Dilute Acid Hydrolysis of Sugarcane Bagasse for ... - ThaiScience

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Chiang Mai J. Sci. 2014; 41(1) Chiang Mai J. Sci. 2014; 41(1) : 60-70 http://epg.science.cmu.ac.th/ejournal/ Contributed Paper

Dilute Acid Hydrolysis of Sugarcane Bagasse for Butanol Fermentation Woranart Jonglertjunya*, Worawatsamon Makkhanon, Tanabodee Siwanta and Paritta Prayoonyong Department of Chemical Engineering, Faculty of Engineering, Mahidol University, Phuttamonthon 4 Road, Salaya, Phuttamonthon, Nakhonpathom 73170, Thailand. *Author for correspondence; e-mail: [email protected] Received: 17 October 2012 Accepted: 3 May 2013

ABSTRACT This research was concerned with butanol production from fermentation of sugarcane bagasse hydrolysate by Clostridium sp. Initially, sugarcane bagasse was treated using a ball mill in order to investigate the effects of mechanical treatment on particle size distribution. Size reduction of sugarcane bagasse by ball milling was carried out for a period of time within a range of 2-8 hours at room temperature. The optimal ball milling time was considered to be 2 hours, which resulted in a yield of 43% for the size range of sugarcane bagasse particles of 1180-212 μm. Then, the sugar hydrolysis was tested at various temperatures (30, 70, 80, 121°C), particle size ranges of sugarcane bagasse, ratios of sugarcane bagasse to solvent (1:10, 1:15, 1:20) and H2SO4 concentrations (0.25-1% by volume). The optimum conditions for acid hydrolysis, which yield the maximum reducing sugar concentration of about 52 g/l, were particle size of 0.212-1.180 mm, sugarcane bagasse-to-solvent ratio of 1:10, H2SO4 concentration of 1% by volume and temperature of 121°C for 1 hour. Finally, the fermentation of sugarcane bagasse hydrolysate by Clostridium sp. affecting yield of butanol, ethanol and acetone production was studied. This work was carried out using various types of Clostridium sp., including Clostridium butyricum (TISTR 1032), Clostridium sporogenes (TISTR 1452), Clostridium beijerinckii (TISTR 1461) and Clostridium acetobutylicum (TISTR 1462). The results showed that 24h-butanol fermentation of sugarcane bagasse hydrolysate by Clostridium beijerinckii (TISTR 1461) gave the highest butanol concentration of 0.27 g/l. Keywords: sugarcane bagasse, acid hydrolysis, fermentation, butanol 1. INTRODUCTION Due to a continuous depletion of petroleum fuel reserves and increased environmental concerns, fuels produced from the fermentation of renewable feedstock have become a global priority. Ethanol has been recognized as an alternative fuel because it can be produced by microbial fermentation from starch and

sucrose [1]. Similarly, butanol can also be utilized as a biofuel. Biobutanol, owing to significant properties such as high energy content, hydrophobicity, blending ability, compatibility to combustion engines, corrosion and octane rating, is increasingly regarded as a renewable source of energy [2]. At present, butanol is produced

Chiang Mai J. Sci. 2014; 41(1)

industrially by chemical synthesis process involving the reaction of propylene, carbon monoxide and hydrogen [3]. Butanol can also be produced via biochemical process by microbial fermentation. Gram-positive spore-forming bacteria of the genera Clostridium (specifically species of C. acetobutylicum and C. beijerinckii) were employed for butanol production using starchy substrates in what was known as Acetone-Butanol-Ethanol (ABE) fermentation. Two pathways have been identified in the metabolism of Clostridia strains: acidogenesis, characterized by substrate conversion into acids (acetic and butyric acids) by high motile cells (acidogenic cells); solventogenesis, characterized by substrate and acid conversion into solvents (ABE) by clostridial-form cells unable to grow (solventogenic cells) [4]. The shift of the two pathways strongly depends on pH and concentration of metabolites [4]. Li et al., [5] observed that pH of 4.5 was the optimal pH for butanol production using Clostridium acetobutylicum ATCC 824. Generally, glucose is a substrate that may give the highest yield for butanol fermentation. For example, Clostridium acetobutylicum ATCC 824 [5], Clostridium beijerinckii BA 101 [6] and Clostridium beijerinckii P260 [7] were grown in media composed of mainly glucose, which are clostridia reactor media, TGY media and glucose and yeast extract media, respectively. However, using glucose is not economically practical. Renewable resources including corn fiber [6], wheat straw [7], corn stover [8], rice straw [9], and other agricultural by products have been promoted as feedstocks for production of butanol. The use of other biomass may give a lower yield because of the presence of inhibitors in the feedstock but may be more economical. Qureshi et al. [6] stated that butanol fermentation from corn starch was

