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. 2025 Apr 4;15(5):114. doi: 10.1007/s13205-025-04281-x

Two stage enzymatic pretreatment of rice straw for its valorisation using Silicase and Laccase

Daljeet Kaur 1, Prabhjot Kaur 1, Amarjit Singh 2, Nitu Mor 1, Sunita Dalal 1, Jitender Sharma 1,✉
PMCID: PMC11971084  PMID: 40191449

Abstract

The present study investigated the cumulative effect of pretreatment of rice straw with two enzymes—Silicase (first report) and Laccase as initial stages. The optimal conditions for silicase pretreatment were determined to be a dosage of 100 U/10 g of rice straw, pH 7.0, temperature 40 ºC, and a treatment duration of 30 h. For laccase pretreatment, the ideal parameters were enzyme dose of 40 U/10 g of rice straw, pH 5.0, temperature 50 ºC, and a treatment time of 30 h. At optimized conditions, the silica reduction of ~ 20% was achieved by Silicase pretreatment, whereas the reduction in lignin was upgraded by ~ 29.8% after two stage pretreatment. A reduction of 28.6% in ash content of rice straw and 29.4% in silica was obtained during two stage enzymatic pretreatment. The FTIR studies of the pretreated and untreated straw also depicted the delignification, ash and silica removal of agro-waste. A peak observed at 1542 cm−1 and 1643 cm−1 suggests cyclic stretching in phenolic lignin, while the absorption band at 1419 cm−1 corresponds to the bending (scissoring) of − OCH3 in the syringyl and guaiacyl units of the phenolic composition. A notable decrease in these vibrations was observed in the silicase + laccase-treated sample, likely resulting from the removal of syringols and guaiacols during the enzymatic pretreatment. Using present outcomes, the study presented that the cumulative impact of Silicase and Laccase was proficient in preparing rice straw for industrial applications and reducing environmental barriers during its conversion.

Keywords: Rice straw, Lignin, Silica, Delignification, Silicase, Laccase, Enzymatic pretreatment

Introduction

Rice straw has been labelled as a chief cereal waste amongst the different lignocellulosic waste released over the Asian continent. Annually, more than 700 million metric tonnes of straw is heaped during rice harvesting and this quantity varied depending upon crop variety and climatic conditions (Kaur et al. 2023). In India and other countries, the sustainable practices to dispose-off rice straw are under question. The major percentage of stubble is unlawfully burned in-field consequently degrading the environmental integrity of the vicinity. It contributed to global greenhouse gas emissions, disturbance of air quality and discharge of noxious pollutants to the troposphere which is a serious concern worldwide. The problems associated with mishandling of rice straw can be overcome by its transformation into value added materials. Goodman (2020) in his review paper presented the potential of agrowastes as a resource for the synthesis of biofertilizer, bioplastic, biofuel, bioenergy, solar panels, nanomaterials, and other specialty chemicals.

Rice straw is potentially rich in cellulose, hemicelluloses, lignin and silica and can contribute to the world’s economy after harnessing its components. The extraction of different constituents and formulation of various products is extremely challenging due to the compact structure of rice straw. Silica which provides strength against animal, environmental and microbial attacks on straw is a major constraint against extracting benefits from it. The lignin in rice straw no doubt shields the polysaccharides from outside pressure but creates hurdles during their conversion. The reduction of silica and removal of recalcitrant lignin from rice straw should keep priority before its conversion. The chemical, physical and physico-chemical pretreatment methods to open rice straw structure have been well-documented by researchers (Dissanayake et al. 2024; Wang et al. 2023; Sharma et al. 2023a, b). The conventional pretreatment methods are uneconomical, energy-intensive, and harmful to environmental quality. In contrast to it, enzymatic pretreatment of lignocellulosic wastes provides a basis for their sustainable and greener management.

The separation of lignin using specific lignolytic enzymes such as Laccase is a powerful way to manage rice straw. Nazar et al. (2023) conducted rice straw pretreatment using microbial consortia of Lactobacillus plantarum and enzymes (Cellulases and Laccase) and resulted in the removal of 4.0% lignin during the study. Terasawat and Phoolphundh (2021) achieved 45.5% of lignin removal during Laccase pretreatment of rice straw at optimized conditions. Another study used the recombinant Trichoderma reesei with Laccase genes of Pycnoporuss anguineus for on-spot production of enzymes under solid-state fermentation (SSF) to dispose off rice straw. Their work builds up an innovative SSF system based on recombinant T. reesei with huge capability for decaying the rice straw and other organic pollutants of soil (Xia and Lin 2022). The lignolytic enzymes can cut down the phenolic lignin up to 20–30% within 2 months (Wulandari et al. 2013). The bio-delignification of lignocellulosic wastes and upgrading their bioconversion efficiency using Laccase pretreatment had been reported in different research (Li et al. 2020; Sherpa et al. 2018). The Silicase are produced by silicate solubilizing bacteria and fungi. Silicase are known for breaking the SiO2 bonding present in the crystalline framework of clay minerals and the Si–C bonding in organo-silicon components of rocks. A recent study by Kaur et al. (2022) revealed that as carbonic anhydrase, the Silicase also expedites the formation of di-oxosilane hydrate by hydration of silicon dioxide. Their study endorsed the enzymatic pretreatment of rice straw with Silicase for reducing the silica from cell wall. Sharma et al. (2023a, b) also isolated the Silicase producing bacteria (Klebsiella quasipneumoniae) from paddy field and urged for treatment of silica containing wastes like cereal straws, microchips, glass scraps and ceramics through a biological and environmentally friendly route. Mor et al. (2024) also isolated the silica solubilizing bacteria from soil and water samples and their Silicase producing ability was analyzed.

