Bioresource Technology 129 (2013) 553560

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Bioresource Technology
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Butyric acid production from sugarcane bagasse hydrolysate by Clostridium tyrobutyricum immobilized in a brous-bed bioreactor
Dong Wei a,b, Xiaoguang Liu c, Shang-Tian Yang a,b,
a

State Key Laboratory of Pulp and Paper Engineering, School of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, PR China William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 140 West 19th Avenue, Columbus, OH 43210, USA c Department of Chemical and Biological Engineering, The University of Alabama, Tuscaloosa, AL 35487, USA
b

h i g h l i g h t s
" We studied butyric acid production from sugarcane bagasse (SCB) hydrolysate. " High sugar yields were obtained from SCB after acid pretreatment and enzymatic hydrolysis. " C. tyrobutyricum was immobilized in a FBB for fed-batch fermentation of SCB hydrolysate. " High butyric acid yield (>0.45 g/g sugar) and productivity (0.51 g/L h) were obtained. " This study demonstrated the feasibility of producing butyric acid from low-cost SCB feedstock.

a r t i c l e

i n f o

a b s t r a c t
A fermentation process using Clostridium tyrobutyricum immobilized in a brous-bed bioreactor (FBB) was developed for butyric acid production from sugarcane bagasse (SCB) hydrolysate. SCB was rst treated with dilute acid and then hydrolyzed with cellulases. The hydrolysate containing glucose and xylose was used as carbon source for the fermentation without detoxication. The bacterium was able to grow at a specic growth rate of 0.06 h1 in media containing 1520% (w/v) SCB in serum bottles. In batch cultures in the FBB, both glucose and xylose in the SCB hydrolysate were simultaneously converted to butyrate with a high yield (0.450.54 g/g sugar) and productivity (0.480.60 g/L h). A nal butyrate concentration of 20.9 g/L was obtained in a fed-batch culture, with an overall productivity of 0.51 g/L h and butyrate yield of 0.48 g/g sugar consumed. This work demonstrated the feasibility of using SCB as a lowcost feedstock to produce butyric acid. 2012 Elsevier Ltd. All rights reserved.

Article history: Received 19 September 2012 Received in revised form 10 November 2012 Accepted 16 November 2012 Available online 29 November 2012 Keywords: Sugarcane bagasse Butyric acid Clostridium tyrobutyricum Fibrous-bed bioreactor Acid pretreatment

1. Introduction Butyric acid, a four-carbon short-chain fatty acid currently produced predominantly via the petroleum-based oxosynthesis of butyraldehyde from propylene, has wide applications in chemical, food and beverage, cosmetic as well as plastic and textile ber industries (Zigov and turdk, 2000). Its use as multiple bioactive and therapeutic compounds in the emerging nutraceutical market is also growing rapidly in the recent years (Kumar et al., 2006). With the rising oil price, the production of butyric acid by anaerobic fermentation from natural resources has become increasingly attractive. Sugarcane bagasse (SCB) is the most abundant byproduct from the sugar renery industry in southern China, Cuba,
Corresponding author at: William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 140 West 19th Avenue, Columbus, OH 43210, USA. Tel.: +1 614 292 6611; fax: +1 614 292 3769. E-mail addresses: fewd304@scut.edu.cn (D. Wei), yang.15@osu.edu (S.-T. Yang).
0960-8524/$ - see front matter 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.11.065

Brazil, Thailand and India. SCB contains 3843% cellulose, 26 28% hemicellulose (xylan, arabinan and galactan) and 25% lignin (Prior and Day, 2008; Rudolf et al., 2008). Considerable amounts of SCB are currently utilized for steam and power generation, and as a feedstock for paper pulp and animal feed production. Using lignoceullulosic biomass such as SCB to produce second-generation biofuels and chemicals has generated large interests in the biorenery industry. Although SCB as a potential feedstock has been studied for production of bioethanol (Martn et al., 2002; Rudolf et al., 2008) and biohydrogen (Pattra et al., 2008), to date there has been no report on the production of butyric acid from sugarcane bagasse. The bacteria in the genera Clostridium are the most used microorganisms in butyric acid fermentation because they can utilize a wide range of substrates, including hexoses, pentoses, oligo- and poly-saccharides derived from various biomass feedstocks (Zigov and turdk, 2000). Batch, fed-batch, continuous and cell-recycled fermentations have been extensively studied

