Abstract
The objective of this study was to develop an effective method for the removal of inhibitory compounds without decreasing oligosaccharides in spent sulfite liquor (SSL). The oligosaccharide fermentation was subsequently conducted by using Pichia stipitis, which is one of the feasible strains that can produce ethanol from oligosaccharides. The effect of inhibitory compounds on ethanol fermentation from cellobiose by P. stipitis was investigated. No ethanol was produced from cellobiose in the presence of more than 5 g/L of acetic acid. At 1 g/L of acetic acid, 2.6 g/L of ethanol was obtained after 40 h of fermentation. The removal of acetic acid in the SSL by the combined CaO and ion exchange resin treatments was also studied. The acetic acid concentration of softwood SSL was decreased from 5.2 to 0.9 g/L without decreasing oligosaccharides concentration by the combined method. Finally, the improvement of ethanol fermentation from oligosaccharides in the SSL by using the combined CaO and ion exchange resin treatments was studied. 1.3 g/L of ethanol was obtained from the SSL treated by the combined methods, while 6.5 g/L of total oligosaccharides were consumed. No ethanol was obtained from the untreated SSL.
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Improvement of Ethanol Fermentation from Oligosaccharides in Spent Sulfite Liquor with Pichia stipitis by Combined Calcium Oxide and Ion Exchange Resin Treatments
Keishi Tanifuji,a,b Shiho Takahashi,b Kevin Shiell,c M. Sarwar Jahan,b Yonghao Ni,b and Hiroshi Ohi a,*
The objective of this study was to develop an effective method for the removal of inhibitory compounds without decreasing oligosaccharides in spent sulfite liquor (SSL). The oligosaccharide fermentation was subsequently conducted by using Pichia stipitis, which is one of the feasible strains that can produce ethanol from oligosaccharides. The effect of inhibitory compounds on ethanol fermentation from cellobiose by P. stipitis was investigated. No ethanol was produced from cellobiose in the presence of more than 5 g/L of acetic acid. At 1 g/L of acetic acid, 2.6 g/L of ethanol was obtained after 40 h of fermentation. The removal of acetic acid in the SSL by the combined CaO and ion exchange resin treatments was also studied. The acetic acid concentration of softwood SSL was decreased from 5.2 to 0.9 g/L without decreasing oligosaccharides concentration by the combined method. Finally, the improvement of ethanol fermentation from oligosaccharides in the SSL by using the combined CaO and ion exchange resin treatments was studied. 1.3 g/L of ethanol was obtained from the SSL treated by the combined methods, while 6.5 g/L of total oligosaccharides were consumed. No ethanol was obtained from the untreated SSL.
Keywords: Ethanol; Fermentation; Pichia stipitis; Spent sulfite liquor; Oligosaccharide; Pretreatment; CaO and ion exchange resin treatment
Contact information: a: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8572 Japan; b: Department of Chemical Engineering and Limerick Pulp and Paper Centre, University of New Brunswick, Fredericton, NB, E3B 5A3; c: Biorefinery Technology Scale-up Centre, New Brunswick Community College, Grand Falls, NB, E3Y 3W3 Canada;
* Corresponding author: oi.hiroshi.gm@u.tsukuba.ac.jp
INTRODUCTION
Sulfite cooking is an established process in the pulp and paper industry. Several currently operational acid sulfite pulp mills can produce various chemicals and materials from wood (Rødsrud et al. 2012). Wood chips as raw materials provide cellulose as pulp. In addition, lignosulfonate, vanillin, and ethanol are also produced from the spent sulfite liquor (SSL). Therefore, acid sulfite cooking is one of the most notable technologies for bio-refinery and bio-ethanol production.
In a previous study (Tanifuji et al. 2011), when acid sulfite cooking was conducted at 150 °C for 1 to 4 h at pH 1.4, gluco-oligosaccharide and manno-oligosaccharide were obtained in softwood SSL. In addition, a softwood sulfite pulp (SP) with a kappa number of 127 had a high resolution ratio (71.4%) by enzyme treatment (Tanifuji et al. 2011), and the resolution ratio of SP with a kappa number 64 was higher than that of soda-anthraquinone and kraft pulps with a kappa number 20 (Takahashi et al. 2010).
Several sulfite pulp mills have utilized SSL for ethanol production (Rødsrud et al. 2012). Saccharomyces cerevisiae can ferment hexose, but it cannot ferment oligosac-charides and pentose. Further hydrolysis steps such as acid or enzyme treatments are required for ethanol fermentation in order to utilize oligosaccharide in SSL, which would require additional stages and equipment.
