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Using trifluoroacetic acid to pretreat lignocellulosic biomass
biomass and bioenergy 33 (2009) 1719–1723
Available at www.sciencedirect.com
http://www.elsevier.com/locate/biombioe
Short communication
Using trifluoroacetic acid to pretreat lignocellulosic biomass Dexian Dong1,*, Jie Sun1, Feiyun Huang, Qian Gao, Yi Wang, Rongxiu Li** MOE Key Laboratory for Microbial Metabolism, College of Life Science and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, PR China
article info
abstract
Article history:
Biomass pretreatment is one of major bottlenecks to convert biomass to bioethanol at
Received 17 July 2008
present. We found that cereal straw could be completely dissolved in a 10-fold volume
Received in revised form
excess of trifluoroacetic acid (99%) (TFA). Pretreatment with TFA completely disrupted the
6 July 2009
dense cellulose crystallinity of the biomass. Further research showed that the TFA
Accepted 9 July 2009
dissolved, but did not degrade, the cellulose in the straw. TFA did, however, degrade 65.65%
Available online 5 August 2009
of xylan in the straw, as well as reduce 20.0–23.3% of the acid-insoluble lignin. Isopropanol could precipitate 92.7% of the material dissolved in the TFA solution. These results led us to
Keywords:
design a procedure for pretreatment of cellulosic biomass involving a dissolving step with
wheat
TFA and a precipitation step with isopropanol. Experiments show that the procedure is
Rice
technically feasible. Moreover, TFA and isopropanol could be completely evaporated off the
Maize
supernatant and precipitate, and recycled back into the process. ª 2009 Elsevier Ltd. All rights reserved.
Sabai grass Straw Stover Xylan Cellulose Lignin Isopropanol Pichea pastoris Trifluoroacetic acid Pretreatment Lignocellulosic Biomass conversion
1.
Introduction
High demands for energy, dwindling petroleum sources, and concern over global climate change have led to the pursuit of alternative energy sources. In response, many countries have
initiated extensive research into cellulosic ethanol, which involves fermenting lignocellulosic biomass to ethanol. Lignocellulosic biomass is a sustainable and renewable source of mixed sugars and includes straw, corn stover, sugarcane residue, wood chip, waste paper, miscanthus, and switch
* Corresponding author. Tel./fax: þ86 21 34204207. ** Corresponding author. E-mail addresses: [email protected] (D. Dong), [email protected] (R. Li). 1 These authors contributed equally to this work. 0961-9534/$ – see front matter ª 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biombioe.2009.07.013
1720
biomass and bioenergy 33 (2009) 1719–1723
grass. Current biomass conversion technology comprises three major steps: thermochemical pretreatment, enzymatic hydrolysis, and sugar fermentation to ethanol. In 2004, Genencor International announced a 30-fold reduction in the cost of enzymes to a range of 26–53 $ m-3 and Novozymes Biotech had also progressed in reducing enzyme costs from 1320 to 79 $ m-3 [1]. This was a breakthrough for the enzymatic hydrolysis step, and it made the pretreatment step the major economic obstacle in cellulosic biomass conversion. Because of the chair conformation of the glucose residues in cellulose and the precise arrangement of cellodextrin chains, the crystalline cellulose core of cell-wall microfibrils [2,3] is very dense, highly structured, and strongly resistant to enzymatic hydrolysis. Many biological, chemical, and physical methods such as steam explosion, ammonia explosion, sulfuric acid, and 80% phosphoric acid [4–6] have been used to disrupt cellulose crystallinity and increase accessibility of the cellulose to hydrolysis by cellulase. However, the steam or ammonia explosion method consumes a lot of energy and is ineffective. Methods involving sulfuric acid or 80% phosphoric acid are unsuitable for industrial production because the acids are difficult to recycle and they would cause severe environmental pollution. By screening a series of organic solvents for dissolving straw, we found trifluoroacetic acid (99%) (TFA) to be highly effective to disrupt cellulose crystallinity. TFA is a volatile liquid solvent used frequently in organic synthesis because of its volatility, acid strength, and miscibility with most organic solvents. The most attractive feature of TFA is that it can be recycled at lower operating temperatures because its boiling point is 72.4 C. There were some reports to indicate that dilute TFA hydrolysis resulted in the highest amount of overall soluble monosaccharides and the least monosaccharide degradation among TFA, HCl, H2SO4, HNO3, H3PO4 and/or KOH (with Loblolly Pine [7], forage grass [8], Arabidopsis thaliana [9] and wheat straw [10]). Morrison and Stewart [11] found that the oat straw in 99% TFA at 37 C did not indicate any major depolymerization of the cellulose molecules during the first 8 days. Fanta et al. [10] found that the wheat straw with 1N TFA for 7 h yielded 80% hemicellulose degradation and 10% lignin degradation. The cellulose component of wheat straw was largely unaffected. In addition, TFA has been used to decrystallize pure cellulose at 0 C [12]. Here we report our results using TFA (99%) to pretreat straw.