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Table 1 Comparison of butyric acid production from various substrates by C. tyrobutyricum. Substrate Pretreatment and hydrolysis methods None Fermentation modea Final titer (g/L) Butyrate 33.0 43.4 49.9 86.9 51.6 10.1 36.0 62.8 55.2 27.5 60.4 29.0 11.0 12.7 14.5 20.9 Acetate 1.0 10.2 8.7 3.0 6.4 2.1 10.0 5.8 2.6 4.9 0.7 9.0 0.9 2.48 7.73 6.33 Reactor productivity (g/L h) 5.3 6.8 1.1 1.1 1.2 0.3 6.8 1.25 3.2 2.75 1.14 2.9 0.1 0.48 0.60 0.51 Yield Reference (g/g) 0.41 0.42 0.44 0.46 0.45 0.40 0.44 0.45 0.46 0.44 0.38 0.47 0.11 0.54 0.45 0.48 Michel-Savin et al. (1990) Wu and Yang (2003) Liu and Yang (2006) Jiang et al. (2011) Liu and Yang (2006) Jiang et al. (2010) Huang et al. (2002) Fayolle et al. (1990) Jiang et al. (2009) Huang et al. (2011) Zhu et al. (2002) Song et al. (2011) This study

Continuous culture with cell recycle Fed-batch in FBB Fed-batch in FBB Repeated fed-batch in FBB Xylose None Fed-batch in FBB Repeated batch in FBB Corn meal Amylase, pH 4.2, 60 C Repeated batch in FBB Wheat our Amylases Fed-batch with controlled substrate feeding Cane molasses Dilute H2SO4, 60 C, 2 h Fed-batch in FBB Jerusalem artichoke 0.01 N H2SO4, 121 C, 30 min Repeated batch in FBB Fed-batch in FBB CFH/CSL 0.25 N HCl, 121 C, 45 min Fed-batch in FBB Brown algae 1.5% H2SO4, 121 C, 1 h Fed-batch Sugarcane bagasse CGM, 121 C, 30 min; Batch in FBB; pH 5.0 cellulases, 50 C, 38 h 0.1 N HCl, 121 C, 15 min; Batch in FBB cellulases, 60 C, 24 h 0.1 N HCl, 121 C, 15 min; Fed-batch in FBB cellulases, 50 C, 24 h CFH: corn ber hydrolysate; CSL: corn steep liquor; FBB: brous-bed bioreactor. a Unless otherwise noted, fermentation was carried out with free cells at pH 6.0.

Glucose

for butyrate production from various substrates, including glucose, xylose, fructose, sucrose, cane molasses, hydrolyzed corn meal, and hydrolysates of corn ber, Jerusalem artichoke and brown algae (see Table 1). In general, butyric acid fermentation with free cells suffered from low product titer, yield and productivity (Michel-Savin et al., 1990; Mitchell et al., 2009; Song et al., 2011), which can be greatly improved with cell immobilization and in situ product removal to alleviate product inhibition (Wu and Yang, 2003). In particular, the brous-bed bioreactor (FBB) with cells immobilized in a brous matrix has been developed for various carboxylic acid fermentations (Huang et al., 2002) with greatly increased reactor productivity and product concentration because its ability to maintain a high viable cell density through continual cell renewal and adaptation to acquire high tolerance of the inhibiting acid products (Jiang et al., 2011; Suwannakham and Yang, 2005; Zhu and Yang, 2003). The objective of this study was to develop a fermentation process for butyrate production from SCB as a low-cost feedstock. Enzymatic hydrolysis of SCB with dilute acid pretreatment was optimized to convert hemicellulose and cellulose to fermentable sugars. The kinetics of batch and fed-batch fermentations by Clostridium tyrobutyricum immobilized in the FBB was studied to evaluate the feasibility of butyric acid production from SCB hydrolysate.

xylanase, 982 U/g protein; Genencor, NY, USA) to hydrolyze cellulose and xylan described below.