The enzymatic pretreatment of rice straw is preferred after the physical/chemical treatments or in combination with them as a biochemical pretreatment. Though, literature on biological delignification is available but removal of silica from rice straw biologically has not been studied till date. The present study emphasized purely on enzymatic pretreatment of rice straw in two stages (Silicase followed by Laccase) and the cumulative effect of Silicase and Laccase on rice straw morphology and chemical composition, a novel approach for rice straw valorisation.

Methodology

Agro-waste

Rice straw was sourced from a village under district Kurukshetra, Haryana, India and cut into 15–20 cm pieces. The cut rice straw was thoroughly cleaned with warm water to remove silica deposits from its surface. After washing, the rice straw was air-dried for 5–6 days before being tightly packed into polyethylene bags. A sample was placed in an oven overnight to determine its moisture content using the ASTM D442 - 20 method. Since all experiments were based on the oven-dry (OD) weight of the rice straw, moisture content was considered to be a critical parameter throughout the study and was measured every 10 days.

Enzymes procurement

The enzymes Laccase and Silicase used in the study were produced in laboratories at two different universities in Haryana, India. Silicase was obtained from research scholars, Department of Biotechnology, Kurukshetra University, while Laccase was generously supplied by Dr. Krishankant Sharma from the Department of Microbiology at Maharshi Dayanand University, Rohtak. The Laccase was produced from Basidiomycetous fungus known as Ganoderma lucidum and obtained Silicase was produced from bacteria Enterobactor asburiae. Although the initial enzyme activity data for both Silicase and Laccase was provided by them, it was re-evaluated during the study.

Materials

High-purity chemicals (primarily AR grade) and borosilicate glassware were employed for the study. Hydrogen fluoride, acetone, glacial acetic acid, sulfuric acid, sodium chlorite, thiourea, p-bromoaniline, nitric acid, xylan, cellulose, and anthrone were utilized in the proximate chemical analysis of rice straw. Guaiacol and galacturonic acid were used for the Laccase enzyme assay. Pure phenol and silicon dioxide (HPLC grade, 99.9%) were used to create standard curves and platinum crucibles were employed to analyze the silica content in rice straw. The instruments like autoclave, water bath, muffle furnace, oven, soxhlet apparatus, UV–visible spectrophotometer, centrifuge and FTIR were used during the study.

Enzyme assay

The Silicase enzyme assay was conducted following the procedure described by Kaur et al. (2022). A tris buffer with a pH of 7.2 was prepared and silicon dioxide was dissolved in it to prepare 100 μg/mL solution. This assay measures the practical correlation between organic acids and acid phosphatase produced during the dissolution of silica. The test is based on 4500 SiO2 C, Molybedosilicate method described by National Environmental Methods Index. During the reaction, ammonium molybedate reacted with silicon dioxide to form heteropoly acids. Oxalic acid was then added to eliminate other organic acids, leaving only yellow colour solution containing molybdosilicic acid. The absorbance of the yellow-coloured reaction mixture was observed at 540 nm. Laccase assay was conducted following the guaiacol oxidation test. Sodium acetate (10 mM, pH 5.0) buffer and 2 mM guaiacol standard were prepared. The reaction mixture, consisting of 1 ml each of the buffer, guaiacol, and enzyme, was incubated in a water bath at 50 ºC for 10 min. Changes in color due to guaiacol oxidation (€ = 26,600 M^− 1 cm^− 1) were observed and measured at 470 nm. Blank assays (without enzyme) were processed similarly to the reaction mixtures. Absorbance values for standard solutions at various concentrations were used to create calibration curves, and enzyme activity (IU/ml) was determined from these standard graphs.

Enzymatic pretreatment

The optimal dose (U/10 g oven-dry rice straw), pH, temperature and time for enzymatic pretreatments were determined during the study. The pretreatment mixture was maintained at a consistency ratio of 1:15. As rice straw possess bulkiness and high-water retention capability, low consistency was chosen to acquire better fiber-enzyme interactions. The One-Factor-At-A-Time (OFAT) approach was used in this study. The optimization process began by focusing on the enzyme dose while maintaining all other parameters constant at levels where the enzyme activity was highest, as indicated by the suppliers and researchers who provided the enzymes. After optimizing the enzyme dose, pH was addressed next, followed by the optimization of time and temperature.

