Pretreatment of rice straw with combined process using dilute sulfuric acid and aqueous ammonia
© Kim et al.; licensee BioMed Central Ltd. 2013
Received: 1 April 2013
Accepted: 26 July 2013
Published: 30 July 2013
Use of lignocellulosic biomass has received attention lately because it can be converted into various versatile chemical compounds by biological processes. In this study, a two-step pretreatment with dilute sulfuric acid and aqueous ammonia was performed efficiently on rice straw to obtain fermentable sugar. The soaking in aqueous ammonia process was also optimized by a statistical method.
Response surface methodology was employed. The determination coefficient (R2) value was found to be 0.9607 and the coefficient of variance was 6.77. The optimal pretreatment conditions were a temperature of 42.75°C, an aqueous ammonia concentration of 20.93%, and a reaction time of 48 h. The optimal enzyme concentration for saccharification was 30 filter paper units. The crystallinity index was approximately 60.23% and the Fourier transform infrared results showed the distinct peaks of glucan. Ethanol production using Saccharomyces cerevisiae K35 was performed to verify whether the glucose saccharified from rice straw was fermentable.
The combined pretreatment using dilute sulfuric acid and aqueous ammonia on rice straw efficiently yielded fermentable sugar and achieved almost the same crystallinity index as that of α-cellulose.
KeywordsRice straw Pretreatment Soaking aqueous ammonia Dilute acid pretreatment Response surface methodology
Biomass pretreatments are key steps in the low-cost bioconversion of cellulosic biomass to sugar because of the rigid and hard-to-degrade structure of the biomass cell walls. Pretreatments are used to release cellulose from amorphous lignin and hemicellulose. Chemical pretreatments using acid and alkali reagents have been widely studied because of their simplicity and efficient performance.
Acid pretreatments hydrolyze plant cell walls, especially their hemicellulose component. H2SO4, HNO3, and HCl are usually used for acid pretreatments in dilute and acidic states [1–4]. The solubilized hemicellulose can be converted to xylose, a monomer, in acidic media, and the xylose can then be overdegraded in a strongly acidic environment [5, 6]. Though glucose and xylose can biologically yield versatile building block products of various biochemicals, they can also be overdegraded and converted to by-products such as furfural and hydroxymethylfurfural (HMF), respectively [3, 7]. Therefore, in order to achieve selective hydrolysis using an acid reagent, an appropriate acid concentration, reaction temperature, and other critical factors must be experimentally determined.
Alkaline pretreatments have also been extensively studied for modifying cell walls. During such pretreatments, solvation and saponification reactions take place . As a result, the biomass swells, and access to its inner space by saccharification enzymes is enhanced [3, 8]. Also, alkali pretreatments selectively remove lignin portion mainly. Xylan and lignin support the cellulose backbone that includes the biomass cell wall [1, 3]. Though ammonia has been widely used in alkali pretreatments, its use leads to many environmental problems because of which recovery and recycling processes must also be used along with this pretreatment. NaOH and KOH have also been evaluated for use in alkali pretreatments because they are cheaper than ammonia [9, 10].
In our previous work, rice straw was pretreated with dilute sulfuric acid and pretreatment using the compounds was analyzed. In addition, to avoid the overdegradation of biomass, the pretreatment was optimized using a statistical model and a computer program . As xylose is obtained from hemicellulose, lignin can also be converted into useful compounds such as organic solvents, aromatic compounds, and fuel additives by chemical and biological methods. Removal of hemicellulose and lignin could dramatically improve enzyme digestibility by enhancing the enzyme accessibility to cellulose. The pretreatment with dilute sulfuric acid followed by aqueous ammonia would then be needed for efficient saccharification and lignin isolation.
Results and discussion
Response surface methodology analysis
Rice straw was pretreated with dilute sulfuric acid under previously optimized conditions: 142°C temperature, 1.21% concentration of sulfuric acid, and 11.6 min reaction time . After the pretreatment, the solid portion comprising the treated rice straw and the liquid portion of hydrolysates containing the solubilized xylose was separated by filtration. The solids were washed with distilled water and subsequently treated with aqueous ammonia. The pretreatment with aqueous ammonia was optimized using a statistical method.
