Acid hydrolysis of sugarcane bagasse for lactic acid production

ARTICLE IN PRESS Bioresource Technology xxx (2009) xxx–xxx

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Acid hydrolysis of sugarcane bagasse for lactic acid production Pattana Laopaiboon a,b,*, Arthit Thani a, Vichean Leelavatcharamas c, Lakkana Laopaiboon a,b a

Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen 40002, Thailand Fermentation Research Center for Value Added Agricultural Products, Khon Kaen University, Khon Kaen 40002, Thailand c Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand b

a r t i c l e

i n f o

Article history: Received 15 May 2009 Received in revised form 21 July 2009 Accepted 24 August 2009 Available online xxxx Keywords: Efficiency of acid hydrolysis Sugarcane bagasse Lactic acid Xylose

a b s t r a c t In order to use sugarcane bagasse as a substrate for lactic acid production, optimum conditions for acid hydrolysis of the bagasse were investigated. After lignin extraction, the conditions were varied in terms of hydrochloric (HCl) or sulfuric (H2SO4) concentration (0.5–5%, v/v), reaction time (1–5 h) and incubation temperature (90–120 !C). The maximum catalytic efficiency (E) was 10.85 under the conditions of 0.5% of HCl at 100 !C for 5 h, which the main components (in g l!1) in the hydrolysate were glucose, 1.50; xylose, 22.59; arabinose, 1.29; acetic acid, 0.15 and furfural, 1.19. To increase yield of lactic acid production from the hydrolysate by Lactococcus lactis IO-1, the hydrolysate was detoxified through amberlite and supplemented with 7 g l!1 of xylose and 7 g l!1 of yeast extract. The main products (in g l!1) of the fermentation were lactic acid, 10.85; acetic acid, 7.87; formic acid, 6.04 and ethanol, 5.24. " 2009 Elsevier Ltd. All rights reserved.

1. Introduction In 2007, Thailand produced approximately 70 million tons of sugarcane and became the world third sugar producer following Brazil and Australia, respectively. Sugarcane bagasse, a byproduct of the sugar production industry, consists of cellulose 43.6%, hemicellulose 33.8%, lignin 18.1%, ash 2.3% and wax 0.8% on a dry weight basis (Sun et al., 2004). It is an abundant source of lignocellulose that can be hydrolysed to yield fermentable sugars for the production of value added bio-products such as lactic acid, thus increasing the economy of the process. Other applications of sugarcane bagasse are as sources of animal feed, energy, pulp, paper and boards (Banerjee and Pandey, 2002). Acids can be used as catalysts for sugarcane bagasse hydrolysis because they can break down heterocyclic ether bonds between sugar monomers in the polymeric chains, which are formed by hemicellulose and cellulose (Aguilar et al., 2002). H2SO4 (Nguyen et al., 1999, 2000; Sun and Cheng, 2002; Kumar et al., 2009) and HCl (Sun and Cheng, 2002; Herrera et al., 2004; Hernández-Salas et al., 2009) are potential acids which can be used to hydrolyse sugarcane bagasse. The hydrolysate of lignocellulosic materials mainly consists of fermentable sugars such as xylose, glucose and arabinose (Rodríguez-Chong et al., 2004). Accompanying these

* Corresponding author. Address: Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen 40002, Thailand. Tel./fax: +66 43 362121. E-mail address: [email protected] (P. Laopaiboon).

monosaccharides, some soluble materials such as lignin, acetic acid and furfural are also produced, which can inhibit both growth and sugar utilisation of microorganisms during the fermentation process (Aguilar et al., 2002; Lavarack et al., 2002; Rodríguez-Chong et al., 2004; Cheng et al., 2008). After pretreatment and hydrolysis of lignocellulosic materials, the hemicellulose fraction is liquefied to make sugars (xylose and glucose) accessible to fermenting microorganisms (Lynd and Wyman, 1999; Aristidou and Penttilä, 2000). However, not all fermenting microorganisms can utilise xylose (5-atom carbon). Laopaiboon (2001) showed that Lactococcus lactis IO-1, a lactic acid producer, could utilise both glucose and xylose via Embden– Meyerhof–Parnas pathway and phosphoketolase pathway, respectively. Lactic acid is widely used as acidulant, flavour and preservative in food, pharmaceutical, leather and textile industries. It is also used for polymerization to biodegradable polylactic acid (PLA), which is used for medical applications such as sutures and clips for wound closure or prosthetic devices. Additionally, it is used for the production of basic chemicals (Hofvendahl and HahnHägerdal, 2000). The aim of this work was to determine the optimum NH4OH concentration for lignin extraction from sugarcane bagasse, and to investigate HCl and H2SO4 hydrolysis of sugarcane bagasse to obtain the hydrolysate containing high fermentable sugar and low inhibitor concentrations for use as a substrate for bioconversion. The feasibility of lactic acid production from the hydrolysate under batch fermentation by L. lactis IO-1 was also studied.