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not economical than that from corn fiber (which is an agricultural residue). One of the most extensively agricultural residues in Thailand is sugarcane bagasse, which is one of the most residues from sugar industry. The most common use of sugarcane bagasse is for combustion energy, which affects the environment from the emissions of CO 2. Utilizing sugarcane bagasse as a biofuel feedstock would be more environmental friendly than disposing it by combustion. Sugarcane bagasse consists mainly of cellulose, hemicellulose and lignin. Cellulose is a linear polysaccharide polymer which consists of long glucose units, whereas hemicellulose contains most of the pentose sugars (such as xylose) and small amounts of hexose sugars (such as glucose). The hydrolysis of such lignocellulosic material by acid solution produces predominantly fermentation sugars, such as glucose, xylose and small amount of arabinose. Besides those fermentable sugars produced during lignocellulose degradation, a wide range of compounds are generated. Unfortunately, some of those compounds are toxic to butanol-producing microorganisms [8]. Acid hydrolysis is a general method for hydrolyzing sugarcane bagasse. Acids can break down heterocyclic ether bonds between sugar monomers in the polymeric chains of cellulose and hemicellulose [10]. Nitric acid, sulfuric acid, phosphoric acid may be used [6, 7, 11]. In general, the hydrolysis uses high acid concentration, temperature and long reaction time [11]. Particle size of material also affects the yield of sugars. An objective of this work was to investigate the yield of glucose and xylose from the acid hydrolysis of sugarcane bagasses using low acid concentration and

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temperature. In addition, the effect of particle size of sugarcane bagasse in hydrolysis was studied. Then, the butanol fermentation utilizing the sugarcane bagasse hydrolysates as substrate without removing inhibitors was preliminary investigated. The microorganisms used were Clostridium butyricum (TISTR 1032), Clostridium sporogenes (TISTR 1452), Clostridium beijerinckii (TISTR 1461) and Clostridium acetobutylicum (TISTR 1462). 2. MATERIALS AND METHODS

2.1 Sugarcane Bagasses Sugarcane bagasses originating from the Kornburi factory producing sugar in Thailand were dried in an oven in temperature range of 50-60°C for 20 hours, then ground in a ball mill, screened into size ranges of 63-53, 106-63, 150-106, 212-150 and 1180-212 μm, and kept in a desiccator prior to testing. Lignin contents were determined as Klason lignin following the ASTM D-1106 standard method [12]. 2.2 Microorganisms and Growth Conditions Clostridium butyricum (TISTR 1032), Clostridium sporogenes (TISTR 1452), Clostridium beijerinckii (TISTR 1461) and Clostridium acetobutylicum (TISTR 1462) were grown separately in cooked meat medium (CMM; Difco Laboratories, Detroit, MI, USA) with composition of 454 g/l beef heart, 20 g/l bactoproteose peptone, 2 g/l bacto dextrose, and 5 g/l NaCl. The cooked meat medium was sterilized by an autoclave at 121°C for 15 minutes. 2.3 Acid Hydrolysis In this step, sugarcane bagasse was hydrolyzed with dilute sulfuric acid and heat treatment. Dried sugarcane bagasses were added to different concentrations of


