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Optimization of pH and acetic acid concentration for bioconversion of hemicellulose from corncobs to xylitol by Candida tropicalis
Biochemical Engineering Journal 43 (2009) 203–207
Contents lists available at ScienceDirect
Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Short communication
Optimization of pH and acetic acid concentration for bioconversion of hemicellulose from corncobs to xylitol by Candida tropicalis Ke-Ke Cheng a , Jian-An Zhang a,∗ , Hong-Zhi Ling b , Wen-Xiang Ping b , Wei Huang a , Jing-Ping Ge b , Jing-Ming Xu a a b
Division of Green Chemistry and Technology, Institute of Nuclear and New Energy Technology, Tsinghua University, Beijing 100084, PR China Key Laboratory of Microbiology, College of Life Sciences, Heilongjiang University, Harbin 150080, PR China
a r t i c l e
i n f o
Article history: Received 19 May 2008 Received in revised form 12 September 2008 Accepted 20 September 2008 Keywords: Corncob Fermentation Hydrolysate Pretreatment Xylitol Xylose
a b s t r a c t Hemicellulose hydrolysate from corncobs, separated by diluted sulfuric acid and sequently detoxed by boiling, overliming and solvent extraction, was used for xylitol production by Candida tropicalis W103. The effect of glucose and acetate in hydrolysate on xylitol production was investigated. It was found that glucose in hydrolysate promoted growth of Candida tropicalis while acetate at high concentration was inhibitory. The acetate inhibition can be alleviated by adjusting pH to 6 prior to fermentation and a substrate feeding strategy. Under these optimum conditions, a maximal xylitol concentration of 68.4 g l−1 was obtained after 72 h of fermentation, giving a yield of 0.7 g g−1 xylose and a productivity of 0.95 g l−1 h−1 . © 2008 Elsevier B.V. All rights reserved.
1. Introduction Lignocellulosic materials represent an abundant and inexpensive source of sugars which can be microbiologically converted to industrial products. Among these products available from lignocellulose-derived sugars, xylitol has received much attention. Xylitol is a natural polyol with particular physico-chemical properties, which permit its use either in foods like chewing gum, bakery and chocolate as a sweetener with anticariogenic property, or in medicines as a sugar substitute for the treatment of diabetes and erythrocytic glucose-6-phosphate dehydrogenase deficiency [1]. Traditional chemical conversion of xylose into xylitol is rather difficult, has a low product yield and consequently its high price has hindered the utilization of xylitol in food or medicine industries. The microbial conversion of xylose to xylitol is particularly attractive in that the process is relatively easy and does not need toxic catalyst [2]. Dilute sulfuric acid hydrolysis is a favourable method for the conversion of lignocellulose to sugars, especially for hemicellulose to hexoses. Hydrolysis of lignocellulosic materials always goes together with the formation of byproducts that inhibit the fer-
mentation process [3]. Hemicellulose hydrolysate can be converted to xylitol by several microorganisms including Pichia and Debaryomyces also Candida [4–7]. Among these, Candida ferments xylose to xylitol in a high yield and productivity. However, due to the inhibitors in hydrolysate, it is difficult to obtain a high xylitol concentration in the fermentation broth. Many studies have been conducted utilizing the hemicellulose portion of agricultural residues like eucalyptus, rice straw, corncob, brewer’s spent grain, sugarcane bagasse, and corn stover for xylitol production [8–13]. Among the various agricultural crop residues, corncob is one of the most abundant agricultural materials in Northeastern China. In this study, corncob was chosen as the raw lignocellulosic material. Candida tropicalis W103, which was screened in our laboratory and can produce 200 g l−1 xylitol at pH 4.5 in fed-batch cultures using xylose as the sole carbon source, was used to covert corncob hydrolysate to xylitol. The effects of glucose and acetate in corncob hydrolysate on xylitol production were investigated to improve both product concentration and productivity. 2. Materials and methods 2.1. Pretreatment
∗ Corresponding author. Tel.: +86 10 89796086; fax: +86 10 62785475. E-mail address: [email protected] (J.-A. Zhang). 1369-703X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2008.09.012
Corncob from Heilongjiang province in Northeastern China was used as raw material. Particles in the size ranged from 0.45 mm
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Table 1 Compositions of corncobs. Component
Values (%)
Methods
Benzene-ethanol extractive (%, w/w) Cellulose (%, w/w)
3.01 ± 0.08 42.69 ± 0.37
Holocellulose (%, w/w)) Hemicellulose Klason lignin (%, w/w) Acid-soluble lignin (%, w/w) Total lignin (%, w/w)
77.11 34.32 17.52 0.91 18.42
GB/T 2677.6-1994 Nitric acid-ethanol method GB/T 2677.10-1995 Calculated value GB/T 2677.8-1994 GB/T 747-2003 GB/T 2677.8-1994, GB/T 10337-1989
± ± ± ± ±
0.55 0.46 0.41 0.04 0.33
The values were the mean of four independent samples. Cellulose content was determined by the nitric acid-ethanol method. Hemicellulose is calculated by difference between hollocellulose and cellulose.
