Food Sci. Biotechnol. 22(2): 441-448 (2013) DOI 10.1007/s10068-013-0099-4
RESEARCH ARTICLE
Production of a High Concentration of Ethanol from Potato Tuber by High Gravity Fermentation Younghoon Lim, Youri Jang, and Keun Kim
Received: 7 September 2012 / Revised: 15 October 2012 / Accepted: 15 October 2012 / Published Online: 30 April 2013 © KoSFoST and Springer 2013
Abstract To produce a high concentration of ethanol from viscous potato tuber mash, potato tuber mash containing high contents of solids (28%) was prepared by grinding the potato tuber without the addition of water. The viscosity of the potato mash was reduced by using Viscozyme (0.1%) at 50oC for 30 min. The potato mash was then liquefied using Liquozyme (0.1%) at 90oC for 30 min and optimal conditions for the simultaneous saccharification and fermentation (SSF) of the potato mash for ethanol production were investigated using statistical methods. Using 24 factorial design, saccharifying-enzyme and incubation temperature were found to be important factors. Using response surface methodology, the optimal saccharifying-enzyme dosage and incubation temperature were determined to be 1.45 AGU/g dry matter and 31.3oC, respectively. Under these optimal conditions for SSF, 14.92%(v/v) ethanol with 91.0% of theoretical yield was produced after 60 h, and all the starch was completely used up. Keywords: potato tuber, ethanol, simultaneous saccharification and fermentation, Saccharomyces cerevisiae, response surface methodology
Introduction A great quantity of fermented industrial ethanol is produced in distilleries worldwide. Recently, demand and production of ethanol has been rapidly increased in many parts of world as sustainable energy source for fuel. Fuel bioethanol manufactured from renewable resources by microbial Younghoon Lim, Youri Jang, Keun Kim () Department of Bioscience and Biotechnology, The University of Suwon, Hwaseong, Gyeonggi 445-743, Korea Tel/Fax: +82-31-220-2344 E-mail:
[email protected]
fermentation is an attractive alternative because it is carbon dioxide neutral (1). Although the ethanol used today is mainly manufactured from sugar cane (Brazil) and corn (USA), ethanol production from other sources such as starch-rich grains, agricultural and forest residues, food processing byproducts, and agricultural energy crops will be required in the future (2,3). According to US Department of Agriculture, the root crop such as sweet potato and cassava produced 1.5 to 2.3 times as much fermentable carbohydrate as field corn (4). If economical harvesting and processing techniques could be developed, the study suggested that the root crops have greater potential as ethanol sources than the present corn systems. In order to compete with grain-based ethanol, the effective and economical processes are necessary for ethanol production from root and tuber crops. One of the methods enhancing the ethanol productivity is the high gravity technology. The technology involves the preparation and fermentation of mash containing highly dissolved solids to yield a high ethanol concentration (5). High ethanol concentration in the fermentation broth has several advantages such as increased fermentor throughput, reduced processing costs, reduced energy cost per liter of ethanol, and reduced risk of bacterial contamination (6). The high gravity technology has been performed in ethanol production mainly from cereal grains due to their low viscous nature. However, using of root or tuber biomass at high solids contents for ethanol production are rarely reported due to their high viscous nature. The limitation of using root and tuber mashes at high solids content is due to their high viscous nature mainly caused by high contents of pectin (7) which has been known as one of the gelling polysaccharides. High viscosity of fresh cassava mash caused resistance to solid-liquid separation and lower ethanol fermentation efficiency (8). High viscosity also causes several handling difficulties
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during processes, and may lead to incomplete hydrolysis of starch to fermentable sugar (9,10). Excessive water could be added to the mash to decrease the viscosity, but the fermentable sugars also diluted and final ethanol concentration will be decreased and more energy is required for distillation. So far the potato mash for ethanol fermentation in other reports are prepared by mixing the ground potato tuber with excessive water, consequently the final ethanol concentration obtained were only 3.9-8.8% (11-14). Ethanol fermentation from starch or cellulose involves 2 main processes: saccharification and fermentation. Simultaneous saccharification and fermentation (SSF) which is enzymatic hydrolysis coupled with yeast fermentation in the same vessel is generally preferred to separated hydrolysis and fermentation (SHF) because it can provide less equipments and fermentation time (15). SSF is especially useful for fermentation of high concentration of dissolved solids, since fermentable sugars released from polysaccharides during SSF are quickly converted to ethanol and therefore low concentration of sugars could be maintained in the fermentation broth, that cause less osmotic stress to the yeast cells. Osmotic stress together with ethanol stresses result in a loss of cell viability, growth, and fermentation performance of yeast (16). The object of this work is to produce high concentration of ethanol from potato tuber with high yield to compete with cereal ethanol. To accomplish the object, potato tuber mash containing high contents of solids was prepared by grounding the potato tuber without addition of water. The viscosity of potato mash with the high solid contents was reduced by cell wall-digesting enzymes containing pectinase.
