Silica Removal from Rice Straw To Improve its Hydrolysis and Ethanol

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Silica Removal from Rice Straw to Improve its Hydrolysis and Ethanol Production Hoori Khaleghian, Maryam Molaverdi, and Keikhosro Karimi Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.7b02830 • Publication Date (Web): 09 Aug 2017 Downloaded from http://pubs.acs.org on August 13, 2017

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Industrial & Engineering Chemistry Research

Organosolv pretreatment

Lignin Silica Rice straw covered by silica layer

Ethanol

Ethanol

Ethanol

Untreated rice straw

Sodium carbonate pretreatment

Lignin

Silica


Simultaneous saccharification and fermentation by Mucor hiemalis

Glucose

Glucose

Glucose

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Na2CO3 pretreated rice straw

Enzymatic hydrolysis

Organosolv followed by Na2CO3 pretreated rice straw


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Silica Removal from Rice Straw to Improve its Hydrolysis and Ethanol Production

Hoori Khaleghiana, Maryam Molaverdia, Keikhosro Karimia,b,∗ a

Department of Chemical Engineering, Isfahan University of Technology, Isfahan 84156-83111, Iran b

Industrial Biotechnology group, Research Institute for Biotechnology and Bioengineering, Isfahan University of Technology, Isfahan 84156-83111, Iran

*

Corresponding author: Tel: +983113915623

Fax: +983113912677 E-mail address: [email protected]

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Abstract Rice straw has high silica content compared with other lignocelluloses. Presence of silica in the outer layer of rice straw reduces enzymatic hydrolysis and ethanol production yield. In this study, silica removal from rice straw was investigated through various alkali treatments. The results showed that sodium carbonate could effectively remove silica from the straw (>91%). To investigate only the effects of silica removal and eliminate the effect of lignin removal on enzymatic hydrolysis, organosolv followed by sodium carbonate pretreatment was conducted. Organosolv pretreatment removed most of the straw lignin, while silica was not separated. Organosolv pretreatment of rice straw, only improved the glucose and ethanol yield from 34.4% and 39.3% for the untreated straw to 52.9% and 51.6%, respectively, whereas further treatment with sodium carbonate increased the yields up to 94.8% and 78.7%. The results indicated that the protecting effect of silica layer was greater than that of lignin in rice straw.

Keywords: Rice Straw, Silica, Sodium Carbonate, Pretreatment, Ethanol

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1. Introduction Achieving sustainable and renewable energy is one of the necessities at global level, and bioethanol is one of the best substitutes for nonrenewable fossil fuels. Considering the availability and price, lignocellulosic materials are the most important resources for ethanol production.1 Agricultural wastes and paper mill wastes are abundantly produced annually around the world. Unfortunately, discarding these materials damage the environment and their burning cause severe air pollution.2,3 One of the most promising strategies for consuming these wastes is converting them into bioethanol.4 However, the yield of ethanol production from the raw form of these substrates is very low due to their complex and recalcitrant structures, making the process uneconomical. Therefore, a pretreatment step is essential to improve the sugar yields.5,6 Selection of the best method and pretreatment conditions highly depend on the type of lignocellulosic material.7 Rice straw is one of the most abundant and available lignocellulosic wastes all around the world.8 This straw has very low applications compared with other cereal straws (e.g., wheat straw). This is due to its high ash content (i.e., 10-17%) with silica of about 75%.

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A layer of

silicified cuticle on the rice straw surface protects the inner layer of the straw. Therefore, silica and lignin protect the carbohydrates in rice straw, making its composting, digestion, and hydrolysis difficult.9,10 Besides being a physical barrier, the silica implies inhibiting effects on the hydrolytic enzymes.9,11 On the other hand, silica, as a valuable byproduct, is consumed in catalyst supports, adsorbents production, molecular sieves, and photocatalysts.12 Thus, it is important to use an appropriate pretreatment method that, in addition to reducing lignin and crystallinity of cellulose, would dissolve silica and remove it from rice straw. To our knowledge, there exists no study on separating silica from rice straw to improve ethanol production yield. 3 ACS Paragon Plus Environment

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The present study was carried out to investigate the effects of silica removal from rice straw on the morphological structure and improvement of hydrolysis and fermentation. To remove silica from the rice straw, pretreatments are made with alkaline material at different concentrations and temperatures.