2.2. Acid pretreatment of SCB Dilute acid pretreatment of SCB was rst carried out in 2-mL cryogenic storage vials (Fisher Scientic, Pittsburgh, PA, USA) to evaluate the effects of acid concentration and treatment time on the breakdown of SCB and sugar release. Each vial containing 50 mg of the SCB powder and 1 mL of a dilute HCl solution (0.1 0.5 N) was autoclaved at 121 C for 10 or 30 min. The undigested SCB was removed by centrifugation and washed twice with distilled water. The supernatant and washed water were collected and the combined solution was adjusted to pH 4.8 with 6 N NaOH, followed by dilution to a metered volume and analyzed for glucose and reducing sugars. To study the effect of dilute acid pretreatment on the subsequent enzymatic hydrolysis, 200 g of SCB were suspended in 3.8 L of 50 mM citrate buffer (pH 4.8) or 0.1 N HCl. After autoclaving at 121 C for 10 min, the solution pH was adjusted to 4.5 with 6 N NaOH, and 200 mL of 1 M citrate buffer (pH 4.8) were added.

2.3. Enzymatic hydrolysis of SCB Enzymatic hydrolysis of SCB was carried out in 2-mL cryogenic storage vials to evaluate the effect of the enzyme dosage on the breakdown of SCB and sugar release. Each vial containing 50 mg sugarcane bagasse powder, 1.0 mL of 50 mM citrate buffer (pH 4.8) and the enzyme at the dosage of 00.5 mL/g SCB was incubated at 50 C, 140 rpm for 24 h. The hydrolysate was collected after centrifugation and analyzed for glucose and reducing sugars. Enzymatic hydrolysis of the pretreated SCB was carried out in sterile 5-L bioreactor (BioFlo 3000, New Brunswick Scientic Co., Inc.) with ACCELLERASE 1000 at the dosage of 0.1 mL/g SCB at 50 C, 140 rpm for 24 h. Samples were taken at appropriate time intervals for analysis of sugar contents. Hydrolysis of SCB at a higher solid loading of 15% and 20% (w/v) were also carried out to prepare SCB hydrolysates for the fermentation study described below.

2. Methods 2.1. Sugarcane bagasse (SCB) Fresh SCB obtained from Jiang-men sugar-renery (Guangdong, China) was dried at 100 C to less than 5% moisture content and stored until use. The dried SCB was milled to ne powder (particle size: 50100 lm) by micro-milling (BFM-6BII Model, Jinan Billionpowder Tech. & Eng. Co., Ltd., Shandong, China) for 50 min. The SCB powder was treated with dilute HCl to solubilize hemicellulose and commercial cellulases (ACCELLERASE 1000: CMCase, 3100 U/g, bglucosidase, 467 pNPG U/g, xylanase, 711 U/g protein; ACCELLERASE 1500: CMCase, 2274 U/g, b-glucosidase, 553 pNPG U/g,

