Biogas is an important renewable fuel that is used as a source of energy for heating, electricity generation and transportation fuel in recent years. Biogas is produced from a wide variety of organic waste, agricultural residuals, and wastewater treatment sludge and energy crops (Kabir et al., 2014; Teghammar et al., 2012). Rice straw is one of the most abundant agricultural wastes around the world. The estimation of annual worldwide rice production in 2017 was about 758.8 million tons, where 1−1.5 kg straw is produced per kg of rice (FAO, 2017). The dominant treatment of rice straw by farmers is to burn it in order to clear the farm for the next crop. Open burning of the straw poses threats to global environmental because of e.g. releasing noxious greenhouse gases and short-lived pollutants (SLPs) into the air (Kaur et al., 2017). Furthermore, rice straw is not suitable as animal feed, due to low digestibility (Rodriguez et al., 2010). It is mainly composed of cellulose, hemicellulose, lignin, silica, and ash (Renu Singh, 2014). In general, in lignocelluloses, the cellulose chains are bound together by hydrogen bonds in a mainly crystalline form in microfibrils. The microfibrils are covered by a gel matrix composed of hemicellulose, lignin, and other carbohydrate polymers, where lignin limits the accessibility of the carbohydrates to the hydrolytic bacteria (Chen et al., 2011).
An extra factor in rice straw is the presence of silica. Most of the silica in rice straw appears to be in the plant cell wall. Silica is probably the first limiting factor in rice straw anaerobic digestion followed by lignin. Rice straw, containing a high amount of silica, the lignin condenses on the silica particles resulting in a higher apparent lignin content (Van Soest, 2006). Lignin does not exist as an independent polymer in the plant cell wall and it is always seen associated with polymers cellulose and hemicelluloses (Abraham et al., 2016). According to these properties, lignin is the main recalcitrant component of the plant cell wall and the high lignin content in rice straw results the greater the resistance to the degradation (Zheng et al., 2014).
Cellulose and hemicellulose of rice straw are not easily accessible by the microorganisms due to the recalcitrant nature of this substrate (Dehghani et al., 2015). Therefore, a pretreatment process is required prior to anaerobic digestion for the disruption of lignin layer and silica for effective digestion of the substrate (Taherzadeh & Karimi, 2008). There are several pretreatment techniques such as physical, chemical, and biological, a key step for removal of lignin, silica and hemicellulose from rice straw is selection of a cheap, efficient and environmentally friendly pretreatment techniques prior to anaerobic digestion (Kabir et al., 2014). Steam explosion is one of the useful pretreatment methods to open up the lignocellulose complex, it involves heating at high temperature combined with a rapid pressure drop that physically disrupts the lignocellulosic structures in the biomass fibers and could effectively cause lignin to be separated from polysaccharides (Kitani et al., 1989; Zhou et al., 2016).
Several studies have reported steam explosion pretreatment of lignocelluloses as an effective method for improving enzymatic hydrolysis and bioethanol production. These pretreatment techniques have also been used to treat several of lignocellulosic biomass for enhancement of biogas production (Zheng et al., 2014). The steam explosion was proved to be effective for increasing methane yield from wheat straw by 20-30%, compared to untreated wheat straw (Bauer et al., 2009, Wang et al., 2010). Optimized steam explosion treatment of bulrush and achieved a maximum methane yield of 205 mL/g VS (24% higher than that of untreated bulrush) at 1.72 MPa steam pressure, 8.14 min residence time, and 11% moisture content. Steam explosion with 2% NaOH and 2% H2O2 was used for pretreatment of paper tube residuals that was observed positive effects on methane production. The best result was obtained at 220 °C, 10 min with methane production 493 N ml/g VS which was 107% more than the unpretreated sample (Teghammar et al., 2010). Zhou et al. (2016) reported that steam explosion pretreatment leads to the distribution of special kind, clear differences in the population and the earlier presence of the bacteria during anaerobic digestion. Pretreatment on the condition of 200 °C/120 s, reached the highest biogas production 328.7 mL g-1 TS (Total Solid) which led to a 51% increase of the methane production than to unpretreated sample. Most of the studies have examined the use of steam explosion in the low retention time with high pressure. However, there is little information on steam explosion as a pretreatment method for modification of rice straw for biogas production.