2. Materials and methods 2.1 Raw material and pretreatment for silica removal Rice straw (Sazandeghi cultivar) was collected from a field at Zarinshahr (Isfahan, Iran). First, the straw was washed and air dried. Then, it was milled by a knife mill grinder (SaeiNikou Co., Isfahan, Iran) and selected to a size of between 177 and 833 µm (between 20–80 meshes). Preliminary experiments were run to find the chemicals that can remove more silica form rice straw. For this purpose, the straw was pretreated in 2 M sodium hydroxide (NaOH), 0.5 and 1 M sodium carbonate (Na2CO3), 2 M sodium acetate (CH3COONa) and 0.3 M sodium dodecyl sulfate (NaC12H25SO4) solutions, separately. In a 500 ml flask, 200 g mixture of straw and the pretreatment solutions (1:20 by mass) were prepared and mixed manually every 10 min for 1 h at 0, 25, and 93 °C (boiling water) in a water bath shaker. Finally, they were rinsed with distilled water under vacuum filter to yield neutral pH. To evaluate only the effect of silica removal on the enzymatic hydrolysis yield, organosolv, pretreatment was performed followed by sodium carbonate treatment. This strategy was adopted to minimize the influence of other parameters on the enzymatic hydrolysis. For organosolv treatment, 50 g straw powder was mixed with 400 g of 75 % (v/v) aqueous ethanol and 1% (w/w) sulfuric acid (based on the straw mass) in a high pressure stainless steel reactor with a working volume of 500 ml (Steel Sanat, Isfahan, Iran). The oil bath was used for the heating 4 ACS Paragon Plus Environment


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media by a rate of 3°C/min (Memmert GmbH, Schwabach, Germany). The mixture was maintained at 180 °C for 1h. Then, it was cooled in an ice bath. 13 Then, the pretreated straw was vacuum filtered and rinsed three times with 100 ml of aqueous ethanol (75% (v/v)) at 60 °C and air-dried. In the next step, 4 g of the organosolv pretreated straw was treated with 76 g of 0.5 M sodium carbonate at 93 °C for 1 h. 2.2. Enzymatic hydrolysis Enzymatic hydrolysis of the pretreated and untreated rice straws was performed at 45 ºC and 120 rpm for 72 h in a shaker incubator. A mixture of 5% w/v solid in 0.05 M sodium citrate buffer (pH 4.8) was autoclaved; next, 0.5 g/l sodium azide and the enzymes mixture were added. The enzyme mixture consisted of two commercial hydrolytic enzymes, the cellulase (Celluclast 1.5 L, Novozyme, Denmark) and β-glucosidase (Novozyme 188, Novozyme, Denmark). The activity and protein content of the cellulase were 52 FPU/ml and 42 mg/ml, as determined according to Adney and Baker (1996)14 and Bradford assay, respectively. The activity of β-glucosidase was 240 IU/ml, as measured by the method presented by Ximenes et al. (1999)15. The enzyme loading was 30 FPU cellulase and 60 IU β-glucosidase per gram of dry substrate. Glucose yield was calculated through Eq. (1)16, results are presented in Fig 3. ( )

( )

(1)

( )

where 1.111 is the factor for hydration of cellulose to glucose. 2.3 Microorganism and biomass production The zygomycetes fungus Mucor hiemalis (CCUG 16148) was obtained from the Culture Collection of Gothenburg University (Gothenburg, Sweden). The fungus was maintained on


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YPD medium, containing 40 g/L glucose, 20 g/L agar, and 10 g/L peptone. This fungus was grown at 32±0.5 °C and stored at 4 °C.17 A 100 ml pre-culture medium, containing (g/L): glucose, 50; yeast extract, 5; (NH4)2SO4, 7.5; MgSO4·7H2O, 0.75; K2HPO4, 3.5; and CaCl2·2H2O, 1 at pH 5.5 ± 0.1, was prepared in a 250 mL Erlenmeyer flask for fungal biomass production.

