inoculum. Parameter
Rice straw
Inoculum
Total solids (%) Volatile solids (% TS) Total carbon (% TS) Total nitrogen (% TS) C/N ratio
97.3 ± 0.3 86.3 ± 0.5 35.2 ± 1.1 0.6 ± 0.0 58.6
2.8 ± 0.0 59.1 ± 0.0 20.5 ± 1.2 3.1 ± 0.1 6.7
Data is expressed as means ± SD (n P 3).
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Table 2 Activities of the commercial enzyme preparations. Enzyme preparation
CMCase (U/mL)
FPA (FPU/mL)
Xylanase (U/mL)
Cellulase Xylanase
263.7 ± 15.8
9.9 ± 3.7
79.7 ± 10.6 82.4 ± 29.6
Data is expressed as means ± SD (n P 3).
cellulase and 0.2 mL of xylanase were added into the tubes, which were incubated at 50 °C with shaking at 190 rpm for 3–72 h. At the end of the hydrolysis, the tubes were incubated in a water bath at 100 °C for 10 min to inactivate the enzymes. When the hydrolysis process was finished, the samples were centrifuged at 12,000 rpm for 15 min, and the supernatants were collected for the reducing sugar, glucose, and cellulose content determinations.
A Fourier transform infrared spectrophotometer (Thermo Fisher Scientific, Nicolet 5700) was used to examine the changes in the functional groups of Ca(OH)2-pretreated and untreated rice straw. The samples were dried and dispersed in KBr, and then approximately 1 MPa pressure was added to the mixture to form sample discs for analysis. Spectra were obtained from 32 scans using a resolution from 4000 to 400 cm 1. The spectral analysis was performed using OMNIC software. Pure KBr was used to calibrate the background. A scanning electron microscope (Phenom, Pro) was used to examine the structural changes in the Ca(OH)2-pretreated and untreated rice straw. The samples were coated with gold and then analyzed with an accelerating voltage of 10 kV. 3. Results and discussion 3.1. Compositional change of pretreated rice straw
2.5. Analytical methods The total solids (TS) and volatile solids (VS) of the extruded rice straw and inoculum were measured according to the standard methods (APHA, 2012). The total carbon and nitrogen content were analyzed using an elemental analyzer (Elementar, Vario EL III). The cellulase and xylanase activities were measured according to the NREL LAP protocol, and the reducing sugar yields were determined by the DNS method (Adney and Baker, 1996). The cellulose and hemicellulose contents in the Ca(OH)2-pretreated and untreated rice straw were analyzed according to the standard NREL LAP protocol (Sluiter et al., 2008). The biogas production was analyzed by calculating the volume and pressure in the headspace of the serum bottle. In the first 10 days, the pressure was tested every day, and from the 11th to the 40th day the pressure was tested every two days. The pressure was determined by a pressure meter (VWR, PS 100-2BAR), and the biogas production was transferred to the normal pressure and temperature. Disposable syringes were used to collect a biogas sample, and then the methane content was analyzed by a gas chromatograph (Agilent, 6890) (Gu et al., 2014). The methane production was calculated by multiplying the methane content by the biogas production. The glucose and xylose yields in the enzyme hydrolysis process were analyzed by an Agilent 1200 high performance liquid chromatography (HPLC) system equipped with a refractive index detector using a SP0810 sugar column (Shodex). The column temperature and the detector temperature were set at 80 and 55 °C, respectively. Degassed HPLC-grade water was used as the mobile phase at a flow rate of 0.6 mL/min. X-ray diffraction was performed on an X-ray diffractometer (Bruker, D8 Advance) with Cu Ka radiation at a wavelength of 0.1542 nm (50 kV, 300 mA). The rice straw was scanned at angles varying from 10° to 50° with an increment of 2°/min. The cellulose crystallinity was calculated as described by Segal et al. (1959).