The factors affecting the statistical analysis were temperature, concentration of aqueous ammonia, and reaction time that were determined from fundamental experiments. In the pretreatment process with aqueous ammonia, the aforementioned three factors strongly and directly affected either the productivity of sugar or the energy cost of the process. Fundamental experiments to determine the range of each of these factors were performed before the experiment using a statistical model. The temperature range, aqueous ammonia concentration, and reaction time were employed as described in the “Methods” section. The amount of glucose recovered from saccharification after SAA for each designed condition was used as the data for the statistical analysis.
Results of analysis of variance (ANOVA)
Sum of squares
Pr > F
Statistical analysis of factors
Pr > F
Optimization, confirmation, and enzyme loading test
The optimal coded values, determined by response surface methodology (RSM), were −0.8625 for temperature (X 1 ), 0.7412 for aqueous ammonia concentration (X 2 ), and 0 for reaction time (X 3 ), whereas the real values were 48.75°C, 20.93%, and 48 h, respectively. In the statistical analysis and optimization, wherein the results were analyzed based on the numerical regression level, the optimal reaction time was approximately 79 h. Though the process lasted for over 80 h, the yield was not notable compared to that obtained with a reaction time of 48 h.
Effect of pretreatments on rice straw composition
Raw rice straw (RR)
Dilute acid pretreated rice straw (DR)
Combined pretreated rice straw (DAR)
39.34 ± 2.35% (TM = 7.86 g/L)
68.38 ± 1.71% (TM = 13.67 g/L)
79.75 ± 3.87% (TM = 15.95 g/L)
28.46 ± 2.07%
10.15 ± 1.05%
10.87 ± 1.56%
30.2 ± 1.68%
21.47 ± 1.52%
9.38 ± 1.01%
Figure 3(B) shows the comparison of the RR, DR and DAR in saccharification using 30 FPU of Celluclast and 10 CBU of Novozyme 188. When dilute sulfuric acid was employed, initial reaction rate was 7.657 × 10-4 g/L · s, which was 6.61 times higher than that of the RR (1.158 × 10-4 g/L · s). When pretreatments with dilute sulfuric acid and aqueous ammonia were employed, the initial reaction rate was approximately 11.94-fold higher (1.384 × 10-3 g/L · s) than the values obtained for RR. Thus, pretreatments with dilute acid and aqueous ammonia were more effective compared to a one-step pretreatment. Ko et al. treated rice straw using aqueous ammonia and obtained 71.1% glucose conversion . Chen et al. suggested the use of a dilute acid and steam explosion to treat rice straw, and as a result, glucose conversion of approximately 85% was obtained . The results of the current study were notable compared to the earlier results. Also, Kim et al. reported a two-stage pretreatment of rice straw using aqueous ammonia and dilute acid in a facilitating apparatus and obtained approximately 89% glucose conversion . Our study employed SAA under atmospheric conditions and yielded results that were significant within the statistical error range.
Analysis of pretreated biomass
Chemically treated rice straw was analyzed by XRD and FTIR to investigate the change in rice straw before and after the pretreatment.
Moreover, correlation of glucose conversion with the CrI values of RR, DR, and DAR were analyzed by the linear regression of the plotted data. Figure 4(B) shows the results of regression between the CrI and glucose conversion. The relation was approximately proportional, which meant that an increase in the CrI corresponded to the removal of barriers for enzymatic accessibility and a relatively high CrI indicated high enzyme digestibility. The value of R2 was 0.7153. Many researchers have examined the relationship between the CrI and enzyme digestibility. Fan et al. (1987) studied this relationship and reported similar results . Kim et al. (2003) studied the effect of ammonia pretreatments on corn stover and found that an increase in CrI roughly corresponded to an increase in enzyme digestibility .
Figure 4(C) shows the results of the FTIR analysis that was performed to prove that the crystalline portion was cellulose. Sun et al. (2002) performed such analysis and reported that the biomass structure was composed of β-glucosidic bonds and carbon hydrates . The control spectrum is no. 1, which corresponded to α-cellulose. The band intensities provided the following information: 3456 cm-1 indicated an O-H group, 2945 cm-1 indicated a CH group, 1381 cm-1 indicated C-CH3 group, 1660 cm-1 indicated a β-glucosidic bond between the sugar monomers, and 1520 cm-1 and 1441 cm-1 indicated an aromatic ring. Spectrum-4 is RR, spectrum-3 is DR, and spectrum-2 is DAR. The peaks in spectrum-4 were blunt and not as sharp as those in spectrum-1, while those in spectrum-3 were less blunt than those in spectrum 4. In contrast, the peaks in spectrum-2 were sharper and clearer. These results indicated that the material became purer after the pretreatment. Thus, these band intensities showed the specific functions and bonds that corresponded to the cellulose structure. By using these pretreatment steps, barriers to enzyme accessibility were removed from the structure of the rice straw, and consequently, the cellulose portion was exposed [20, 21].