Materials and Methods Microorganism and culture condition Saccharomyces cerevisiae ATCC 26603 was used in this study. The cells were grown on a YPD agar plate that consisted of 1% yeast extract (Y), 2% peptone (P), and 2% dextrose (D) solidified with 2% agar, at 30oC for 2 days. For liquid culture, cells were inoculated into a 250-mL Erlenmeyer flask containing 100 mL YPD was incubated at 30oC in a rotary shaking incubator operated at 200 rpm for 18 h. Potato tuber Potato tubers of cultivar ‘Haryeong’ were obtained from Highland Agriculture Research Center, National Institute of Crop Science, Rural Development Administration, Korea. Fresh unpeeled tubers were washed, cut into approximate 2×2 cm cubes, and mashed using Grow-mill grinder (DA-388; Green mix, Seoul, Korea). Initial pH and average moisture content were 5.6 and 72%, respectively.
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Enzyme Enzyme preparations including cellulase [Celluclast® 1.5 L, 91 filter paper unit (FPU)/mL], pectinase [Pectinex® Ultra SP-L, 10,292 polygalacturonase unit (PGU)/mL], hemicellulase [Viscozyme L, 112 fungal-glucanase unit (FBG)/mL], thermo-stable α-amylase [Liquozyme SC, 167 kilo Novo α-amylase unit (KNU)/mL], and glucoamylase [Spirizyme® Fuel, 953 Novo glucoamylase unit (AGU)/ mL] were supplied from Novozyme A/S (Bagsvaerd, Denmark). Viscosity reduction of potato mash by enzymatic hydrolysis Prior to liquefaction, the viscosity of potato mash was reduced with the addition of cell wall-digesting enzymes such as Celluclast 1.5 L, Pectinex Ultra SP-L, or Viscozyme L, which were provided from Novozyme. The final slurry concentration of potato mash was approximately 28%(w/v). Viscosity reduction of potato mash was monitored using a viscometer (LVDV-E; Brookfield Engineering Laboratories Inc, Middleborough, MA, USA) at 50oC for 60 min with a paddle speed of 150 rpm. The efficiency and optimum concentration of enzyme on viscosity reduction were determined. Liquefaction Potato mash treated with the viscosityreducing enzyme was then liquefied with Liquozyme SC (240 KNU/g, Novozyme) at different enzyme dosages (0.03-0.9%), incubation times (10-150 min), and temperatures (85-100oC). The initial mash pH was 5.6. The extent of liquefaction was determined by liquefaction ratio (%) and dextrose equivalent (DE, g reducing sugars/100 g dry matter). The liquefied mash was centrifuged (2,000×g) and the % of liquid volume of upper layer versus total volume was expressed as the liquefaction ratio. The effects of enzyme dosage, incubation time, and heating temperature on liquefaction ratio and DE value were evaluated. Simultaneous saccharification and fermentation (SSF) SSF experiments were carried out in a sterilized 250-mL Erlenmyer containing 100 mL of potato mash. The viscosity of the potato mash was reduced by using 0.1% Viscozyme at 50oC for 30 min. The potato mash was then liquefied using Liquozyme (0.1%) at 90oC for 30 min after liquefaction, the liquefied mash was cooled to room temperature, and simultaneously added with 0.1% saccharifying enzyme (glucoamylase) and yeast inoculum to achieve a final dry matter of 28%(w/v). Three types of glucoamylase tested were Spirizyme Fuel (750 AGU/g), Dextrozyme DX 1.5x (255 AGU/g), and AMG 300 L (300 AGU/mL). For all experiments, 1 loopful cells of yeast culture was inoculated to the potato mash and fermentation was conducted for 60 h. Optimal condition for SSF of the potato mash for ethanol production was investigated using statistical methods. Experiments are carried out in 2 stages. At the 1st stage, the
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24 factorial design was used to find the significant factors affecting ethanol production. At the 2nd stage, the central composite rotatable design (CCRD) was used. A 3-level 2 factors of CCRD and response surface method (RSM) was designated to find the optimal condition. Concentrations of ethanol and residual glucose were determined as described below. The theoretical yield of 0.511 g of ethanol is produced from 1 g of glucose. Analytical methods Ethanol concentrations were determined by using HPLC (Shiseido Nanospace SI-2, SUGAR SC1011, column size 8.0 mm i.d.×300 mm length) equipped with RI detector (RI 101; Shoko Korea Co., Seoul, Korea). The chromatogram was run at 50oC oven temperature and 4oC injecting temperature using a flow rate of 0.6 mL/min. All the experiments were replicated 3 times and the average values are presented. A variation of about 5% was seen between the 3 experiments. Residual reducing sugar released from starch during SSF was determined by the 3,5 dinitrosalycilic acid method (17) using glucose as a standard. Starch content was determined according to the method of Kim and Hamdy (18). For the analysis of solid content, 10 g ground potato tuber was dried at 105oC for 24 h and the dried potato was weighed. Statistical analysis The experimental data were analyzed according to RSM to fit the second-order polynomial equation (Eq. 1): Y=Ao +AlX1 +A2X2+A3X12+A4X22+A5X1X2
(1)
Y is the predicted response (ethanol yield, %, v/v), Ao is the intercept term, Al and A2 are the linear coefficients, A3 and A4 are the quadratic coefficients, A5 is the cross-product coefficient, and Xi is the coded independent variable. All the statistical analysis was performed using SAS (Statistical Analysis Systems Institute, Cary, NC, USA). The level of significance of all coefficients was 0.05.