2.
Experimental section
2.1.
Materials
Microcrystalline cellulose Avicel PH105 was from FMC, USA. Xylan (degree of polymerization 2–7) was from Longlive Biotech Ltd. (China); its composition was 89.78% xylose, 6.47% arabinose, and 3.75% glucose. Its mean molecular weight was calculated to be 89.78% 150.13 þ 6.47% 150.13 þ 3.75% 180.16 ¼ 153.26. TFA (99%) was from Shanghai Jiachen Chemical Ltd. (China). Other chemicals were AR grade and were from Sinopharm Chemical Reagent Co. Ltd.
The wheat straw and rice straw were from the general cultivars grown in Danling county of Sichuan Province, China. The corn straw was from the general cultivar grown in Nantong county of Jiangsu Province, China. The sabai grass was the wild grass grown in Shanghai, China. All the straws were sundried and shipped to lab. All the stems and leaves were cut into 2.5-cm fragments and then dried at 50 C for further application.
2.2.
Determination of reducing sugar amount
The molar amount of reducing sugar was determined using the classical method involving 3,5-dinitrosalicylic acid (DNS) [13]. DNS reagent (2.0 ml) was added to 1.0 ml of test sample in a microcentrifuge tube. The mixture was heated at 90 C for 5 min to develop the red-brown color. After cooling to room temperature, the mixture was diluted with 9.0 ml of water. Absorbance at 540 nm was measured with a spectrophotometer. The molar amount of reducing sugar was calculated using a glucose calibration curve.
2.3.
Determination of acid-insoluble lignin
Lignin is mostly insoluble in mineral acids. Acid-insoluble lignin can be analyzed gravimetrically after hydrolyzing the cellulose and hemicellulose fractions with sulfuric acid [14]. Air-dried test sample (100 mg) was mixed with 1.00 ml of 72% (v/v) H2SO4. The mixture was placed in a water bath at 30 C and stirred frequently. After 2 h the samples were mixed with 28 ml of water and autoclaved at 121 C for 1 h. The residue was filtered and washed with water to remove acid. The samples were dried at 105 C, weighed, and acid-insoluble lignin content was expressed as mass percent of the original sample.
2.4.
Measurement of TFA
The lanthanum–alizarin complexone (La3þ–ALC) method is known to be one of the most reliable techniques for detecting fluoride ion in aqueous solutions [15]. To determine TFA amount in the supernatant and precipitate, 0.05 g of the residue from the supernatant or the precipitate was put into a bottle containing oxygen, burned up with Whatman No. 1 filter paper, and dissolved in 37.5 ml of deionized water. Sample solution (15.0 ml) was added, together with 10.0 ml of alizarin complexone, 3.0 ml of 12% (w/w) NaAc and 10 ml of La(NO3)3. After 1 h, the absorbance of the mixture (pH 4.5) was measured at 620 nm. The standard curve was determined using 0.3, 0.6, 0.9, 1.2, and 1.5 mg ml1 NaF solution.
3.
Results and discussion
3.1.
Dissolving wheat straw with TFA
The wheat straw was cut into 2.5-cm fragments and dried at 50 C. Straw fragments (10.0 g) were added to a round-bottom flask containing 100 ml of trifluoroacetic acid (TFA) and incubated for 16 h at 60 C in a paraffin oil bath. Unexpectedly, we found that the mixture became black and sticky and the
biomass and bioenergy 33 (2009) 1719–1723
1721
wheat straw was completely dissolved. No pellet was obtained after centrifugation at 10,000 rpm. This phenomenon indicated that TFA completely dissolved all the cellulose, hemicellulose, and lignin components of wheat straw. To confirm this new finding, 1.0 g of microcrystalline cellulose Avicel PH105 and 1.0 g of xylan were added to TFA. Both the cellulose and xylan dissolved completely.