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2.4. Culture and media A mutant strain of C. tyrobutyricum ATCC 25755 with inactivated pta gene that produced a high yield of butyric acid (Zhu et al., 2005) was used in this study. The seed culture was grown in the Reinforced Clostridium Medium (RCM; Difco, Detroit, MI) at 37 C over night, and then transferred into a synthetic Clostridium Growth Medium (CGM) with glucose or SCB hydrolysate as the carbon source for the fermentation study. The CGM contained (g/L): 5 trypticase, 5 yeast extract, 6 NaHCO3, 0.31 K2HPO43H2O, 0.24 KH2PO4, 0.24 (NH4)2SO4, 0.48 NaCl, 0.1 MgSO47H2O, 0.0064 CaCl22H2O, and trace metals and vitamins (Zhu and Yang, 2003). 2.5. Effect of SCB hydrolysate on cell growth The inhibition effect of SCB hydrolysate on cell growth was investigated in serum bottles with CGM containing SCB hydrolysate as the carbon source. SCB hydrolysate (15% and 20% w/v) was prepared by treating with 0.1 N HCl at 121 C for 10 min and ACCELLERASE 1500 (0.6 mL/g SCB) at 50 C, pH 5.0 for 48 h. After centrifugation to remove solid residues, the supernatant (SCB hydrolysate) was mixed with an appropriate amount of CGM (without glucose and NaCl, pH adjusted to 6.0) to prepare SCB media, which were then dispensed in serum bottles, purged with nitrogen gas to remove dissolved oxygen. The serum bottles were then capped with butyl rubber septa and sealed with aluminum crimp seals, and autoclaved at 121 C for 30 min. After cooling, these bottles were inoculated with fresh seed culture grown in RCM overnight, and cell growth was monitored by measuring the optical density at 600 nm (OD600). 2.6. Batch fermentation The production of butyric acid from SCB hydrolysate was investigated in the fermentation system consisting of a 2-L stirred-tank fermentor (Spinner ask 1967-02000, Bellco Glass, Inc. USA) and a 0.5-L brous-bed bioreactor (FBB) with medium recirculation similar to the one previously described (Zhu et al., 2002). The FBB was made of a glass column packed with a spiral wound cotton towel (185 mm 300 mm; 5 mm in thickness; with >95% porosity). Before use, the system was autoclaved twice at 121 C for 60 min, and then brought to anaerobiosis by sparging the 2-L medium with N2 for about 1 h. The system containing 2 L of the CGM with 50 g/L glucose was inoculated with 100-mL of a seed culture grown overnight in RCM, and cells were allowed to grow and be immobilized in the cotton towel by circulating the medium through the brous bed at a ow rate of 25 mL/min. When the glucose concentration had been reduced to lower than 5.0 g/L, the CGM medium was replaced with a fresh medium containing SCB hydrolysate as the carbon source. Batch fermentation kinetics was then studied with the medium recirculation at 100 mL/min. The fermentation was operated at 37 C, 200 rpm with pH control by adding concentrated NH4OH. The rst batch was carried out at pH 5.0 with the medium prepared from 20% (w/v) SCB in CGM (pH 5.0) autoclaved at 121 C for 30 min and then treated with ACCELLERASE 1000 (0.6 mL/g SCB) at 50 C, 200 rpm for 38 h. The second batch was carried out at pH 6.0 in the medium prepared from 15% (w/v) SCB in 0.1 N HCl autoclaved at 121 C for 15 min, neutralized to pH 5.0 with 6 N NaOH, and then treated with ACCELLEASE 1500 (0.4 mL/g SCB) at 60 C, 200 rpm for 24 h. The hydrolysate with solid residues was used to prepare the medium for the fermentation. 2.7. Fed-batch fermentation Fed-batch fermentation was also carried out with cells immobilized in the FBB system. After the initial growth of cells in the

medium containing SCB hydrolysate prepared from 5% (w/v) SCB treated with 0.1 N HCl at 121 C for 15 min and then ACCELLERASE 1500 (0.1 mL/g SCB) at 50 C for 24 h, additional glucose (60 g/L) was pulse-fed into the stirred-tank fermentor three times to increase the cell density in the FBB. Then, the fermentation broth was replaced with 2 L of sterile CGM to start a new batch, with pulse feeding of the medium containing concentrated SCB hydrolysate (210.9 g/L reducing sugar; 80.8 g/L glucose) when the glucose concentration in the fermentation broth was below 2.0 g/L. This fed-batch process continued until the fermentation ceased to produce butyric acid. Before each feeding, equal volume of the fermentation broth was removed from the stirred-tank fermentor. The concentrated SCB hydrolysate used in the feeding was prepared from 5% (w/v) SCB as described above, with the solid residues removed by centrifugation, and concentrated about 3 times by evaporation in a rotary evaporator under vacuum. The fermentation was operated at 37 C with pH controlled at 6.0 with concentrated NH4OH. 2.8. Analytical methods Cell density was determined by measuring the optical density (OD600) of the cell suspension at 600 nm with a spectrophotometer (SequoiaTurner, Model 340) after centrifuging and washing cells twice. The glucose concentration was analyzed with a glucose analyzer (YSI 2700, Yellow Spring, OH). An HPLC system was used to analyze the organic compounds, including glucose, xylose, arabinose, fructose, butyrate, and acetate, in the fermentation broth and SCB hydrolysate following the procedures previously described (Liu and Yang, 2006). Microassay for reducing sugar was carried out in 96-well microplates using the modied BCA method (Zhang and Lynd, 2005) with glucose (050 lM) as the standard. All assays were performed in triplicate. 3. Results and discussion 3.1. Hydrolysis of SCB Micro-milling was used to produce superne powder of SCB with the particle size of 50100 lm conducible for acid and enzymatic hydrolysis because of the high specic surface area (Hendriks and Zeeman, 2009). Acid pretreatment is commonly used for the solubilization of hemicelluloses, removing lignin, and decreasing the crystallinity of cellulose in the lignocellulosic biomass, and thus increasing the enzymatic hydrolysis efciency (Jrgensen et al., 2007). However, acid treatment also could cause undesirable oxidation and degradation of sugars and phenolic compounds released in the hydrolysis, which not only reduce sugar yield but also generate toxic products that could inhibit the fermentation. The optimal concentration of HCl for the acid pretreatment thus needs to be determined. Fig. 1 shows the sugar yields from SCB from acid hydrolysis with various HCl concentrations and treatment times. Compared to the control (no HCl), the reducing sugar yield increased to 0.31 g/g SCB with 0.1 N HCl, suggesting that 0.1 N HCl for 10 min was sufcient to solubilize and hydrolyze hemicellulose in the SCB. Further increasing the acid concentration to 0.4 N and treatment time to 30 min increased the reducing sugar yield to 0.40 g/g SCB. However, the higher acid concentration would increase the risk of producing toxic sugar degradation products such as furfural and Hydroxymethylfurfural (HMF) (Mosier et al., 2002; Hendriks and Zeeman, 2009) and the resulting hydrolysate would require more caustic salt for neutralization (Kumar et al., 2009), both could inhibit the fermentation. Therefore, acid pretreatment with 0.1 N HCl at 121 C for 1015 min was recommended and used in the subsequent experiments in this study.

