Abstract
Acid sugarcane bagasse hydrolyzate was submitted to pH shifts in order to remove toxic compounds from the medium. The hydrolyzate was treated with bases containing mono-, di- or tri-valent cations and H2SO4, and its performance as a fermentation medium was evaluated by the production of xylitol by Candida guilliermondii FTI 20037. The use of bases containing mono-valent cations was not an efficient method of detoxification, and the use of a tri-valent cation did not show any detectable improvement in detoxification. The treated hydrolyzate recovery (in volume) is greatly affected by the utilized base. Treatment using Al(OH)3 and NaOH showed the best hydrolyzate recovery (87.5%), while the others presented a recovery of about 45% of the original hydrolyzate volume. Considering the whole process, best results were achieved by treatment using Al(OH)3 and NaOH which allowed 0.55 g of xylitol produced from each gram of xylose in the raw hydrolyzate.
Sugarcane bagasse hydrolyzate; xylitol production; Candida guilliermondii
EVALUATION OF SUGARCANE BAGASSE ACID HYDROLYZATE TREATMENTS FOR XYLITOL PRODUCTION
P.V. GURGEL1, S.A. FURLAN2,3** To whom correspondence should be addressed. To whom correspondence should be addressed., S.E.R. MARTINEZ2 and I.M. MANCILHA1,2
1Departamento de Tecnologia de Alimentos, UFV, 36570-000, Viçosa, MG, Brazil
2Centro de Biotecnologia, FAENQUIL, 12600-000, Lorena, SP, Brazil
3Current address: Pró-reitoria de Pós-graduação, Pesquisa e Extensão - UNIVILLE, C.P. 1361,
89201-972, Joinville - SC, Tel / Fax (047) 473-0200 (219)
(Received: January 13, 1998; Accepted: June 20, 1998)
Abstract - Acid sugarcane bagasse hydrolyzate was submitted to pH shifts in order to remove toxic compounds from the medium. The hydrolyzate was treated with bases containing mono-, di- or tri-valent cations and H2SO4, and its performance as a fermentation medium was evaluated by the production of xylitol by Candida guilliermondii FTI 20037. The use of bases containing mono-valent cations was not an efficient method of detoxification, and the use of a tri-valent cation did not show any detectable improvement in detoxification. The treated hydrolyzate recovery (in volume) is greatly affected by the utilized base. Treatment using Al(OH)3 and NaOH showed the best hydrolyzate recovery (87.5%), while the others presented a recovery of about 45% of the original hydrolyzate volume. Considering the whole process, best results were achieved by treatment using Al(OH)3 and NaOH which allowed 0.55 g of xylitol produced from each gram of xylose in the raw hydrolyzate.
Keywords: Sugarcane bagasse hydrolyzate, xylitol production, Candida guilliermondii.
INTRODUCTION
The sugars present in the hemicellulose can be easily obtained from sugarcane bagasse using mild acid hydrolysis. Nevertheless, during this process, several toxic compounds are produced. These compounds can inhibit the total utilization of the released sugars, like D-xylose, D-glucose and L-arabinose, by fermentative microorganisms, decreasing the productivity in ethanol or xylitol (Magee and Kosaric, 1985; Tran and Chambers, 1985).
The inhibitory substances of microbial growth could be removed, partial or totally, by prior treatment of the acid hydrolyzate (Sjolander et al., 1938). Leonard et al. (1947) showed that the hydrolyzate treatment with activated carbon increases the microorganisms fermentation ability. Fein et al. (1984) studied different pretreatments like ether extraction and ion exchange chromatography. Best results were obtained by treating acid hydrolyzate with ether and strain acclimatization. Cell acclimatization was also studied in sugarcane bagasse hemicellulose hydrolyzate fermentation to xylitolby Chen and Gong (1985).
Some investigators (Tran and Chambers, 1985) used treatments such as chromatography and molecular sieving, but due to their high costs, alternative processes were developed to increase ethanol (Strickland and Beck, 1984; Van Zyl et al., 1988) and xylitol (Roberto et al., 1991) production by pH changing. Trials were made to determine the best base to neutralize the hydrolyzate. Neutralization of hydrolyzate with mono-valent (KOH) or di-valent cations (CaO or Ca(OH)2) is not na effective technique (Roberto et al., 1991). Nevertheless, raising pH to 10 using Ca(OH)2 showed higher yields in xylose consumption.