The dose range of enzymes was selected according to preliminary lab studies. The pH ranges (4.0, 5.0, 6.0, 7.0, 8.0, and 9.0) were tested to achieve maximum delignification, ash and silica removal while preserving the polymeric content of straw. The effect of temperature on enzyme efficiency was examined with pretreatments at 30 ºC, 40 ºC, 50 ºC, 60 ºC, and 70 ºC. The period of 4, 8, 12, 24, 36, and 48 h was evaluated to find the optimal duration during pretreatment study. The experiments were carried out in polythene bags and were tightly packed after putting the mixture in it. The contents were kept in water bath and its intermittent mixing was carried out manually.

After each pretreatment, the rice straw was washed with warm water, air-dried, and stored in an airtight container for further analysis of lignin, hemicelluloses, cellulose, ash, and silica content. The cumulative effect of enzymes on rice straw composition was studied by conducting a two-stage pretreatment process: Silicase followed by Laccase at their optimized conditions. All results were compared to untreated straw, and the percentage reduction in various components was calculated based on the collected data. The experiments were conducted in triplicates and for their statistical analysis, standard errors/bars of the data were calculated and given in all figures and tables. The one-way ANOVA for the final results obtained at final treatment was conducted and the significance of the results was presented in the subsequent section.

Raw material processing and proximate analysis

Rice straw was ground into a powder for its compositional analysis by standard TAPPI method (T 257 cm- 85). It was processed in a Wiley mill, and coarser particles were sieved through a 0.40 mm mesh. This process was repeated to obtain a fine powder, with any particles that did not pass through the mesh being discarded. The raw material was then treated to remove extractives using TAPPI method T 204 om- 88 to prevent interference in the analysis. The extractive-free material was air-dried and stored in a Mason jar to maintain moisture levels during handling and weighing. This method was applied to all rice straw samples (untreated and pretreated) and a portion of the extractive-free powder was used to determine moisture content using ASTM D442 - 20. The rice straw components—holocellulose, hemicellulosic fractions, cellulosic fibers, lignin, ash, and silica in ash were quantified. Holocellulose was determined by delignifying the straw with sodium chlorite-acetic acid at 75 ºC, following Le's (1946) method, and weighing the lignin-free residue. Hemicelluloses were analyzed using Deschatelets et al. (1986) method, which involved hydrolyzing the straw with 3% sulfuric acid, filtering the hydrolysate, adjusting the pH to 7.0, converting pentoses to furfural in acetic acid with thiourea at 70 ºC, and measuring the pink product formed with p-bromoaniline at 540 nm. Cellulose content was measured using Updegraff's (1971) method. Lignin content (acid-insoluble) was determined using TAPPI method T 222 om- 85, by treating the sample with 72% sulfuric acid, refluxing the residue in 3% sulfuric acid, and weighing the lignin after drying on pre-weighed filter paper. The ash percentage in rice straw was analyzed by combusting rice straw powder in muffle furnace at 575 ± 25 °C (T 211 om- 88), acid-digesting the ash with HCl, filtering, and then heating in platinum crucibles at 575 ± 25 °C. The remaining contents were digested in HF, combusted at 850 ± 25 °C, and the silica content was calculated.

FTIR analysis

The untreated and pretreated (Silicase and two stage Silicase + Laccase) rice straw were analyzed through FTIR. The FTIR spectrum obtained for different samples depicted the compositional variation that occurred during pretreatments. For the purpose, the analysis was made using Frontier MIR LiTa/KBr/AI spectrometer (PerkinElmer, UK). All the samples and KBr were oven dried for 4–5 h and then mixed properly using a pestle and mortar. The finely ground powder of samples and KBr was pelleted. The spectrum of rice straw samples was acquired by performing 64 scans, from 4000 to 400 cm−1 wavelength, at a resolution of 8 cm−1 to identify the impacts of pretreatment.

Results and discussions

Untreated raw material characteristics

The powder derived from washed rice straw was analyzed, and the observations are presented in Fig. 1. The analysis indicated that rice straw is a valuable source of polysaccharides, making it highly useful for industries such as pulp and paper, textiles, biopolymers, biocomposites, and nanofibers. For instance, Jayanti et al. (2023) successfully created pulp from rice straw and enhanced it with other natural polymers to increase the hydrophobicity of rice straw paper. The total holocellulosic content of rice straw was found to be 70.2%. Cellulose content was determined to be 33.5%, but extracting this polymer is challenging due to its close association with hemicelluloses and lignin. The results are in agreement with study of Kaur et al. (2019). Rice straw contains a high number of fines and short fibers, which are primarily composed of five-carbon chains; their fermentation for biofuel and bioenergy is well-supported in the literature (Nasr and Shehata 2023). Although, rice straw contains less lignin in comparison to other lignocellulosic wastes, delignifying rice straw remains difficult due to its complex structure, and chemical or physical delignification methods can often damage the fibers. The ash content in rice straw was approximately 18%, with a significant portion being silica 87.2%, which poses a major challenge for its use in both domestic and industrial applications. Several studies have detailed the issues related to silica in rice straw (Kaur and Singh 2024; Kaur et al. 2023; Van Hung et al. 2019).