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It is noted that a small amount of acetate, as a byproduct derived from acetylated cellulose (Mosier et al., 2002), was also produced in the SCB hydrolysate. However, the low-concentration acetate should not have any negative effect on C. tyrobutyricum and the butyric acid fermentation. 3.2. Effect of SCB hydrolysate on cell growth Since SCB hydrolysates would be used as the carbon source for fermentation, its potential inhibition effect on cell growth was investigated rst in serum bottles. In general, C. tyrobutyricum grew well by utilizing both glucose and xylose present in the SCB hydrolysate. The specic growth rates for cells grown in the media prepared from 15% and 20% (w/v) dilute acid-pretreated SCB hydrolysates were 0.060 h1 and 0.058 h1, respectively, comparable to those from growth on xylose (0.048 h1) and glucose (0.095 h1) as the carbon source (Liu and Yang, 2006). It is noted that a higher specic growth rate was reported for cells grown on dilute acid-pretreated cane molasses (0.073 h1), which mainly contained glucose and fructose as the carbon source (Jiang et al., 2009), and corn ber hydrolysate (CFH), which contained mainly glucose and xylose, supplemented with corn steep liquor (CFH) (0.118 h1) (Zhu et al., 2002) in bioreactors controlled at pH 6.0. A rich medium such as the one supplemented with CFH could increase cell growth and alleviate the inhibition caused by NaCl and the degradation products (furfural, HMF, and phenolic compounds) present in the SCB hydrolysate (Mosier et al., 2002; Kumar et al., 2009). Nevertheless, the growth study demonstrated that SCB hydrolysates were suitable for C. tyrobutyricum without detoxication. 3.3. Butyric acid production from SCB hydrolysate in batch fermentation Fig. 4 shows the batch fermentation kinetics with SCB hydrolysate as carbon source. In general, both glucose and xylose were consumed at similar rates with a steady production of butyrate until the fermentation stopped when the butyrate concentration reached 13 g/L in Batch 1 and 14.5 g/L in Batch 2. Butyrate yield and reactor productivity (based on the FBB volume) were 0.54 g/g sugar and 0.48 g/L h in Batch 1 and 0.45 g/g sugar and 0.60 g/L h in Batch 2. The fermentation stopped before all sugars were consumed, probably due to butyric acid inhibition. Because the fermentation is inhibited mainly by the undissociated butyric acid (Wu and Yang, 2003), a lower butyrate productivity was obtained at pH 5.0 (Batch 1) than at pH 6.0 (Batch 2). On the other hand, the lower pH gave a higher butyrate yield of 0.54 g/g, which was higher than the theoretical maximum value of 0.489 g/g (Jiang et al., 2009), indicating the presence of additional carbon sources in the medium that were also used in the fermentation. It is likely that additional sugars (glucose, xylose, arabinose, etc.) were released from the SCB solid residues, which were not removed from the hydrolysate, during the fermentation at pH 5.0 and thus also contributed to the higher butyric acid yield. It should be noted that the two SCB hydrolysates used in the batch fermentations had different amounts of glucose (22.0 and 12.4 g/L) and xylose (9.1 and 21.8 g/L), and also contained some arabinose (0.28 and 1.55 g/L) and acetate (1.96 and 6.13 g/L). Apparently, acid pretreatment resulted in more pentoses (xylose and arabinose) and acetate in the resulting SCB hydrolysate used in Batch 2. The different resulting sugar contents in the SCB hydrolysates clearly indicated that pretreatment would affect the subsequent enzymatic hydrolysis and need to be optimized as already discussed earlier. Nevertheless, these different hydrolysates were used in the batch fermentations to study the kinetics and demonstrate the feasibility of simultaneously converting glucose and