Fermentation of glucose
Also, trace amounts of the pretreatment reagent could have remained in the fermentation media. Ammonia is a strong and effective reagent for alkali pretreatments, but it is environmentally harmful and could affect the growth of microorganism. Thus, it needs to be recovered and recycled. In practice, SAA is a batch process. The current work deals with the optimization of the pretreatment conditions and could be utilized in processes involving the recycling and percolation of chemical reagents.
Rice straw was pretreated in a two-step sequential process using dilute sulfuric acid and aqueous ammonia. Statistical studies were performed for the pretreatment process and the results indicated that the model and experiments were reliable and significant. The optimal conditions were found to be a temperature of approximately 42.74°C, ammonia concentration of approximately 20.93%, and a reaction time of 48 h. When the pretreatment was performed using the optimal conditions, approximately 13.91 g/L (approximately 87.24% of the theoretical maximum) of fermentable glucose was recovered. Fermentation process using the recovered glucose yielded ethanol at approximately 83% of the theoretical maximum. The combined pretreatment of rice straw with dilute acid and aqueous ammonia was effective, and this was supported by saccharification, fermentation, XRD, and FTIR analyses.
Feedstock and chemicals
Rice straw was obtained from the Biochemical Engineering Laboratory in Kyonggi University, Suwon, Korea, and stored at 20°C with a relative humidity of 70% in the dark. The rice straw was ground and homogenized using a sieve of 40–60 mesh. Sulfuric acid (H2SO4) and aqueous ammonia (NH3∙H2O) were utilized for the pretreatment. Both were purchased from Dea-jung Chemical, Korea.
Assay method involving enzymes
Celluclast 1.5 L (cellulase) from Trichoderma reesei (Novozymes, Denmark) and Novozyme188 (β-glucosidase) from Aspergillus niger (Novozymes, Denmark) were used for the enzymatic hydrolysis of biomass. FPU for cellulase and CBU units for β-glucosidase were employed to measure the activity of enzymes. To measure FPU, enzymes were diluted at several levels. Then, 1.0 mL of the diluted Celluclast and 1.0 mL of 0.05 M citrate buffer (pH 4.8) were transferred into test tubes containing 50 mg of filter paper (Whatman No. 1, 1.1 × 6 cm). An assay reaction was performed accurately at 50°C for 60 min. Thereafter, dinitrosalicylic acid (DNS) was added to stop the reaction. The tubes were transferred to a water bath kept at 100°C, and the enzymes were deactivated for 5 min, followed by the addition of 10 mL of distilled water. The colored content of the tube was analyzed using a UV spectrophotometer at 575 nm. The β-glucosidase activity was assayed in 1.0 mL of a reaction mixture containing 0.1 mL of the diluted enzyme solution and 0.9 mL of 1 mM p-nitrophenyl-β-D-glucopyranoside (p NPG) in 0.05 M citrate buffer (pH 4.8) at 50°C for 30 min. Then, 1 M Na2CO3 solution was added to the mixture and allowed to develop a color. Later, 10 mL of distilled water was added and the release of p-nitrophenol was confirmed at 400 nm. The activities of Celluclast and Novozyme188 were 60 FPU and 30 CBU, respectively.
Pretreatment processes of biomass
The pretreatment of rice straw with dilute sulfuric acid was performed in an oil bath using a well-sealed tube reactor that was 1.2 cm in diameter and 18 cm in length. Preheating, reaction, and cooling were performed in the oil bath. The temperature of the preheating bath was maintained at 210°C for faster heat transfer whereas the cooling bath was kept at room temperature. The temperature, sulfuric acid concentration, and reaction time were similar to those reported in our previous work: 142°C, 1.21% and 11.6 min, respectively . After the pretreatment, solid–liquid separation was conducted, and the solids (rice straw) were extracted, washed, and dried. Later, the pretreatment with aqueous ammonia was carried out at 26.36–93.64°C, ammonia concentrations of 1.54–28.45%, and reaction times of 7.63–88.36 h. The agitation speed and solid–liquid ratio were 250 rpm and 1:12, respectively. This pretreatment step was performed in a 100-mL capped bottle. The ranges of the aforementioned parameters were determined by the fundamental experiments based on other reports [20, 24]. After the pretreatment, solids separated by filtration were washed with distilled water to remove the residual ammonia and establish a neutral pH followed by drying at 50°C until the weight became constant [25, 26].