Results and Discussion Viscosity reduction of potato mash by enzymatic hydrolysis The potato mash contained 28% solid and 23% starch. To reduce the viscosity of the potato mash, hydrolysis of the potato mash by cell wall-degrading enzymes such as Celluclast, Pectinex, and Viscozyme was examined. The results (Fig. 1) showed that the Cellulclast had the least effect to reduce the viscosity. While both of Pectinex and Viscozyme were effective in the viscosity reduction, Viscozyme showed faster viscosity reduction than Pectinex. The viscosity reduction of potato mash seems to be mainly caused by pectin degradation (19), since both Pectinex and Viscozyme contain pectinase while
Celluclast is mainly composed of cellulase. Viscozyme contains multiple pectin-degrading enzymes in addition to cellulase and xylanase and arabinase (20), therefore it seems that pectin is more efficiently degraded by mixture of pectinase and other cell wall-digesting enzymes than only pectinase. Xylanase also contributed the reduction of viscosity of sweet potato (21, 22). According to the authors (22), the viscosity obtained by treatment of xylanase in industrial scale of ethanol production from sweet potato was 500 cp which will make the very high gravity fermentation to realize and eliminate the follow-through processing such as pipeline transmission and solid-liquid seperation. The synergistic action among cell wall-digesting enzymes for polysaccharide degradation was also observed in other studies (19,23,24). Figure 1B shows the effect of Viscozyme concentration on the viscosity reduction of potato mash. Herelein, after 20 min, 0.40 and 0.80 FBG reached to the lowest viscosity and further incubation did not decrease the viscosity. With 0.20 FBG, the viscosity was decreased continuously for about 30 min and the viscosity did not decrease with further incubation. Since the final viscosity obtained by the 0.20 FBG was close to the one obtained by the 0.40 or 0.80 FBG, we decided to use 0.20 FBG of Viscozyme for the viscosity reduction of potato mash for further experiments. Liquefaction For ethanol production from starch by yeast, the starch is liquefied and then saccharified to fermentable sugar. Liquefaction process involves a partial hydrolysis of starch to maltodextrins at high temperature and the reduction of starch-paste viscosity by the action of thermostable α-amylase (19). High temperature for starch gelatinization is required to increase the enzyme digestibility of starch. The potato mash was treated with Viscozyme for viscosity reduction and then subjected to liquefaction. Four commercial liquefaction enzymes were tested for their liquefaction power and the results (Fig. 2A) showed that Liquozyme exhibited the highest liquefaction ratio of 34.16, whereas activity of BAN480L was the lowest as 16.66% and significantly inferior compared to the other enzymes. Liquozyme also showed the highest DE value. Therefore Liquozyme was selected for further study. Various concentrations of the selected Liquozyme were tested for liquefaction of potato mash and the results are shown in Fig. 2B. The liquefaction ratio of 25% at 0.03% Liquozyme was significantly increased to 36.7% at 0.1% Liquozyme and further increase of enzyme concentration did not result in any significant increase of liquefaction ratio. The trend was same with the DE. Therefore, Liquozyme concentration of 0.1% was chosen for the further experiments. The effect of temperature on the liquefaction of potato mash was examined and the results are shown in Fig. 2C.