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This medium was autoclaved for 20 min at

121 °C. After cooling to room temperature, 25 ml of 6 × 106 spore/ ml of M. hiemalis was added. Next, this medium was incubated at 32±0.5 °C for 24 h. The biomass of M. hiemalis was removed by centrifugation at 4000 rpm for 15 min (Hettich-Universal 320R, Tuttlingen, Germany) and used for ethanolic fermentation. 2.3. Simultaneous saccharification and fermentation (SSF) Simultaneous saccharification and fermentation of pretreated and untreated rice straw was run at 37 °C and 130 rpm under anaerobic conditions for 72 h. This SSF medium contained (g/l): pretreated or untreated rice straw, 50; yeast extract, 5; (NH4)2SO4, 7.5; K2HPO4, 3.5; MgSO4·7H2O, 0.75; and CaCl2·2H2O, 1, was prepared in 0.05 M buffer citrate (pH 5.5). This mixture was autoclaved at 121 °C for 20 min. After cooling to room temperature, 1 g/l of prepared biomass (based on the dry weight) and 2.5 g/l tween 20 were added.17 The enzyme mixture and loading were the same as that of the enzymatic hydrolysis experiments. Ethanol yield was compared to theoretical yield, Fig (4), which were calculated through Eq. (2)16: ( )

( (

)

(2)

)

2.5. Analytical methods The silica content of untreated and treated straws was analyzed using the method provided by AOAC (1999).19 First 250 mg of rice straw was burned in a furnace at 500 °C for 2 h. Next, the 6 ACS Paragon Plus Environment


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burned straw was added to 5 ml sodium hydroxide (50% w/w) and 2.5 ml hydrogen peroxide (30% w/w) in a falcon 50 mL conical centrifuge tube and mixed gently. The tube was autoclaved at 138 kPa for 1 h and diluted up to 250 ml with distilled water. Then, 0.1 ml ammonium molybdate was mixed with one ml of this solution in another tube. After 10 min, 0.4 ml tartaric acid was mixed with solution by swirling. The prepared solution was added to the tube and diluted to 10 ml with distilled water. Finally, to determine the silica content of this solution, the absorbance of solution was measured by spectrophotometer at 650 nm.19,20 The composition of lignin and carbohydrates of untreated and treated rice straws was determined by the standard method of Sluiter et al. (2008)21. High performance liquid chromatography (HPLC) that was equipped with RI detector (Jasco International Co., Tokyo, Japan) was used for the analysis of the liquid samples from enzymatic hydrolysis, SSF, and the carbohydrates analysis. Ethanol and glycerol were separated on an Aminex HPX-87H column (Bio-Rad, Richmond, CA, USA), eluted through 5 mM H2SO4 with 0.6 ml/min at 60°C. Sugars were separated in an Aminex HPX-87P column (Bio-Rad, Richmond, CA, USA), eluted through 0.6 ml/min deionized water at 60°C. To evaluate the structural changes of the rice straw by the pretreatment, scanning electron microscopy (SEM) was used. The dried samples of untreated and treated straws were coated with gold (BAL-TECSCD 005) and analyzed by SEM (XL30, PHILIPS) at 15 kV. 2.6. Statistical analysis ANOVA test using Tukey method was conducted by SAS (Version 9.2, SAS Institute, Inc., 1999, Cary, NC, USA) to identify significant differences among the means of results at a 5% probability level (P < 0.05). The same lettered group did not have significant differences at a 5% probability level.