0960-8524/$ - see front matter " 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.08.091

Please cite this article in press as: Laopaiboon, P., et al. Acid hydrolysis of sugarcane bagasse for lactic acid production. Bioresour. Technol. (2009), doi:10.1016/j.biortech.2009.08.091

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P. Laopaiboon et al. / Bioresource Technology xxx (2009) xxx–xxx

2.1.2. Lignin extraction NH4OH (8%, 10% and 12% v/v) was used to extract lignin in the sugarcane bagasse particles (Domínquez et al., 1997). The extraction was performed at 25 !C for 24 h using a liquor/solid ratio (LSR) of 100 ml liquor/g dry weight of sugarcane bagasse (modified from Sun et al. (1995)). The lignin content in the soluble fraction was measured by spectrophotometer at the absorbance of 649 nm. The solid fraction was washed twice with distilled water and dried at 90 !C for 18 h or until weight was constant. 2.1.3. Acid hydrolysis After the lignin extraction, the remaining solid fraction was hydrolysed by 0.5%, 2%, 3% and 5% (v/v) of HCl or H2SO4. The temperature of the hydrolysis was controlled at 90, 100, 110 and 120 !C, and the reaction time was varied at 0, 2, 3 and 5 h. All conditions were carried out using a LSR of 15 ml liquor/g dry weight of sugarcane bagasse (modified from Aguilar et al. (2002), Herrera et al. (2004) and Rodríguez-Chong et al. (2004)). Scale up of acid hydrolysis under the conditions giving the highest acid hydrolysis efficiency (E) was performed in a 50 l reactor at an agitation rate of 60 rpm to obtain the hydrolysate for lactic acid fermentation. 2.1.4. Analytical methods and acid hydrolysis efficiency After centrifugation of the reaction mixtures under various acid hydrolysis conditions, the supernatants were determined for sugars (xylose, glucose and arabinose), acetic acid and furfural concentrations using HPLC (Shimadzu, Japan) with an AminexHPX 87H column and a refractive index detector (oven temperature, 50 !C; isocratic elution, flow rate of 0.57 ml min!1; mobile phase, 5 mM H2SO4). The catalytic efficiencies of HCl and H2SO4 for the hydrolysis of sugarcane bagasse were calculated using the following equation (modified from Rodríguez-Chong et al. (2004)):

P S P E¼ 1þ I P

where S is the sum of sugar concentrations in the hydrolysates P (glucose, xylose and arabinose) and I is the sum of inhibitor concentrations in the hydrolysates (acetic acid and furfural). 2.2. Lactic acid fermentation 2.2.1. Basal medium and inoculum preparation The basal medium was composed of (in g l!1) yeast extract (Oxoid, England), 5; peptone (Oxoid, England), 5 and NaCl (BDH, UK), 5. The medium was supplemented with 22 or 30 g l!1 of xylose (Fluka, Switzerland) and adjusted to pH 6.0 with 2 M HCl. The stock culture of L. lactis IO-1 (JCM 7638) was rejuvenated by incubation in 5 ml of thioglycolate medium (Difco, USA) for 16 h at 37 !C in a static incubator. One ml of the culture was transferred into 150 ml of the basal medium and incubated at 37 !C, 150 rpm for 3 h. An inoculum (5%, v/v) was used to initiate the batch cultures.

2.2.3. Determination of cell growth, sugar and product concentrations Bacterial growth was monitored by spectrophotometric measurement at 562 nm (Shimadzu, Japan) and converted to dry cell weight from a standard calibration curve. Xylose, glucose, arabinose, lactic acid, formic acid, acetic acid and ethanol were deter-

16

(a)

glucose arabinose xylose acetic acid furfural efficiency

14 12 10 8

16 14 12 10 8

6

6

4

4

2

2

0 16

(b)

14

0 16 14

12

12

10

10

8

8

6

6

4

4

2

2

0 16

(c)

14

0 16 14

12

12

10

10

8

8

6

6

4

4

2

2

0 16

0 16

(d)

14

14

12

12

10

10

8

8

6

6

4

4

2

2

0

0

1

2

3

Time (h)

4

5

Efficiency

2.1.1. Raw material and pretreatment Sugarcane bagasse (cv. Supunburi 50, Thailand) was dried at 90 !C for 18 h or until weight was constant. Then, it was milled to obtain small particles (less than 5 mm). The particles were kept at 4 !C until use.