A xylose fermentable strain Pichia stipitis has the genes for seven -glucosidases, two -mannosidases, and one endoxylanase, thus facilitating oligosaccharide utilization (Jeffries 2006; Jeffries et al. 2007). It has been reported that P. stipitis can produce ethanol from cellobiose (Parekh and Wayman 1986; Parekh et al. 1988) and xylan (Lee et al. 1986). Parekh and Wayman (1986) reported that P. stipitis CBS 5776 gave 10.3 g/L of ethanol from 25 g/L cellobiose within 48 h. Lee et al. (1986) reported that P. stipitis CBS 5775 can lead to the production of 2 g/L of ethanol from 2% larch wood xylan solution.
The SSL also contains furfural, acetic acid, and sulfite ion (Sixta 2006), which inhibit the ethanol production from monosaccharides with microorganisms (Agbogbo and Coward-Kelly 2008). P. stipitis is more sensitive to inhibitory compounds than S. cerevisiae (Delgenes et al. 1996; Palmqvist and Hahn-Hagerdal 2000). However, the effect of inhibitory compounds on oligosaccharide fermentation is not well understood. Moreover, it is necessary to remove inhibitory compounds from SSL to achieve effective ethanol fermentation.
The removal of inhibitory compounds such as lignin, furfural, and acetic acid from pulping waste liquors e.g. prehydrolysis liquor (PHL), based on the concepts of flocculation and adsorption, has been reported (Liu et al. 2011, 2012; Shen et al. 2011, 2012; Shi et al. 2011, 2012; Takahashi et al. 2012, 2013).
As an example of the flocculation concept, Shen et al. (2012), by using lime to treat PHL, found that the concentration of furfural and lignin in PHL can be decreased by 100% and 25-30%, respectively, by lime treatment. Shi et al. (2011, 2012) focused on the combined acidification/poly ethylene oxide (PEO) flocculation process of PHL; this process was found to be effective in removing lignin from PHL.
Activated carbon has been used as adsorbent to treat PHL and SSL. Takahashi et al. (2012) studied the simultaneous detoxification and fermentation (in-situ detox-ification) of SSL using activated carbon; half of the acetic acid in model softwood SSL was removed by activated carbon treatment. Liu et al. (2011) reported that the peroxide- or sulfuric acid-modified activated carbons have increased adsorption capacities for hemicellulose, lignin, and furfural present in the PHL.
In this study, we followed the concept of removing acetic acid by the combination of CaO and ion exchange resin treatments. In the previous study, we treated hardwood SSL, which contained monosaccharides but did not contain oligosaccharides, by the combined methods, and found that the combined treatments of CaO, CO2, and two-stage strong base ion exchange resin (OH– form) for 4 min decreased acetic acid concentration from 11.2 g/L to 0.9 g/L (Takahashi et al. 2013).
The objective of this study was to apply the same concept to SSL, which contains oligosaccharides, that is, to develop an effective method to improve the ethanol production from oligosaccharides in SSL. This was achieved by the removal of inhibitory compounds with effect on oligosaccharides so that the ethanol production is maximized. Firstly, the effect of inhibitory compounds on ethanol fermentation from cellobiose by using P. stipitis was clarified. Secondly, the removal of acetic acid in softwood SSL containing oligosaccharides by the combined CaO and ion exchange resin treatment was studied. Thirdly, the improvement of ethanol production from oligosaccharides in softwood SSL by using the combined CaO and ion exchange resin treatments was studied.
EXPERIMENTAL
SSL and Waste Liquor Samples
The softwood SSL and the hardwood waste liquor from alkaline sulfite pulp mill were obtained from pulp mills in Eastern Canada. The softwood SSL was prepared as followed: pH; 3.5 to 5.5, maximum temperature; 164 ºC for 2 h, wood to liquor ratio; 4.5 L/kg, and magnesium bisulfite charge; 24% as SO2. The waste liquor was derived from xylanase treatment of hardwood alkaline sulfite pulp and alkaline extraction of that. The chemical composition of waste liquor and SSL are shown in Table 1 and Table 2, respectively.
Ion Exchange Resins
The strong base ion exchange resin (Diaion PA408 Cl– form) was obtained from Mitsubishi Chemical and used for the experiments. The OH– form of PA408 was prepared as follows; the Cl– form resin was soaked in 2 N NaOH for 30 min, and then filtered. This treatment was repeated 5 times; subsequently the resin was washed by distilled water 5 times. The OH– form of PA408 was kept in distilled water to prevent oxidation and denaturation (Takahashi et al. 2013).