3.2.
Isopropanol precipitation of the TFA solution
Using the microcrystalline cellulose Avicel PH105, we examined the ability of different solvents to precipitate cellulose dissolved in TFA. Different volumes of methanol, 1-propanol, isopropanol, 1-butanol, and 2-butanol were tested. For each precipitation trial, 2.0 g of microcrystalline cellulose Avicel PH105 were dissolved in 20.0 ml of TFA, the organic solvent was added, and the mixtures were centrifuged at 10,000 rpm for 10 min. Pellets were dried at 50 C and weighed. Results showed that both isopropanol and 2-butanol could precipitate more than 77% of the dissolved cellulose. Almost all (92.7%) of the cellulose could be precipitated with isopropanol at a concentration of 0.5 volumes. Since the boiling points of isopropanol (82 C) and TFA (72.4 C) are lower than that of 2-butanol (98 C), we chose to use isopropanol because it would allow for more energy-efficient fractional distillation.
3.3.
Ability of TFA to degrade cellulose, xylan, and lignin
To determine whether TFA degraded cellulose and xylan, 10.0 ml of TFA was mixed with the following materials: 1.0 g of microcrystalline cellulose Avicel PH105, 1.0 g of xylan, 1.0 g of wheat straw, 1.0 g of rice straw, 1.0 g of corn stover, or 1.0 g of sabai grass (Eulaliopsis binata). After addition of 5.0 ml of isopropanol, the mixtures were centrifuged at 10,000 rpm for 10 min. Tris (2 molL1, 0.9 ml) was added to a portion (0.1 ml) of the supernatant, and this mixture was used as the test sample to determine the molar amount of reducing sugar. Results are shown in Fig. 1A. The molar amount of reducing sugar in the supernatant of TFA-treated cellulose was 0.037 mmolL1. According to the molecular weight of glucose (180.16), the degradation extent of the microcrystalline cellulose was calculated to be 0.66%. These results show that TFA dissolved, but did not degrade, the cellulose. The molar amount of reducing sugar in the supernatant of TFA-treated xylan was 4.34 mmolL1. Given the mean molecular weight of xylan (151.26), the degradation extent of xylan was calculated to be 65.65%. The possible presence of some xylan disaccharide in the TFA solution may explain why the xylan was not completely degraded to reducing sugar. These results show that xylan was partially degraded by TFA. The molar amounts of reducing sugar of rice straw, wheat straw, corn stover and sabai grass were, respectively, 0.932 mmolL1, 0.878 mmolL1, 1.153 mmolL1, and 0.823 m molL1. Hemicellulose is the major component in cereal straws that TFA degrades, and it is composed of xylose, arabinose, glucose, and mannose. Taking into account the molecular weights of xylose (150.13), arabinose (150.13), glucose (180.16), and mannose (180.16), the extent of degradation of rice straw, wheat straw, corn stover, and sabai grass were calculated, respectively, to be 14–17%, 13–16%, 17–21%, and 12–15% [the first percentage (14, 13, 17, 12) was multiplied
Fig. 1 – The ability of TFA to degrade cellulose, xylan, and lignin. (A) Degradation rates of TFA-pretreated cellulose, xylan, rice straw, wheat straw, corn stover, and sabai grass. The tops of upper stack histograms (sparse patterns) stand for the percentages calculated by the molecular weight 180.16 and the bottoms of upper stack histograms stand for the percentages calculated by the molecular weight 150.13. The degradation rates (vertical axis) were calculated by the molar amount of reducing sugar 3 the molecular weight of reducing sugar (180.16 or150.13) O sample weight (1.0 g); (B) Acid-insoluble lignin content of rice straw, wheat straw, corn stover, and their precipitates after TFA pretreatment and isopropanol precipitation. Pr, Pw, Pc: precipitates of rice straw, wheat straw, and corn stover, respectively.