-1

2.1. Hydrolysis of sugarcane bagasse

2.2.2. Fermentation medium and conditions The hydrolysate from sugarcane bagasse with and without detoxification through amberlite weak-base anion-exchange resins (IRA-67, Fluka, France) was supplemented with 7 g l!1 of yeast extract and used as a fermentation medium for lactic acid production. The medium was adjusted to pH 6.0 with 2 M HCl. Lactic acid fermentation was carried out in a batch system in a Biostat B (B. Braun, Germany) 2 l fermenter with a working volume of 1 l at 37 !C. The culture was agitated at 100 rpm. The pH of the culture was kept at 6.0 by automatic addition of 2.5 M NaOH.

Sugar, acetic acid and furfural concentrations (g l )

2. Methods

0

Fig. 1. The main components of sugarcane bagasse hydrolysate and acid hydrolysis efficiency (E) under 0.5% of HCl and different temperatures: (a) 90, (b) 100, (c) 110 and (d) 120 !C.


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mined by HPLC with a refractometer detector (Shimadzu, Japan) using an Aminex-HPX 87H column (300 mm $ 78 mm, Bio-Rad Lab, CA, USA) under the following conditions: a temperature of 50 !C, mobile phase 5 mM of H2SO4 and a flow rate of 0.57 ml min!1 (Thani et al., 2006). 3. Results and discussion 3.1. Effects of ammonium hydroxide on lignin extraction The NH4OH treatment had a marked effect on lignin removal from the sugarcane bagasse. The lignin concentration in the soluble fraction after extraction by 0%, 8%, 10% and 12% of NH4OH were 38.01 ± 1.02, 303.39 ± 4.48, 322.19 ± 0.93 and 277.46 ± 4.26 mg l!1, respectively. This indicates that NH4OH could break the lignin seal in the sugarcane bagasse. The highest lignin concentration was achieved when applying 10% of NH4OH. The lignin concentration was approximately 8.5 times of that under the control condition (without NH4OH treatment). It was found that the lignin concentration was decreased at 12% of NH4OH; however, the reason is unknown. Therefore, 10% of NH4OH was used for lignin extraction from the sugarcane bagasse in all subsequent experiments. 3.2. Acid hydrolysis of sugarcane bagasse Main components of the sugarcane bagasse solutions hydrolysed by 0.5% of HCl at different temperatures are shown in Fig. 1. The results showed that xylose was the main product. The profiles of the main components of the sugarcane bagasse solutions hydrolysed by 2%, 3% and 5% of HCl were similar to those of 0.5% of HCl (data not shown). At 90–110 !C, it was observed that the longer the reaction time was, the higher xylose concentration was obtained (Fig. 1a–c). However, at the highest temperature (120 !C), the maximum xylose concentration was obtained at the reaction time of 4 h (Fig. 1d). A slight decrease in xylose concentration at 5 h might be due to the occurrence of decomposition reactions from xylose to furfural as shown in Fig. 1d and previously described by Rodríguez-Chong et al. (2004). Table 1 shows soluble fraction of sugarcane bagasse hydrolysed at different hydrolysis temperatures and HCl concentrations giving the maximum xylose concentration. The highest xylose concentration (15.16 g l!1) was obtained under 0.5% of HCl at 120 !C for 4 h. Under these conditions, glucose, 2.85; arabinose, 1.35; acetic acid, 0.04 and furfural, 0.66 (in g l!1) were detected. The amount of