Combined Treatment of CaO and Strong Base Ion Exchange Resin Treatments
The CaO treatment was conducted as follows: the pH of softwood SSL and hardwood waste liquor were adjusted to 10.5 with 100 g/L of CaO slurry and then treated at 70 ºC for 15 min in a water bath (Howard 1932; Kuroishi 1983). The mixture was separated with filtration, and the filtrate was used for further experiments. The CaO treated samples were neutralized with CO2 to pH of 6.8 and were subsequently treated with the ion exchange resin for up to 5 min at 30 ºC with 150 rpm. The resin dosage was 20% on sample in all the experiments (Takahashi et al. 2013) .
Microorganism
P. stipitis CBS 6054 was obtained from the USDA. Stock cultures were kept on a YPDX agar plate which contained 10 g/L of yeast extract, 20 g/L of peptone, 10 g/L of D-glucose, 20 g/L of D-xylose, and 10 g/L of agar (Amartey and Jeffries 1994).
Inoculum Preparation
Inoculum was prepared by transferring a loopful of colonies from the agar plate into 50 mL of media which contained peptone, 3.5 g/L; yeast extract, 3 g/L; KH2PO4, 2 g/L; MgSO4, 1 g/L; (NH4)2SO4, 1 g/L; and xylose, 80 g/L, pH 5.0 in 125 mL of Erlenmeyer flask. Incubation was conducted at 30 ºC and 150 rpm for 48 h. The yeast was collected by centrifugation at 5000 rpm for 5 min and washed with sterile distilled water twice (Amartey and Jeffries 1994).
Fermentation
In the model fermentation, cellobiose (15 g/L) solution in the presence of various acetic acid, furfural, and SO2 concentrations (0.01, 0.1, 1, 5, and 10 g/L) were prepared. In the fermentation of SSL, the untreated, CaO-treated, and ion exchange resin-treated SSLs were used. These SSL samples were concentrated by a vacuum evaporator to make the total oligosaccharides concentration 15 g/L. The peptone, yeast extract, and ions, which were the same amount as those used for the inoculum preparation, were added to samples. About 1 g/L of dry cell weight was used. All of the fermentation experiments were conducted at 30 ºC with 225 rpm, and the initial pH was adjusted to 5.0. (Takahashi et al. 2013).
Analytical Methods
Monosaccharides concentrations were measured using an ion chromatography unit equipped with CarboPac® PA1 column (Dionex-300, Dionex Corporation, USA) and a pulsed amperometric detector (PAD) (Shen et al. 2011). Oligosaccharides were hydrolyzed by 4% H2SO4 at 121 ºC for 1 h and then measured by the ion chromatography unit. Oligosaccharides concentration was calculated by the subtract sugar concentration before hydrolysis from that after hydrolysis. The furfural, acetic acid, and ethanol concentrations were determined by the 1H-NMR method as described in the literature (Saeed et al. 2011). The area of signal at 1.2 ppm was used for ethanol determination. The lignosulfonate concentration was determined by the absorbance at 205 nm with UV spectrometry (Browning 1967). A calibration curve was prepared by using commercial lignosulfonate. Sulfite and sulfate ions analyses were conducted according to TAPPI test method T699 OM-87. The cellobiose concentration was determined using an HPLC equipped with RI detector (Shimadzu). Separations were performed at 65 ºC on Rezex ROA-organic Acid H+ column (phenomenex). Injection volume was 20 L. The mobile phase was 5 mM H2SO4. The flow rate was 0.6 mL/min.
RESULTS AND DISCUSSION
Effect of Inhibitory Compounds on Ethanol Production from Cellobiose by Using P. stipitis
We first used cellobiose with or without the inhibitory compounds (acetic acid, furfural, sulfite ions) on the ethanol fermentation using P. stipitis, and the results are shown in Fig. 1 (ethanol production) and Fig. 2 (cellobiose consumption). The maximum ethanol concentration from the control (without inhibitory compounds) during fermentation was 2.9 g/L. It was found that the presence of more than 5 g/L of acetic acid in media totally inhibited ethanol production from cellobiose and no ethanol was produced (Fig. 1 A).