to 0.9 mm (20–40 mesh) were used in the experiments. The compositions of corncob were determined according to corresponding Chinese National standards [14]. The data were shown in Table 1. The pretreatment was carried out in 1000 ml glass flasks. 40 g corncobs at a solid loading of 10% (w/w) was mixed with dilute sulfuric acid (1% (w/w)) and pretreated in an autoclave at 120 ◦ C with residence time of 1 h. The liquid fraction was separated by filtration and the unhydrolysed solid residue was washed with 40 ml warm water (60 ◦ C). The filtrate and wash liquid were pooled together. The composition of the corncob hemicellulose acid hydrolysate 5.4 g glucose l−1 , 3.7 g arabinose l−1 , was 28.7 g xylose l−1 , −1 −1 0.5 g cellobiose l , 0.7 g galactose l , 0.4 g mannose l−1 , 2 g acetic acid l−1 , 0.8 g furfural l−1 , 0.2 g 5-hydroxymethylfurfural l−1 . 2.2. Detoxification Hemicellulose acid hydrolysate was heated to 100 ◦ C, and maintained for 15 min to reduce the volatile components. The hydrolysate was then overlimed with solid Ca(OH)2 up to pH 10.0, in combination with 0.1% sodium sulfite, filtered to remove the insoluble. The filtrate was adjusted to pH 7 with 3 M H2 SO4 and extracted with ethyl acetate to remove the phenolics derived from lignin according to González [15]. The water phase was used for xylitol production. The detoxed hydrolysate was concentrated under vacuum at 50–75 ◦ C to achieve 10–11% (w/v) of xylose concentration for storage and later usage.
xylose fermentation medium. The pH was adjusted at 4.5 by addition of 3 M NaOH before inoculation and pH was maintained at 4.5 throughout the cultivations. The cultivation period was 50 h and the experimental values were the means of three independent samples. In xylitol production under different substrate, the detoxed and concentrated hydrolysate was diluted to prepare the hydrolysate fermentation medium, which contained 60 g xylose l−1 , 10 g glucose l−1 , 7 g arabinose l−1 and 2.6 g acetate l−1 . In other comparative fermentation tests, analytic pure reagents were used for substrate. The cultivation periods under different substrate were varied so that xylitol concentration in broth reached the highest respectively. In all experiments of this section, pH was adjusted to 4.5 before inoculation and maintained at 4.5 throughout the cultivations. The experimental values were the means of three independent samples. In xylitol production with hydrolysate fermentation medium under different pH, pH was adjusted to designed value before inoculation and maintained at designed value throughout the cultivations. The cultivation periods were also varied so that xylitol concentration in broth reached the highest respectively. The experimental values were the means of three independent samples. In xylitol fed-batch fermentation, pH was adjusted to 6 before inoculation and maintained at 6 throughout the cultivations. Sterilized concentrated hydrolysate was continuously droped to the bioreactor with a peristaltic pump in 15–45 h. The experimental values were the means of two independent samples. 2.4. Analytical methods The liquid samples were analyzed by HPLC, equipped with UV and RI detectors. The concentrations of glucose, xylose, galactose, mannose and arabinose were determined using refractive index detector and Aminex HPX-87P column at 85 ◦ C with H2 O as mobile phase at 0.8 ml min−1 . Cellobiose, acetic acid and ethanol were analyzed using refractive index detector and Aminex HPX-87H column at 65 ◦ C with 5 mM H2 SO4 as mobile phase at 0.8 ml min−1 . Furfural was detected on UV chromatograms at 250 nm. Cell growth was monitored at 600 nm and converted to cell dry weight (CDW) by an appropriate calibration curve. 3. Results
2.3. Microorganism and fermentation experiments
3.1. Effects of acetic acid and substrate on the xylitol formation
Candida tropicalis W103 was grown on the preculture medium containing 2 g KH2 PO4 l−1 , 5 g (NH4 )2 SO4 l−1 , 4 g yeast extract l−1 , 0.5 g MgSO4 ·7H2 O l−1 , 20 g xylose l−1 . The pretreated corncob hemicellulose hydrolysate or prepared xylose, supplemented with 2 g KH2 PO4 l−1 , 5 g (NH4 )2 SO4 l−1 , 0.5 g MgSO4 ·7H2 O l−1 , 1 g peptone l−1 , 5 g yeast extract l−1 , was used as the fermentation medium. The seed cells for the bioreactor were prepared in 500 ml flasks containing 100 ml preculture medium. The flasks were incubated at 35 ◦ C and 250 rpm for 14 h and subsequently inoculated into the bioreactor at 5% (v/v). The biomass concentration in bioreactor at the beginning of the fermentation ranged from 0.29 g l−1 to 0.31 g l−1 . The batch or fed-batch cultivations were conducted in a 1000 ml stirred-vessel bioreactor (Biostat Q1000, B.Braun, Germany) containing 750 ml fermentation medium under 0.6 vvm air flow. All fermentation experiments were carried out at 35 ◦ C and 500 rpm and the broth was sampled in every 6–12 h to monitor the xylitol concentration. In acetic acid addition batch fermentation test, acetic acid of different concentration (0.5–4 g l−1 ) was added to the prepared
After detoxification by boiling, overliming and solvent extraction, volatile compounds, such as furfural and 5hydroxymethylfurfural, were not detected. However, detoxification also resulted in the loss of glucose by 6%, xylose by 9.7% and arabinose by 10.1%. Similar observations were reported during the neutralization of sugarcane bagasse and corncob acid-catalysed hemicellulose acid hydrolysate with lime [16,17]. In order to evaluate the effect of acetic acid on the formation of xylitol in Candida tropicalis, cells were grown in synthetic medium containing acetic acid at different levels. Xylose consumption was not affected by acetic acid until its concentration was higher than 2 g l−1 . The consumption of xylose depended on the amount of acetic acid. When the acetic acid reached 4 g l−1 , the final xylitol concentration in broth was 72% lower than in the medium without acetic acid addition (Table 2). The acetic acid in the medium was consumed completely if its concentration was lower than 2 g l−1 . However, only 2 g l−1 acetic acid was used when its concentration was higher than 2 g l−1 . Similar observations were reported by Lima et al. [18].