sulfuric acid solution (0.25-1 % v/v). The pulp was prepared at different solid/liquor ratios of 1:10, 1:15 and 1:20 (g dry weight of sugarcane bagasse: ml of sulfuric acid solution) in a conical flask with screw cap. These experiments were carried out at 30, 70, and 80°C using a water-bath shaker for 1 hour and at 121°C using an autoclave for 15 minutes and 1 hour. All experiments were replicated three times. The hydrolyzed solution was adjusted its pH to about 7 with NaOH, then passed through a filter paper and microfiltration (Clyde filter, 0.2 μm) and subsequently diluted with sterile distilled water prior to the analysis of glucose and xylose. 2.4 Fermentation All batch experiments of Clostridium sp. were carried out in a conical flask with screw cap containing 90 ml of previously sterilized medium for 48 hours of fermentation time at 37°C under anaerobic and static conditions. The medium that contained glucose and xylose from sugarcane bagasse hydrolysate was chosen for the growth of Clostridium sp. The medium was autoclaved at 121°C for 15 min followed by cooling under oxygen free nitrogen gas environment which was created by sweeping the gas across medium surface. This anaerobic condition in the fermenter followed the method of Qureshi et al. [7]. The 24 hr-bacterial cell measured by a microscope counting chamber (hemocytometer) was about 2 × 109 cells/ml. A 10 % (v/v) inoculum of the bacteria was added to a series of flasks. Two flasks were removed to provide duplicate samples for each data point. The fermentation broth was passed through a microfiltration (Clyde filter, 0.2 μm) and subsequently analyzed the concentration of fermentation products viz., acetone, butanol and ethanol by HPLC.


2.5 Analytical Methods The total reducing sugar was measured by the dinitrosalicylic acid (DNS) method [13]. Glucose and xylose concentration was determined by a high performance liquid chromatography system (Model YL 9100, YOUNG LIN INSTRUMENT Co., Ltd, Korea), equipped with a SofTA ELS detector (Model 1400, SofTA Corporation, USA). A HPLC column (VertisepTM sugar LMP) was used with deionized water as a mobile phase at a flow rate of 0.4 ml/min and column temperature was maintained at 85°C. Acetone, butanol and ethanol concentrations were determined by a high performance liquid chromatography system (Perkin Elmer series 200), equipped with a Reflective Index Detector. A HPLC column (VertiSepTM GES C18) was used with the eluent being acetonitrile at a flow rate of 0.8 ml/min and column temperature was maintained at 30°C. 3. RESULTS AND DISCUSSION

3.1 Effect of Grinding Time on Particle Size Distribution of Ground Sugarcane Bagasse The ball milling and sieving operations of this work allowed the size reduction of sugarcane bagasse, yielding a large range of particle sizes from 1180 to 53 μm. Figure 1 shows the size distribution curves of the sample sugarcane bagasse ground for different periods of time up to 8 hours. It can be seen from the figure that size reduction proceeds continuously as grinding time increases within the scope of this experiment. The ball milling method exerts

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a great influence on the particle within a given coarse particles (1180 μm). Therefore, 1180-212 μm was the optimum particle size for acid hydrolysis.


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Figure 2. The reducing sugar contents of sugarcane bagasse hydrolyzed at sulfuric acid solution of 0.5% (v/v), solid/liquor ratio of 1:15, temperature of 121°C, reaction time of 1 hour and different particle sizes. The hydrolysis of sugarcane bagasse using sulfuric acid concentration of 0.5% (v/v) at varied temperatures (30, 70, 80 and 121°C) were carried in order to find the temperature that gives the highest yield of glucose and xylose. Not surprisingly, the extent of hydrolysis increases with temperature and time. Reducing sugars released during hydrolysis at 30, 70 and 80°C had low

concentrations, only 1.6, 1.7 and 2.8 g/l, even after a long treatment time of 60 minutes (Figure 3). At the higher temperature, reducing sugars released from acid hydrolysis at 121°C for 60 minutes increased substantially up to 24.2 g/l. However, reducing sugars obtained from the same temperature hydrolysis had a slightly lower concentration at a shorter treatment time.

Figure 3. The reducing sugar contents of sugarcane bagasse hydrolyzed at sulfuric acid solution of 0.5% (v/v), solid/liquor ratio of 1:15, particle size range of 1180-212 μm and different temperatures and reaction times.

