The rice straw used as the substrate for anaerobic digestion was acquired from Rice Research Institute

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The 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%. Then the straw was cut into 3 mm sections by a scissor. The inoculum used in this case study was obtained from 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.

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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. In order to determine 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 then 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 bottle, 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) 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, 60×1,8? OD, 80/100, Mesh) and a thermal conductivity detector (Perkin Elmer) with an injection temperature of 150 ºC. The carrier gas used 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 K? radiation). Radial scans of intensity were recorded at ambient condition over, scattering 2? angles from 5? to 40? (step size = 0.02?, scanning rate = 2 s/step) using a Ni-filtered Cu K? radiation (?=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 2? 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 2? between 18° and 19° representing amorphous part of lignocelluloses.


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