The compositions of the rice straw before and after Ca(OH)2 pretreatment are presented in Table 3. A significant degradation in hot water extraction (HWE) and ethanol extraction (EE) was observed in all Ca(OH)2-pretreated samples and this led to the incensement of total lignocellulose. The structural carbohydrates contained in the different samples were analyzed to study the impact of Ca(OH)2 pretreatment. The glucose content, which refers to the remaining cellulose, was the highest and most important component in the substrate. In the 5% and 8% samples, the glucose content was in a relatively high level and reached 40.6% and 38.1%, respectively. This indicates superior biodegradability and higher fermentable sugar content in these two samples. Compared with glucose, hemicellulose, mainly composed of xylan, is more easily solubilized in Ca(OH)2. This partially contributes to the spatial structure of lignocellulose, in which cellulose is covered by hemicellulose and lignin (Chen et al., 2014a). The xylose content, which refers to the xylan content, decreased sharply when the Ca(OH)2 loading was higher than 8%. Although lower xylan content will accelerate the cellulose hydrolysis, xylose loss will also decrease the potential biogas production. Galactose, arabinose and mannose, as a part of hemicellulose, were also significantly removed in the high Ca(OH)2 loading pretreatment, but their affections to biogas production were limited. Lignin has been regarded as a cellulase adsorbent, which reduces the cellulase accessibility in lignocellulosic material hydrolysis (Zhang and Lynd, 2004). After the Ca(OH)2 pretreatment, the acid insoluble lignin (AIL) was decreased in all pretreated samples, and its removal rate increased with the Ca(OH)2 loading rate. The original lignin content was 13.7%, and the lowest content, observed in the 15% Ca(OH)2 treated sample, was 10.6%. Considering the compositional change caused by the decreases in HWE and EE, the AIL removal efficiency increased slightly in the high Ca(OH)2 loading rate. Although less lignin was contained in the high Ca(OH)2 loading samples, the potentially fermentable sugar content (mainly glucose and xylose)
Table 3 Chemical composition of different Ca(OH)2 pretreated extruded rice straw. Ca(OH)2 addition
Untreated 5% 8% 10% 12% 15%
HWE (%)
6.7 ± 0.6 0.7 ± 0.1 0.3 ± 0.0 0.2 ± 0.0
Data is expressed as means ± SD (n P 3).
EE (%)
5.3 ± 0.8 3.1 ± 0.3 0.8 ± 0.1 0.3 ± 0.0 0.1 ± 0.0
Structural carbohydrates content (%)
AIL (%)
Glu
Xyl
Gal
Ara
Man
36.8 ± 0.4 40.6 ± 1.2 38.1 ± 0.6 34.6 ± 1.1 34.1 ± 0.9 31.0 ± 0.4
17.8 ± 0.2 18.0 ± 0.6 13.0 ± 0.3 10.8 ± 0.2 10.5 ± 0.0 9.3 ± 0.3
0.8 ± 0.1 0.7 ± 0.0 0.3 ± 0.0
6.2 ± 0.1 6.5 ± 0.2 6.9 ± 0.1 4.4 ± 0.0 3.6 ± 0.4 4.0 ± 0.1
0.3 ± 0.0
13.7 ± 0.1 12.8 ± 0.1 11.7 ± 0.2 11.3 ± 0.0 10.9 ± 0.0 10.6 ± 0.2
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also decreased with the Ca(OH)2 loading rate increase. Considering the combined effects of these two factors, an 8% Ca(OH)2 loading would be the best choice. 3.2. Biogas production Inoculated rice straw was digested at 35°C for 40 days to investigate the effect of Ca(OH)2 pretreatment on biogas production. The daily biogas production, cumulative biogas production and methane production are depicted in Fig. 1. Compared with untreated rice straw, the biogas production of the Ca(OH)2 pretreated rice straw was much more severe. It is obvious that the Ca(OH)2-pretreated rice straw had a higher daily biogas production, and peaks appeared much earlier. There is therefore no doubt that the Ca(OH)2-pretreated rice straw is more suitable for biogas production. Similar trends of biogas production were found in different Ca(OH)2-pretreated samples. The biogas was generated immediately after the rice straw was seeded, and it continued to increase until reaching the first peak. Before the fermentation ceased, there were several peaks during the process. The peak value is the main difference between the different pretreated substrates. The daily biogas production of the 5–15% Ca(OH)2 pretreated rice straws reached their first peaks between day 4 and day 6 with 135.8, 156.4, 159.9, 188.6 and 174.6 mL/g VS biogas productions, respectively. The second peaks appeared between day 12 and day 20, with 136.4, 178.7, 160.7, 152.0 and 141 mL/g VS biogas productions, respectively. It can be concluded that the higher Ca(OH)2 loading pretreated rice straw produced