The main aim of this study was to modify the structure of rice straw using steam explosion pretreatment techniques. This aims at enhancing the digestibility of rice straw in order to improve the methane yield.
Rice straw used as the substrate for anaerobic digestion was acquired from Rice Research Institute (Amol, Iran). Rice straw as the blank sample was dried in an oven with temperature 105 °C for 24 hours to reduce the moisture content to less than 5%. The straw was cut into 3 mm sections by a scissor. The inoculum used in this case study was obtained from the Municipal Wastewater treatment plant (south of Tehran, Iran). The inoculum was centrifuged in order to increase the total solid from 9 to 19.83 and thereafter activated at 37 °C for 4 days prior to use.
2.2 Steam explosion Pretreatment
The steam explosion reactor was constructed with operating pressure up to 40 bar and temperature of 280 °C. It consisted of a steam generator, pressure vessel and vacuum tank. A steam generator was used with the capacity of 20 L for pressure and heat production. The pressure vessel had capacity of 3 L and in each cycle housed up to 100 g of rice straw. Using a solenoid valve control key on the control panel steam was directed to the pressure vessel until desirable pressure was reached. Parameters such as Pressure 5-15 bar, moisture 0-70% and time 1-15 min were studied during pretreatment. After high pressure pretreatment by opening the angle valve, the pressure was released through vacuum tank (200 L) creating a sudden pressure drop. During the process, the steam is penetrated into the pores of the rice straw. After sudden pressure drop and creation of pressure difference between that of inside the fibers and surrounding environment the rice straw was destructed.
2.3 Anaerobic Digestions
Anaerobic digestions were carried out using mesophilic bacteria (inoculum) at 37.5 °C in batch digesters. The digesters were glass bottles with 118 mL total volume, closed with butyl rubber seals and aluminium caps (Hansen et al., 2004). Pretreated rice straw used as the substrate for the biogas production. The volatile solids percentage (%VS average) was 60.9 % and 10.37 % for rice straw and inoculum respectively. Each reactor contained 1.144 gVS inoculum and 0.609 gVS of pretreated rice straw, and by certain deionized water added, the total volume of the reactor reached to 25 ml. For determining the methane production from the inoculum, the blank sample containing only inoculum and deionized water was and mixture of pretreated rice straw and inoculum was as the control sample. In order to obtain anaerobic conditions, the headspace of the reactor was flushed with N2 for 2 min. All experimental setups were performed in triplicates, and the reactors were incubated at 37.5 ºC for 60 days. During this experimental period, the reactors were shaken once per day. Gas samples were withdrawn regularly from the headspace of each reactor, and the accumulated methane production was determined using gas chromatography.
2.4 Analytical methods:
2.4.1 Measuring the chemical composition of rice straw
The identified components are extractive cellulose, hemicellulose, lignin and ash of the unpretreated and pretreated rice straw was determined according to Laboratory Analytical Procedures from the National Renewable Energy Laboratory (NREL, USA) and their values were compared to another. The total solids (TS) and volatile solids (VS) and ash contents of the samples were determined by drying at 105 °C for 3 h and subsequent heating at 550 °C for 2 h, according to the standard method presented (Sluiter et al., 2008b, Sluiter et al., 2008a). The amount of biogas produced in each reactor was measured on daily basis using a syringe. The methane produced during the anaerobic digestion was measured using a gas chromatograph (Auto System Perkin Elmer, Waltham, MA), equipped with a packed column (Perkin Elmer, 60×1,8ʺ OD, 80/100, Mesh) and a thermal conductivity detector (Perkin Elmer) with an injection temperature of 150 ºC. The carrier gas was nitrogen, with a flow rate of 23 ml/min at 60 ºC. A 250 µl pressure-tight gas syringe (VICI, Precision Sampling Inc., LA) was used for the gas sampling. Data analysis was performed as described by (Teghammar et al., 2010).