Fig. 1.

Fig. 1

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Chart depicting the major constituents of rice straw

Effect of process parameter during pretreatment

Effect of Silicase on rice straw properties

The silicase activity of the enzyme procured was found to be 0.8796 IU/mL. During pretreatment of rice straw using Silicase, it was found that enzyme did not destroy the polysaccharide components. Initially, the effect of doses (U/10 g) ranging from 5 to 50 was investigated, but no significant effect was observed up to 20U/10 g. Therefore, the dose range for pretreatment study was expanded and 25U/10 g, 50U/10 g, 75U/10 g, 100U/10 g, and 150U/10 g were taken into consideration. The reduction in cellulose across these doses varied 0.9%–10.1%. The hemicelluloses were reduced by 3.6% at 25U/10 g of Silicase and the reduction rate was upgraded by 17.8% at 75U/10 g in comparison to control. Lignin reductions were 7.9%, 12.3%, 14.5%, 16.7% and 17.5% for doses of 25, 50, 75, 100, and 150 U/10 g, respectively. Silicase significantly impacted the reduction of ash and its silica proportions in rice straw. The percentage of ash was decreased by 17.4% at 25U/10 g and 21.4% at 50U/10 g. At higher doses (75U/10 g, 100U/10 g, and 150U/10 g), the reduction ranged from 25.8% to 26.0%, as shown in Fig. 2. Silicase pretreatment was efficient in achieving prominent reduction in silica content, ranging from 17 to 20%.

Fig. 2.

Fig. 2

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Optimization of enzyme dose for silicase pretreatment of rice straw and its impact on a Cellulose and hemicellulose b Lignin and ash c Silica

Silicase demonstrated the high efficiency for pretreating rice straw at pH 7.0 (Fig. 3). At pH below 6.0, no remarkable differences in the composition of straw were observed. The most substantial reductions in cellulose and hemicelluloses, measuring 9.9% and 16.9% respectively, were achieved at pH 7.0. Lignin removal rates were similar at both pH 7.0 and 8.0. The ash content decreased by 21.3% at pH 7.0, but higher pH levels negatively impacted the results. Silica removal was 15.4% at neutral pH, though its efficiency declined under alkaline conditions. This might be attributed to the high catalytic activity of Silicase at neutral pH, which allows it to efficiently convert silica to solubilized form. Therefore, pH 7.0 was selected as the optimal condition for Silicase pretreatment based on these findings. The studies conducted for isolation of silica solubilizing bacteria also showed that these microbes have high silicase activity at neutral pH (Kaur et al. 2022; Sharma et al. 2023a, b).

Fig. 3.

Fig. 3

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Optimization of pH for Silicase pretreatment of rice straw and its effect on a polysaccharides b Lignin and ash content c Silica content

Silicase did not exhibit any prominent impact on the chemical composition of rice straw between 4 and 16 h. Hemicelluloses in rice straw were reduced by 15.6%, 16.1%, 19.1%, and 20.8% after 24, 30, 36, and 42 h, respectively. Lignin content decreased progressively with longer pretreatment times from 24 to 42 h, with a maximum reduction of 21% observed at 42 h, though this was not significantly different from results obtained at 30 and 36 h. In comparison to untreated rice straw, the ash reduction in pretreated rice straw was 19.7%, 21.3%, 22.5%, and 23.0% after 24, 30, 36, and 42 h. A significant effect on silica reduction was observed when the pretreatment time was extended from 12 to 16 h. After 16 h, ash content continued to decrease, but silica levels remained unchanged. The greatest silica reduction, 18.5%, occurred after 36 h of pretreatment as shown in Table 1. Overall, pretreatment of rice straw was most effective at 36 h, with no significant further reductions in lignin, ash, or silica observed with extended treatment times.

Table 1.