Based on this, the present work intended to evaluate the performance of Candida guilliermondii FTI 20037 in conversion of xylose to xylitol using sugarcane bagasse acid hydrolyzate, treated with bases containing mono-, di- or tri-valent cations.
MATERIALS AND METHODS
Hemicellulosic Hydrolyzate
Sugarcane bagasse was hydrolyzed by adding 141 mL H2SO4 to 2 Kg (dry weight) mixed to 13 L water. This mixture was heated at 140 oC for 20 minutes. After this period the reactor was cooled, and the mixture was filtrated. Filtrate was vacuum concentrated at a ratio of 4:1 and at a temperature lower than 70 oC.
Hydrolyzate Treatments
The hydrolyzate was submitted to one of the following treatments:
(I) raising pH to 10.0 with Ca(OH)2, decreasing pH to 6.5 with H2SO4;
(II) raising pH to 3.5 with Mg(OH)2, and then to 10.0 with NaOH, decreasing pH to 6.5 with H2SO4;
(III) addition of 20 g/L Al(OH)3, and raising pH to 10.0 with NaOH, decreasing pH to 6.5 with H2SO4;
(IV) raising pH to 3.5 with Mg(OH)2, and then to 10.0 with Ca(OH)2, decreasing pH to 6.5 with H2SO4;
(V) addition of 20 g/L Al(OH)3, and raising pH to 10.0 with Ca(OH)2, decreasing pH to 6.5 with H2SO4;
In all treatments the precipitate resulting from pH adjustment was removed by centrifugation at 1500 x g for 15 minutes.
Fermentation Conditions
Treated hydrolyzates were autoclaved for 15 min. at 0.5 atm. All other components of the medium were autoclaved at 1 atm for 20 min., and were added to yield the indicated concentrations: (NH4)2SO4, 5 g/L; CaCl2.2H2O, 0.1 g/L; rice bran, 20.0 g/L. A volume of 500 mL of each treated media was then inoculated with 5 mL of a one-day-old Candida guilliermondii cell suspension. Flasks were incubated in a shaker at 30 oC and 200 min-1. Samples of 5 mL were removed at 0, 7, 22, 31, 46, 55, 70, 118, 127, 142, 151 and 166 hours of fermentation.
Analytical Methods
Glucose, xylose, xylitol, ethanol and acetic acid concentrations were measured by high performance liquid chromatography (Hewlett-Packard 1084B) using a refractive index detector and an HPX-87H column and employing the following conditions: H2SO4 (0.01 N) eluant, flow rate of 0.6 mL/min., column temperature 45 oC, sample volume 20 µL. The pH values were evaluated potentiometrically.
RESULTS AND DISCUSSION
Based on the results obtained by Roberto et al (1991) which showed that overtitration has advantageous effects over simple titration for treatment of steam explosion bagasse hemicellulosic hydrolyzate, in the present study the overtitration by different bases was investigated for prior treatment of acid hydrolyzate.
The pH adjustment has two purposes: firstly, to set the pH of the growth medium at 6.5 in order to keep acetate and acidic molecules in their unprotonated and less toxic form, and secondly, to precipitate the toxic compounds like furfural, heavy metals, terpenes, tannins, and phenolics (Lee and McCaskey, 1989). Besides, overtitration seems to be involved in the removal of inorganic ions (Sjolander et al., 1938; Strickland and Beck, 1984) and furfural (Tran and Chambers, 1985; Strickland and Beck, 1984).
The treatments of the sugarcane bagasse hemicellulosic acid hydrolyzate were evaluated based on their fermentative performances, whose results are shown in Table 1.
Table 1: Fermentative parameters of xylose fermentation by Candida guilliermondii FTI 20037, after different hydrolyzate pretreatments. Treatment as described in methodology
concentration of xylose (n ), glucose (l ), xylitol (s ), ethanol (m ), and acetic acid (u ) during the fermentation of hemicellulosic hydrolyzate submitted to treatment III.