Fig. 1. The yields of reducing sugars and glucose released from SCB treated with HCl at various concentrations at 121 C for 10 min or 30 min.

Fig. 2. The yields of reducing sugars and glucose from SCB hydrolyzed with ACCELLERASE 1000 at various dosages at 50 C for 24 h. No acid pretreatment was done on the SCB.

Enzymatic hydrolysis of untreated SCB was rst studied with ACCELLERASE 1000 at 50 C for 24 h. As shown in Fig. 2, the reducing sugar yield increased with increasing the enzyme loading, reached 0.35 g/g SCB at 0.1 mL/g SCB and 0.43 g/g SCB) at 0.25 mL/ g SCB. Further increasing the enzyme loading did not increase the sugar yield. The relative low sugar yield could be attributed to the limited accessibility of cellulose to enzymatic hydrolysis. To increase the sugar yield, acid pretreatment is needed. SCB (5% w/v) with or without the pretreatment with 0.1 N HCl was hydrolyzed with ACCELLERASE 1000 at the dosage of 0.1 mL/g SCB to study the effect of acid pretreatment on the enzymatic hydrolysis of SCB. As shown in Fig. 3, acid pretreatment increased the reducing sugar yield to 0.58 g/g SCB, from 0.43 g/ g SCB for the control. To increase the sugar concentration in the hydrolysate, SCB at a higher solid loading of 15% (w/v) was treated with 0.1 N HCl and ACCELLERASE 1000 (0.2 mL/g SCB) and the results are shown in Fig. 3c. Although the total reducing sugar concentration increased to 37.1 g/L (18.3 g/L glucose and 14.1 g/L xylose), the reducing sugar yield was only 0.25 g/g SCB. The low sugar yield indicated that the enzymes (cellulases and xylanase) did not work as effectively at the higher solid loading probably because of glucose inhibition and their binding to lignin in the SCB (Jrgensen et al., 2007). To increase the sugar yield, a higher enzyme loading is needed, which, however, would greatly increase the enzyme cost.

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Fig. 3. Production of reducing sugar, glucose, xylose, and acetic acid from SCB by enzymatic hydrolysis with ACCELLERASE 1000. (a) 5% (w/v) SCB without acid pretreatment, 0.1 mL enzyme per gram SCB at 50 C for 24 h; (b) 5% (w/v) SCB with dilute acid pretreatment, 0.1 mL enzyme per gram SCB at 50 C for 24 h; (c) 15% (w/v) SCB with dilute acid pretreatment, 0.2 mL enzyme per gram SCB at 50 C for 48 h.