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3. Results and discussion Rice straw, having strong and compact structure, covered with silica layer, is highly resistant to enzymatic hydrolysis; therefore, the efficiency of sugar production yields and consequently ethanol is low. To increase the efficiency of ethanol production from this straw, a pretreatment process, with the objective of eliminating the silica from rice straw, was applied. First, to investigate the best silica removal result, various alkaline substances at different concentrations and temperatures were used. Then, the effects of temperature and concentration of the applied pretreatment on silica removal, hydrolysis and ethanol production yield, and straw composition were evaluated. To investigate the sole effect of silica removal and eliminate other factors affecting the hydrolysis, e.g., lignin removal, organosolv pretreatment followed by the pretreatment for silica removal was conducted. Moreover, SEM analysis of the samples was run to investigate the effect of pretreatment on the morphology of the straw. 3.1. Silica removal from rice straw by alkali pretreatment This process involves pretreatments with various alkaline substances at different concentrations and temperatures. The silica content and recovery percentages of untreated and treated straws are tabulated in Table 1. The rice straw used in these experiments contained 5.2% silica (5.9% ash). As observed in Table 1, sodium carbonate and sodium hydroxide treatments had the best efficiency in silica removal at 93°C (boiling water temperature at our laboratory). This can be explained by an increase in the solubility of silica at high pH.22 Sodium carbonate at 93 ºC removed 91% of silica from rice straw, while sodium carbonate at room temperature removed a minor amount of silica.



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Silica of soil is absorbed by rice plant as silicic monoacid, Si(OH)4, and penetrated into the leaves, stalks, and husks. Evaporation and transpiration of the plant makes Si(OH)4 condensed up to its saturation point, thereby polymerizing it into insoluble polysilicic acid, deposited in the outer layer of plant, for protecting the plant from physical and biological attacks.9,11 The silica (SiO2) available in rice straw is changed into sodium silicate in an alkaline medium in the presence of sodium ions at high temperature. Sodium silicate is solubilized in alkaline medium through an endothermic reaction23; therefore, the silica in straw is dissolved and removed from rice straw through alkaline pretreatments. This could be the reason for removal of more than 90% of silica by consuming sodium carbonate and sodium hydroxide solutions at high temperature (Table 1). Inefficient dissolution of silica at low temperatures could be related to the endothermic nature of the reaction. As observed in Table 1, sodium dodecyl sulfate and sodium acetate were not able to remove silica from rice straw, even at high temperatures. Aujla et al.23 evaluated the effect of pH on silica solubility. Their results showed silica turned into sodium silicate only at pH >10 that could be dissolved in the medium, whereas sodium silicate was changed into silicic acid at pH 10 is required for silica to be dissolved. Thus, the inefficacy of sodium dodecyl sulfate and sodium acetate in silica removal, despite having sodium ion even at high temperatures, could be due to their low pH. Pretreatment with 0.5 M sodium carbonate for one hour at 93 °C resulted in 91% silica removal from the straw, which was equal to that by 1M sodium carbonate. It should be noticed that this pretreatment agent should be recovered and reused, otherwise the process consumes a huge


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amount of Na2CO3, resulting in severe environmental problems. Therefore, sodium carbonate concentration higher than 0.5 M may not recommended for silica removal purpose. To produce paper from wheat straw through a preliminary process, Pekarovic et al.

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removed

wheat straw ash by applying sodium carbonate. They eliminated 75% of wheat straw ash at 100 °C. In another study, Pekarovic et al.26 used rice and wheat straws to produce paper in a twophase process and removed 51% silica from rice straw. They used sodium carbonate solution at 90 and 95°C for 30 minutes. Lynamet al.12 pretreated rice husk and eliminated 35% of rice husk ash through ionic HMIMCl and AMIMCl liquids at 110 °C for 8 h. They also removed 60% of rice hull ash by EMIMAC at 90 °C during 8 h. As noticed in this study, the silica removal was more than 90% that is higher than that of the ones obtained when ionic liquids were consumed. The pretreatments carried out on rice straw, in addition to eliminating silica, led to a reduction in lignin; thus, the sole effect of silica removal on improving enzymatic hydrolysis could not be observed. To analyze the impact of silica removal on sugar production from rice straw through enzymatic hydrolysis and eventually ethanol production, first the rice straw was preprocessed with an organic solvent to remove the major part of lignin, and next it was pretreated by sodium carbonate. As observed in Table 1, in the solvent pretreatment, just a small portion of silica was removed from the rice straw, while in the following alkaline pretreatment, 82.3% of silica was removed. As indicated, sodium hydroxide and sodium carbonate were able to remove silica from rice straw at high temperature(s). However, relatively low solid recovery (36.15%) was achieved by sodium hydroxide treatment at 93 °C, in which lignin and other materials dissolves in a significant manner in addition to silica. This fact indicates that the effect of silica on improving 10 ACS Paragon Plus Environment