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Fig. 1. Viscosity reduction of potato mash by enzymatic hydrolysis. (A) Viscosity reduction by different enzyme treatments, (B) effect of viscozyme concentration on the viscosity reduction
Among the various temperatures tested, 95oC showed the highest liquefaction ratio of 35.8% and DE of 8.7%. The increase of temperature to 100oC exhibited the lowest liquefaction ratio of 35.8% and DE 7.2% probably due to the decrease of enzyme activity at 100oC. Therefore the 95oC was chosen for further study. The effect of incubation time on the liquefaction was examined and the result (Fig. 2D) shows that the liquefaction ratio of 25.8±1.4% at 10 min was significantly increased to 35.8±1.4 and 8.0±0.2% at 30 min and further incubation did not result in any significant increase of liquefaction ratio and DE. Therefore, 30 min of incubation time was chosen. Ethanol production by SSF Saccharifying enzyme: To select the most efficient saccharifying enzyme for the SSF, each of 3 different enzymes was added into liquefied potato mash together with yeast cells and fermentation was conducted for various time. The results (Fig. 3) showed that Spirizyme Fuel showed the best performance in ethanol production and was selected for further study. Optimization of SSF by statistical methods: To find factors
significantly affecting SSF, experiment was performed according to 24 factorial design. Four factors considered were temperature (25 and 33oC), yeast inoculum size (106 and 108/mL), added concentration of glucoamylase (Spirizyme Fuel, 0.4 and 1.65 AGU/g wet substrate), amount of ammonium sulfate (5 and 30 mM) as nitrogen source. The resulting data are shown in Table 1. Table 2 is the ANOVA table for the above 24 factorial data. The ANOVA table shows that temperature, ammonium sulfate, and Spirizyme except inoculum size was the significant factors for the ethanol production by SSF. As shown in Table 3, the average concentrations of ethanol were produced at the different levels of temperature, ammonium sulfate, and glucoamylase. Since the higher amount of ammonium sulfate resulted in the decrease of ethanol produced, ammonium sulfate was eliminated from further RSM study. Therefore, from this first statistical experimental analysis, 2 factors such as Spirizyme concentration and temperature were found to be the ones which affected the ethanol production significantly and need to be studied further in detail. This study showed that the addition of nutrients was not needed for the high concentration of ethanol production from potato tuber
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Fig. 2. Effect of different parameters on liquefaction of potato mash. (A) Enzyme type, (B) Liquozyme concentration (90oC, 1 h). (C) temperature (0.1% Liquozyme, 90oC, 1 h), (D) incubation time (0.1% Liquozyme, 95oC). Before liquefaction, the potato mash was treated with Viscozyme at 50oC for 30 min. For experiment A, each liquefaction enzyme (final concentration, 0.1%) was added into the potato mash treated with Viscozyme and the mash was incubated at 90oC for 1 h.
Fig. 3. Effect of different glucoamylases on ethanol production by simultaneous saccharification and fermentation (SSF).
mash. The improvement of ethanol production by supplementation of nitrogen sources such as yeast extract or ammonium sulfate were observed in very high gravity fermentation of wheat mashes (25), sweet sorghum juice (26), and rice wine cake (27). However, for the ethanol fermentation of sweet potato with very high gravity, there was no need for supplementing additional nutrients, since
sweetpotato provided necessary nutrient elements for the fermentation (21,22). In other study of ethanol production from potato flour, supplementation of nitrogen sources did not contribute to ethanol yield, suggesting that potato may be a good substrate for ethanol fermentation (14). To investigate the optimal Spirizyme concentration and temperature for the highest ethanol production by SSF,
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Table 1. Data from 24 factorial design Run no.
Inoculum size (CFU/mL)
Temperature (oC)
Ammonium sulfate (mM)
Spirizyme (AGU/g)
Ethanol yield (%, v/v)
Glucose (%, w/v)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
106 106 106 106 106 106 106 106 108 108 108 108 108 108 108 108
25 25 25 25 33 33 33 33 25 25 25 25 33 33 33 33
5 5 30 30 5 5 30 30 5 5 30 30 5 5 30 30
0.4 1.65 0.4 1.65 0.4 1.65 0.4 1.65 0.4 1.65 0.4 1.65 0.4 1.65 0.4 1.65
12.54 13.46 9.69 13.07 13.74 14.54 12.97 14.04 12.77 13.33 11.75 13.26 14.09 14.62 13.44 13.87
0.00 0.13 0.61 0.17 0.00 0.03 0.11 0.58 0.00 0.00 0.52 0.00 0.00 0.05 0.19 0.44
Table 2. Analysis of variance table of the 24 factorial data
Table 4. Experimental data according to central composite rotatable design (CCRD)
1)
Factor
F-value
p-value
Inoculum size Ammonium sulfate conc. Spirizyme conc. Temperature
1.249 -2.838 3.729 4.637
0.23775 0.01615* 0.00333** 0.00072***
Run no. 1 2 3 4 5 6 7 8 9 10
1)
Level of significance=0.05*, 0.01**, and 0.001***
experiment was conducted according to CCRD and the results are shown in Table 4. SAS were used for RSM. ANOVA results of the above data shows that Spirizyme concentration significantly affected the ethanol production with p