more biogas in the initial period but had a lower subsequent potential. Fig. 1(b) shows the cumulative biogas production, which was calculated based on the daily biogas production. The total methane production is shown in Fig. 1(c). The total biogas and methane in the Ca(OH)2-pretreated rice straws were significantly higher than those in the untreated straw. There was no significant difference between the 8%, 10%, and 12% Ca(OH)2 pretreated rice straws in the final results, with their total methane productions reaching 330.9, 337.8, and 322.6 mL/g VS, respectively. The biogas productions in the 5% and 15% Ca(OH)2 pretreated samples were lower. Although the 5% Ca(OH)2 pretreated rice straw had the highest cellulose content, its methane production was still 10% lower than that of the 8% Ca(OH)2 pretreated straw. This might be caused by its high lignin content, which was the main reason for the biomass recalcitrance. When the Ca(OH)2 loading rate was 15%, the total biogas production decreased, and the methane production was less than 300 mL/g VS. This phenomenon might be caused by the over dissolution of cellulose and hemicellulose in the high Ca(OH)2 process. These results indicate that 8% is the best Ca(OH)2 loading rate for extruded rice straw. The Ca(OH)2 was insufficient in lower loading rates, and overloading was also inappropriate. Compared with the combined mill-alkali pretreatment process (Cheng et al., 2010; He et al., 2008), the extruded rice straw in this study achieved higher biogas productions in both the Ca(OH)2-pretreated and untreated samples. The optimal alkali addition was also lower than that of milled rice straw (Song et al., 2013). This may be attributed to its larger specific surface area and looser spatial structure (Chen et al., 2014b; Chen et al., 2008).
Fig. 1. Biogas production for Ca(OH)2 pretreated rice straw: (a) daily biogas production, (b) cumulative biogas production, (c) methane production.
3.3. Enzymatic hydrolysis The enzymatic digestibility of untreated and Ca(OH)2-pretreated rice straws was tested by cellulase and xylanase hydrolysis for 72 h. Fig. 2(a) and (b) plots the glucose and xylose conversion rates in enzymatic hydrolysis as functions of the incubation time for different samples. The glucose and xylose conversion rates were defined as the percentages of glucose and xylose in the pretreated rice straw that were solubilized during
the hydrolysis process. The glucose and xylose produced during the pretreatment were not included. The untreated rice straw was resistant to enzymatic hydrolysis, so the conversion rates of untreated rice straw were low for both cellulase and xylanase hydrolysis. The glucose and xylose conversion rates increased dramatically in the Ca(OH)2-pretreated samples. All of the curves had the same trend, with most of the
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experiments. Although the alkali pretreatment caused the lignocellulose to dissolve, its effect on the fiber size was limited. Samples with lower lignin contents achieved higher reducing sugar production. Delignification was considered as a more important reason for the enhanced glucose and xylose conversion rates. The hydrolysis is the rate-limiting step in the anaerobic digestion. Cellulose and hemicellulose (mainly xylan) are converted to glucose and xylose by enzymes and their conversion efficiencies greatly affects the biogas production. The enhanced conversion efficiency usually led to more efficient biogas production, but the 15% Ca(OH)2 pretreated rice straw that achieved the highest conversion rates produced less biogas than the 8% Ca(OH)2 pretreated sample. This was due to the different structural carbohydrates contained in the different samples. Because there was more glucose and xylose, the 8% Ca(OH)2 pretreated rice straw could release more fermentable sugars for biogas production than the 15% Ca(OH)2 pretreated rice straw. Other studies also found that a high alkali loading rate would decrease the total sugar release in enzyme hydrolysis (Saha and Cotta, 2007; Sills and Gossett, 2011). When using both the structural carbohydrate content and conversion rate to estimate the effect of Ca(OH)2 pretreatment on the extruded rice straw, 8% Ca(OH)2 should be the best option. 3.4. Structural change analysis
Fig. 2. The effect of different Ca(OH)2 pretreatment on glucose and xylose conversion rate during 72 h hydrolysis.