by 150.13 and the last percentage (17, 16, 21, 15) was multiplied by 180.16]. The lignin in cereal straws is composed of acid-insoluble lignin, which is the major component (about 29.1–33.5%), and acid-soluble lignin [1.8% in rice straw, 1.0% in wheat straw, 1.3% in corn stover measured by the OD205 method [14]. To determine whether TFA degraded acid-insoluble lignin, samples of rice straw, wheat straw, corn stover and their precipitates after TFA pretreatment and isopropanol precipitation were dried at 50 C. Their content of acid-insoluble lignin was measured by the method described above, and the
1722
biomass and bioenergy 33 (2009) 1719–1723
results are shown in Fig. 1B. TFA pretreatment caused the loss of 6.2% of acid-insoluble lignin (31.0% 24.8%) in rice straw, 7.5% (33.5% 26.0%) in wheat straw, and 6.8% (29.1% 22.3%) in corn stover. These results indicate that TFA degraded 20.0–23.3% of the acid-insoluble lignin.
3.4. The possibility of TFA pretreatment of cellulosic biomass To utilize this TFA procedure for the pretreatment of cellulosic biomass, we designed the process shown in Fig. 2. After the cereal straw is broken into short fragments using a cracker, the straw fragments are dissolved in 10 volumes of TFA. Then 0.5 volumes of isopropanol are added to the TFA solution to precipitate the materials dissolved in the TFA. The supernatant and precipitate are separated by filtration. After evaporating the TFA and isopropanol from the mixture, the supernatant and precipitate are used for further enzymatic hydrolysis to produce bioethanol or cellulosic products. The mixture of TFA and isopropanol can be fractionally distilled and recycled back into the process. Fig. 3 shows the precipitation of isopropanol, the dry precipitate obtained by filtration, and the residue left after the TFA and isopropanol were evaporated off the supernatant. The results indicate that using isopropanol to precipitate material dissolved in TFA is feasible. The key step in this process is that the TFA and isopropanol must be completely evaporated off the supernatant and precipitate. Using the La3þ–ALC method, the residual TFA amounts in the supernatant and precipitate were measured to be 0.0041% and 0.0032%, respectively. Their pH values (6.02, 6.04) were very close to that of tap water (6.12). These results confirmed that TFA can be completely evaporated off the supernatant and precipitate, which can then be used safely in downstream processes. Using the M9 mineral plates plus the TFA reaction supernatant, we successfully cultured the Pichia pastoris GS115 (data not shown). Thus, utilizing TFA for the pretreatment of cellulosic biomass is feasible.
Fig. 2 – Technical process of TFA pretreatment of cellulosic biomass.
Fig. 3 – Precipitation of isopropanol, the residue, and precipitate products of TFA-pretreated wheat straw.
Acknowledgement This work was supported by the Municipal Research Program of Shanghai Nos. 072312048, 0839191100, 08DZ1204400, the High-Tech Research and Development Program of China (863 Program) Nos. 2006AA02Z228, 2007AA02Z215, 2009ZX09306008, 2007AA100506, National Basic Research Program of China (973 Program) No. 2007CB936004, the Key Program of Natural Science Foundation of China No.30630012 and Shanghai Leading Academic Discipline Project No.B203.
references