K.-K. Cheng et al. / Biochemical Engineering Journal 43 (2009) 203–207
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Table 2 Xylitol production in batch cultures by Candida tropicalis W103 from prepared xylose containing acetic acid at different levels. Acetic acid −1
Initial (g l
Xylose )
0 0.5 1 2 3 4
−1
Residual (g l
)
0 0 0 0 1.0 ± 0.1 2.0 ± 0.1
Xylitol production −1
Initial (g l 75.4 74.8 79.2 75.5 78.9 75.5
± ± ± ± ± ±
)
0.7 1.4 2.1 0.9 1.3 1.1
−1
Residual (g l 2.1 1.0 1.0 7.8 11.6 49.4
± ± ± ± ± ±
)
0.9 0.2 0.4 1.9 1.4 1.7
Concentration (g l−1 ) 52.8 53.9 54.7 48.8 44.3 14.4
± ± ± ± ± ±
0.9 0.4 0.7 1.3 1.5 1.7
Productivity (g l−1 h−1 ) 1.05 1.07 1.09 0.98 0.89 0.29
± ± ± ± ± ±
0.02 0.03 0.02 0.04 0.05 0.03
Yield (g g−1 xylose) 0.72 0.73 0.70 0.72 0.66 0.55
± ± ± ± ± ±
0.01 0.02 0.01 0.02 0.03 0.02
The biomass concentration at the beginning of the fermentation ranged from 0.29 g l−1 to 0.31 g l−1 .All fermentation experiments were carried out at 35 ◦ C and pH 4.5. The cultivation period was 50 h and the experimental values were the means of three independent samples.
Table 3 Xylitol production in batch cultures by Candida tropicalis W103 with treated corncob hydrolysate as substrates. pH
Fermentation period (h)
Xylose
Xylitol production −1
Initial (g l 4.5 5 5.5 6 6.5
64 70 76 64 64
65.8 66.3 66.7 65.9 66.2
± ± ± ± ±
0.9 1.8 2.4 1.9 1.3
)
−1
Residual (g l 12.7 3.6 3.4 2.0 2.9
± ± ± ± ±
)
1.9 0.2 1.0 0.7 0.4
Concentration (g l−1 ) 22.3 30.1 40.5 45.4 44.3
± ± ± ± ±
0.9 0.4 1.7 1.5 1.5
Productivity (g l−1 h−1 ) 0.34 0.43 0.54 0.71 0.69
± ± ± ± ±
0.04 0.03 0.06 0.03 0.01
Yield (g g−1 xylose) 0.42 0.48 0.63 0.71 0.70
± ± ± ± ±
0.03 0.02 0.06 0.02 0.01
The biomass concentration at the beginning of the fermentation ranged from 0.29 g l−1 to 0.31 g l−1 .All fermentation experiments were carried out at 35 ◦ C. The values were the means of three independent samples.
To examine if the hydrolysate after detoxification can be used directly in xylitol production, a batch fermentation was conducted firstly using a sterilized hydrolysate. For comparison, batch xylitol production was also carried out using prepared xylose medium, xylose and glucose medium, xylose, glucose and acetate medium (Fig. 1A–D). The results showed that both the cell growth rate and xylitol productivity were lower in hydrolysate batch culture than those in prepared xylose batch culture. Fig. 1A, C and D showed that the glucose prior to xylose was used for cell growth. The consumption rate of glucose in xylose and glucose medium was 0.78 g l−1 h−1 in 0–6 h, much higher than that of xylose (0.1 g l−1 h−1 ). During the early stages of prepared glucose/xylose mixture fermentation, the cell densities were higher than those in xylose fermentation. The maximal growth rate was 0.27 h−1 in the prepared xylose and glucose medium, while it was 0.19 h−1 in the xylose medium. No xylitol but ethanol formation was observed when glucose was used as substrate. However, the xylitol productivity under prepared xylose and glucose medium was higher than that under prepared xylose medium. This phenomenon can be explained by higher biomass in prepared xylose and glucose medium. With prepared xylose, glucose and acetate as substrates, both cell growth and xylitol formation were slower than those with prepared xylose medium. These further validated that high concentration acetate had a negative effect on xylitol production.
3.3. Xylitol production by fed-batch fermentation In view of the high cost of xylitol recovery from aqueous solution, an economical production of xylitol from corncob hydrolysate requires the improvement of both product concentration and productivity. Xylitol batch fermentation was inhibited by high acetate concentration, so fed-batch cultivations with low initial acetate concentration and at optimized pH were studied further in order to increase the final xylitol concentration in broth. In the subsequent experiments, concentrated hydrolysate containing 117.7 g xylose l−1 , 21.1 g glucose l−1 , 15.3 g arabinose l−1 , 6.8 g acetate l−1 was added during 15–45 h to maintain xylose concentration within 15–40 g l−1 . Fig. 2 showed typical profiles of cell growth, substrate consumption and product formation. Higher assimilation rate of glucose was observed compared with that of xylose. Glucose was not detected after the initial glucose in the fermentation medium was exhausted. Besides xylose, a small concentration of arabinose (3 g l−1 ) was present in the hydrolysate, being partially consumed (72.4%) throughout the fermentative process. With the feeding of hydrolysate, the acetate in broth increased and then decreased after the ending of feeding. During the fermentation process, the concentration of acetate was lower than 2.5 g l−1 . The final concentration of xylitol was 68.4 g l−1 with a yield of 0.7 g g−1 xylose and productivity of 0.95 g l−1 h−1 .