Effect of pretreatment time on rice straw characteristics

Components (%) Pretreatment time (hours)
16 24 30 36 42
Cellulose 31.4 ± 0.05 30.3 ± 0.04 30.1 ± 0.06 30.3 ± 0.05 30.2 ± 0.09
Hemicellulose 32.0 ± 0.04 30.9 ± 0.05 30.7 ± 0.06 29.6 ± 0.06 29.0 ± 0.05
Acid insoluble lignin (ash corrected) 10.7 ± 0.04 9.4 ± 0.06 9.2 ± 0.05 9.1 ± 0.04 9.0 ± 0.03
Holocellulose 64.3 ± 0.03 61.4 ± 0.03 60.3 ± 0.05 60.2 ± 0.06 59.0 ± 0.09
Ash 15.6 ± 0.05 14.3 ± 0.04 14.0 ± 0.05 13.8 ± 0.07 13.7 ± 0.03
Silica 79.5 ± 0.05 71.6 ± 0.09 71.5 ± 0.07 71.0 ± 0.08 71.1 ± 0.06
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In regard to temperature, Silicase showed the best results for delignification, ash and silica removal at 40 ºC (Fig. 4). At lower temperatures, cellulose reduction was between 3 and 4%, but this reduction decreased to 1.8–2.0% at higher temperatures, indicating that Silicase did not target the cellulosic component of rice straw across different temperature ranges. This enzyme may be particularly valuable for industries focused on cellulose. Hemicelluloses were also minimally affected by Silicase pretreatment at various temperatures, with the greatest reduction of 11.5% occurring at 50 ºC. A total reduction of 8.1% in holocellulosic fractions was also observed at 50 ºC. In contrast, lignin, ash, and silica content was cut down significantly at 40 ºC and the highest lignin reduction of 16.7% was achieved at this temperature. The ash reduction was markedly impacted by temperature, with a removal of 11.8% at 30 ºC and an increase to 20.2% at 40 ºC. Silica content in the ash was maximally reduced by 11.4% at 40 ºC, and no further changes in silica content were observed with higher temperatures. It was attributed that the degree of enzymatic response enhanced up to a threshold temperature range and beyond that reaction slowed down. This happened as thermal inactivation of enzymes and their specificity towards optimum temperature (Sharma et al. 2019). Kaur et al. (2024a, b) showed that the modification of silica and associated processes depend on silica dissolution, which is facilitated by enzyme silicase. The study further revealed that a novel silicolytic bacterium identified as Staphylococcus gallinarum CON2 offered a promising solution by facilitating the breakdown of silica based complex materials through silica solubilization have been added.

Fig. 4.

Fig. 4

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Optimization of temperature during silicase pretreatment of rice straw and the % reduction observed in its proximate components

According to obtained results, the optimized process conditions for Silicase pretreatment were found to be pH 7.0, an enzyme dose of 100U/10 g of oven-dried rice straw, a time period of 36 h, and a temperature of 40 ºC. Under these conditions, the process was most effective in reducing ash and silica in rice straw ash, with reductions of 25.8% and 19.7%, respectively. Garg et al. (2017) reported a reduction of approximately 43.7% in lignin and 31.1% in silica during pretreatment of rice straw with silicases. Silicase had the least impact on cellulose and hemicelluloses as reductions of only 3.0% and 12.3%, respectively, were achieved.

Effect of laccase on rice straw properties

Laccase enzymes are the most accessible and effective enzyme for lignin removal due to their multiple copper sites, which facilitate the oxidation of phenolic components in lignin using oxygen as an electron acceptor (Aza and Camarero 2023). One of the main advantages of using Laccase for delignification is its specificity; it targets only the phenolic components of lignin, leaving the polysaccharides in raw materials unaffected (Paraschiv et al. 2022). Due to specificity towards lignin, it is preferred over chemical pretreating methods (Ezike et al. 2021). During this study, the effects of Laccase on rice straw composition were examined, and the pretreatment conditions were optimized to achieve the maximum lignin removal. The Laccase activity was observed to be 94 IU/mL. During pretreatment at different doses of Laccase, it was observed that the enzyme degraded the cellulosic fibers, minimally. Even at the highest dose of 50 U/10 g of oven-dried rice straw, cellulose reduction was limited to 9.8%. At lower doses ranging from 5 to 30 U/10 g, hemicellulose reduction was between 10%—12%. On increasing the dose to 40 U/10 g, 22.4% reduction in hemicelluloses was achieved, which pointed towards maximum (26.5%) at 50 U/10 g. Laccase showed high efficacy for lignin removal as reductions of 15.8%, 22.8%, and 23.7% corresponding to increasing doses from 5 U/10 g, 10 U/10 g and 20 U/g, respectively. Gao et al. (2011) noted that Laccase-induced lignin degradation facilitates the subsequent hydrolysis of hemicelluloses. The highest observed reduction in hemicelluloses was 28.1% at 50 U/10 g, though reductions at 30 and 40 U/10 g were comparable. Laccase markedly decreased the ash and silica content in rice straw. At a concentration of 5 U/10 g, the ash content dropped by 18.5%, and this reduction increased to 25.8% with 10 U/10 g. Higher doses did not produce further significant reductions in ash content. Silica content decreased notably across all doses: by 10.3% at 5 U/10 g, 16.3% at 10 U/10 g, 17.3% at 20 U/10 g, and 18.6% at 40 U/10 g. Therefore, a Laccase dose of 40 U/10 g was identified as the most effective for pretreatment. According to Rencoret et al. (2017), during Laccase-facilitated lignin depolymerization, the enzyme first targeted aliphatic side chains in lignin, then removed hydroxyphenyl, guaiacyl, and syringyl monomeric units, and ultimately broke down the ester bonds in coumarates and ferulates within lignin-carbohydrate complexes. Detailed results on Laccase dosage and rice straw composition are shown in Table 2.