[1] Greer D. Spinning straw into fuel. BioCycle 2005;46:61–4. [2] Nishiyama Y, Langan P, Chanzy H. Crystal structure and hydrogen bonding system in cellulose ibeta from synchrotron X-ray and neutron fiber diffraction. J Am Chem Soc 2002;124:9074–82. [3] Matthews JF, Skopec CE, Mason PE, Brady JW. Computer simulation studies of microcrystalline cellulose Ib. Carbohydr Res 2006;341:138–52. [4] Wyman CE, Dale BE, Elander RT, Holtzapple M. Coordinated development of leading biomass pretreatment technologies. Bioresour Technol 2005;96:1959–66. [5] Chandra RP, Bura R, Saddler JN. Substrate pretreatment: the key to effective enzymatic hydrolysis of lignocellulosics? Adv Biochem Engin Biotechnol 2007;108:67–93. [6] Zhang YHP, Cui J, Lynd LR. A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid: evidence from enzymatic hydrolysis and supramolecular structure. Biomacromolecules 2006;7:644–8. [7] Marzialetti T, Valenzuela Olarte MB, Sievers C, Hoskins TJC, Agrawal PK, Jones CW. Dilute acid hydrolysis of loblolly pine: a comprehensive approach. Ind Eng Chem Res 2008;47:7131–40. [8] De Ruiter JM, Burns JC. Characterization of trifluoroacetic acid hydrolyzed subtropical forage grass cell walls. J. Agric. Food Chem. 1987;35:308–16. [9] Gardner SL, Burrell MM, Fry SC. Screening of Arabidopsis thaliana stems for variation in cell wall polysaccharides. Phytochemistry 2002;60:241–54. [10] Fanta GF, Abbott TP, Herman AI, Burr RC, Doane WM. Hydrolysis of wheat straw hemicellulose with trifluoroacetic
biomass and bioenergy 33 (2009) 1719–1723
acid. Fermentation of xylose with Pachysolen tannophilus. Biotechnol. Bioeng 1984;26:1122–5. [11] Morrison IM. Hydrolysis of plant cell walls with trifluoroacetic acid. Phytochemistry 1988;27:1097–100. [12] Zhao HB, Holladay JE, Kwak JH, Zhang ZC. Inverse temperature-dependent pathway of cellulose decrystallization in trifluoroacetic acid. J Phys Chem B 2007; 111:5295–300. [13] Sengupta S, Jana ML, Sengupta D, Naskar AK. A note on the estimation of microbial glycosidase activities by
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dinitrosalicylic acid reagent. Appl Microbiol Biotechnol 2000; 53:732–5. [14] Ganesh KA, Sekaran G, Krishnamoorthy S. Solid state fermentation of Achras zapota lignocellulose by Phanerochaete chrysosporium. Bioresour Technol 2006;97: 1521–8. [15] Wada H, Mori H, Nakagawa G. Spectrophotometric determination of fluoride with lanthanum/alizarin complexone by flow injection analysis. Anal Chim Acta 1985; 172:297–302.
Available at www.sciencedirect.com
http://www.elsevier.com/locate/biombioe
Short communication
Using trifluoroacetic acid to pretreat lignocellulosic biomass Dexian Dong1,*, Jie Sun1, Feiyun Huang, Qian Gao, Yi Wang, Rongxiu Li** MOE Key Laboratory for Microbial Metabolism, College of Life Science and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, PR China
article info
abstract
Article history:
Biomass pretreatment is one of major bottlenecks to convert biomass to bioethanol at
Received 17 July 2008
present. We found that cereal straw could be completely dissolved in a 10-fold volume
Received in revised form
excess of trifluoroacetic acid (99%) (TFA). Pretreatment with TFA completely disrupted the
6 July 2009
dense cellulose crystallinity of the biomass. Further research showed that the TFA
Accepted 9 July 2009
dissolved, but did not degrade, the cellulose in the straw. TFA did, however, degrade 65.65%
Available online 5 August 2009
of xylan in the straw, as well as reduce 20.0–23.3% of the acid-insoluble lignin. Isopropanol could precipitate 92.7% of the material dissolved in the TFA solution. These results led us to
Keywords:
design a procedure for pretreatment of cellulosic biomass involving a dissolving step with
wheat
TFA and a precipitation step with isopropanol. Experiments show that the procedure is
Rice
technically feasible. Moreover, TFA and isopropanol could be completely evaporated off the
Maize
supernatant and precipitate, and recycled back into the process. ª 2009 Elsevier Ltd. All rights reserved.
Sabai grass Straw Stover Xylan Cellulose Lignin Isopropanol Pichea pastoris Trifluoroacetic acid Pretreatment Lignocellulosic Biomass conversion
1.