3.2. Xylitol production under different pH 4. Discussion It had been found for some microorganisms that the inhibition of acetate was due to its undissociated form [19]. With the increase of pH, the undissociated form of acetic acid would decrease. Xylitol fermentations with corncob hydrolysate as substrate were performed at different pH to alleviate the inhibition of acetate. Total xylose consumption at pH 4.5 was lower than one half the starting xylose concentration, while it was almost complete after an average time of only 64 h at pH 6. An increase in pH from 4.5 to 6.0 led to dramatic increases in both xylitol productivity and yield, while the highest yield of biomass on consumed xylose was observed at pH 6.0. It indicated that acetate inhibition could be alleviated to some extend by pH adjustment (Table 3).
In our previous study, Candida tropicalis W103 can produce 200 g l−1 xylitol in fed-batch cultures using xylose as the sole carbon source. However, the fermentation parameters for these treated corncob hemicellulose acid hydrolysate are much lower than those obtained with a synthetic medium. This shows that there were some leftover toxic components in the treated hemicellulose acid hydrolysate that negatively affected the fermentation performance of C. tropicalis. To further improve the fermentation efficiency, proper culture conditions and detoxification techniques should be developed to alleviate the inhibitions. Lima studied the effect of acetic acid present in bagasse hydrolysate on the activ-
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Fig. 2. Time course of xylitol fed-batch fermentation by Candida tropicalis W103 with treated corncob hydrolysate as substrates at pH 6: xylose (), glucose (), arabinose (), xylitol (䊉), acetate (), ethanol (), CDW ().
ities of xylose reductase and xylitol dehydrogenase in Candida guilliermondii FTI 20037. They reported the maximum values of xylitol batch production (25 g l−1 ), productivity (0.42 g l−1 h−1 ) and xylitol yield (0.7 g g−1 ) after 58 h fermentation. The cell growth of C. guilliermondii FTI 20037 decreased 22% when it grew in medium containing 5 g acetic acid l−1 at pH 5.5, which corresponded to 0.75 g undissociated acetic acid l−1 . Rodrigues studied xylitol fed-batch production by C. guilliermondii FTI 20037 in a sugarcane bagasse hydrolysate medium. The values achieved for xylitol concentration, yield and volumetric productivity were 44 g l−1 , 0.78 g g−1 and 0.62 g l−1 h−1 [20]. In this study, higher xylose concentration of 68.4 g l−1 and productivity of 0.95 g l−1 h−1 were obtained. Xylose consumption and xylitol formation were not affected by acetic acid at pH 4.5 if its concentration was not higher than 2 g l−1 , corresponding to 1.29 g undissociated acetic acid l−1 at pH 4.5. Candida tropicalis W103 was more tolerant to the inhibition of undissociated acetic acid than Candida guilliermondii FTI 20037 [18]. In view of the high cost of product recovery, a high final product concentration with low impurity is desirable. To achieve this, the complex sugar, including xylose, glucose, cellobiose, mannose, galactose, and arabinose should be controlled at the minimum level in the final broth. The utilization of glucose and xylose by C. tropicalis was simultaneous but the consumption rate of glucose is much higher than that of xylose. Cellobiose, mannose, galactose, and arabinose were consumed after glucose was depleted. In our study, glucose, cellobiose, mannose, and galactose were consumed completely and residual xylose was lower than 1 g l−1 . Though arabinose was not fermented to a satisfied extent, there was only 3 g arabinose l−1 left in broth. The low residual arabinose was very advantageous to xylitol recovery. In Ernesto’s study, only 92–94% purity xylitol product was obtained. One of the main impurities was arabinose, which was left in broth at 6 g l−1 [21]. To further improve the xylitol purity during product recovery process, metabolic engineering can be very helpful in manipulating the pathways so that arabinose will be totally used for cell growth. Fig. 1. Time course of xylitol batch fermentation by Candida tropicalis W103 under different substrates: A (treated corncob hydrolysate), B (xylose), C (xylose and glucose), D (xylose, glucose and acetate), xylose (), glucose (), arabinose (), xylitol (䊉), acetate (), ethanol (), CDW ().
Acknowledgements This study was supported by ‘863’ Hi-Tech Research and Development Program of China (2007AA100702-3), Science Fund of Young Scholars (Heilongjiang University) and Research Fund of Key Laboratory of Microbiology (College of Life Sciences, Heilongjiang University, China).
