Detoxification of sago trunk hydrolysate using activated charcoal for xylitol production

Procedia Food Science 1 (2011) 908 913 11 th International Congress on Engineering and Food (ICEF11) Detoxification of sago trunk hydrolysate using activated charcoal for xylitol production Siti M. Mustapa Kamal aa *, Nurul L. Mohamad a, Abdul G. Liew Abdullah b, Norhafizal Abdullah b a Department of Process and Food Engineering, b Department of Chemical and Environmental Engineering Faculty of Engineering, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. Email 1* : siti@eng.upm.edu.my Abstract Xylitol is one of the alternative natural sweeteners; belong to a group of sugar alcohol. It can be derived from D- xylose which mainly contains in lignocellulose materials. In this study sago trunk cortex was chosen as lignocellulose source due to its availability and abundant in the sago starch processing industry. The production of xylitol includes hydrolysis which breaks the cellulose and hemicellulose polymers to fermentable sugar, mainly xylose, followed by fermentation process which converts the sugars to xylitol. However, some by-products such as furfural and phenolic are released during chemical hydrolysis and inhibit fermentation process. Detoxification procedures were carried out over sago trunk hydrolysates. Powdered activated charcoal was mixed with the hydrolysate at 1% and 2.5% (w/v) and stirred for 30 and 60 minutes at room temperature. The recovery of xylitol was performed using yeast Candida tropicalis in sago trunk cortex hydrolysate. This study describes detoxification methods of sago trunk cortex hydrolysates to improved xylitol production by Candida tropicalis. The effects of 1% and 2.5% (w/v) activated charcoal were identified to the growth and xylitol concentration. This study found that with the application of activated charcoal method, it enabled a reduction of furfural (58%) and total phenolics (78%) compounds. The best conditions was achieved with 2.5% activated charcoal at adsorption time of 60 minutes and the maximum xylitol concentration, xylitol yield and volumetric productivity obtained were 19.53 g l -1, 0.78 g g -1 and 0.37 g l -1 h -1. The value of xylitol yield using the detoxification hydrolysate medium was higher when compared to non-treated medium (0.307 g g -1 ). This strongly suggests that detoxification method using activated charcoal has a significant impact in xylitol production. 2011 Published by Elsevier B.V. Open access under CC BY-NC-ND license. Selection and/or peer-review under responsibility of 11th International Congress on Engineering and Food (ICEF 11) Executive Committee. Keywords: Sago trunk hydrolysate; activated charcoal; xylitol; detoxification; Candida tropicalis * Corresponding author. Tel.: 006-03-89466294; fax: +006-03-8946440. E-mail address: siti@eng.upm.edu.my. 2211 601X 2011 Published by Elsevier B.V. Open access under CC BY-NC-ND license. Selection and/or peer-review under responsibility of 11th International Congress on Engineering and Food (ICEF 11) Executive Committee. doi:10.1016/j.profoo.2011.09.137

Siti M. Mustapa Kamal et al. / Procedia Food Science 1 (2011) 908 913 909 1. Introduction Xylitol is five carbon sugar alcohols used as an alternative sweetener due to the same sweetness of the conventional sugar [1]. Xylitol can be derived from D-xylose which mainly contains in lignocellulose materials. In this study sago trunk cortex was chosen as lignocellulose source due to its availability and abundant in the sago starch processing industry. The production of xylitol includes hydrolysis which breaks the cellulose and hemicellulose polymers to fermentable sugar, mainly xylose, followed by fermentation process which converts the sugars to xylitol. The main drawback of lignocelluloses synthesis from diluted acid hydrolysis processes is the degradation of sugars in hydrolysis processes and formation of undesirable by-products, which inhibit the fermentation process [2]. The by-products in diluted acid concentrations are divided into three main groups: (1) weak acids, e.g. acetic acids and formic acids, (2) furan derivatives, e.g. furfural and 5- hydroxymethylfurfural, and (3) phenolic compound [3]. Several detoxification methods have been studied to convert inhibitors to inactive compounds or to reduce their concentrations. The most common methods employed are neutralization and overliming [4,5], biological adaptation [6], extraction with organic solvent [7], adsorption with activated charcoal [8,9] and ion-exchange resins. The effectiveness of the methods depends on the types of hemicellulose hydrolysate and the species of microorganisms employed because different types of hydrolyzate have different degrees of toxicity and each microorganism with different degrees of tolerance to inhibitors [10]. Adsorption with activated charcoal is widely used to remove compounds from the hydrolysate. The effectiveness of this treatment depends on several variables used for adsorption process, namely; ph, temperature, contact time and charcoal concentration [10]. However, charcoal concentration and contact time strongly influence the compound removal [11]. The sugarcane bagasse treated with activated charcoal in varying proportions varying 1% to 30% [12]. In addition, they observed that 1% of charcoal was sufficient to remove 94% of phenolic compound. The main aim of this study was to investigate the effects of detoxification process using activated charcoal for reducing inhibitors from acid hydrolysis of sago trunk hydrolysate. To achieve the aim, two objectives were outline which to identify the effects of the two levels of activated charcoal concentration on the adsorption time and to verify the effectiveness to the xylitol concentration, yield and productivity in the sago trunk hydrolysate medium. 2. Materials and Methods Raw material Sago bark was collected from a local plantation in Melaka. The cortex (outer layer) was removed from the bark and the pith (inner portion) was used for starch production. The sago trunk cortex was subjected to hot and cold water extraction, air-dried and ground to a particle size <1 mm. The homogenized sago trunk cortex was oven-dried at 105 C overnight and considered dried when it reach moisture content less than 10% by weight and the dried sago trunk cortex was analyzed for determinations of its main compositions 2.2. Diluted Acid Hydrolysis Conditions Sago trunk ground chips were hydrolyzed by using 8% of H 2 SO 4 at 121 C for 60 minutes. The slurry was filtered through a vacuum filtration to separate the solid contains of cellulose and lignin with the liquid portion, which primarily contained hemicellulose. The hydrolysate contained the original concentration for each treatment.