arabinose under various conditions was not different (Table 1). Aguilar et al. (2002) found that acetic acid in hydrolysates was derived from the hydrolysis of the acetyl groups bound to the hemicellulosic monomers. The acid could be an inhibitor of microbial growth because it entered the cell membrane and decreased intercellular pH, thus affecting the metabolism of the microorganisms (Rodríguez-Chong et al., 2004). In this study; however, acetic acid was not produced in most conditions tested. The profiles of the main components of the sugarcane bagasse solutions hydrolysed by 0.5%, 2%, 3% and 5% of H2SO4 at different temperatures were similar to those of HCl treatment (data not shown). Table 2 summarises the soluble fraction of sugarcane bagasse hydrolysed at different hydrolysis temperatures and H2SO4 concentrations giving the maximum xylose concentration. The highest xylose concentration of 12.64 g l!1 was obtained when the bagasse was treated by 0.5% of H2SO4 at 110 !C for 4 h. Under these conditions, glucose, 2.28; arabinose, 1.33 and acetic acid, 0.06 (in g l!1) were detected whereas furfural was not detected. The highest xylose concentration obtained under H2SO4 hydrolysis was lower than that under HCl hydrolysis (Table 1), implying that HCl could break hemicellulose in sugarcane bagasse better than H2SO4. Gupta et al. (2009) reported that the release in sugar increased with increase in acid concentration and it declined thereafter. They explained that any further increase in acid concentration caused the increase in release of some toxic compounds or inhibitors, resulting in a decrease of sugar concentration. Under some acid hydrolysis conditions in the present study, the increase in acid concentration caused a decrease in xylose concentration without any increase in the inhibitors (furfural and acetic acid). However, the reason is not clear. 3.3. Catalytic efficiency (E) of acid hydrolysis and scale up acid hydrolysis of sugarcane bagasse The E values of HCl and H2SO4 hydrolysis at various conditions were calculated to compare the efficiency of the acids for sugarcane bagasse hydrolysis (Table 3 and Fig. 1). The E values of all acid hydrolysis conditions were higher than 1.0, indicating that these conditions gave the hydrolysates containing high concentrations of sugars and low concentrations of growth inhibitors (Rodríguez-Chong et al., 2004). The higher the E value was, the better acid hydrolysis occurred. The maximum E value of HCl hydrolysis (16.01) was obtained under the condition of 0.5% of HCl at 100 !C for 5 h, whereas that of H2SO4 hydrolysis (15.33) was obtained under the conditions of 0.5% of H2SO4 at 110 !C for 4 h. HCl gave

Table 1 Soluble fraction of sugarcane bagasse hydrolysed at different temperatures and HCl concentrations giving the maximum xylose concentration. Temperature (!C)

HCl (%, v/v)

Reaction time (h)

Xylose (g l!1)

Glucose (g l!1)

Arabinose (g l!1)

Acetic acid (g l!1)

Furfural (g l!1)

90

0.5 2 3 5 0.5 2 3 5 0.5 2 3 5 0.5 2 3 5

5 5 5 3 5 4 4 2 4 3 2 1 4 2 1 2

7.55 ± 0.38 7.33 ± 0.37 9.46 ± 0.47 8.56 ± 0.43 13.09 ± 0.65 7.56 ± 0.38 8.97 ± 0.45 8.94 ± 0.45 13.33 ± 0.67 9.40 ± 0.47 8.93 ± 0.45 11.18 ± 0.56 15.16 ± 0.76 7.69 ± 0.38 10.50 ± 0.53 11.89 ± 0.59

0.21 ± 0.01 1.21 ± 0.06 1.64 ± 0.08 1.18 ± 0.06 2.66 ± 0.13 1.67 ± 0.08 2.23 ± 0.11 2.00 ± 0.10 3.28 ± 0.16 1.87 ± 0.09 1.79 ± 0.09 1.20 ± 0.06 2.85 ± 0.14 1.41 ± 0.07 1.69 ± 0.08 5.47 ± 0.27

0.93 ± 0.05 1.04 ± 0.05 1.16 ± 0.06 1.13 ± 0.06 1.22 ± 0.06 1.23 ± 0.06 1.43 ± 0.07 1.11 ± 0.06 1.12 ± 0.06 1.24 ± 0.06 1.31 ± 0.07 1.30 ± 0.07 1.35 ± 0.07 1.22 ± 0.06 1.22 ± 0.06 1.11 ± 0.06

0 0 0 0 0.06 ± 0.00 0 0 0 0 0 0 0 0.04 ± 0.00 0 0 0

0 0 0 0 0 0 0.20 ± 0.01 0 0.28 ± 0.01 0 0 0 0.66 ± 0.03 0 0 0.75 ± 0.04

100

110

120

The experiments were performed in duplicate.


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Table 2 Soluble fraction of sugarcane bagasse hydrolysed at different temperatures and H2SO4 concentrations giving the maximum xylose concentration. Temperature (!C)

H2SO4 (%, v/v)

Reaction time (h)

Xylose (g l!1)

Glucose (g l!1)

Arabinose (g l!1)

Acetic acid (g l!1)

Furfural (g l!1)

90

0.5 2 3 5 0.5 2 3 5 0.5 2 3 5 0.5 2 3 5

4 5 5 5 5 5 4 3 4 2 3 2 4 2 2 1

7.62 ± 0.38 6.02 ± 0.30 5.84 ± 0.29 11.63 ± 0.58 10.96 ± 0.55 6.25 ± 0.31 6.12 ± 0.31 7.59 ± 0.38 12.64 ± 0.63 6.86 ± 0.34 7.91 ± 0.40 11.48 ± 0.57 10.12 ± 0.51 6.48 ± 0.32 8.87 ± 0.44 8.44 ± 0.42