K.-K. Cheng et al. / Biochemical Engineering Journal 43 (2009) 203–207
References [1] S.I. Mussatto, I.C. Roberto, Kinetic behaviour of Candida guilliermondii yeast during xylitol production from highly concentrated hydrolysate, Process Biochem. 39 (2004) 1433–1439. [2] T. Walther, P. Hensirisak, F.A. Agblevor, The influence of aeration and hemicellulosic sugars on xylitol production by Candida tropicalis, Bioresour. Technol. 76 (2001) 213–220. [3] P. Eva, H. Bärbel, Fermentation of lignocellulosic hydrolysates. I. Inhibition and detoxification, Bioresour. Technol. 74 (2000) 17–24. [4] T.D. Leathers, S.C. Gupta, Xylitol and riboflavin accumulation in xylose-grown cultures of Pichia guilliermondii, Appl. Microbiol. Biotechnol. 47 (1997) 58– 61. [5] C.S. Fábio, M.C.A. Virgínia, C. Attilio, M.L.P. Flávia, L.C.C. Jorge, Influence of cultivation conditions on xylose-to-xylitol bioconversion by a new isolate of Debaryomyces hansenii, Bioresour. Technol. 99 (2008) 502–508. [6] M. Azuma, T. Ikeuchi, R. Kiritani, J. Kato, H. Ooshima, Increase in xylitol production by Candida tropicalis upon addition of salt, Biomass Bioener. 19 (2000) 129–135. [7] M.L.M. Villarreal, A.M.R. Prata, M.G.A. Felipe, J.B.E.S. Almeida, Detoxification procedures of eucalyptus hemicellulose hydrolysate for xylitol production by Candida guilliermondii, Enzyme Microb. Technol. 40 (2006) 17–24. [8] L. Canilha, S.J.B. Almeida, A.I.N. Solenzal, Eucalyptus hydrolysate detoxification with activated charcoal adsorption or ion-exchanger resins for xylitol production, Process Biochem. 39 (2004) 1909–1912. [9] W.C. Liaw, C.S. Chen, W.S. Chang, K.P. Chen, Xylitol production from rice straw hemicellulose hydrolyzate by polyacrylic hydrogel thin films with immobilized Candida subtropicalis WF79, J. Biosci. Bioeng. 105 (2008) 97–105. [10] L. Farooq, I.R. Mohammad, Production of ethanol and xylitol from corn cobs by yeasts, Bioresour. Technol. 77 (2001) 57–63. [11] I.M. Solange, D. Giuliano, C.R. Inês, Influence of the toxic compounds present in brewer’s spent grain hemicellulosic hydrolysate on xylose-to-xylitol bio-
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Contents lists available at ScienceDirect
Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Short communication
Optimization of pH and acetic acid concentration for bioconversion of hemicellulose from corncobs to xylitol by Candida tropicalis Ke-Ke Cheng a , Jian-An Zhang a,∗ , Hong-Zhi Ling b , Wen-Xiang Ping b , Wei Huang a , Jing-Ping Ge b , Jing-Ming Xu a a b
Division of Green Chemistry and Technology, Institute of Nuclear and New Energy Technology, Tsinghua University, Beijing 100084, PR China Key Laboratory of Microbiology, College of Life Sciences, Heilongjiang University, Harbin 150080, PR China
a r t i c l e
i n f o
Article history: Received 19 May 2008 Received in revised form 12 September 2008 Accepted 20 September 2008 Keywords: Corncob Fermentation Hydrolysate Pretreatment Xylitol Xylose
a b s t r a c t Hemicellulose hydrolysate from corncobs, separated by diluted sulfuric acid and sequently detoxed by boiling, overliming and solvent extraction, was used for xylitol production by Candida tropicalis W103. The effect of glucose and acetate in hydrolysate on xylitol production was investigated. It was found that glucose in hydrolysate promoted growth of Candida tropicalis while acetate at high concentration was inhibitory. The acetate inhibition can be alleviated by adjusting pH to 6 prior to fermentation and a substrate feeding strategy. Under these optimum conditions, a maximal xylitol concentration of 68.4 g l−1 was obtained after 72 h of fermentation, giving a yield of 0.7 g g−1 xylose and a productivity of 0.95 g l−1 h−1 . © 2008 Elsevier B.V. All rights reserved.
1. Introduction Lignocellulosic materials represent an abundant and inexpensive source of sugars which can be microbiologically converted to industrial products. Among these products available from lignocellulose-derived sugars, xylitol has received much attention. Xylitol is a natural polyol with particular physico-chemical properties, which permit its use either in foods like chewing gum, bakery and chocolate as a sweetener with anticariogenic property, or in medicines as a sugar substitute for the treatment of diabetes and erythrocytic glucose-6-phosphate dehydrogenase deficiency [1]. Traditional chemical conversion of xylose into xylitol is rather difficult, has a low product yield and consequently its high price has hindered the utilization of xylitol in food or medicine industries. The microbial conversion of xylose to xylitol is particularly attractive in that the process is relatively easy and does not need toxic catalyst [2]. Dilute sulfuric acid hydrolysis is a favourable method for the conversion of lignocellulose to sugars, especially for hemicellulose to hexoses. Hydrolysis of lignocellulosic materials always goes together with the formation of byproducts that inhibit the fer-
mentation process [3]. Hemicellulose hydrolysate can be converted to xylitol by several microorganisms including Pichia and Debaryomyces also Candida [4–7]. Among these, Candida ferments xylose to xylitol in a high yield and productivity. However, due to the inhibitors in hydrolysate, it is difficult to obtain a high xylitol concentration in the fermentation broth. Many studies have been conducted utilizing the hemicellulose portion of agricultural residues like eucalyptus, rice straw, corncob, brewer’s spent grain, sugarcane bagasse, and corn stover for xylitol production [8–13]. Among the various agricultural crop residues, corncob is one of the most abundant agricultural materials in Northeastern China. In this study, corncob was chosen as the raw lignocellulosic material. Candida tropicalis W103, which was screened in our laboratory and can produce 200 g l−1 xylitol at pH 4.5 in fed-batch cultures using xylose as the sole carbon source, was used to covert corncob hydrolysate to xylitol. The effects of glucose and acetate in corncob hydrolysate on xylitol production were investigated to improve both product concentration and productivity. 2. Materials and methods 2.1. Pretreatment
∗ Corresponding author. Tel.: +86 10 89796086; fax: +86 10 62785475. E-mail address: [email protected] (J.-A. Zhang). 1369-703X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2008.09.012
Corncob from Heilongjiang province in Northeastern China was used as raw material. Particles in the size ranged from 0.45 mm
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K.-K. Cheng et al. / Biochemical Engineering Journal 43 (2009) 203–207
Table 1 Compositions of corncobs. Component
Values (%)
Methods
Benzene-ethanol extractive (%, w/w) Cellulose (%, w/w)
3.01 ± 0.08 42.69 ± 0.37
Holocellulose (%, w/w)) Hemicellulose Klason lignin (%, w/w) Acid-soluble lignin (%, w/w) Total lignin (%, w/w)
77.11 34.32 17.52 0.91 18.42
GB/T 2677.6-1994 Nitric acid-ethanol method GB/T 2677.10-1995 Calculated value GB/T 2677.8-1994 GB/T 747-2003 GB/T 2677.8-1994, GB/T 10337-1989
± ± ± ± ±
0.55 0.46 0.41 0.04 0.33
The values were the mean of four independent samples. Cellulose content was determined by the nitric acid-ethanol method. Hemicellulose is calculated by difference between hollocellulose and cellulose.