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One of parameter factors affecting acid hydrolysis includes solid/liquor ratio. The solid/liquor ratios of 1:10, 1:15 and 1:20 were studied because it became difficult to keep the solid suspending in the solution at a ratio higher than 1:10 due to less liquid being


present. The reducing sugar contents were enhanced by the increase fraction of liquid solution (figure 4). The reducing sugar content was increased from 15.0 to 35.5 g/l when the solid/liquor ratio was increased from 1:20 to 1:10 respectively.

Figure 4. The reducing sugar contents of sugarcane bagasse hydrolyzed at sulfuric acid solution of 0.5% (v/v), particlesize range of 1180-212 μm, temperature of 121°C, reaction time of 1 hour and different solid/liquor ratios.

Figure 5. Glucose and xylose contents of sugarcane bagasse hydrolyzed at solid/liquor ratio of 1:10, particlesize range of 1180-212 μm, temperature of 121°C, reaction time of 1 hour and different sulfuric acid solutions. Glucose and xylose contents in sugarcane bagasse hydrolysate at different H 2SO 4 concentrations were shown in Figure 5. An increase in glucose and xylose concentrations were observed when H2SO4 concentration was increased from 0.25 to 1% (v/v) but the xylose concentration slightly decreased

when 1% (v/v) acid was used. The highest glucose content obtained was 7.8 g/l when 1% (v/v) H2SO4 was used to hydrolyze sugarcane bagasse whereas the highest xylose content was 45.8 g/l when 0.75% (v/v) H2SO4 was used. An increase in acid concentration in the acid hydrolyzing could


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provide a strong or complete reaction for breaking down the chemical bonds inside sugarcane bagasse achieving sugars in the

hydrolysate [14]. This result shows that xylose released was much higher than glucose released from acid hydrolysis.

Table 1. Comparison of main components of sugarcane bagasse hydrolysates reported in literature. Conditions of acid hydrolysis Acid 2%HNO3 (w/w) 0.25%H2SO4(v/v) 0.5%H2SO4(v/v) 1.25% H2SO4(v/v) 1%H2SO4(v/v)

Temp (°C) 122 121 120 121 121

Reaction time (h) 0.9 1 4 2 1

The highest yield of sugar hydrolysate obtained in this work was compared with that from works of several researchers, as shown in Table 1. While the glucose content was comparable, the amount of xylose obtained in this work was much higher than those from other work. The merit of the acid hydrolysis method used in this work was that low acid concentration and reaction time were used leading to energy and production cost savings. The hydrolysis condition (i.e. sulfuric acid solution of 1% (v/v), solid/liquor ratio of 1:10, particlesize range of 1180-212 μm, temperature of 121°C and reaction time of 1 hour) was selected for butanol fermentation as it gave high glucose and low xylose concentrations of hydrolysate. The high glucose concentration is preferable because microorganisms generally prefer growing in the medium containing glucose. The low xylose concentration is favorable because xylose may generate furfural and 5-hydroxymethylfurfural [14]. Those degradation products from xylose are known as inhibitors in butanol fermentation [19]. Therefore, high glucose and low xylose concentrations of

Main components of hydrolysates Xylose Glucose (g/l) (g/l) 17.6 1.2 8.6 6.7 10.1 1.8 17.1 7.2 44.1 7.8

Ref. [18] [14] [10] [15] This work

sugarcane bagasse hydrolysate achieved from acid hydrolysis were selected as an appropriate condition for further fermentation. 3.3 Butanol Fermentation from Sugarcane Bagasses Hydrolysate Genus Clostridia embracing a vast variety of biobutanol producing bacteria (e.g. Clostridium butyricum, Clostridium sporogenes, Clostridium beijerinckii and Clostridium acetobutylicum) and Clostridium acetobutylicum was the first microorganism that was employed in industrial fermentation from sugar and starchy grains for production of acetone and butanol [2]. For the growth of Clostridium sp., the medium that contained glucose and xylose from sugarcane bagasse hydrolyzed using sulfuric acid solution of 1% (v/v), solid/liquor ratio of 1:10, particle size range of 1180-212 μm, temperature of 121°C and reaction time of 1 hour was chosen. In the beginning of the fermentation, 8 g/l glucose and 45 g/l xylose were present in sugarcane bagasse hydrolysate. The hydrolysate was filtered to remove sugarcane bagasse, and then its pH was adjusted to 6.8. A 10 % v/v inoculum of the bacterial culture