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enzymatic hydrolysis cannot be detected by pretreatment with sodium hydroxide. Furthermore, alkali pretreatment, particularly with NAOH and KOH at high concentrations and temperatures, typically accompanied with high carbohydrate loss, which is one of the disadvantages of these pretreatments. However, sodium carbonate in comparison to sodium hydroxide causes much lower carbohydrate loss. As observed in Table 2, the lignin and ash content of rice straw were reduced by sodium carbonate pretreatment and the solid recovery of 61% was mainly related to lignin and ash (silica) removal. For this reason, in this study pretreatment with sodium carbonate is of concern. 3.2. Effects of pretreatments on the carbohydrates and lignin content of rice straw The content of glucan (44.9%), xylan (19.9%), and lignin (18.1%) prevail in the rice straw used in this study. According to Table 3, sodium carbonate pretreatment reduced ash and lignin content in rice straw. The straw compositions were also analyzed after the organosolv and organosolv followed by sodium carbonate pretreatment (Table 2). Organosolv pretreatment effectively reduced the lignin content of the straw. An increasing ash percentage was related to the lignin removal; therefore, the organosolv treatment did not affect ash content in the straw. The ash content of the straw was significantly reduced by the secondary pretreatment with sodium carbonate after organosolv pretreatment, while the lignin content was changed only from 11.1% to 10.8%. However, in secondary pretreatment, only silica was removed, indicating the observance of the effects of silica removal on enzymatic hydrolysis and fermentation. These results are consistent with the findings of silica analysis. 3.3. Effect of pretreatment on the straw morphological structure SEM is used to investigate the lignocelluloses morphology, characterization and structures at nanoscale. These images show the cell walls and microfibrils.27 The SEM images of the straw


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treated with 0.5 M Na2CO3 at 93 °C for 1 h as well as untreated straw are obtained to investigate the effects of pretreatment with sodium carbonate on the straw surface characteristics and morphological features, Fig. (1). By comparing the images, the structural destruction of rice straw through the pretreatment is observed, where sodium carbonate pretreatment has removed the silica available on the outer layer of the straw and loosened the compact structure of rice straw, increasing the accessible surface of cellulose for hydrolytic enzymes. 3.4. Enzymatic hydrolysis To determine the temperature effect on silica removal from rice straw and improvement of enzymatic hydrolysis, pretreatment was run on rice straw by sodium carbonate (1 M) at 0, 25, and 93 °C. The glucose yield after 24 and 72 hours of enzymatic hydrolysis are illustrated in bar charts in Figure 2. The best result was reported to be of the high-temperature pretreatment. These results were corresponded to that of the silica analysis results of the pretreated straw. Sodium carbonate pretreatment at 93 °C removed 91% of silica from the straw. The temperature of pretreatment had a significant effect on silica removal and enzymatic hydrolysis. 3.5. Effect of silica removal from rice straw on enzymatic hydrolysis In addition to silica removal, the lignin content of rice straw was reduced by sodium carbonate pretreatment; therefore, to investigate the effect of silica removal on enzymatic hydrolysis, lignin was first removed by organosolv pretreatment. This pretreatment removed most of the straw lignin, while silica was not separated. This was followed by the pretreatment with 0.5 M sodium carbonate at 93 °C for 1 h. This strategy was used to minimize the effects of other straw properties’ changes and highlight the effects of silica removal. Glucose production via the hydrolysis was increased from 8.6 (untreated straw) to 16.2 g/l by the organosolv pretreatment,