Introduction
High demands for energy, dwindling petroleum sources, and concern over global climate change have led to the pursuit of alternative energy sources. In response, many countries have
initiated extensive research into cellulosic ethanol, which involves fermenting lignocellulosic biomass to ethanol. Lignocellulosic biomass is a sustainable and renewable source of mixed sugars and includes straw, corn stover, sugarcane residue, wood chip, waste paper, miscanthus, and switch
* Corresponding author. Tel./fax: þ86 21 34204207. ** Corresponding author. E-mail addresses: [email protected] (D. Dong), [email protected] (R. Li). 1 These authors contributed equally to this work. 0961-9534/$ – see front matter ª 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biombioe.2009.07.013
1720
biomass and bioenergy 33 (2009) 1719–1723
grass. Current biomass conversion technology comprises three major steps: thermochemical pretreatment, enzymatic hydrolysis, and sugar fermentation to ethanol. In 2004, Genencor International announced a 30-fold reduction in the cost of enzymes to a range of 26–53 $ m-3 and Novozymes Biotech had also progressed in reducing enzyme costs from 1320 to 79 $ m-3 [1]. This was a breakthrough for the enzymatic hydrolysis step, and it made the pretreatment step the major economic obstacle in cellulosic biomass conversion. Because of the chair conformation of the glucose residues in cellulose and the precise arrangement of cellodextrin chains, the crystalline cellulose core of cell-wall microfibrils [2,3] is very dense, highly structured, and strongly resistant to enzymatic hydrolysis. Many biological, chemical, and physical methods such as steam explosion, ammonia explosion, sulfuric acid, and 80% phosphoric acid [4–6] have been used to disrupt cellulose crystallinity and increase accessibility of the cellulose to hydrolysis by cellulase. However, the steam or ammonia explosion method consumes a lot of energy and is ineffective. Methods involving sulfuric acid or 80% phosphoric acid are unsuitable for industrial production because the acids are difficult to recycle and they would cause severe environmental pollution. By screening a series of organic solvents for dissolving straw, we found trifluoroacetic acid (99%) (TFA) to be highly effective to disrupt cellulose crystallinity. TFA is a volatile liquid solvent used frequently in organic synthesis because of its volatility, acid strength, and miscibility with most organic solvents. The most attractive feature of TFA is that it can be recycled at lower operating temperatures because its boiling point is 72.4 C. There were some reports to indicate that dilute TFA hydrolysis resulted in the highest amount of overall soluble monosaccharides and the least monosaccharide degradation among TFA, HCl, H2SO4, HNO3, H3PO4 and/or KOH (with Loblolly Pine [7], forage grass [8], Arabidopsis thaliana [9] and wheat straw [10]). Morrison and Stewart [11] found that the oat straw in 99% TFA at 37 C did not indicate any major depolymerization of the cellulose molecules during the first 8 days. Fanta et al. [10] found that the wheat straw with 1N TFA for 7 h yielded 80% hemicellulose degradation and 10% lignin degradation. The cellulose component of wheat straw was largely unaffected. In addition, TFA has been used to decrystallize pure cellulose at 0 C [12]. Here we report our results using TFA (99%) to pretreat straw.
2.
Experimental section
2.1.
Materials
Microcrystalline cellulose Avicel PH105 was from FMC, USA. Xylan (degree of polymerization 2–7) was from Longlive Biotech Ltd. (China); its composition was 89.78% xylose, 6.47% arabinose, and 3.75% glucose. Its mean molecular weight was calculated to be 89.78% 150.13 þ 6.47% 150.13 þ 3.75% 180.16 ¼ 153.26. TFA (99%) was from Shanghai Jiachen Chemical Ltd. (China). Other chemicals were AR grade and were from Sinopharm Chemical Reagent Co. Ltd.
The wheat straw and rice straw were from the general cultivars grown in Danling county of Sichuan Province, China. The corn straw was from the general cultivar grown in Nantong county of Jiangsu Province, China. The sabai grass was the wild grass grown in Shanghai, China. All the straws were sundried and shipped to lab. All the stems and leaves were cut into 2.5-cm fragments and then dried at 50 C for further application.
2.2.
Determination of reducing sugar amount
The molar amount of reducing sugar was determined using the classical method involving 3,5-dinitrosalicylic acid (DNS) [13]. DNS reagent (2.0 ml) was added to 1.0 ml of test sample in a microcentrifuge tube. The mixture was heated at 90 C for 5 min to develop the red-brown color. After cooling to room temperature, the mixture was diluted with 9.0 ml of water. Absorbance at 540 nm was measured with a spectrophotometer. The molar amount of reducing sugar was calculated using a glucose calibration curve.
2.3.
Determination of acid-insoluble lignin
Lignin is mostly insoluble in mineral acids. Acid-insoluble lignin can be analyzed gravimetrically after hydrolyzing the cellulose and hemicellulose fractions with sulfuric acid [14]. Air-dried test sample (100 mg) was mixed with 1.00 ml of 72% (v/v) H2SO4. The mixture was placed in a water bath at 30 C and stirred frequently. After 2 h the samples were mixed with 28 ml of water and autoclaved at 121 C for 1 h. The residue was filtered and washed with water to remove acid. The samples were dried at 105 C, weighed, and acid-insoluble lignin content was expressed as mass percent of the original sample.