glucan converted in 24 h. For instance, the glucose yields after 72 h hydrolysis, from the untreated rice straw to the 15% Ca(OH)2 treated rice straw, were 24.7%, 35.7%, 45.2%, 50.2%, 53.7% and 58.4%, which represent 68.6%, 76.4%, 75.8%, 83.1%, 85.2% and 87.4% of the total converted glucan, respectively. This indicates that higher Ca(OH)2 pretreated rice straw had a higher digestibility and also digested more quickly. The same phenomenon could also be observed in xylan hydrolysis. The xylan conversion rate represents the hemicellulose digestibility because xylan is the main component of the hemicellulose in rice straw (especially in the Ca(OH)2-pretreated samples). Although hemicellulose can be transformed to a fermentable sugar and used to produce biogas, it exists as a type of physical barrier for cellulose (Hu et al., 2011). Therefore, the better hemicellulose digestibility would not only affect the xylose conversion but also had a positive effect on the glucan conversion. The results also prove that a higher glucose conversion always accompanies a high xylose conversion. The high hydrolysis rate observed in Ca(OH)2-pretreated rice straws indicated better enzyme accessibility and lower biomass recalcitrance. The enzyme accessibility was affected by many facts, such as fiber size and lignin content. As previously described, the extruded rice straw was sieved to a uniform size before the
3.4.1. Changes in cellulose crystallinity The cellulose structure and cellulose crystallinity was measured by X-ray diffraction. The X-ray diffraction pattern did not change between the different Ca(OH)2 pretreated samples, indicating that the cellulose style was not obviously changed during the Ca(OH)2 pretreatment. This might indicate that the Ca(OH)2 could only reach the surface of the cellulose, and the same phenomenon was also observed in the NaOH-pretreated rice straw (He et al., 2008). According to previous study, the peak observed at 22.0° represents the presence of crystalline cellulose and the peak at 18.0 represents amorphous cellulose (Segal et al., 1959). The crystallinity, which is defined as the ratio of the crystalline regions to the total cellulose (including crystalline and amorphous cellulose), have been calculated. The crystallinity in the untreated rice straw was 33.2%, and it increased to 36.2%, 38.8%, 40.4%, 39.4% and 34.9% in the 5–15% Ca(OH)2 pretreated samples, respectively. The slightly increase in crystallinity after pretreatment had also been reported (Liang et al., 2014). The possible reasons for the increased crystallinity are (1) the crystalline cellulose being more resistant to the alkali solution than the amorphous cellulose (Himmel et al., 2007); (2) the decreased contents of lignin and hemicellulose, both of which are amorphous (Zhao et al., 2012). Wu et al. reported that the crystalline cellulose increased after the lignin removal (Wu et al., 2014). Although the crystallinity increased slightly after the alkali pretreatment, the final value was still much lower than that of the combined milled-alkali pretreated samples reported elsewhere (He et al., 2008; Liang et al., 2014; Yang et al., 2009). The crystallinity affects the cellulose digestion speed but not the limitation. The low crystallinity in the extruded rice straw promises an efficient digestion, and the increased crystallinity after the Ca(OH)2 pretreatment had no obvious negative effect on the biogas production and enzymatic hydrolysis. 3.4.2. Changes in functional groups The changes in functional groups of Ca(OH)2 pretreated rice straw were analyzed by Fourier transform infrared (FTIR) spectroscopy. The untreated rice straw was compared with the 8% Ca(OH)2 pretreated rice straw, which was believed to have