2.4.2 Analysis of structural changes
The functional group changes occurred in unpretreated and pretreated rice straw was examined using Fourier transform infrared spectroscopy (Impact 410, Nicolet Instrument Corp., Madison, WI), and the analyzing software was Nicolet OMNIC 4.1. The FTIR spectra were recorded in the absorption band mode in the range of 600–4000 cm-1 with a resolution of 4 cm-1 and 32 scans.
2.4.3 Crystal structure measurements
The overall crystallinity of phases of samples was determined by XRD (PW 1710, copper K𝛼 radiation). Radial scans of intensity were recorded at ambient condition over, scattering 2θ angles from 5◦ to 40◦ (step size = 0.02◦, scanning rate = 2 s/step) using a Ni-filtered Cu Kα radiation (λ=1.54A˚), an operating voltage of 45 kV, and a filament current of 40 mA. Crystallinity index (CrI) of each sample was calculated by following empirical equation: (27)
CrI (%) = [(I002-Iam)/I002] × 100 (1)
where I002 is the intensity of diffraction at 2θ between 22° and 23° for cellulose I, and IAm is the intensity, above the baseline, between the 020 and 110/110 peaks of diffraction at 2θ between 18° and 19° representing amorphous part of lignocelluloses.
2.5 Statistical analysis
The values shown were the means of triplicates ± the standard deviation. The data were Statistical analyzed, for 95% confidence intervals using the Minitab software package version 17. The response surface model was expressed by a general linear model ANOVA to quantify the parameter impact on the anaerobic digestion (methane yield) after the steam explosion pretreatment. The effect of pressure and moisture variables at three levels and the effect of time variable at four levels have examined a total of experimental runs.
Rice is cultivated in the large area of different parts of the world especially in Asia, and its straw is mainly burned by farmers. Rice straw contains cellulosic and hemicellulosic, which are suitable as a source for renewable fuel production. The cellulose and hemicellulose of the rice straw are not easily accessible to biodegradation, that it is related to the compact structure, presence of high lignin and silica. In the present study, steam explosion pretreatment in different cycles (pressure, time and moisture) was used to modify the structure of rice straw. Finally, the effects of the pretreatment on the composition and structure of rice straw were evaluated by performing anaerobic batch digestion and comparing the accumulated methane yields.
3.1 steam explosion pretreatment conditions
The effects of main parameters pressure, time and moisture of the steam explosion pretreatment were examined on rice straw structure modification. Rice straw was pretreated in a steam explosion reactor at pressures 5-15 bar. The straw was kept at each pressure inside pressure vessel for 1-15 min. Rice straw was investigated in three levels of primary moisture (0-70%), resulting in a total of 36 pressure/time/moisture combinations. Typically pretreatment pressure and time are within the ranges of 5-40 bar, 160-280 °C, and several seconds to a few minutes, respectively. Most studies have examined a limited range of steam explosion parameters, also primary moisture parameter of feedstock has been poorly considered in steam explosion pretreatment. Kobayashi et al. (2004) studied Methane production from steam-exploded bamboo under various steaming times of 1, 3, 5, 10, and 20 min, at a steam pressure of 35 bar (243 °C). Teghammar et al. (2010) has examined pretreatment of paper residuals for improved biogas. Steam explosion pretreatment conditions were varied 15 and 20 bar (190–220 °C) within 10 or 30 min. Vivekanand et al. (2012) reported biogas production from the brown seaweed Saccharina latissima. Thawed SW was steam exploded at 130 and 160 °C for 10 min. Similarly, milled WS were subjected to the steam explosion at 210 °C for 10 min.