The presence of 1 g/L of acetic acid inhibited ethanol production until 24 h, but after 40 h of fermentation, 2.6 g/L of ethanol was obtained, which was almost the same as that of the control. At this time, the amount of cellobiose consumption increased significantly from 24 h to 40 h (Fig. 2 A). The above results indicated that the inhibition of cellobiose fermentation caused by acetic acid is negligible in the late stage of fermentation at the presence of 1 g/L acetic acid. Glucose was not found from all of fermented samples.
The maximum ethanol concentrations with 1 g/L of furfural and 0.1 g/L of sulfite ion were 1.9 g/L and 1.3 g/L, respectively (Figs. 1 B and C). Both still show strong inhibition even at the late stage of fermentation (40 h). Also, cellobiose in 1 g/L of furfural and 0.1 g/L of sulfite ion model solutions was not completely assimilated at the late stage of fermentation and its concentrations were 2.2 g/L and 3.3 g/L, respectively; while all of the cellobiose in the control was assimilated (Figs. 2 B and C).
Figure 3 shows the change of acetic acid and furfural concentrations during fermentation. The acetic acid concentration was reduced after 16 h of fermentation. On the other hand, furfural concentration only changed marginally. Implied here is that P. stipitis consumed acetic acid during fermentation. This is the reason why the inhibitory effect of acetic acid was reduced in the late stage of fermentation.
In addition, the ethanol production from 1 g/L acetic acid model solution was higher than that from 1 g/L furfural solution (Figs. 1 A and B), and less furfural was assimilated than acetic acid (Fig. 3). Even 0.1 g/L of sulfite ion inhibited ethanol production (Fig. 1 C). These results indicate that the negative effect of acetic acid on the inhibition of cellobiose fermentation is less than that of furfural or sulfite ion.
Fig. 1. Ethanol production from cellobiose by P. stipitis in the presence of inhibitory compounds: (A) acetic acid; (B) furfural; (C) sulfite ion. Legends: ( 
) control; (×) 0.01 g/L; ( 
) 0.1 g/L; (
) 1 g/L; (
) 5 g/L; ( 
) 10 g/L
Fig. 2. Cellobiose consumption during fermentation in the presence of inhibitory compounds: (A) acetic acid; (B) furfural; (C) sulfite ion. Legends: ( ) control; (×) 0.01 g/L; ( ) 0.1 g/L; ( ) 1 g/L; ( ) 5 g/L; ( ) 10 g/L
Fig. 3. Change of acetic acid and furfural concentrations during cellobiose fermentation with P. stipitis. Legends: (○) furfural; (■) acetic acid
Removal of Inhibitory Compounds by Combination of CaO and Ion Exchange Resin Treatments
In the previous study hardwood SSL was treated by the combined methods; such liquor contained monosaccharides but did not contain oligosaccharides. It was found that the combined treatments of CaO, CO2, and two-stage strong base ion exchange resin (OH- form) for 4 min decreased acetic acid concentration from 11.2 g/L to 0.9 g/L. However, total monosaccharides concentration also decreased to 15.9 g/L from 35.9 g/L (Takahashi et al. 2013).
In this study, we followed the same concept for removing acetic acid from softwood SSL, which contained oligosaccharides, and studied the effect of this method on decrease of acetic acid and oligosaccharides concentrations. The results are shown in Fig. 4, and it can be found that the acetic acid concentration in softwood SSL was decreased from 5.2 g/L to 0.9 g/L by the combined CaO treatment and one-stage ion exchange treatment for 2 min. The total monosaccharides concentration was also decreased by this treatment (1.8 g/L). However, there was little change in total oligosaccharides concentration.
Fig. 4. Time dependence of removal of acetic acid and sugars in softwood SSL by strong base ion exchange resin (PA408) treatment: (A) acetic acid; (B) oligosaccharides; (C) monosaccharides
Table 1 shows the results of the combined treatment of hardwood waste liquor that contained 23.1 g/L of acetic acid and 7.9 g/L of xylo-oligosaccharide. The acetic acid content of the waste liquor treated by combined CaO and the first-stage of ion exchange resin treatments was 14.9 g/L, and further ion exchange treatments were required to remove more acetic acid. The third-stage of ion exchange resin treatment decreased acetic acid content in the waste liquor to 5.9 g/L; furthermore the decrease of oligosaccharides with the third-stage treatment was marginal.
It was reported that the OH– form of strong base anion exchange resin could adsorb monosaccharides and consequently decompose them (Koizumi and Okada 1980; Phillips and Pollard 1953; Turton and Pacsu 1955). Oligosaccharides could hardly be adsorbed to ion exchange resin, thus their decomposition was minimum. It was found that the acetic acid can be removed by the ion exchange resin treatment without decreasing oligosaccharide from softwood SSL and hardwood waste liquor.