Table 2.

Effect of Laccase dose on rice straw composition

Rice straw composition (%) Control Laccase dose (U/10 g)
5 10 20 30 40 50
Cellulose 33.5 ± 0.04 32.6 ± 0.05 32.4 ± 0.05 32.3 ± 0.04 30.2 ± 0.05 30.3 ± 0.05 30.2 ± 0.05
Hemicelluloses 36.6 ± 0.05 32.9 ± 0.04 32.5 ± 0.04 32.2 ± 0.06 32 ± 0.09 28.4 ± 0.06 26.9 ± 0.04
Holocellulose 70.2 ± 0.05 65.8 ± 0.06 64.9 ± 0.05 64.4 ± 0.07 62.2 ± 0.08 58.7 ± 0.07 59 ± 0.06
Lignin 11.4 ± 0.04 9.6 ± 0.04 8.8 ± 0.05 8.7 ± 0.05 8.4 ± 0.04 8.3 ± 0.05 8.2 ± 0.07
Ash 17.8 ± 0.03 14.5 ± 0.04 13.2 ± 0.06 13.3 ± 0.06 13.1 ± 0.05 13 ± 0.04 13.2 ± 0.05
Silica in Ash 87.2 ± 0.04 78.2 ± 0.05 73 ± 0.04 72.1 ± 0.09 72.6 ± 0.08 71 ± 0.09 72.3 ± 0.09
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During pretreatment, it was found that Laccase performed optimally at pH 5.0, as illustrated in Table 3. Although cellulose reduction peaked at pH 6.0 with a 9.2% decrease, the highest levels of delignification and ash removal occurred at pH 5.0. At this pH, reductions of 27% in lignin and 19.7% in ash content were achieved. Further increase in pH did not significantly result into silica removal, as reductions were about 13% at pH levels 5.0, 6.0, and 7.0. The reduction in hemicelluloses was similar at pH 5.0 and 6.0. The study is in agreement with Freitas et al. (2021) as their study also showed that pH 5.0 was optimal for Laccase derived from L. sajorcaju.

Table 3.

Effect of pH on rice straw composition during Laccase pretreatment

Rice straw composition (%) Control pH during Laccase pretreatment
4.0 5.0 6.0 7.0
Cellulose 33.5 ± 0.04 32.1 ± 0.05 30.9 ± 0.04 30.4 ± 0.07 32.3 ± 0.05
Hemicelluloses 36.6 ± 0.05 33.4 ± 0.03 30.2 ± 0.06 30.1 ± 0.06 30.4 ± 0.05
Holocellulose 70.2 ± 0.05 65.4 ± 0.06 61.2 ± 0.08 60.5 ± 0.09 62.7 ± 0.07
Lignin 11.4 ± 0.04 11.2 ± 0.04 8.3 ± 0.06 8.5 ± 0.05 9.3 ± 0.07
Ash 17.8 ± 0.03 15.6 ± 0.05 14.3 ± 0.06 14.6 ± 0.07 14.5 ± 0.03
Silica in Ash 87.2 ± 0.09 82.3 ± 0.08 75.6 ± 0.09 75.3 ± 0.08 75 ± 0.06
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Similar to Silicase, Laccase also had no significant influence on the chemical composition of rice straw up to 12 h, as shown in Fig. 5. After 24 h of treatment, degradation of cellulose and hemicelluloses was observed. However, Laccase did not notably affect cellulose content even after 42 h of pretreatment. The hemicelluloses reduction had been increased with the duration of pretreatment, reaching a maximum of 19.2% after 36 h. The reductions in hemicelluloses were comparable for time periods of 24, 30, 36, and 42 h. The lignin content decreased gradually between 24 to 42 h, with a comparable reduction of about 21.1%. The lowest ash and silica contents as 27% and 14.1% respectively, were achieved after 30 h of pretreatment. The results showed that changes in rice straw composition were minimal beyond the 30 h pretreatment time period so, this duration was chosen for the study.

Fig. 5.