3.2 Effects of Pretreatments on the Chemical Composition of Rice Straw
The untreated rice straw consisted of cellulose (45.15%), hemicellulose (22.87%), lignin (18.61%), and ash (10.33%). Results of chemical composition changes of pretreated rice straw are presented in Table 1. According to results with increasing pressure and retention time, the percentage of cellulose content increased in pretreated rice straw compared with that of the unpretreated. However, other components such as lignin, hemicellulose, and ash have been reduced significantly. The analysis demonstrated that in the cycle 10 bar and 10 min with blank moisture, followed by 15 bar and 1 min, 15 bar and 5 min, resulted in more lignin and ash removal. The content of hemicellulose was generally reduced in the pretreated rice straw. Pretreatment in the cycle 5 bar and low retention time approximately led to the same amount of the lignin, ash, and carbohydrate as the unpretreated sample (Table 1). These results indicate that the pretreatment technique is capable of removing lignin and hemicellulose under high pressure condition.
Pressure is the most important parameter in steam explosion pretreatment compared to the other parameters such as reaction time and moisture ratio. During the steam explosion pretreatment, the hemicellulose is first degraded followed by the lignin when the temperature >150 °C (Ma et al., 2014; Xiao et al., 2014). In addition to the removal of the hemicellulosic, the steam explosion induces important modifications in the structure of lignins. Lignin degrades in the temperature range of 120-200 °C and it divides into smaller particles and is separated from celluloses (Fan et al., 2016). It damaged the cell wall of rice straw by disrupting the lignin structure.
The analysis show that pretreated rice straw contained 91.8% total solids and 60.9% volatile solids on a wet weight basis that is increased by different cycles of pretreatment. Table 2 shows the percentage of VS and TS of the pretreated rice straws. These results also show that increase pressure of the pretreatment led to the increase of VS content. This is an indication of higher carbohydrate contents in the pretreated rice straw (Dehghani et al., 2015).
3.3 Effects of Pretreatment on Rice Straw Structure
Results of changes in the structure and the functional groups of rice straw are shown in Table 3. Bands have been found in all of the samples in the range of 450-3,600 cm-1. The band between 3,600 and 3,100 cm-1 caused by the presence of alcoholic and phenolic hydroxyl groups involved in hydrogen bonds that is related to OH stretching vibrations present in the cellulose, hemicellulose, and lignin (Rahnama et al., 2013). The intensity of band 3415 cm-1 was decreased, as a result after the steam explosion pretreatment in the high-pressure cycles 10 bar for 5-10 min and 15 bar for 1-5 min with blank moisture, 15 bar for 10-15 min with moisture 35% and 10 bar, 10 min and 15 bar for 5-10 min with moisture 70%. Therefore it indicates that the partial hydrogen bond in cellulose was destroyed. This is a key step toward increasing the accessibility of enzymes and microorganisms to cellulose (He et al., 2008). The intensity of peaks at 2920 and 2861 cm-1 was related to C-H stretching vibrations. The intensity of both peaks indicates the distinguished features of cellulose (methyl and methylene) and hemicellulose. The intensity of bands was reduced after the pretreatment pressure increased (Kazeem et al., 2017). Structural changes in lignin and loss of aromatic units were shown by changes of intensity in the 1,649 cm-1 band, and the intensity of bands at 1516 cm-1 is related to C=C stretching of the aromatic ring of lignin (Isroi et al., 2012). The intensity of both peaks decreased with increase in pretreatment pressure in the cycle 10 bar for 5-10 min and 15 bar for 1-5 min with blank moisture, 10 bar and 10 min and 15 bar for 10-15 min with moisture 35% and 10 bar for 5-10 min and 15 bar for 5-10 min with moisture 70%. This was an indication of lignin structure changes.