0.17 ± 0.00 0.66 ± 0.03 0.71 ± 0.04 1.43 ± 0.07 2.01 ± 0.10 1.08 ± 0.05 0.71 ± 0.04 0.86 ± 0.04 2.28 ± 0.11 1.54 ± 0.08 1.59 ± 0.08 1.34 ± 0.07 1.76 ± 0.09 0.88 ± 0.04 1.22 ± 0.06 1.23 ± 0.06

1.03 ± 0.05 0.70 ± 0.04 0.65 ± 0.03 1.34 ± 0.07 1.18 ± 0.06 1.06 ± 0.05 1.20 ± 0.06 0.51 ± 0.03 1.33 ± 0.07 0.98 ± 0.05 1.13 ± 0.06 1.17 ± 0.06 0.86 ± 0.04 1.06 ± 0.05 0.93 ± 0.05 1.08 ± 0.05

0 0 0.04 ± 0.00 0 0 0 0 0 0.06 ± 0.00 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0.18 ± 0.00 0 0 0 0 0 0 0 0

100

110

120


Table 3 Catalytic efficiencies of acid hydrolysis (E) under various conditions. Temperature (!C)

Acid (%, v/v)

90

0.5 2 3 5 0.5 2 3 5 0.5 2 3 5 0.5 2 3 5

100

110

120

HCl hydrolysis

H2SO4 hydrolysis

Reaction time (h)

E

Reaction time (h)

E

5 5 5 3 5 4 4 2 4 3 2 1 4 2 1 2

8.69 ± 0.43 9.58 ± 0.48 12.26 ± 0.61 10.87 ± 0.54 16.01 ± 0.40 10.46 ± 0.52 10.53 ± 0.53 12.05 ± 0.60 13.85 ± 0.69 12.51 ± 0.63 12.03 ± 0.60 13.68 ± 0.68 11.39 ± 0.57 10.32 ± 0.52 13.41 ± 0.67 10.55 ± 0.53

4 5 5 5 5 5 4 3 4 2 3 2 4 2 2 1

8.82 ± 0.44 7.38 ± 0.37 6.92 ± 0.35 14.40 ± 0.72 14.15 ± 0.71 8.39 ± 0.42 8.03 ± 0.40 7.59 ± 0.38 15.33 ± 0.20 9.38 ± 0.47 10.63 ± 0.53 13.99 ± 0.70 12.74 ± 0.64 8.42 ± 0.42 11.02 ± 0.55 10.75 ± 0.54

higher hydrolysis efficiency and the hydrolysis temperature of HCl for sugarcane bagasse hydrolysis was lower than that of H2SO4. Moreover, HCl could generate more sugars than H2SO4 (Tables 1 and 2). Therefore, sugarcane bagasse hydrolysis using 0.5% of HCl at 100 !C for 5 h was the optimum conditions performed in all subsequent experiments. When the acid hydrolysis under the optimum conditions was carried out in the 50 l reactor at agitation rate of 60 rpm, a marked increase in xylose concentration (approximately 73%) was detected compared to that in the 30 ml test tube (Table 4). This might be due to the effects of agitation during the reaction. However, glucose and arabinose concentrations were similar in both reactors. In addition, agitation also promoted the production of the inhibitors, acetic acid and furfural, resulting in lower E value (10.85) in

Table 4 Comparison of the main components of the hydrolysates of sugarcane bagasse using 0.5% of HCl at 100 !C for 5 h in a 30 ml test tube and a 50 l reactor. Components (g l!1)

30 ml Test tube

50 l Reactor

Xylose Glucose Arabinose Acetic acid Furfural E

13.09 ± 0.65 2.66 ± 0.13 1.22 ± 0.06 0.06 ± 0.00 0 16.01 ± 0.40

22.59 ± 1.13 1.50 ± 0.08 1.29 ± 0.06 0.15 ± 0.06 1.19 ± 0.02 10.85 ± 0.50

the 50 l reactor. Under the optimum conditions, yields of xylose, glucose and arabinose were 254.91, 16.93 and 14.56 mg g!1 dry biomass. The yield of xylose in this study was approximately 92% of the theoretical xylose available from the sugarcane bagasse (Lavarack et al., 2002). According to the lignin content in sugarcane bagasse (18.1% on a dry weight basis) (Sun et al., 2004), the highest percentage of lignin removal in this work was approximately 21% (by weight). Han et al. cited in Ko et al. (2009) reported that removal of 20–65% of lignin in a sample was sufficient to increase in accessibility of cellulose for hydrolysis by enzymes. In this study, the delignified bagasse was further hydrolysed by HCl not enzymes. To our knowledge, there has been no report regarding the amount of lignin that should be removed before the acid hydrolysis. However, 92% of the theoretical xylose available from the sugarcane bagasse was occurred in the present study. This finding implied that 21% of lignin removal would be sufficient for accessibility of cellulose to the acid. The main components of the acid-treated hydrolysates of sugarcane bagasse and the E values from the literature were compared in Table 5. Xylose concentration obtained from this work was the highest compared to those from the other works, whereas total sugar concentration was approximately 1–5 g l!1 lower than those reported by Chandel et al. (2007) and Cheng et al. (2008). This was mainly due to lower glucose concentration in our work. Table 5 also suggests that the decomposition of xylan in sugarcane