Experimental design and statistical analysis
Independent variables and coded values
(X 1 )
Concentration of aqueous ammonia (%, v/v)
(X 2 )
Reaction time (h)
(X 3 )
Design of experiment (DOE), and results of DOE and statistical predictions
Observed glucose (%)
Predicted glucose (%)
95% confidence limits for mean predicted value
A CCD for three independent variables, each at five levels, was employed to fit a second-order polynomial model, which required 20 experiments [27, 28]. The Design-Expert® 6.0 package program (Stat-Ease, USA) was used for experimental design, regression analysis of data, and estimation of the coefficients of the regression equation.
Saccharification and fermentation
Enzymatic hydrolysis for enzyme digestibility was investigated according to the NREL standard procedure . The reactions were performed at 50°C in 0.05 M of citrate buffer (pH 4.8) at 150 rpm. Fermentation with glucose, produced by saccharification, was performed using Saccharomyces cerevisiae K35 [22, 29]. The cells were inoculated with the YM broth medium and incubated at 30°C and 200 rpm for 24 h. For the main culture, 0.5 mL of inoculums in the main medium was prepared in 250 mL Erlenmeyer flasks. The main medium was 25 mL of distilled water containing 0.5 g yeast extract, 0.5 g peptone, 0.1 g MgSO4∙7H2O, and 0.1 g K2HPO4, to which 25 mL of a saccharified liquid containing 62 g/L glucose was added. The initial pH and temperature of the fermentation process were kept at 5.0 and 30°C, respectively, for 12 h. On the completion of the fermentation process, solid–liquid separation by centrifugation was performed at 8000 rpm for 30 min, and the solids were separated and dried to measure the dry cell weight.
Analysis of the solid biomass was performed to determine its absolute composition, according to the standard procedures of the National Renewable Energy Laboratory (NREL, USA) . For acid hydrolysis, biomass was incubated with sulfuric acid (72%, w/w) at 25°C. After the primary hydrolysis, the solution was diluted to 4% and heated to 121°C in an autoclave. On cooling, the mixture was neutralized with calcium carbonate. The supernatant of the biomass composition was then analyzed by high-performance liquid chromatography (HPLC) using an Aminex HPX-87H ion exclusion column (Bio-Rad) and a refractive index detector. The HPLC conditions included a column at 50°C, a mobile phase of 0.005 N H2SO4, and a flow rate of 0.8 mL/min. The amount of glucose after saccharification and ethanol production yield were also measured by HPLC. All calculations of production and mass balance were performed by considering the biomass composition. This method was based on the solid biomass analysis of NREL . While executing the experimental steps, the biomass was carefully manipulated to prevent weight loss.
where I θ° is intensity at the corresponding θ.
An FTIR spectroscopy analysis was also performed. The transmission the FTIR spectra were obtained using the FTIR spectrometer on an ambient atmosphere bench (Perkin-Elmer, Spectrum GX). The instrument was equipped with liquid-nitrogen-cooled mercury cadmium tellurium (MCT). The resolution of the spectra was 4 cm-1, and 256 scans were included to increase the signal-to-noise ratio [20, 21].
Central composite design
Coefficient of variance
Dilute acid and aqueous ammonia combined pretreated rice straw
Design of experiment
Dilute-acid-pretreated rice straw
Filter paper unit
Fourier transform infrared
High-performance liquid chromatography
Mercury cadmium tellurium
National Renewable Energy Laboratory
Raw rice straw
Response surface methodology
Soaking in aqueous ammonia
Xylose, mannan and galactan
This study was supported by the Technology Development Program (309016–5) for Agriculture and Forestry from the Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea, and Creative Allied Project (CAP) of the Korea Research Council of Fundamental Science and Technology (KRCF)/Korea Institute of Science and Technology (KIST).
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