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was added to a series of flasks of the sterile hydrolysate (previously autoclaved at 121°C for 15 minutes). Figure 6 shows the results of butanol fermentation of the sugarcane bagasse hydrolysate using Clostridium sp. About 0.27 g/l butanol was produced in 24 hours from C. beijerinckii fermentation, whereas the other bacteria had a lower productivity. However, ethanol and acetone were observed to be very low (< 0.05 g/l). Generally, the ratio of acetone, butanol and ethanol in the fermentation broth is found to be 3:6:1, where the culture produced 2.72 g/l acetone, 6.05 g/l butanol and 0.59 g/l ethanol in 50 hours [7]. It can be seen that the use of sugarcane bagasse hydrolysate with high sugar content (52 g/l) did not bring benefit to the butanol production. This low acetone-butanol-ethanol productivity was presumably due to the fact that low cell growth occurred in the experiments. The


initial free cell concentration of each Clostridium sp. in these experiments was found to be about 3 x 108 cells/ml, compared to about only 4 x 108 cells/ml after 48 hours fermentation. The reason for this low cell growth may be that the bacteria prefer to use only glucose as a substrate rather than xylose [20]. In addition, this sugarcane bagasse hydrolysate may have an inhibitive effect on bacterial growth. This low in growth rate is consistent with the low concentration/yield in the following fermentation products i.e. acetone, butanol and ethanol. Several researchers have reported the effect of inhibitors observed in biomass hydrolysateon fermentation products [6, 8, 19]. The findings of this study may lead to future research with particular attention to the increase in butanol productivity. A presence of inhibitors may be toxic to the bacterial cells. Therefore, future studies may focus on butanol production by removal of fermentation inhibitors.

Figure 6. Butanol production by C. butyricum, C. sporogenes, C. beijerinckii and C. acetobutylicum in batch fermentation of sugarcane bagasse hydrolysate. 4. CONCLUSION

The use of sugarcane bagasses for acid hydrolysis obtained valuable fermentation products. Glucose and xylose were the main fermentable sugar achieved from the dilute

acid hydrolysis. Maximum glucose yield of 8 g/l and xylose yield of 45 g/l were obtained when sugarcane bagasse with particle size range of 0.212-1.180 mm and the ratio of sugarcane bagasse to solvent


of 1:10 was hydrolyzed by 1% (v/v) sulfuric acid solution at 121°C for 1 hour. Clostridium beijerinckii (TISTR 1461) gave the highest butanol concentration of 0.27 g/l amongst those bacteria considered. 5. ACKNOWLEDGEMENT

This work was supported by the undergraduate research grant from Energy Policy and Planning office, Ministry of Energy, Thailand (2011-2012). REFERENCES [1] Liu S. and Qureshi N., How microbes tolerate ethanol and butanol, New Biotechnol., 2009; 26: 117-21. [2] Kumar M. and Gayen K., Developments in biobutanol production, Appl. Energ., 2011; 88: 1999-2012. [3] Garcia V., Pakkila J., Ojamo H., Muurinen E. and Keiski R.L., Challenges in biobutanol productions: How to improve the efficiency? Renew. Sust. Energ. Rev., 2011; 15: 964-980. [4] Napoli F., Olivieri G., Russo M.E., Marzocchella A. and Salatino P., Continous lactose fermentation by Clostridium acetobutylicum-Assessment of acidogenesis kinetics, Bioresource Technol., 2011; 102: 1608-1614. [5] Li S.Y., Srivastava R., Suib S., Li Y. and Parnas R.S., Performance of batch, fed-batch, and continuous A-B-E fermentation with pH control, Bioresource Technol., 2011; 102: 4241-4250. [6] Qureshi N., Ezeji T.C., Ebener J., Dien B.S., Cotta M.A. and Blaschek H.P. Butanol production by Clostridium beijerinckii. Part I: Use of acid and enzyme hydrolyzed corn fiber, Bioresource Technol., 2008; 99: 5915-5922.

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