to 0.9 mm (20–40 mesh) were used in the experiments. The compositions of corncob were determined according to corresponding Chinese National standards [14]. The data were shown in Table 1. The pretreatment was carried out in 1000 ml glass flasks. 40 g corncobs at a solid loading of 10% (w/w) was mixed with dilute sulfuric acid (1% (w/w)) and pretreated in an autoclave at 120 ◦ C with residence time of 1 h. The liquid fraction was separated by filtration and the unhydrolysed solid residue was washed with 40 ml warm water (60 ◦ C). The filtrate and wash liquid were pooled together. The composition of the corncob hemicellulose acid hydrolysate 5.4 g glucose l−1 , 3.7 g arabinose l−1 , was 28.7 g xylose l−1 , −1 −1 0.5 g cellobiose l , 0.7 g galactose l , 0.4 g mannose l−1 , 2 g acetic acid l−1 , 0.8 g furfural l−1 , 0.2 g 5-hydroxymethylfurfural l−1 . 2.2. Detoxification Hemicellulose acid hydrolysate was heated to 100 ◦ C, and maintained for 15 min to reduce the volatile components. The hydrolysate was then overlimed with solid Ca(OH)2 up to pH 10.0, in combination with 0.1% sodium sulfite, filtered to remove the insoluble. The filtrate was adjusted to pH 7 with 3 M H2 SO4 and extracted with ethyl acetate to remove the phenolics derived from lignin according to González [15]. The water phase was used for xylitol production. The detoxed hydrolysate was concentrated under vacuum at 50–75 ◦ C to achieve 10–11% (w/v) of xylose concentration for storage and later usage.
xylose fermentation medium. The pH was adjusted at 4.5 by addition of 3 M NaOH before inoculation and pH was maintained at 4.5 throughout the cultivations. The cultivation period was 50 h and the experimental values were the means of three independent samples. In xylitol production under different substrate, the detoxed and concentrated hydrolysate was diluted to prepare the hydrolysate fermentation medium, which contained 60 g xylose l−1 , 10 g glucose l−1 , 7 g arabinose l−1 and 2.6 g acetate l−1 . In other comparative fermentation tests, analytic pure reagents were used for substrate. The cultivation periods under different substrate were varied so that xylitol concentration in broth reached the highest respectively. In all experiments of this section, pH was adjusted to 4.5 before inoculation and maintained at 4.5 throughout the cultivations. The experimental values were the means of three independent samples. In xylitol production with hydrolysate fermentation medium under different pH, pH was adjusted to designed value before inoculation and maintained at designed value throughout the cultivations. The cultivation periods were also varied so that xylitol concentration in broth reached the highest respectively. The experimental values were the means of three independent samples. In xylitol fed-batch fermentation, pH was adjusted to 6 before inoculation and maintained at 6 throughout the cultivations. Sterilized concentrated hydrolysate was continuously droped to the bioreactor with a peristaltic pump in 15–45 h. The experimental values were the means of two independent samples. 2.4. Analytical methods The liquid samples were analyzed by HPLC, equipped with UV and RI detectors. The concentrations of glucose, xylose, galactose, mannose and arabinose were determined using refractive index detector and Aminex HPX-87P column at 85 ◦ C with H2 O as mobile phase at 0.8 ml min−1 . Cellobiose, acetic acid and ethanol were analyzed using refractive index detector and Aminex HPX-87H column at 65 ◦ C with 5 mM H2 SO4 as mobile phase at 0.8 ml min−1 . Furfural was detected on UV chromatograms at 250 nm. Cell growth was monitored at 600 nm and converted to cell dry weight (CDW) by an appropriate calibration curve. 3. Results
2.3. Microorganism and fermentation experiments
3.1. Effects of acetic acid and substrate on the xylitol formation
Candida tropicalis W103 was grown on the preculture medium containing 2 g KH2 PO4 l−1 , 5 g (NH4 )2 SO4 l−1 , 4 g yeast extract l−1 , 0.5 g MgSO4 ·7H2 O l−1 , 20 g xylose l−1 . The pretreated corncob hemicellulose hydrolysate or prepared xylose, supplemented with 2 g KH2 PO4 l−1 , 5 g (NH4 )2 SO4 l−1 , 0.5 g MgSO4 ·7H2 O l−1 , 1 g peptone l−1 , 5 g yeast extract l−1 , was used as the fermentation medium. The seed cells for the bioreactor were prepared in 500 ml flasks containing 100 ml preculture medium. The flasks were incubated at 35 ◦ C and 250 rpm for 14 h and subsequently inoculated into the bioreactor at 5% (v/v). The biomass concentration in bioreactor at the beginning of the fermentation ranged from 0.29 g l−1 to 0.31 g l−1 . The batch or fed-batch cultivations were conducted in a 1000 ml stirred-vessel bioreactor (Biostat Q1000, B.Braun, Germany) containing 750 ml fermentation medium under 0.6 vvm air flow. All fermentation experiments were carried out at 35 ◦ C and 500 rpm and the broth was sampled in every 6–12 h to monitor the xylitol concentration. In acetic acid addition batch fermentation test, acetic acid of different concentration (0.5–4 g l−1 ) was added to the prepared
After detoxification by boiling, overliming and solvent extraction, volatile compounds, such as furfural and 5hydroxymethylfurfural, were not detected. However, detoxification also resulted in the loss of glucose by 6%, xylose by 9.7% and arabinose by 10.1%. Similar observations were reported during the neutralization of sugarcane bagasse and corncob acid-catalysed hemicellulose acid hydrolysate with lime [16,17]. In order to evaluate the effect of acetic acid on the formation of xylitol in Candida tropicalis, cells were grown in synthetic medium containing acetic acid at different levels. Xylose consumption was not affected by acetic acid until its concentration was higher than 2 g l−1 . The consumption of xylose depended on the amount of acetic acid. When the acetic acid reached 4 g l−1 , the final xylitol concentration in broth was 72% lower than in the medium without acetic acid addition (Table 2). The acetic acid in the medium was consumed completely if its concentration was lower than 2 g l−1 . However, only 2 g l−1 acetic acid was used when its concentration was higher than 2 g l−1 . Similar observations were reported by Lima et al. [18].