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bagasse to xylose by our conditions occurred better than that of the other works as reported by Lavarack et al. (2002), while the conditions of Chandel et al. (2007) and Cheng et al. (2008) enhanced the formation of glucose. In addition, the inhibitors (acetic acid and furfural) detected in this work were significantly lower than those in the others, resulting in higher E value. The lower inhibitors might be due to lower acid concentration and temperature used in this study. Based on the obtained E value, the acid treatment conditions in our study gave the maximum efficiency of acid hydrolysis.

Although the reaction time of acid hydrolysis in this work was longer than those in the other works (Table 5), the incubation temperature was lower. Moreover, acetic acid was rarely occurred compared to that of the others. Therefore, acetic acid removal from the hydrolysate was not required. In some studies, high concentration of acetic acid in hydrolysate was removed by electrodialysis (Cheng et al., 2008) or ion exchange treatment (Chandel et al., 2007) before it could be used as a substrate for a bio-conversion. This causes an increase in the total cost of acid hydrolysis of lignocellulosic materials.

Table 5 Comparison of the main components of the acid-treated hydrolysates of sugarcane bagasse and the E values under various conditions from several studies. Xylose (g l!1)

Glucose (g l!1)

Arabinose (g l!1)

Total sugar (g l!1)

Acetic acid (g l!1)

Furfural (g l!1)

E

0.5% of HCl, 100 !C, 5 h (this work) 2% of H2SO4, 122 !C, 24.1 min (Aguilar et al. (2002)) 6% of HNO3, 122 !C, 9.3 min (Rodríguez-Chong et al. (2004)) 4% of H3PO4, 122 !C, 5 h (Gámez et al. (2006)) 2.5% of HCl, 140 !C, 30 min (Chandel et al. (2007)) 1.25% of H2SO4, 121 !C, 2 min (Cheng et al. (2008))

22.59 21.6 18.60

1.50 3.00 2.87

1.29 ND* 2.04

25.38 24.60 23.51

0.15 3.65 0.90

1.19 0.52 1.32

10.85 4.76 7.30

17.60 21.50 17.10

3.00 5.84 7.20

2.60 2.95 2.00

23.20 30.29 26.30

4.00 5.45 4.00

1.20 ND 1.40

3.74 4.70 4.11

Not determined.

2.0 1.8

7

1.6

1.0

2.0 1.8 1.6 1.4 1.2 1.0 .8 .6 .4 .2 0.0

2

5

1 0

0 0

10

20

30

40

50

60

70

80 25

8 7

(b)

20

6 5

-1 )

10

3

-1 Product concentrations (g l )

-1 ) Dry cell weight (g l

0.0

15

4

.8

.2

20

5

1.2

.4

(a)

6

1.4

.6

25

8

Sugar concentrations (g l

*

Acid hydrolysis conditions (references)

15

4 10

3 2

5

1 0

0 0

10

20

30

40

50

60

70

80

Time (h) Fig. 2. Batch culture profiles of L. lactis IO-1 grown on the non-detoxified hydrolysate (a) and the detoxified hydrolysate (b) of sugarcane bagasse supplemented with 7 g l!1 of yeast extract: lactic acid (d), formic acid (s), acetic acid (.), ethanol (4), dry cell weight (j), glucose (h), xylose (}), arabinose (q).


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14

30

(a)

12

25

10

20

8 15 6

5

2 0

0

10

14

20

30

40

50

60

70

30

(b)

12

0

25

10

20

-1

10

4

Sugar concentrations (g l )