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while it was increased to 31.9 g/l by further treatment with sodium carbonate. The glucose conversion and concentration were 88.0% and 24.4 g/l, respectively, from the rice straw pretreated with 0.5M sodium carbonate for 1 h.28 Silica in the outer layer of rice straw, protects the internal components, like armor, thereby making the straw difficult to hydrolysis.12 Higher improvement of hydrolysis conversion results after secondary pretreatment by sodium carbonate indicated the effect of silica removal was greater than that of lignin. Silica removal from rice straw in the secondary treatment causing more enzyme accessibility to cellulose, resulting in more glucose yield. As observed in Fig (3), organosolv pretreatment increased the hydrolysis yield from 34.4% for untreated straw to 52.9%, while further sodium carbonate treatment increased it to 94.8%. Obama et al.13 pretreated a lignocellulosic material by organosolv and reported 45% glucose production. The conditions used in the current study were the same as the ones in that study. Furthermore, Salehi et al.29 pretreated rice straw in a high-pressure reactor at high temperature with sodium carbonate and obtained over 90% hydrolysis efficiency. 3.6. Simultaneous saccharification and fermentation (SSF) To investigate the effect of silica removal from rice straw on bioethanol production, SSF was run by M. hiemalis. For the untreated straw, ethanol yield was only 39.3%. This yield was increased to 51.6% by organosolv treatment, whereas a higher yield of 78.7% was achieved by secondary treatment with sodium carbonate. In the previous study, the ethanol yield and concentration were 90.5 % and 12.8 g/lf rom the straw pretreated with 0.5 M sodium carbonate for 1 h, respectively. 28

The results of SSF correspond to those of enzymatic hydrolysis.


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4. Conclusions Silica is a protective layer in rice straw and attempt was made in this study to remove silica and determine its effects on hydrolysis and ethanol production. Sodium carbonate and sodium hydroxide were able to efficiently remove silica from rice straw, particularly at high temperatures (91% silica removal). It was found that removing silica from rice straw led to a significant increase in glucose and ethanol production. After organosolv pretreatments for lignin removal followed by sodium carbonate pretreatment especially for silica removal, a high ethanol yield of 78.7% was obtained while ethanol yield for untreated and treated straw subject to organosolv pretreatment only increased from 39.3 to 51.6%, respectively. Thus, it was concluded that silica layer play an important role in protecting rice straw from enzymatic hydrolysis. Supporting information For comparison of silica analysis, in addition to method provided by AOAC (1999), a selected number of samples were analyzed for silica content using ICP (Inductively Coupled Plasma) method. References 1. Taherzadeh, M. J., and Karimi, K., "Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: A review, Int. J. Mol. Sci., 2008, 9, 1621-1651. 2. Karimi, K.; Kheradmandinia, S.; Taherzadeh, M.J. Conversion of rice straw to sugars by diluteacid hydrolysis. Biomass. Bioenerg. 2006, 30, 247-253. 3. Karimi, K.; Emtiazi, G.; Taherzadeh, M.J. Production of ethanol and mycelial biomass from rice straw hemicellulose hydrolyzate by Mucor indicus. Process. Biochem. 2006, 41, 653-658. 4. Clark A.J. Biodegradation of cellulose: Enzymeology and Biothechnology, Technomic Publishing Co., Lanchester, 1997. 5. Sun, Y.; Cheng, J. Hydrolysis of lignocellulosic materials for ethanol production: review. Bioresource. Technol. 2002, 83,1,1-11. 6. Nag, A. Biofuels refining and performance, Mc Graw Hill: United States, 2008.