The CaO treatment of SSL was applied for the recovery of lignosulfonate in pulp mills (Howard 1932). The sulfate and sulfite ions can also be removed from the SSL by the CaO treatment (Howard 1932; Kuroishi 1983). In this study, the sulfite ion content of softwood SSL was 12.7 g/L, and no sulfite ion was found after CaO treatment of SSL (Table 2). Other studies have also shown that furfural in the pre-hydrolyzates or SSL can be removed by lime treatment (Nigam 2001; Purwadi et al. 2004), and the furfural in the hardwood waste liquor (0.7 g/L) was removed by CaO treatment (Table 1). On the other hand, in this study we found that the combined CaO and ion exchange resin treatments can decrease the acetic acid level to a fermentable level, while having minimum effect on oligosaccharides.
Table 1. Change of Chemical Composition of Hardwood Waste Liquor with the Combined Method
Enhanced Ethanol Production from Oligosaccharides in Softwood SSL by Combined CaO and Ion Exchange Resin Treatments
Table 2 shows the chemical composition of SSL samples before and after fermen-tation. The fermentation results are shown in Fig. 5. When the fermentation with P. stipitis was conducted for 24 h, 1.3 g/L of ethanol was obtained in the SSL treated by the combined CaO and ion exchange resin treatments. All of monosaccharides were consumed at 16 h of fermentation. In addition, 3.3 g/L manno-oligosaccharides, 1.9 g/L gluco-oligosaccharides, and 0.2 g/L of xylo-oligosaccharides were consumed (Table 2). P. stipitis has the genes for seven -glucosidases, two -mannosidases, and one endoxylanase (Jeffries 2006; Jeffries et al. 2007). Oligosaccharides were hydrolyzed by these enzymes and consequently consumed by the strain. Several reports have shown that P. stipitis can produce ethanol from cellobiose (Parekh and Wayman 1986; Parekh et al. 1988) and xylan (Lee et al. 1986), however limited results are available on utilization of manno-oligosaccharides by P. stipitis. The present results confirmed that P. stipitis has an ability to metabolize manno-oligosaccharide. As shown in Fig. 5, further oligosaccharides consumption from the SSL treated with combined CaO and ion exchange resin did not occur after 16 h of fermentation. -glucosidases and -mannosidases, which are produced by P. stipitis, have high specificities to hydrolyze cellobiose and mannobiose.
Table 2. Chemical Composition of Softwood SSL Samples Before and After Fermentation
Fig. 5. Effect of ion exchange resin treatment on ethanol production from oligosaccharides in softwood SSL with P. stipitis: (A) ethanol; (B) total oligosaccharides; (C) total monosaccharides. Legends: ( ) CaO and ion exchange resin treated; ( ) CaO treated; (■) untreated
The ethanol concentration from the CaO-treated SSL after 40 h fermentation was 0.5 g/L, and no ethanol was obtained from the untreated SSL. Based on these results, it can be concluded that the combined CaO and ion exchange treatments of SSL are effective for improving ethanol production from oligosaccharides in softwood SSL.
CONCLUSIONS
- From the result of model experiments using cellobiose solution, 2.6 g/L of ethanol was obtained with 1 g/L of acetic acid present, while 2.9 g/L of ethanol was produced from the control with no acetic acid present. The acetic acid concentration was decreased during the P. stipitis fermentation; thus, the inhibitory effect of acetic acid was lowered in the late stage of fermentation.
- It was found that inhibitory compounds such as acetic acid, furfural, and sulfite ions in the softwood spent sulfite liquor (SSL) and the hardwood waste liquor can be removed by the combined CaO and ion exchange resin treatments with minimum effect on the oligosaccharides. The acetic acid concentration in the softwood SSL decreased from 5.2 g/L to 0.9 g/L by the combined method.
- The combined method was effective for improving ethanol production from oligosac-charides in softwood SSL with P. stipitis. 38% of total oligosaccharides (initial concentration; 14.8 g/L) were consumed and 1.3 g/L of ethanol was obtained from the SSL treated by the combined method, while no ethanol was obtained from the untreated SSL.
ACKNOWLEDGMENTS
One of the authors, Keishi Tanifuji, was supported by JSPS Program for Young Researcher Overseas Visits.
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Article submitted: January 14, 2013; Peer review completed: March 24, 2013; Revised version received: May 17, 2013; Accepted: May 30, 2013; Published: June 4, 2013.