The base conjugation utilized to raise pH shows a marked effect in fermentation performance. The combination of Mg(OH)2 and NaOH (treatment II) was not an effective treatment in xylose to xylitol conversion, while the combination of Mg(OH)2 or Al(OH)3 with Ca(OH)2 (treatments IV and V, respectively) showed the same performance of Ca(OH)2 alone (treatment I) in relation to volumetric productivity. Nevertheless, treatment I presented a higher yield in xylitol production when compared to the results of treatments IV and V. A combination of Al(OH)3 with NaOH (treatment III) resulted in the same yields of treatment I but with lower productivity. These results suggest that the use of mono-valent cations are not an effective treatment in the removal of toxic compounds from the hydrolyzate, since treatment II showed the poorer performance, and treatment IV showed good results. This behavior was also observed by Van Zyl et al. (1988)and Roberto et al. (1991). The use of a tri-valent cation (treatment III) did not show any detectable effect in improving detoxification.
Ethanol was the only by-product detected in considerable amounts in the fermentations. Treatment III showed an ethanol production 30% lower than the others did what is an advantage, since a lower carbon amount was deviated from the respiratory to ethanol production. Ethanol production is comparable to those reported in the literature (Van Zyl. et al., 1988; Tran and Chambers, 1986). Acetic acid showed the same profile in all fermentation tests, with a concentration of 5 to 8 g/L during the fermentations.
Considering the evaluated parameters, treatments I, III, IV and V showed good performances. Xylose consumption rate is higher in the fermentation using treatments I, IV and V. Xylitol production was higher in treatments I and III when compared to those results obtained in treatments IV and V. These results suggest that there is a different effect of the inhibiting compounds, which remained in the media after treatment, on the cell growth and on xylitol production, since the results of treatment III showed a lower xylose consumption when compared to the other treatments, but with a similar xylitol formation rate. This was also observed by Fein et al. (1984), who studied the fermentative performance of 37 yeast strains growing in acid wood-derived hemicellulose hydrolyzate.
Another factor that should be considered in choosing the best treatment is hydrolyzate recovery. The volume loss in treatment I is 51.7%, while in treatments IV and V the losses were 54.2 and 57.5%, respectively, of the original volume. These losses are due to the high amount (approximately 75 g/L) of Ca(OH)2 and/or Mg(OH)2 that were added to the hydrolyzate to reach pH 10. The low solubility of the bases increases the filtration cake, leading to a higher hydrolyzate retention by capillarity, and thus, reducing the amount of liquid recovered. The hydrolyzate retention diminishes the xylose total amount in the process, reducing the global yield. Treatment III uses a small amount of Al(OH)3 (20 g/L) in association with NaOH, that stands in solution, without any hydrolyzate retention. The filtration step becomes easier in treatment III because of the lower amount of solids in suspension in the medium. These effects lead to a volume loss of 12,5% in treatment III.
Considering the whole process, treatment I allowed the production of 0.3 g of xylitol for each gram of xylose in raw hydrolyzate, while treatments IV and V produced 0.23 and 0.22 g of xylitol, respectively. Treatment III allowed 0.55 g of xylitol and Figure 1 shows the xylose, glucose, xylitol, ethanol and acetic acid concentration profiles for treatment III.
CONCLUSIONS
Despite the higher rates of xylose consumption and xylitol production obtained by treatments I, IV and V, and taking into account the lower level of ethanol production and the higher percentage of hydrolyzate recovery obtained by treatment III, this method was considered the most effective in treating sugarcane bagasse acid hydrolyzate for xylitol production by fermentation.
ACKNOWLEDGEMENTS
P. V. Gurgel, S. A. Furlan, and S.E.R. Martinez were supported by fellowships from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil.
NOMENCLATURE
Efin final ethanol concentration (g.L-1)
QP volumetric rate of xylitol production (g.L-1.h-1)
QS rate of xylose concumption (g.L-1.h-1)
Sres residual xyloseconcentration (g.L-1)
S0 initial xylose concentration (g.L-1)
tf fermentation time (h)
Vhvd volume of hydrolyzate recovery (%)
YP/S yield factor of product on substrate (g.g-1)
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Publication Dates
-
Publication in this collection
27 Oct 1998 -
Date of issue
Sept 1998
History
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Accepted
20 June 1998 -
Received
13 Jan 1998