K.-K. Cheng et al. / Biochemical Engineering Journal 43 (2009) 203–207
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Table 2 Xylitol production in batch cultures by Candida tropicalis W103 from prepared xylose containing acetic acid at different levels. Acetic acid −1
Initial (g l
Xylose )
0 0.5 1 2 3 4
−1
Residual (g l
)
0 0 0 0 1.0 ± 0.1 2.0 ± 0.1
Xylitol production −1
Initial (g l 75.4 74.8 79.2 75.5 78.9 75.5
± ± ± ± ± ±
)
0.7 1.4 2.1 0.9 1.3 1.1
−1
Residual (g l 2.1 1.0 1.0 7.8 11.6 49.4
± ± ± ± ± ±
)
0.9 0.2 0.4 1.9 1.4 1.7
Concentration (g l−1 ) 52.8 53.9 54.7 48.8 44.3 14.4
± ± ± ± ± ±
0.9 0.4 0.7 1.3 1.5 1.7
Productivity (g l−1 h−1 ) 1.05 1.07 1.09 0.98 0.89 0.29
± ± ± ± ± ±
0.02 0.03 0.02 0.04 0.05 0.03
Yield (g g−1 xylose) 0.72 0.73 0.70 0.72 0.66 0.55
± ± ± ± ± ±
0.01 0.02 0.01 0.02 0.03 0.02
The biomass concentration at the beginning of the fermentation ranged from 0.29 g l−1 to 0.31 g l−1 .All fermentation experiments were carried out at 35 ◦ C and pH 4.5. The cultivation period was 50 h and the experimental values were the means of three independent samples.
Table 3 Xylitol production in batch cultures by Candida tropicalis W103 with treated corncob hydrolysate as substrates. pH
Fermentation period (h)
Xylose
Xylitol production −1
Initial (g l 4.5 5 5.5 6 6.5
64 70 76 64 64
65.8 66.3 66.7 65.9 66.2
± ± ± ± ±
0.9 1.8 2.4 1.9 1.3
)
−1
Residual (g l 12.7 3.6 3.4 2.0 2.9
± ± ± ± ±
)
1.9 0.2 1.0 0.7 0.4
Concentration (g l−1 ) 22.3 30.1 40.5 45.4 44.3
± ± ± ± ±
0.9 0.4 1.7 1.5 1.5
Productivity (g l−1 h−1 ) 0.34 0.43 0.54 0.71 0.69
± ± ± ± ±
0.04 0.03 0.06 0.03 0.01
Yield (g g−1 xylose) 0.42 0.48 0.63 0.71 0.70
± ± ± ± ±
0.03 0.02 0.06 0.02 0.01
The biomass concentration at the beginning of the fermentation ranged from 0.29 g l−1 to 0.31 g l−1 .All fermentation experiments were carried out at 35 ◦ C. The values were the means of three independent samples.
To examine if the hydrolysate after detoxification can be used directly in xylitol production, a batch fermentation was conducted firstly using a sterilized hydrolysate. For comparison, batch xylitol production was also carried out using prepared xylose medium, xylose and glucose medium, xylose, glucose and acetate medium (Fig. 1A–D). The results showed that both the cell growth rate and xylitol productivity were lower in hydrolysate batch culture than those in prepared xylose batch culture. Fig. 1A, C and D showed that the glucose prior to xylose was used for cell growth. The consumption rate of glucose in xylose and glucose medium was 0.78 g l−1 h−1 in 0–6 h, much higher than that of xylose (0.1 g l−1 h−1 ). During the early stages of prepared glucose/xylose mixture fermentation, the cell densities were higher than those in xylose fermentation. The maximal growth rate was 0.27 h−1 in the prepared xylose and glucose medium, while it was 0.19 h−1 in the xylose medium. No xylitol but ethanol formation was observed when glucose was used as substrate. However, the xylitol productivity under prepared xylose and glucose medium was higher than that under prepared xylose medium. This phenomenon can be explained by higher biomass in prepared xylose and glucose medium. With prepared xylose, glucose and acetate as substrates, both cell growth and xylitol formation were slower than those with prepared xylose medium. These further validated that high concentration acetate had a negative effect on xylitol production.