2.4.
Measurement of TFA
The lanthanum–alizarin complexone (La3þ–ALC) method is known to be one of the most reliable techniques for detecting fluoride ion in aqueous solutions [15]. To determine TFA amount in the supernatant and precipitate, 0.05 g of the residue from the supernatant or the precipitate was put into a bottle containing oxygen, burned up with Whatman No. 1 filter paper, and dissolved in 37.5 ml of deionized water. Sample solution (15.0 ml) was added, together with 10.0 ml of alizarin complexone, 3.0 ml of 12% (w/w) NaAc and 10 ml of La(NO3)3. After 1 h, the absorbance of the mixture (pH 4.5) was measured at 620 nm. The standard curve was determined using 0.3, 0.6, 0.9, 1.2, and 1.5 mg ml1 NaF solution.
3.
Results and discussion
3.1.
Dissolving wheat straw with TFA
The wheat straw was cut into 2.5-cm fragments and dried at 50 C. Straw fragments (10.0 g) were added to a round-bottom flask containing 100 ml of trifluoroacetic acid (TFA) and incubated for 16 h at 60 C in a paraffin oil bath. Unexpectedly, we found that the mixture became black and sticky and the
biomass and bioenergy 33 (2009) 1719–1723
1721
wheat straw was completely dissolved. No pellet was obtained after centrifugation at 10,000 rpm. This phenomenon indicated that TFA completely dissolved all the cellulose, hemicellulose, and lignin components of wheat straw. To confirm this new finding, 1.0 g of microcrystalline cellulose Avicel PH105 and 1.0 g of xylan were added to TFA. Both the cellulose and xylan dissolved completely.
3.2.
Isopropanol precipitation of the TFA solution
Using the microcrystalline cellulose Avicel PH105, we examined the ability of different solvents to precipitate cellulose dissolved in TFA. Different volumes of methanol, 1-propanol, isopropanol, 1-butanol, and 2-butanol were tested. For each precipitation trial, 2.0 g of microcrystalline cellulose Avicel PH105 were dissolved in 20.0 ml of TFA, the organic solvent was added, and the mixtures were centrifuged at 10,000 rpm for 10 min. Pellets were dried at 50 C and weighed. Results showed that both isopropanol and 2-butanol could precipitate more than 77% of the dissolved cellulose. Almost all (92.7%) of the cellulose could be precipitated with isopropanol at a concentration of 0.5 volumes. Since the boiling points of isopropanol (82 C) and TFA (72.4 C) are lower than that of 2-butanol (98 C), we chose to use isopropanol because it would allow for more energy-efficient fractional distillation.
3.3.
Ability of TFA to degrade cellulose, xylan, and lignin
To determine whether TFA degraded cellulose and xylan, 10.0 ml of TFA was mixed with the following materials: 1.0 g of microcrystalline cellulose Avicel PH105, 1.0 g of xylan, 1.0 g of wheat straw, 1.0 g of rice straw, 1.0 g of corn stover, or 1.0 g of sabai grass (Eulaliopsis binata). After addition of 5.0 ml of isopropanol, the mixtures were centrifuged at 10,000 rpm for 10 min. Tris (2 molL1, 0.9 ml) was added to a portion (0.1 ml) of the supernatant, and this mixture was used as the test sample to determine the molar amount of reducing sugar. Results are shown in Fig. 1A. The molar amount of reducing sugar in the supernatant of TFA-treated cellulose was 0.037 mmolL1. According to the molecular weight of glucose (180.16), the degradation extent of the microcrystalline cellulose was calculated to be 0.66%. These results show that TFA dissolved, but did not degrade, the cellulose. The molar amount of reducing sugar in the supernatant of TFA-treated xylan was 4.34 mmolL1. Given the mean molecular weight of xylan (151.26), the degradation extent of xylan was calculated to be 65.65%. The possible presence of some xylan disaccharide in the TFA solution may explain why the xylan was not completely degraded to reducing sugar. These results show that xylan was partially degraded by TFA. The molar amounts of reducing sugar of rice straw, wheat straw, corn stover and sabai grass were, respectively, 0.932 mmolL1, 0.878 mmolL1, 1.153 mmolL1, and 0.823 m molL1. Hemicellulose is the major component in cereal straws that TFA degrades, and it is composed of xylose, arabinose, glucose, and mannose. Taking into account the molecular weights of xylose (150.13), arabinose (150.13), glucose (180.16), and mannose (180.16), the extent of degradation of rice straw, wheat straw, corn stover, and sabai grass were calculated, respectively, to be 14–17%, 13–16%, 17–21%, and 12–15% [the first percentage (14, 13, 17, 12) was multiplied
Fig. 1 – The ability of TFA to degrade cellulose, xylan, and lignin. (A) Degradation rates of TFA-pretreated cellulose, xylan, rice straw, wheat straw, corn stover, and sabai grass. The tops of upper stack histograms (sparse patterns) stand for the percentages calculated by the molecular weight 180.16 and the bottoms of upper stack histograms stand for the percentages calculated by the molecular weight 150.13. The degradation rates (vertical axis) were calculated by the molar amount of reducing sugar 3 the molecular weight of reducing sugar (180.16 or150.13) O sample weight (1.0 g); (B) Acid-insoluble lignin content of rice straw, wheat straw, corn stover, and their precipitates after TFA pretreatment and isopropanol precipitation. Pr, Pw, Pc: precipitates of rice straw, wheat straw, and corn stover, respectively.