Our preliminary study revealed that L. lactis IO-1 could not grow and produce in the sugarcane bagasse hydrolysate without nutrient supplementation (data not shown). Similar results were observed by Nancib et al. (2001) who found that nitrogen supplementation in dates hydrolysate had a significant effect on growth and lactic acid production of L. casei subsp. rhamnosus. Olsson and Hahn-Hägerdal (1996), Garde et al. (2002) and Martin et al. (2002) also found that lignocellulosic hydrolysates contained some inhibitors of bacterial growth e.g. furfural, 5-hydroxymethylfurfural (HMF), levulunic acid and phenol compounds. In addition, Mussatto and Roberto (2004) reported that anion-exchange resins removed high percentages of the toxic compounds (96% of acetic acid, 91% of phenolic compounds, 73% of furfural and 70% of HMF) from lignocellulosic hydrolysates. Therefore, in this study the sugarcane bagasse hydrolysate was detoxified by passing the broth through the amberlite (modified from Domínquez et al. (1997)) before use. It was found that the concentrations of xylose glucose, arabinose and acetic acid before and after detoxification were equal, whereas that of furfural was decreased from 1.19 to 0.09 g l!1. The effects of nitrogen supplementation were investigated by the addition of 0, 3, 5 and 7 g l!1 of yeast extract, peptone or ammonium sulphate or both 5 g l!1 of yeast extract and 5 g l!1 of peptone (as used in the basal medium) into the detoxified hydrolysate. The results showed that 7 g l!1 of yeast extract gave the highest yield of lactic acid (0.26 g lactic per g sugar utilised), while microbial growth and lactic acid production were not detected in the broth containing ammonium sulphate at all concentrations (data not shown). The absence of ammonium assimilation by L. lactis IO-1 suggests that small peptides may be required by this microorganism. Fig. 2 shows the batch culture profiles of L. lactis IO-1 grown on the non-detoxified and the detoxified hydrolysate containing 7 g l!1 of yeast extract. The results clearly showed that L. lactis could slightly grow in the non-detoxified broth but lactic acid was not produced (Fig. 2a). This might be due to the effects of some inhibitors in the medium as previously described. When the hydrolysate was detoxified, the results clearly showed that the detoxification improved the fermentability of the hydrolysate by L. lactis IO-1 compared to the non-detoxified hydrolysate, since without detoxification there was no lactic acid production (Fig. 2a). Xylose utilisation was started after glucose was completely consumed (Fig. 2b). Xylose was almost completely utilised, while L. lactis was not able to utilise arabinose. Products from the fermentation were increased against time and relatively constant at 40 h. The highest main product concentrations detected were acetic acid, formic acid, lactic acid and ethanol, respectively. In Fig. 2a, a weak growth is observed, however without any substrate consumption. This phenomena was explained by Benthin and Villadsen (1996), Loubière et al. (1997), Novák and Loubière (2000), who reported that for lactic acid bacteria, sugar is only a catabolic substrate leading to catabolic end-products whereas the biomass is built from anabolic precursors, e.g., amino acids, nucleotides, etc., present in yeast extract and peptone in the medium. Similar results were observed by Plihon et al. (1995) who performed batch experiments with Leuconostoc mesenteroids in de Man, Rogosa, and Sharpe (MRS) medium and concluded that from the carbon balance, biomass was not created from sugar metabolism. Thani et al. (2006) revealed that in xylose batch cultures, the molar product (lactic acid, acetic acid, formic acid and ethanol) yields were dependent on the initial xylose concentration. At high initial xylose concentrations of 30 and 50 g l!1, the molar product yield of lactic acid was the highest with the values of 0.71 and 0.87 mol product mol!1 xylose utilised, respectively while at low

initial xylose concentrations of 5 and 10 g l!1, the molar product yield of acetic acid was highest with the values of 1.21 and 0.95 mol product mol!1 xylose utilised, respectively. To increase molar product yield of lactic acid in this study, xylose concentration in the hydrolysate at 22 g l!1 was increased by the addition of xylose to 30 g l!1. Fig. 3a shows sugar consumption and product formation in the detoxified hydrolysate containing 30 g l!1 of xylose. The pattern of sugar consumption was similar to that of the detoxified hydrolysate without xylose addition (Fig 2b). The rates of xylose uptake in both media (Figs. 2b and 3a) were similar with approximately 0.51–0.52 g l!1 h!1 in the first 30 h. The xylose uptake in the detoxified hydrolysate with xylose addition continued at the same rate until the end of the fermentation (64 h) with complete sugar consumption. The lactic acid production in the detoxified hydrolysate with and without xylose addition were markedly observed in 64 and 40 h, respectively. The longer fermentation time giving the highest product concentration in the detoxified hydrolysate with xylose addition was due to higher initial sugar concentration. The rate of lactic acid production in the medium with xylose addition (0.14 g l!1 h!1) was slightly higher than that in the medium without xylose addition (0.11 g l!1 h!1). In the medium with xylose addition (Fig. 3a), lactic acid was produced at the highest concentration (10.85 g l!1) compared to other organic acids (Table 6). This observation suggested that xylose concentration might have a controlling effect on pyruvate metabolism in L. lactis IO-1 (CocaignBousquet et al., 1996). Arabinose was not utilised at all conditions