3.3. Xylitol production by fed-batch fermentation In view of the high cost of xylitol recovery from aqueous solution, an economical production of xylitol from corncob hydrolysate requires the improvement of both product concentration and productivity. Xylitol batch fermentation was inhibited by high acetate concentration, so fed-batch cultivations with low initial acetate concentration and at optimized pH were studied further in order to increase the final xylitol concentration in broth. In the subsequent experiments, concentrated hydrolysate containing 117.7 g xylose l−1 , 21.1 g glucose l−1 , 15.3 g arabinose l−1 , 6.8 g acetate l−1 was added during 15–45 h to maintain xylose concentration within 15–40 g l−1 . Fig. 2 showed typical profiles of cell growth, substrate consumption and product formation. Higher assimilation rate of glucose was observed compared with that of xylose. Glucose was not detected after the initial glucose in the fermentation medium was exhausted. Besides xylose, a small concentration of arabinose (3 g l−1 ) was present in the hydrolysate, being partially consumed (72.4%) throughout the fermentative process. With the feeding of hydrolysate, the acetate in broth increased and then decreased after the ending of feeding. During the fermentation process, the concentration of acetate was lower than 2.5 g l−1 . The final concentration of xylitol was 68.4 g l−1 with a yield of 0.7 g g−1 xylose and productivity of 0.95 g l−1 h−1 .
3.2. Xylitol production under different pH 4. Discussion It had been found for some microorganisms that the inhibition of acetate was due to its undissociated form [19]. With the increase of pH, the undissociated form of acetic acid would decrease. Xylitol fermentations with corncob hydrolysate as substrate were performed at different pH to alleviate the inhibition of acetate. Total xylose consumption at pH 4.5 was lower than one half the starting xylose concentration, while it was almost complete after an average time of only 64 h at pH 6. An increase in pH from 4.5 to 6.0 led to dramatic increases in both xylitol productivity and yield, while the highest yield of biomass on consumed xylose was observed at pH 6.0. It indicated that acetate inhibition could be alleviated to some extend by pH adjustment (Table 3).
In our previous study, Candida tropicalis W103 can produce 200 g l−1 xylitol in fed-batch cultures using xylose as the sole carbon source. However, the fermentation parameters for these treated corncob hemicellulose acid hydrolysate are much lower than those obtained with a synthetic medium. This shows that there were some leftover toxic components in the treated hemicellulose acid hydrolysate that negatively affected the fermentation performance of C. tropicalis. To further improve the fermentation efficiency, proper culture conditions and detoxification techniques should be developed to alleviate the inhibitions. Lima studied the effect of acetic acid present in bagasse hydrolysate on the activ-
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Fig. 2. Time course of xylitol fed-batch fermentation by Candida tropicalis W103 with treated corncob hydrolysate as substrates at pH 6: xylose (), glucose (), arabinose (), xylitol (䊉), acetate (), ethanol (), CDW ().
ities of xylose reductase and xylitol dehydrogenase in Candida guilliermondii FTI 20037. They reported the maximum values of xylitol batch production (25 g l−1 ), productivity (0.42 g l−1 h−1 ) and xylitol yield (0.7 g g−1 ) after 58 h fermentation. The cell growth of C. guilliermondii FTI 20037 decreased 22% when it grew in medium containing 5 g acetic acid l−1 at pH 5.5, which corresponded to 0.75 g undissociated acetic acid l−1 . Rodrigues studied xylitol fed-batch production by C. guilliermondii FTI 20037 in a sugarcane bagasse hydrolysate medium. The values achieved for xylitol concentration, yield and volumetric productivity were 44 g l−1 , 0.78 g g−1 and 0.62 g l−1 h−1 [20]. In this study, higher xylose concentration of 68.4 g l−1 and productivity of 0.95 g l−1 h−1 were obtained. Xylose consumption and xylitol formation were not affected by acetic acid at pH 4.5 if its concentration was not higher than 2 g l−1 , corresponding to 1.29 g undissociated acetic acid l−1 at pH 4.5. Candida tropicalis W103 was more tolerant to the inhibition of undissociated acetic acid than Candida guilliermondii FTI 20037 [18]. In view of the high cost of product recovery, a high final product concentration with low impurity is desirable. To achieve this, the complex sugar, including xylose, glucose, cellobiose, mannose, galactose, and arabinose should be controlled at the minimum level in the final broth. The utilization of glucose and xylose by C. tropicalis was simultaneous but the consumption rate of glucose is much higher than that of xylose. Cellobiose, mannose, galactose, and arabinose were consumed after glucose was depleted. In our study, glucose, cellobiose, mannose, and galactose were consumed completely and residual xylose was lower than 1 g l−1 . Though arabinose was not fermented to a satisfied extent, there was only 3 g arabinose l−1 left in broth. The low residual arabinose was very advantageous to xylitol recovery. In Ernesto’s study, only 92–94% purity xylitol product was obtained. One of the main impurities was arabinose, which was left in broth at 6 g l−1 [21]. To further improve the xylitol purity during product recovery process, metabolic engineering can be very helpful in manipulating the pathways so that arabinose will be totally used for cell growth. Fig. 1. Time course of xylitol batch fermentation by Candida tropicalis W103 under different substrates: A (treated corncob hydrolysate), B (xylose), C (xylose and glucose), D (xylose, glucose and acetate), xylose (), glucose (), arabinose (), xylitol (䊉), acetate (), ethanol (), CDW ().
Acknowledgements This study was supported by ‘863’ Hi-Tech Research and Development Program of China (2007AA100702-3), Science Fund of Young Scholars (Heilongjiang University) and Research Fund of Key Laboratory of Microbiology (College of Life Sciences, Heilongjiang University, China).
K.-K. Cheng et al. / Biochemical Engineering Journal 43 (2009) 203–207
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