910 Siti M. Mustapa Kamal et al. / Procedia Food Science 1 (2011) 908 913 2.3. Detoxification Procedures Powdered activated charcoal was mixed with the ph 5.5 hydrolysate at 1% and 2.5% (w/v) and stirred for 30 and 60 minutes at room temperature as this range was enough to remove the inhibitors [9]. The complete detoxified hydrolysates were recovered by using a vacuum filtration. The ph of the hydrolysate was adjusted to 7 with NaOH or H 2 SO 4 addition prior to fermentation. 2.4. Microorganism The strain of C. tropicalis was selected in this work due to its ability to ferment xylose and it is known as a promising xylitol producer [13]. The cells were maintained in the Saboroad Dextrose Agar plates and subcultured for twice a month at 4 C. 2.5. Inoculum Preparation and Production Media The hydrolysates of ph 5.5 (non-treated medium) and detoxified medium were used for fermentation experiments. The medium containing 25 g L -1 D-xylose, 30 g L -1 yeast extract, 45 g L -1 KH 2 PO 4, 9 g L -1 (NH 4 ) 2 HPO 4, and 3 g L -1 MgSO 4 7H 2 O was used for inoculum preparation, and the sago trunk cortex hydrolysate was used for the production medium. 2.6. Fermentation The recovery of xylitol was performed using yeast Candida tropicalis in sago trunk cortex hydrolysate. The strain of Candida tropicalis was selected in this work due to its ability to ferment xylose. The fermentations were placed in a platform shaker at 200 rpm with temperature of 30 C for 72 h without controlling ph. Fermentations were performed to compare the fermentability of detoxification hydrolysate medium and non-treated medium and the effect of activated charcoal on removing inhibitors (furfural and phenolic compounds). 2.7. Analysis The samples from the production medium were centrifuged at 4000 rpm for 15 min. The decant supernatant was then separated and used for analyzing sugar concentration. Xylose, glucose, xylitol and furfural compounds were analyzed by using the High Performance Liquid Chromatography (HPLC) Class VP Shimadzu, Japan and the method used as described in Chapter 3. On the other hand, the total amount of phenolic compound was detected by employing the spectral analysis using the UV-VIS spectrophotometer with the absorbance at 280 nm [14] using distilled water as standard. Meanwhile, the cell concentration was estimated by measuring the absorbance at 600 nm using the UV-VIS spectrophotometer. The dry cell weight was determined by drying in the oven at 95 C until a constant weight was achieved. All analyses were done in duplicate. 3. Results and Discussion The sago trunk hydrolysate after going through the diluted acid hydrolysis contained about 21% of total fermentable sugars (xylose and glucose). Furfural and total phenolic compounds were also found in the hydrolysate. The furfural compound was detected as the main degradation compound of pentoses (xylose), since pentoses are the main monosaccharides presented in the hydrolysates [8]. Table 1 shows the data obtained of the compounds in the hydrolysates for the non-treated medium and detoxified medium with activated charcoal effects. The ph was adjusted to ph 5.5 (non-treated medium) used for raising the acidic ph of raw hydrolysates to the suitable conditions for yeast growth. The

