# Steam Explosion-Based Method for the Extraction of Cellulose and Lignin from Rice Straw Waste
## 2 Materials and Methods
### Extraction of Cellulose Fibres from Rice Straw  
In this study, a multistep method, based on the sequential execution of several chemical and physical processes, was employed for the isolation of cellulose fibres from rice straw. The methodological framework, adapted from [PERSON] et al. [32], was comprehensively optimised to enhance the extraction efficacy while striving for a reduction in the number of stages, duration, and reagent use. Figure 2 shows the schematic representation of the process, illustrating the sequential steps followed, tailored specifically for rice straw. The process begins with the collection of rice straw, a plentifal agricultural residue from rice (Step 0), which undergoes preparatory treatments to facilitate the subsequent extraction phases (Step 1-4). In order to maximise the surface area in contact with the treatment solutions, rice straw is initially cut longitudinally to 10 cm in size, as shown (in solution) in Step 1. Each stage is described in detail below, as well as its function. **Step 1: Alkaline Pretreatment**: This process involves immersing the rice straw in an aqueous sodium hydroxide (NaOH) solution, which acts on the fibre structure of the straw. The primary purpose of this treatment is to modify the hydrogen bonds within the cellulose, increasing the surface roughness and facilitating the removal of the lignin, waxes, and oils [42; 43; 44]. This is chemically represented by the reaction:  
\[\mathrm{Fibre-OH+NaOH\to Fibre-O-Na+H_{2}O} \tag{1}\]  
Figure 1: Rice waste: (**a**) fresh harvested rice straw and (**b**) wet chopped rice straw. In our experimental approach, 10 g of dried chopped rice straw were introduced in a jacketed glass reactor of 1 L with 600 mL distilled water and heated to 70 \({}^{\circ}\)C, with a pH maintained at 12.5 using NaOH for a duration of 3 h and stirring at 500 rpm. This step has been traditionally considered essential for enhancing the mechanical properties of the extracted fibres. The solid residue is filtered and washed several times in order to standardise the condition of the biomass before the steam explosion stage, ensuring consistency in our experimental results. **Step 2: Steam Explosion and Supercritical CO2 Integration:** In this phase of the study, the rice straw, regardless of having undergone the pretreatment or not, is subjected to a steam explosion process within a high-temperature/high-pressure reactor (HPHT) (novoclave, Buchi AG--Pilot Plant & Reactor Systems; Uster, Switzerland). In this process, a water-to-raw material ratio of 10:1 is employed, with 10 g of rice straw added in every instance. The reactor, equipped with a thermal jacket of 600 mL, is sealed and heated to maintain an internal temperature of 200 \({}^{\circ}\)C, ensuring that no pressure is released. To assess the impact of time on the effectiveness of lignin depolymerisation and hemicellulose degradation, the reactor's temperature is sustained at 200 \({}^{\circ}\)C for varying durations, ranging from 30 min up to 4 h. Following this treatment, the reactor is methodically depressurised and left to cool to an ambient temperature over the course of the night. This step involves both mechanical action through shear forces caused by rapid pressure release and chemical action resulting from autohydrolysis, primarily the hydrolytic attack by acetic acid formed from acetyl groups in the hemicelluloses at high temperatures [9]. The innovative inclusion in this step is the application of supercritical carbon dioxide (ScCO2). ScCO2 is commonly utilised for cellulose modification and as a pretreatment for enzymatic hydrolysis [45; 46; 47]. The diffusion of ScCO2 can be penetrative, impacting the structural and physicochemical properties of cellulose derived from rice straw. Therefore, in this study, ScCO2 has been chosen as an alternative medium for cellulose acetylation to explore its potential to improve the process.