-1

3.4. Lactic acid fermentation from acid hydrolysate

Dry cell weight and product concentrations (g l )

6

8 15 6 10

4

5

2 0

0

10

20

30

40

Time (h)

50

60

70

0

Fig. 3. Batch culture profiles of L. lactis IO-1 grown on the detoxified hydrolysate of sugarcane bagasse containing 30 g l!1 of xylose and 7 g l!1 of yeast extract (a) and the basal medium containing 30 g l!1 of xylose (b): lactic acid (d), formic acid (s), acetic acid (.), ethanol (4), dry cell weight (j), glucose (h), xylose (}), arabinose (q).


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P. Laopaiboon et al. / Bioresource Technology xxx (2009) xxx–xxx Table 6 Main products in batch culture of L. lactis IO-1 grown on fermentation media. Fermentation medium

Production concentration (g l!1)

Fermentation time (h)

Dry cell weight

Lactic acid

Formic acid

Acetic acid

Ethanol

Detoxified hydrolysate containing 30 g l!1 of xylose Basal medium containing 30 g l!1 of xylose

1.34 ± 0.20 0.74 ± 0.13

10.85 ± 0.51 12.67 ± 0.62

6.04 ± 0.30 4.72 ± 0.23

7.87 ± 0.44 7.66 ± 0.38

5.24 ± 0.61 3.36 ± 0.33

64 37


tested (Figs. 2b and 3a). This might be due to the deficiency of nitrogen source for the sugar consumption of this bacterium. The reason was supported by Garde et al. (2002) who found that apart from glucose and xylose, arabinose (1.1 g l!1) in wheat straw hemicellulose hydrolysate supplemented with components of MRS medium were depleted within 12–24 h by Lactobacillus pentosus and Lactobacillus brevis. This might be due to the enrichment of the MRS medium which contained (in g l!1) casein peptone, 10; meat extract, 10; yeast extract, 5; tween 80, 1; K2HPO4, 2; sodium acetate, 5; diammonium citrate, 2; MgSO4%7H2O, 0.2 and MnSO4%H2O, 0.05. In this study, not only lactic acid but also acetic acid, formic acid and ethanol were the main products in the fermentation broth. These results indicated that xylose, the main sugar in the hydrolysate, was metabolised to lactic acid via heterolactic fermentation pathway or mixed acid fermentation (Cocaign-Bousquet et al., 1996; Liu, 2003). Batch culture profiles of L. lactis IO-1 grown on the lactic acid production medium (basal medium) containing 30 g l!1 of xylose are shown in Fig. 3b. Comparison between both media (Fig. 3a and b) revealed that the xylose uptake rate of the detoxified hydrolysate (0.51 g l!1 h!1) was 45% lower than that of the basal medium (0.92 g l!1 h!1), indicating that the inhibitors might not be completely removed by detoxification and/or some essential nutrient might be deficient. HPLC analysis showed that the amount of furfural significantly decreased from 1.19 to 0.09 g l!1 after detoxification. Acetic acid, an inhibitor from the acid hydrolysis, was not removed in this study because its concentration was very low (0.15 g l!1). Table 6 compares the product formation of L. lactis IO-1 on the detoxified hydrolysate and the basal medium containing 30 g l!1 of xylose. Growth of L. lactis IO-1 and formic acid and ethanol production in the detoxified broth was higher than those in the basal medium. On the other hand, lactic acid production was 14% lower than that in basal medium and the fermentation time was 27 h longer. Therefore, the rate of lactic acid production in the hydrolysate (0.14 g l!1 h!1) was 61% lower than that in the basal medium (0.36 g l!1 h!1). The yield of lactic acid was dependent on initial xylose concentration and the molar product yield of lactic acid was highest when the initial xylose concentration was at least 30 g l!1 as described previously (Thani et al., 2006). However, in this study the initial xylose concentration in the acid hydrolysate was only 22 g l!1. To raise xylose content in the hydrolysate to the desired level, concentrating the hydrolysate by evaporation prior to use it for lactic acid production is recommended. 4. Conclusions Sugarcane bagasse could be hydrolysed by dilute acid (0.5% of HCl) and xylose was obtained as the main fermentable sugar (89%). Lignin extraction by NH4OH was an essential pretreatment process prior to the acid hydrolysis, and the detoxification was required for removing the inhibitory compounds found in the hydrolysate. The detoxified hydrolysate of sugarcane bagasse supplemented with yeast extract was found to be a potential substrate for lactic acid production by L. lactis IO-1. As yeast extract

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