Fig. 5

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Optimization of time during Laccase pretreatment of rice straw and the % reduction achieved in its proximate components

Temperature significantly influenced the pretreatment of rice straw. Laccase yielded the best results at temperatures between 50 and 60 ºC. Cellulose content decreased by 6.3% at 50 ºC and 7.5% at 60 ºC. This suggests that Laccase is a target-specific enzyme, as it did not affect cellulose levels across different pH, temperature, and time conditions. The enzyme is particularly suited for applications in industries such as polymers, pulp, and paper. Hemicelluloses reduction reached its maximum at 50 ºC, with a reduction of 21.5%. The most significant lignin reduction, 17.5%, was observed at 50 ºC compared to untreated straw, with the rate of reduction decreasing at higher temperatures. Freitas et al. (2021) reported that crude Laccase remained stable between 40 and 50 ºC, but its activity declined rapidly above 50 ºC. Similarly, ash content reduction was greatest at 50 ºC, with a reduction of 14%, while silica reduction was 11% at both 50 ºC and 60 ºC. Therefore, 50 ºC was determined to be the optimal temperature for Laccase pretreatment of rice straw, as illustrated in Fig. 6.

Fig. 6.

Fig. 6

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Optimization of temperature during Laccase pretreatment based on % reduction observed in its proximate composition

Cumulative effect of two stage enzymatic pretreatment at optimized conditions

Silicase, though not yet commonly used for pretreating lignocellulosic wastes, demonstrated strong effectiveness in reducing ash and silica levels in rice straw. In the initial stage of Silicase pretreatment at optimized conditions, 25.8% reduction in ash and 19.7% reduction in silica were achieved. This finding aligned with Garg et al. (2017), who reported reductions of approximately 43.7% in lignin and 31.1% in silica from rice straw during Silicase pretreatment. Silicase showed minimal effect on cellulose and hemicelluloses and resulted in reduction by 3.0% in long fibers and 12.3% in hemicelluloses, respectively, in comparison to the untreated straw. It clearly indicated the great efficacy of Silicase for modifying cell wall composition rather than internal structure of rice straw. The lignin content of Silicase pretreated rice straw was found to be 14% less than untreated rice straw. The Silicase-pretreated rice straw was then washed thoroughly and subjected to the next enzymatic stage using Laccase under optimized conditions. After second enzymatic pretreatment, the cellulose content was further reduced by 8.0% in comparison to Silicase pretreated straw. Laccase pretreatment resulted in a substantial reduction of 8.7% in hemicelluloses in comparison to Silicase pretreated rice straw. The overall reduction in hemicelluloses was 19.9% which may likely be due to fibrillation of rice straw during the initial stage, which facilitated Laccase's interaction with lignin and shorter fibers. Kaur et al. (2025) reported a of 26.5% in lignin and 20.8% in ash content of rice straw during laccase pretreatment. Similar study also revealed a reduction of 19.7% in silica during silicases pretreatment. Laccase imposed a dramatic impact on lignin content of rice straw and after this second pretreatment, lignin content of rice straw was decreased by 18.4% in comparison to the Silicase-pretreated straw, achieving an overall reduction of 29.8% compared to untreated straw. Giacobbe et al. (2018) reported that Laccase could achieve delignification rates of up to 89% without need of additional enzymes or chemicals. The ash and silica contents were also significantly reduced after Laccase pretreatment and decreases of 3.8% in ash and 11.1% in silica were achieved. After two stages of pretreatment, the rice straw had an ash content of 12.7% and silica content of 61.6% which indicated that two stage pretreatment is highly effective for enhancing the value of rice straw for various applications. The cumulative effect of two stage enzymatic pretreatment was presented in Table 4. A study by Anu et al. (2021) found that alteration in chemical composition of biomass via biological pretreatment weakened the interaction between polysacchrides and phenolic constituents which exaggerated the reduction of lignin in rice straw (Fig. 7).

Table 4.

Cumulative effect of two stage enzymatic pretreatment

Rice straw composition (%) Control Silicase treated Silicase + Laccase treated
Cellulose 33.5 ± 0.04 32.5 ± 0.05 29.9 ± 0.04
Hemicelluloses 36.6 ± 0.05 32.1 ± 0.03 29.3 ± 0.06
Holocellulose 68.5 ± 0.05 64.4 ± 0.06 60.2 ± 0.08
Lignin 11.4 ± 0.04 9.8 ± 0.04 8.0 ± 0.06
Ash 17.8 ± 0.03 13.2 ± 0.05 12.7 ± 0.06
Silica in Ash 87.2 ± 0.09 69.3 ± 0.08 61.6 ± 0.09
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Fig. 7.

Fig. 7

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FTIR analysis of different rice straw samples showing cumulative effect of enzymatic pretreatment

The statistical analysis of the date presented in Table 4 was conducted using one way ANOVA which resulted that the P value for each rice straw characteristics is less than 0.05. The means of different parameters for control, Silicase treated, and Silicase + Laccase treated rice straw were significantly different. The variance, F and P value for each parameter is given in Table 5.

Table 5.