The intensity of band obtained at 1064 cm-1 is usually attributed to the structural specifications of cellulose and hemicelluloses (Rahnama et al., 2013). It was clear that the absorption band at 1064 cm-1 was lower compared to the unpretreated rice straw. The drop observed in this band is related to decreased hemicellulose content after steam explosion pretreatment in the cycles 10 bar for 5-10 min and 15 bar for 1-5 min with blank moisture, 10 bar, 10-15 min and 15 bar for 10-15 min with moisture 35% and 10 bar for 10-15 min and 15 bar for 1-5-10 min with moisture 70%. The increase in the intensity of this band might be dependent to the dissolution of non-cellulose components that causes the increase of cellulose content in the rice straw (Ang et al., 2012). The absorption band 1430 cm-1 is related to amorphous cellulose (Kazeem et al., 2017). The absorption band by 1430 cm-1 has reduced after the steam explosion pretreatment and it means the degraded of the amorphous part structure, especially the lignin structure changes has occurred. Lignin structure was degraded in the cycles 10 bar for 5-10 min and 15 bar for 1-5 min with blank moisture and 15 bar for 10-15 min with moisture 35% and 15 bar for 5-10 min with moisture 70%.
Crystallinity index of the lignocelluloses is determined by two parts amorphous and crystalline. The amorphous part consists of hemicelluloses and lignin, while the crystalline constituent part includes cellulose (Sakdaronnarong & Jonglertjunya, 2012). The crystallinity index [CrI (%)] unpretreated and pretreated rice straws are shown in Table 4. Two peaks were observed at 2θ of between 18°-19° and between 22°-23°, that relating amorphous and crystalline regions of the rice straw. According to results, the crystallinity index increased in the pretreated rice straw than to the unpretreated rice straw. The highest crystallinities of pretreated rice straw were 28.8%, 28.58% and 29.3% in the cycles 10 bar and 10 min with blank moisture, 15 bar and 15 min with moisture 35% and 15 bar and 10 min with moisture 70%, respectively, than to the unpretreated rice straw 22.9%. In all pretreated samples, the region of crystalline cellulose was higher than to the unpretreated rice straw (Table 4). During the steam explosion pretreatment, Hemicellulose and lignin are hydrolyzed with the increase of pressure, which mainly constitutes amorphous regions of rice straw. This led to the Increase of crystallinity index after pretreatment.
Previous research has also suggested that the crystallinity index of rice straw could enhance by other thermal pretreatments, and this increase not have negative effect on fermentation (Kshirsagar et al., 2015). Increase in crystallinity index due to the effect of pretreatment was more in the amorphous region than to the crystalline region. Rahnama et al. (2013) reported an enhancement in the crystallinity index from 50.81% to 61.41% in the alkali pretreated rice straw. Crystallinity index increased in the pretreated rice straw with microwave and acid to 61.36% compared with pretreated rice straw 52.2% (Renu Singh, 2014).
3.3 Effect of Steam Explosion Pretreatment on Biogas Production
The cumulative biogas production from the pretreated rice straw after 60 days of incubation is presented in Table 5. Retention time, pressure and moisture level were considered as pretreatment variable parameters, and the effects of the pretreatments were evaluated using the accumulated methane production as the response variable. The initial production rates were measured as the average of the produced methane per day during the first ten days of anaerobic digestion process, and are shown in Table 5.
The yield of methane produced by inoculum was deducted from the total of produced methane from each sample. The highest initial production rate was after pretreatment of rice straw in the cycle 10 bar-10 min with an increase of 59% from 92 to 146 N ml/g VS and followed by 144 N ml/g VS, compared with that of the unpretreated (Fig 1c). The unpretreated rice straw resulted in almost the same initial production rate in methane production as compared to some the pretreated rice straw in the blank moisture with low pressure and time (Fig. 1a). The highest initial production rate was obtained in pretreated rice straw in the cycle 15 bar-10 min with moisture 35%, with an increase of 166% from 92 to 245 N ml/g VS followed by 244 N ml/g VS (Fig. 2c). The maximum initial production rate was after the pretreatment of rice straw in the cycle 10 bar-15 min with moisture 70% with an increase of 141% from 92 to 222 N ml/g VS followed by 221 N ml/g VS (Fig. 3b).