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7. Alvira, P.; Tomas-Pejo, E.; Ballesteros, M.; Negro, M.J. Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: A review. Bioresource. Technol, 2010, 101, 4851-4861. 8. Saha, B.C. Hemicellulose bioconversion. J. Ind. Microbiol. Biot. 2003, 30, 279-291. 9. Van Soest, P.J. Review: Rice straw, the role of silica and treatments to improve quality. Anim. Feed. Sci. Tech. 2006, 130, 137-171. 10. Binod, P.; Sindhu, R.; Singhania, R.R.; Vikram, S.; Devi, L.; Nagalakshmi, S.; Kurien, N.; Sukumaran, R.K.; Pandey, A. Bioethanol production from rice straw: An overview. Bioresource. Technol. 2010, 101, 4767-4774. 11. Agbagla-Dohnani, A.; Noziere, P.; Gailard-Martine, B.; Puard, M.; Doreau, M. Effect of silica content on rice straw ruminal degradation. J. Agrl. Sci. 2003, 140, 183-192. 12. Lynam, J.G.; Reza, M.T.; Vasquez V.R.; Coronella, C.j. Pretreatment of rice hulls by ionic liquid dissolution. Bioresource Technol. 2012, 114, 629-636. . 13. Obama, P.; Ricochon, G.; Muniglia, L.; Brosse, N. Combination of enzymatic hydrolysis and ethanol organosolv pretreatments: Effect on lignin structures, delignification yieldsand cellulose-toglucose conversion. Bioresource. Technol. 2012, 112, 156-163. 14. Adney, B.; Baker, J. Measurement of cellulase activities. National Renewable Energy Laboratory, Laboratory Analytical Procedure (LAP), 1996. 15. Ximenes, E.A.; Felix, C.R.; Ulhoa, C.J. Production of cellulases by Aspergillusfumigatus and characterization of one -glucosidase. Curr. Microbiol. 1996, 32,119–123. 16. Poornejad, N.; Karimi, K.; Behzad, T. Improvement of saccharification and ethanol production from rice straw by NMMO and [BMIM][OAc] pretreatments. Ind. Crop. Prod. 2013, 41, 408–413. 17. Karimi, K.; Emtiazi, G.; Taherzadeh, M.J. Ethanol production from dilute-acid pretreated rice straw by simultaneous saccharification and fermentation with Mucor indicus, Rhizopus oryzae, and Saccharomyces cerevisiae. Enzyme Microb Technol. 2006, 40, 138–144. 18. Goshadrou, A.; Karimi, K.; Taherzadeh, M.J. Bioethanol production fromsweet sorghum bagasse by Mucor hiemalis. Ind. Crop. Prod. 2011, 34, 1219–1225. 19. AOAC, Official Methods of Analysis, 16th ed. Association of Official AnalyticalChemists: Washington DC, 1999. 20. Elliott, C. L.; Snyder, G.H. Autoclave-Induced digestion for the colorimetricdetermination of silicon in rice straw. J. Agric. Food Chem. 1991, 39, 1118–1119. 21. Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Templeton, D.; Sluiter, J.; Crocker, D. Determination of structural carbohydrates and lignin in biomass. Lab. Anal. Proced. 2008, 510–42618, NREL/TP. 22. Minu, K.; K.Kurian, J.; Kishore, V.V.N. Isolation and purification of lignin and silica from the black liquer generated during the production of bioethanol from rice straw. Biomass Bioenergy. 2012, 39, 210-217. 23. Aujla, M.I.; Rahman, I.; Javaid, A. Mechanism of silica precipitation by lowering pH in chemithermomechanical pulping black liquors. 1st WSEAS International Conference on Computational Chemistry: USA. 2007, 58-62. 24. Inglesby, M.K.; Gray, G.M.; Wood, D.F.; Gregorski, K.S.; Robertson, R.G.; Sabellano, G.P. Surface characterization of untreated and solvent-extracted rice straw. Colloid. Surface. B. 2005, 43, 8394.


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25. Pekarovic, J.; Pekarovicova, A.; Joyce, T.W. Desilication of agricultural residues - the first step prior to pulping. Appita J. 2005, 58, 130-134. 26. Pekarovic, J.; Pekarovicova, A.; Fleming III, P.D. Two-step straw processing, a new concept of silica problem solution. Engineering, Pulping, and Environmental Conference: Atlanta, USA. 2006. 27. Karimi, K., Taherzadeh, M.J., A critical review of analytical methods in pretreatment of lignocelluloses: composition, imaging, and crystallinity, Bioresource. Technol. , 2016, 200, 1008–1018. 28. Khaleghian, H., K. Karimi, and T. Behzad. Ethanol production from rice straw by sodium carbonate pretreatment and Mucor hiemalis fermentation. Ind. Crop. Prod, 2015. 76, 1079-1085. 29. Salehi, S.M.A.; Karimi, K.; Behzad, T.; Poornejad, N. Efﬁcient conversion of rice straw to bioethanol using sodium carbonate pretreatment. Energy. Fuels. 2012, 26, 7354–7361.