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achieved the best performance. The spectra were analyzed according to previous studies (Liang et al., 2014; Popescu et al., 2010; Yang et al., 2009). Although the results showed same trend and had almost the same peaks, it is obvious that all of the peaks in the Ca(OH)2-pretreated rice straw shifted relative to the untreated straw. This implies that the inter- and intramolecular bonds had changed during the Ca(OH)2 pretreatment process. Compared with untreated rice straw, the O–H stretching absorbance and C–H stretching absorbance in Ca(OH)2-pretreated rice straw decreased significantly, indicating an influence of pretreatment on the hydroxyl-stretching region (Popescu et al., 2010). The lower absorbance of the hydroxyl-stretching region observed in Ca(OH)2-pretreated rice straw indicates a removal of hydrogen bonds during the pretreatment process. In the fingerprint region, there were remarkable differences in the observed O–H and conjugated C–O, C–C of aromatic ring, and C–O stretching, which reflect modifications of lignin in the pretreated rice straw. The C–O stretching in different samples did not change much, indicating the low effect of Ca(OH)2 on the crystalline structure, which agrees with the XRD results. These changes in the functional groups demonstrate the lignin removal and cellulose depolymerization after the Ca(OH)2 pretreatment. The results indicate a better biological and enzymatic digestibility of 8% Ca(OH)2 pretreated rice straw, which explains the increased biogas production efficiency and enzyme hydrolysis in the pretreated samples. 3.4.3. Physical surface structural changes The physical surface structural changes also had been analyzed in this study. The 8% Ca(OH)2 pretreated rice straw was chosen for comparison with the untreated rice straw for its superior digestibility and higher biogas production. Compared with other physically pretreated lignocellulosic materials (Hu and Wen, 2008; Zeng et al., 2007), the extruded rice straw seemed to have a gullied surface rather than a smooth surface. The water holding capacity also increased according to previous study (Chen et al., 2014b). These changes create a larger accessible surface for the alkali to react. Although the extrusion could partially tear the rice straw surface and expose the cellulose fiber in part (Chen et al., 2014b), the surface was still covered with the wax layer and lignin. After the Ca(OH)2 pretreatment, the rice straw was further damaged, and the compact surface was destroyed. The wax layer was also destroyed and removed during the pretreatment process. Moreover, inner voids, which are important for the enzymes and microbes to approach the internal cellulose fibrils, were exposed after the pretreatment, as the outer structure had been broken. It is obvious that more cellulose fibrils were exposed after the alkali pretreatment, which increased the cellulose accessibility. The changes observed in the surface structure could well explain the enhanced biogas production and enzymatic hydrolysis in Ca(OH)2-pretreated rice straw. Compared with other physical pretreatments such as the mill pretreatment, extrusion seemed to cause a lower consumption in the following alkali pretreatment. This might suggest a synergistic effect of extrusion in enhancing the effect of the alkali pretreatment.
4. Conclusions The results of this study show that using a Ca(OH)2 pretreatment could significantly enhance the extruded rice straw biogas production rate and enzymatic hydrolysis efficiency. Lignin and fermentable sugar contents changed during the Ca(OH)2 pretreatment process. The physical and chemical structures of the extruded rice straw also have changed after the pretreatment, which lead to better biodegradability. Positive effects were observed in all Ca(OH)2 loading rates, but overloading Ca(OH)2
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would cause a greater fermentable carbohydrate loss, which decreased the total biogas production. The optimal Ca(OH)2 loading rate was 8%, which achieved both efficient hydrolysis and high biogas production. Acknowledgement This research was funded by the National key technologies R&D program of China (No. 2012BAJ25B04).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2015.07. 004.
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