xylose to butyric acid and to evaluate any potential inhibition effect from the hydrolysates. However, the different fermentation results were caused mainly by the different pH values used in these fermentations. The pH effect on butyric acid fermentation has been well documented (Zhu and Yang, 2004). Also, only a small amount of acetate was produced in these fermentations because the ptadeleted mutant used in this study had its acetate biosynthesis pathway partially inactivated (Zhu et al., 2005). 3.4. Fed-batch fermentation Fed-batch fermentations with glucose and SCB hydrolysate as the carbon source were studied with cells immobilized in the FBB, and the results are shown in Fig. 5. In the rst fed-batch fermentation with glucose feeding, the butyrate concentration reached 29.87 g/L in 138 h (Fig. 5a), with an average butyrate yield of 0.40 g/g and overall reactor productivity (based on the FBB volume) of 0.88 g/L h (see Fig. 6). When the medium containing concentrated SCB hydrolysate was used in the feeding, the fed-batch fermentation stopped producing butyric acid when its concentration reached 21 g/L (Fig. 5b), with an average butyrate yield of 0.48 g/g and reactor productivity of 0.51 g/L h (see Fig. 6). It should

be noted that both sugar consumption and butyric acid production rates decreased consecutively after each feeding due to the increased butyric acid concentration in the fed-batch fermentation. As shown in Fig. 6, the reactor productivity decreased with increasing the butyric acid concentration. However, at the same butyric acid concentration, the fermentation with glucose feeding had a much higher productivity than that fed with the medium containing concentrated SCB hydrolysate. Apparently, the fermentation was inhibited by butyric acid as well as the salts and the degradation products (e.g., furfural, HMF, and phenolic compounds) present in the concentrated SCB hydrolysate (Mosier et al., 2002; Kumar et al., 2009). Nevertheless, a higher butyrate yield was obtained from the SCB hydrolysate than from glucose in the fed-batch fermentation, which could be attributed to the reduced cell growth. Acetate production was about 0.11 g/g in the beginning but reduced to less than 0.03 g/g when the butyric acid concentration was above 15 g/L in the fed-batch fermentation (Fig. 6a). Compared to the batch fermentation at the same pH 6.0, more butyrate was produced in the fed-batch fermentation, indicating increased butyric acid tolerance resulting from adaptation in the FBB (Zhu and Yang, 2003). It is clear that butyric acid production from SCB hydrolysate is feasible but the fermentation is limited by butyric

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a
Concentration (g/L)

35
Fed-batch with Glucose

30
Glucose

Butyric acid

25 20 15 10 5 0 0 25 50 75 100 125 150

Acetic acid Xylose

Time (h)

b
Concentration (g/L)

25
Fed-batch with SCB Hydrolysate
Butyric acid

20

15
Glucose

10
Acetic acid

5
Fig. 4. Batch fermentation kinetics for butyric acid production from SCB hydrolysate by C. tyrobutyricum immobilized in the FBB. (a) Batch 1 at pH 5.0; 20% (w/v) SCB in CGM (pH 5.0) at 121 C for 30 min and hydrolysis with ACCELLERASE 1000 at 0.6 mL/g SCB at 50 C for 38 h; (b) Batch 2 at pH 6.0; 15% (w/v) SCB in 0.1 N HCl at 121 C for 15 min and hydrolysis with ACCELLERASE 1500 at 0.4 mL/g SCB at 60 C for 24 h.

Xylose

0 0 50 100 150 200 250 300 350 400

Time (h)
Fig. 5. Fed-batch fermentation for butyric acid production by C. tyrobutyricum immobilized in the FBB at pH 6.0. (a) Fermentation start-up with SCB hydrolysate (5% w/v in CGM) followed with three pulse-feedings of glucose; (b) Fed-batch fermentation for butyric acid production from SCB hydrolysate. The medium containing concentrated SCB hydrolysate was used in the feeding.