Siti M. Mustapa Kamal et al. / Procedia Food Science 1 (2011) 908 913 911 detoxification treatments of all activated charcoal treatments had minimal effects on sugar recovery, with about 4% of sugar loss for xylose and glucose. The Eucalyptus hydrolysates treated with 5% activated charcoal, resulted in 4.5% of sugar losses [15]. Table 1. Effects of activated charcoal treatments on the composition of sago trunk cortex hydrolysate Detoxification Treatment Xylose Glucose Furfural Phenolic Compound Non treated medium (ph 5.5) 25.74 3.72 2.53 2.15, 1%, 30 min 24.95 3.57 1.70 1.13, 1%, 60 min 24.71 3.16 1.72 0.989, 2.5%, 30 min 24.43 3.19 1.34 0.66, 2.5%, 60 min 24.27 3.05 1.18 0.47 The detoxification method applied enabled a reduction of furfural and total phenolic compounds. However, the most important reductions of 53% and 78% of the furfural and phenolic compounds were obtained after the treatment with 2.5% activated charcoal at the reaction time of 60 min. It can be seen that the activated charcoal treatment was effective in reducing the phenolic compound. The activated charcoal was a very efficient method for the total removal (100%) of furan derivatives (furfural) in eucalyptus residue hydrolysates [15]. In order to ensure the effectiveness of the detoxification, Candida tropicalis was grown in media made from raw sago trunk hydrolysate (non-treated medium) and detoxified medium. All hydrolysates were supplemented with nutrients for the purpose of exclude growth limitation due to nutritional deficiency. Figure 1 shows the time courses of xylose consumption, xylitol production and cell growth in media made from detoxified hydrolysate. Table 2 presents the fermentative parameters of fermentation assays. Results were calculated for fermentation times leading to the maximum xylitol concentrations. Table 2. Kinetic parameters of Candida tropicalis growth and product formation in the sago trunk cortex hydrolysates Detoxification Treatment Non-treated medium (ph 5.5) 72 7.128 0.307 0.099 0.040 0.383 0.003 72 12.130 0.649 0.168 0.06 0.934 0.021 1%, 30 min 60 13.080 0.683 0.218 0.08 0.821 0.021 1%, 60 min 48 16.840 0.67 0.350 0.124 0.684 0.023 2.5%, 30 min 52 19.530 0.78 0.370 0.113 1.179 0.027 2.5%, 60 min t, time required to achieve maximum xylitol concentration; P m, maximum xylitol production; Y P/S, xylitol yield on the consumed xylose; Q P, volumetric productivity; q P, specific productivity, Xylitol yield on consumed biomass, μ,specific growth rate

912 Siti M. Mustapa Kamal et al. / Procedia Food Science 1 (2011) 908 913 A B C D Fig. 1. Effect of detoxification methods on Candida tropicalis fermentative behaviour for xylitol production after treatment with activated charcoal: (A) 1% (w/v), 30 min, (B) 1% (w/v), 60 min, (C) 2.5% (w/v),30 min, (D) 2.5% (w/v), 60 min. The symbols ( ) xylose ( ) glucose, ( ) xylitol and ( ) cell concentration Based on the results in Table 2, xylitol production was strongly inhibited in non-treated medium where Candida tropicalis produced only 7.128 g L -1 after 72 h. Low volumetric productivity and poor product yield achieved. The fermentation results of detoxified hydrolysates indicate that detoxification method was capable of improving the Candida tropicalis performance. Xylitol was the main product while the production ranged from 7 g L -1 to about 20 g L -1 at different time requirements. The activated charcoal treatment at 2.5% and 60 min exhibited the highest xylitol productivity (0.37 g L -1 h -1 ) and a rather high xylitol yield on the consumed xylose (0.780 gg -1 ) if compared to the non-treated medium (0.307 g g -1 ). This strongly suggests that detoxification method using activated charcoal has a significant impact on xylitol production. The overall results obtained for the fermentability of Candida tropicalis in the sago trunk cortex hydrolysate showed that there was a greater significant difference in the yield and productivity between the nontreated medium and the detoxification medium. Xylitol productivity was improved by three times compared to the non-treated medium. Table 2 also shows the effects of detoxified and non-detoxified media on the speficic growth rate of Candida tropicalis on the sago trunk hydrolysates. The values of specific growth rate point out the ability of strain to grow in the hydrolysates [16]. The detoxification of activated charcoal resulted in an increase in the specific growth rate. Activated charcoal method had improved the biomass yield up to 1.179 g g -1 and the specific growth rate was quite high. 4. Conclusion From the results, it is evident that activated charcoal method could reduce some inhibitory compounds present in the studied sago trunk hydrolysate. The results also indicate that a good fermentability could be

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