by 150.13 and the last percentage (17, 16, 21, 15) was multiplied by 180.16]. The lignin in cereal straws is composed of acid-insoluble lignin, which is the major component (about 29.1–33.5%), and acid-soluble lignin [1.8% in rice straw, 1.0% in wheat straw, 1.3% in corn stover measured by the OD205 method [14]. To determine whether TFA degraded acid-insoluble lignin, samples of rice straw, wheat straw, corn stover and their precipitates after TFA pretreatment and isopropanol precipitation were dried at 50 C. Their content of acid-insoluble lignin was measured by the method described above, and the
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results are shown in Fig. 1B. TFA pretreatment caused the loss of 6.2% of acid-insoluble lignin (31.0% 24.8%) in rice straw, 7.5% (33.5% 26.0%) in wheat straw, and 6.8% (29.1% 22.3%) in corn stover. These results indicate that TFA degraded 20.0–23.3% of the acid-insoluble lignin.
3.4. The possibility of TFA pretreatment of cellulosic biomass To utilize this TFA procedure for the pretreatment of cellulosic biomass, we designed the process shown in Fig. 2. After the cereal straw is broken into short fragments using a cracker, the straw fragments are dissolved in 10 volumes of TFA. Then 0.5 volumes of isopropanol are added to the TFA solution to precipitate the materials dissolved in the TFA. The supernatant and precipitate are separated by filtration. After evaporating the TFA and isopropanol from the mixture, the supernatant and precipitate are used for further enzymatic hydrolysis to produce bioethanol or cellulosic products. The mixture of TFA and isopropanol can be fractionally distilled and recycled back into the process. Fig. 3 shows the precipitation of isopropanol, the dry precipitate obtained by filtration, and the residue left after the TFA and isopropanol were evaporated off the supernatant. The results indicate that using isopropanol to precipitate material dissolved in TFA is feasible. The key step in this process is that the TFA and isopropanol must be completely evaporated off the supernatant and precipitate. Using the La3þ–ALC method, the residual TFA amounts in the supernatant and precipitate were measured to be 0.0041% and 0.0032%, respectively. Their pH values (6.02, 6.04) were very close to that of tap water (6.12). These results confirmed that TFA can be completely evaporated off the supernatant and precipitate, which can then be used safely in downstream processes. Using the M9 mineral plates plus the TFA reaction supernatant, we successfully cultured the Pichia pastoris GS115 (data not shown). Thus, utilizing TFA for the pretreatment of cellulosic biomass is feasible.
Fig. 2 – Technical process of TFA pretreatment of cellulosic biomass.
Fig. 3 – Precipitation of isopropanol, the residue, and precipitate products of TFA-pretreated wheat straw.
Acknowledgement This work was supported by the Municipal Research Program of Shanghai Nos. 072312048, 0839191100, 08DZ1204400, the High-Tech Research and Development Program of China (863 Program) Nos. 2006AA02Z228, 2007AA02Z215, 2009ZX09306008, 2007AA100506, National Basic Research Program of China (973 Program) No. 2007CB936004, the Key Program of Natural Science Foundation of China No.30630012 and Shanghai Leading Academic Discipline Project No.B203.
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