One-way ANOVA for statistical evaluation

Parameters SS df MS Variance F P-value
Cellulose 20.86222 2 10.43111 0.003056 284.4848 1.14E- 06
Hemicelluloses 82.50987 2 41.25493 0.002333 3519.378 6.18E- 10
Holocellulose 109.3689 2 54.68444 0.004629 108.167 1.97E- 05
Lignin 27.60333 2 13.80167 0.002128 96.89158 2.65E- 09
Ash 58.40167 2 29.20083 0.003467 162.7291 8.6E- 08
Silica in ash 1066.309 2 533.1544 0.008321 2856.185 1.16E- 09
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FTIR results of pretreated and untreated straw

The structural changes after enzymatic pretreatment of rice straw were examined using FTIR spectra. The FTIR results of three samples (untreated, Silicase-pretreated, and Silicase + Laccase- pretreated rice straw) displayed a prominent band between 3250 and 3400 cm−1, indicating hydroxyl bond (− OH) vibrations associated with polysaachrides and lignin (Kaur et al. 2024a, b). Specific bands at 3255 cm−1, and 3335 cm−1 corresponded to various intramolecular and intermolecular hydrogen bonds, while spectra obtained at 3417 cm−1 was related to hydrogen bonding between molecules in the 101 plane. A spectrum at 2900–2930 cm−1 was evident across all samples, representing the functional group C-H alkanes with their irregular distending in − CH3, − CH2, and − CH groups. The peak formed at 1743 cm−1 corresponded to C = O bonding which is formed due to free carbonyl species. This showed typical hemicellulose fractions in rice straw (Malik et al. 2023). The spectrum at wavelengths 1300 cm−1–1650 cm−1 represented the presence of lignin. A peak found at 1542 cm−1 and 1643 cm−1 indicated cyclic stretching in phenolic lignin, and absorption spectra at 1419 cm−1 reflected bending (scissoring) of -OCH3 in syringyl and guaiacyl units of phenolic composition (Zhang et al. 2020). A significant reduction in these vibrations was noted in the silica + Laccase-treated sample, likely due to the removal of syringols and guaiacols during enzymatic pretreatment. The absorption spectrum showing peaks between 1041 and 1164 cm−1 were conforming the C–O–C linkage in rice straw (RP et al. 2022).

The spectrum obtained at 800 cm−1 to 950 cm−1 represented the glycosidic bonding of cellulose and hemicelluloses in rice straw (Sun et al. 2018). A slight increase in the peak at 933 cm−1 in the Silicase + Laccase-treated rice straw indicated the cleavage of cellulosic chains and the degradation of carbohydrates following the enzymatic treatment. The peaks from at 660–786 cm−1 are related to bending vibration of silicate bonding (Si–O) in silanol (Sakhiya et al. 2021; Wynn 2019). The FTIR spectra from the various samples supported the findings of the chemical investigation of straw where removal of lignin and ash was observed.

Conclusions

Rice straw has a complex and tough morphology that limits its effective use in various industrial applications. This study focused on reducing lignin and ash content through novel(enzymatic) pretreatment method and adding value to this waste. The study concluded that a two-stage enzymatic pretreatment involving Silicase followed by Laccase is highly efficient in opening up the rice straw structure while preserving its polymeric content, which could facilitate its conversion into biopolymers. Enzymatic pretreatment significantly decreased the lignin content by 29.8%, potentially reducing further need of chemicals during rice straw processing for industrial production. The study also achieved approximately 29.0% reduction in both ash and silica percentage of rice straw, which could help in minimizing the corrosion of industrial equipment during its conversion to value added products. The removal of silica and lignin before valorisation of rice straw is highly fruitful in reducing the chemical usage, energy requirement and environmental damages. The enzymatically pretreated rice straw can be used to extract pure biopolymer (cellulose) and its derivatives. The study provided a basis for novel approach to pretreat rice straw biologically.

Acknowledgements

The first author acknowledges the Science and Engineering Board for providing financial assistance through their National Postdoctoral Scheme vide approval number PDF/2022/000288. The author is also deeply grateful to Dr. Krishankant Sharma from Maharishi Dayanand University, Rohtak, Haryana for supplying the essential enzymes that made this study possible.

Author contributions

Dr. DK conducted all the experiments and wrote the manuscript. Prof. JS guided, reviewed the content and provided all essential resources at Kurukshetra University. Dr. PK assisted with editing and organizing the manuscript. Dr. AS provided valuable feedback that helped in shaping the research. Ms. NM carried the research part and provided the enzymes used in the study. Dr. SD paid valuable time in arranging the raw data to reach positive outcomes.

Data availability

The data generated during research has been included in the manuscript and available with the first author which can be made available on request.

Declarations

Conflict of interest

The authors declare that there are no conflicts of interest regarding this study.

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Associated Data

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Data Availability Statement

The data generated during research has been included in the manuscript and available with the first author which can be made available on request.

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