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Page 18 of 24

Figure captions Fig. 1. SEM images: (a) untreated rice straw and (b) the straw treated with 0.5 M Na2CO3 for 1 h Fig. 2. Glucose yield after 24 h (

) and 72 h (

) enzymatic hydrolysis of untreated rice straw

and the straw treated with sodium carbonate at 0, 25, and 93 °C Fig. 3. Enzymatic hydrolysis yield of untreated rice straw, organosolv treated straw, and organosolv followed by sodium carbonate treated straw after 24 (

) and 72 hours (

enzymatic hydrolysis. Fig. 4. SSF yield of untreated rice straw, organosolv treated straw, and organosolv followed by sodium carbonate treated straw at 93 °C for 1 h.


)

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. Solid recovery in the pretreatments and silica content of the pretreated rice straw No.

Pretreatment chemical

Temperature (ºC)

Time (h)

Solid recovery (%)

SiO2 content (%)

Silica removal (%)

-

-

-

5.2±0.3

-

Sodium carbonate (1M)

93

1

61

0.8±0.3

91

3


25

1

82.5

5.5±0.2

10

4


0

1

80.5

6.0±0.4

4

5

Sodium hydroxide (2M)

93

1

36.2

1.3±0.2

90

6


25

1

50.8

5.0±0.2

50

7


0

1

62.3

5.3±0.3

34

8

Sodium dodecyl sulfate(0.3M)

93

1

80.9

6.3±0.5

0

9


25

1

85.3

5.9±0.5

0

10


0

1

88.1

5.9±0.3

0

11

Sodium carbonate (0.5M)

93

1

61.0

0.8±0.2

91

12

Sodium acetate (1M)

93

1

83.8

5.4±0.4

10

1


2

13

Sodium acetate (2M)

93

1

83.5

5.3±0.4

13

14

Organosolv

180

1

67.7

6.9±0.2

7.4

93

1

45.7

1.8±0.5

82.3

Organosolv + 15 sodium carbonate 0.5 M



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 24

Table 2 . Compositional analysis of untreated and pretreated rice straw Pretreatments

Other carbohydrates Ash (%)

Lignin (%)

Conditions

Glucan (%)

Xylan (%) (%)

Untreated

5.9±0.3A 18.1±0.6A

44.9±0.5 A 19.7±0.5A

3.7±0.6 A

Sodium carbonate

1.3±0.4B 13.1±0.5B

49.5±0.9 B

20.1±0.8A

4.7±0.3 A

7.7±0.3 C

55.5±0.6 C

21.3±0.5 A

0.5M, 93 ºC, 1h Organosolv Organosolv + sodium

1.1±0.1

B

11.1±0.2 B 10.8±0.5

B

60.6±0.7

D

20.1±0.6

carbonate pretreatment


A

5.6±0.5 A 3.9±0.6

A

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Fig. 1.



100

A A

90 80 Glucose yield (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 24

70 60

B

50 40

B C

B

BC

B

30 20 10 0 Untreated

0

25

Temperature (°C )

Fig. 2.


93

Page 23 of 24

A

100

A

90 80 Glucose yield (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


70

B

60

B

50 40

B

C

30 20 10 0 Untreated

Organosolv

Fig. 3.


Organosolv+ Sodium carbonate 0.5 M


90

A

80 70 Ethanol yield (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

B

60 50

C

40 30 20 10 0 Untreated

Organosolv

Organosolv+ Sodium carbonate 0.5 M

Fig. 4.


Page 24 of 24

Silica Removal from Rice Straw To Improve its Hydrolysis and Ethanol

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