acid inhibition, which could be alleviated with in situ product removal such as in the extractive fermentation previously reported by Wu and Yang (2003). 3.5. Comparison with other studies Acid or alkali pretreatment is usually used to facilitate the hydrolysis of lignocellulosic biomass (Fuentes et al., 2010; Hendriks and Zeeman, 2009). However, the inhibitors (e.g., HMF, furfural, phenolic compounds, acetate and salts) present in the hydrolysate could reduce fermentation productivity (Mosier et al., 2002; Kumar et al., 2009). For example, sono-assisted alkaline pretreatment and acid hydrolysis gave a high sugar yield, recovering 99% of cellulose, 78.95% of hemicelluloses, and 75.44% of lignin from SCB, but the hydrolysate gave a low ethanol yield of 0.17 g/g in ethanol fermentation (Velmurugan and Muthukumar, 2011). To avoid the inhibitors generated in alkali or acid pretreatment, pretreatment with a weak organic acid (e.g., 1% phosphoric acid, 10 min) at a higher temperature of 160190 C was used to assist SCB hydrolysis. However, the high temperature pretreatment also generated inhibitors that signicantly reduced ethanol production (Geddes et al., 2011). The inhibitors present in the hydrolysate can be removed by adsorption using macroporous activated carbon or resins (Cantarella et al., 2004; Zhang et al., 2002), which however, would also remove some sugars and increase costs. Recently, ionic liquids (IL) as green solvents were used in the process of SCB pretreatment to alleviate the inhibitor problem (Zhang et al., 2012; Zhu et al., 2012). How-

ever, the IL process suffers from the high cost of IL, low biomass loading, and extra operation of IL recycle. The inhibitors problem can also be partially alleviated by reducing the acid concentration and treatment time in the dilute acid pretreatment process. In our study, a high reducing sugar yield, up to 0.61 g/g, was achieved with 5% (w/v) SCB pretreated with 0.1 N HCl at 121 C for 15 min followed by enzymatic hydrolysis with commercial enzymes, ACCELLERASE. Compared to other pretreatment processes, the dilute acid process is simple, cost effective and can produce SCB hydrolysate with low toxicity compatible for direct use in fermentation without requiring detoxication. Table 1 compares butyrate production from various sugars and biomass hydrolysates. In general, higher production rate (productivity) and nal product titer can be obtained from glucose or sugar and starch based feedstocks in fermentations with immobilized cells in the FBB, compared to fermentations with free cells and biomass hydrolysates, which usually contained inhibitors generated in the acid pretreatment. Concentrated SCB hydrolysate was used as the substrate in fed-batch fermentation in this study, yielding a nal butyrate concentration of 20.9 g/L, butyrate productivity of 0.51 g/L h, and yield of 0.48 g/g sugars, which was comparable or better than those from sugars and other biomass feedstocks. However, the productivity and nal butyrate titer were relatively low compared to those from corn

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a
Product Yield (g/g)

0.6

Acknowledgements This work was supported in part by the Ohio Department of Development-Third Frontier Advanced Energy Program (Tech 08036) and Open Funding of State Key Laboratory of Pulp and Paper Engineering (Grant Nos. 200937 and 2012TS03), South China University of Technology. References

0.5
Butyrate

0.4

0.3

0.2
Acetate

0.1

0.0 0 5 10 15 20 25 30

Butyrate Concentration (g/L)

b
Productivity (g/L/h)

2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6

Acetate Butyrate

0.4 0.2 0.0 0 5 10 15 20 25 30

Butyrate Concentration (g/L)

Fig. 6. Product yields and productivities in fed-batch fermentation as affected by the butyric acid concentration accumulated in the fermentation broth. (a) Product yield with dash lines indicating the average value; (b) productivity. Open symbols: fermentation with glucose feeding, lled symbols: fermentation with SCB hydrolysate. Data shown are the average values from each feeding period shown in Fig. 5.

ber hydrolysate, indicating the presence of inhibitors in the concentrated SCB hydrolysate. Detoxication of SCB hydrolysate is thus warranted for further improving the fermentation process. Cell immobilization and adaptation in the FBB also could increase cell tolerance to the inhibitors and butyrate (Zhu and Yang, 2003). In addition, extractive fermentation can also be applied to increase butyrate titer and productivity (Wu and Yang, 2003). Therefore, it is possible to further improve the fermentation to achieve the economical production of butyrate from SCB as a low-cost feedstock. 4. Conclusions A fed-batch fermentation was developed to produce butyric acid with a high yield from a low-cost feedstock, sugarcane bagasse, a byproduct of the sugar renery industry. Dilute acid pretreatment was effective in increasing the sugar yield in enzymatic hydrolysis of SCB by cellulases. High butyrate concentration, productivity and yield can be obtained in fed-batch fermentation with C. tyrobutyricum immobilized in a brous bed bioreactor. This is the rst study demonstrating the feasibility of butyric acid production from SCB hydrolysate.

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