The highest methane production was obtained for pretreated rice straw in the cycles 10 bar-10 min and 15 bar-5 min with blank moisture 418 and 370 N ml/g VS compared with that of the unpretreated rice straw 196 N ml/g VS (Table 5) and this means increased by 114% of the methane yield. The pretreated rice straw in the cycles 15 bar for 15-10 min with moisture 35% was obtained the highest methane production 399 and 387 N ml/g VS, respectively. Methane yield increased by 196 to 399 N ml/g VS, and this means increased by 104% compared to the unpretreated rice straw. The highest methane production was reached for the pretreated rice straw after pretreatment in the cycles 15 bar for 10-5 min with moisture 70% to 496 and 450 N ml/g VS, respectively (Fig. 3c). The methane yield of rice straw increased by 196 to 496 N ml/g VS, and this means increased by 154% compared to the unpretreated sample.
According to the improvement of methane production yield, structural modification of rice straw was performed by steam explosion pretreatment, and methane production in primary days of the anaerobic digestion process was increased for the pretreated rice straw. According to the initial production rate of pretreated rice straw in the cycles 10 bar-10 min, 15 bar-15 min with moisture 35% and 15 bar-10 min with moisture 70%, were about 35%, 57% and 37% of methane production yield occurred in 10 days of incubation, respectively.
The lowest initial production rate was observed, in the cycles 5 bar-15 min with blank moisture, leading to a methane level of 91 N ml/g VS than to the unpretreated rice straw 92 ml/g VS (Fig. 1b). The lowest methane production was in the cycle 10 bar-15 min with blank moisture, leading to a methane production of 178 N ml/g VS that the less was than to the unpretreated rice straw 196 ml/g VS (Fig. 1d). The lowest initial production rate and methane production was observed, for pretreated rice straw in the cycle 5 bar-1 min with moisture 35%, leading to a methane production of 166 and 267 N ml/g VS compared with that of the unpretreated 92 and 196 ml/g VS, respectively (Fig. 2a and 2b). Pretreated rice straw in the cycle 15 bar-10 min with moisture 70% resulted in the lowest initial production rate of 180 N ml/g VS (Fig. 3c). The lowest methane production was in cycle 5 bar-5 min with moisture 70% leading to a methane production of 272 N ml/g VS (Fig. 3a).
Parameters effects, confidence intervals of steam explosion pretreatment were calculated on the anaerobic digestion experiments. The response surface after steam explosion pretreatment was with two levels of each parameter. The parameters were used as covariates to be able to measure the size of the effects of the pretreatments. The parameters were considered significant when the probability (p-value) was less than 0.05. The ANOVA analysis of the response surface design model (Table 6) showed pressure and moisture as significant factors in increasing methane yield, while the time was not statistically significant (Fig. 4).
According to the results of fits and diagnostics for unusual observations, two pretreatments in the cycles 10 bar-10 min and 15 bar-10 min with the moisture level of the blank were the best and worst pretreatment conditions considering standard residual 2.50 and -2.91 and fit 413.6 and 447.5 respectively.
4 Conclusions
Steam explosion is suitable pretreatment method for improving biogas production efficiency from rice straw. The initial production rates were increased by 59%, 166%, and 141% compared with that to the unpretreated rice straw. The methane gas was obtained after 60 days 535, 516 and 613 N ml/g VS that each has increased 114%, 104%, and 154% than to the rice straw unpretreated, respectively. Steam explosion pretreatment was effective in remove lignin and hemicellulose and also opening up the crystalline structure of the cellulose of rice straw prior to anaerobic digestion. This demonstrates the ability of the steam explosion pretreatment to modify the structure of rice straw.