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Effects of nitrogen source on ethanol production in very high gravity fermentation of corn starch
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Effects of nitrogen source on ethanol production in very high gravity fermentation of corn starch Zhaofeng Li a,b, Donghai Wang c, Yong-Cheng Shi a,∗ a
Department of Grain Science and Industry, Kansas State University, Manhattan, KS 66506, USA School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, PR China c Department of Biological and Agricultural Engineering, Kansas State University, Manhattan, KS 66506, USA b
a r t i c l e
i n f o
Article history: Received 3 July 2016 Revised 25 October 2016 Accepted 27 October 2016 Available online xxx Keywords: Nitrogen source Starch Ethanol Saccharomyces cerevisiae Very high gravity fermentation
a b s t r a c t Nitrogen sources, the critical media component, were optimized to enhance ethanol production by Saccharomyces cerevisiae in very high gravity (VHG) fermentation of corn starch (340 g/l). Screening experiments revealed yeast extract as an ideal nitrogen source for ethanol production. When yeast extract concentration was controlled at 2%, ethanol yield and fermentation efficiency reached approximately 20.3% and 84.5%, respectively, after 72 h of fermentation. To reduce ethanol production cost, yeast extract supplementation was partially replaced with less expensive nitrogen sources, namely urea and ammonium sulfate. Combined effects of the three nitrogen sources on ethanol production were determined through central composite design. The optimum combination of nitrogen sources (0.6% yeast extract, 69 mM urea, and 26 mM ammonium sulfate) enabled ethanol yield and fermentation efficiency comparable to those supplemented with 2% yeast extract, indicating that urea and ammonium sulfate synergistically enhanced ethanol production by S. cerevisiae in VHG fermentation. © 2016 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction Fuel ethanol is a sustainable energy source that is intended to provide a more environmentally friendly alternative to fossil fuels such as diesel and gasoline [1–3]. World ethanol production for transport fuel has increased more than four-fold in the past decade and reached about 93 billion liters in 2014. In conventional alcoholic fermentation, a substrate containing 180– 220 g/l total sugars is used to achieve ethanol concentration of 10–14% (v/v) [4]. Very high gravity (VHG) fermentation technology can considerably increase both fermentation productivity and ethanol concentration while consuming less water and energy [5,6]. VHG is defined as ‘‘the preparation and fermentation to completion of mashes containing 27 g or more dissolved solids per 100 g mash’’ [7]. An important consideration for VHG fermentation is that a high final ethanol concentration subjects yeast to ethanol stress, which decreases its growth and cell viability [8]. Saccharomyces cerevisiae (S. cerevisiae), the budding yeast, is used universally for industrial production of fuel ethanol because of its ability to produce high ethanol concentrations [9–14]. Yeast
∗
Corresponding author. Fax: +1 7855327010. E-mail address: [email protected] (Y.-C. Shi).
requires an adequate supply of nutrients to grow. In addition to fermentable sugars and inorganic sources, the nitrogen source is an essential component of yeast growth media [15]. Lack of nitrogen source leads to a significant reduction in ethanol yield, and such negative effect cannot be omitted particularly in VHG fermentation [16,17]. S. cerevisiae is able to use a wide variety of nitrogen sources for growth, such as organic nitrogen, inorganic nitrogen, or a combination of both. Previous studies have shown that ammonium ion [18], urea [18,19], peptone [20], yeast extract [20], corn steep liquor [19], free amino acid [21], spent brewer’s yeast [22], and other nitrogen sources [23], could improve the growth of yeast cells and increase ethanol production; however, not all nitrogen sources contribute to yeast growth equally well [24,25]. In addition, the nitrogen source is usually the most expensive component of microbial growth media [26,27]. Because the nitrogen source is one of the main contributors in the total material cost, there is great need to replace costly nitrogen sources with less expensive ones for ethanol production. In this study, we investigated VHG fermentation of corn starch (340 g/l, w/v) and compared the effects of the readily available and often-used nitrogen sources (ammonium sulphate, urea, peptone, and yeast extract) on ethanol production by S. cerevisiae. Furthermore, a central composite design (CCD) was used to investigate the possibility of replacing high-cost nitrogen sources with more economical ones.
http://dx.doi.org/10.1016/j.jtice.2016.10.055 1876-1070/© 2016 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Z. Li et al., Effects of nitrogen source on ethanol production in very high gravity fermentation of corn starch, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2016.10.055
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2. Materials and methods 2.1. Materials Normal corn starch was obtained from National Starch Food Innovation, now Ingredion Inc. (Bridgewater, NJ). Active dry yeast (S. cerevisiae, Red Star Ethanol Red) was obtained from Lesaffre Yeast Corp. (Milwaukee, WI). The yeast contained > 20 × 109 viable cells/g [22,28]. Thermostable α -amylase (Liquozyme SC DS) and glucoamylase (Spirizyme SC DS) were obtained from Novozymes (Franklinton, NC). Enzyme activity of Liquozyme SC DS was 240 kilo Novo units (KNU)/g, and one KNU was defined as the amount of enzyme that hydrolyzed 5.26 g of starch (soluble starch) per hour under Novo Nordisk’s standard conditions for α -amylase determination (37 ± 0.05 °C, 0.3 mM Ca2+ , and pH 5.6). Enzyme activity of Spirizyme SC DS was 750 AGU/g, and one AGU is defined as the amount of enzyme that hydrolyzed 1 μM maltose/min under standard conditions (37 ± 0.05 °C, pH 4.3, 23.2 mM maltose, and reaction time of 5 min). Yeast extract (5.1% of assimilable nitrogen), the water-soluble portion of autolyzed fresh yeast, and peptone (3.7% of assimilable nitrogen), a mixture of peptides and free amino acids from pancreatic digest of casein, were purchased from Thermo Fisher Scientific (Santa Clara, CA). Ammonium sulfate (21.0% of assimilable nitrogen), urea (46.5% of assimilable nitrogen), and other general chemicals were also purchased from Thermo Fisher Scientific. 2.2. Liquefaction of normal corn starch A pressure reactor from Parr Instrument Company (Moline, IL) was used for the liquefaction of normal corn starch. Corn starch was mixed with distilled water to obtain starch slurry at 340 g/l (w/v). Corn starch slurry was adjusted to about pH 5.8 with 0.1 N HCl and thermostable α -amylase (Liquozyme SC DS, 9 KNU/100 g dry starches) was then added to the corn starch slurry. The mixture of corn starch and enzyme was placed in a beaker in the pressure reactor. The temperature of the mixture was ramped from 25 to 90 ± 2 °C with continuous agitation at 200 rpm in about 30 min. After the temperature reached 90 °C, the mixture was kept at this temperature and agitating rate of 200 rpm for 90 min for starch liquefaction. The liquefied solution was then used for ethanol fermentation. 2.3. Reducing sugar analysis
(20 g/l), peptone (5 g/l), yeast extract (3 g/l), KH2 PO4 (1 g/l), and MgSO4 ·7H2 O (0.5 g/l) and incubated at 38 °C for 30 min in an incubator shaking at 200 rpm. The fermentation broth containing the liquefied sample (100 g), activated yeast culture (1 ml), glucoamylase (Spirizyme SC DS, 2 U/g dry starches), K2 HPO4 (1 g/l), CaCl2 (0.2 g/l), and a nitrogen source were adjusted to pH 4.2 with 2 M HCl and added to each flask, which was subsequently sealed with an S-shaped airlock filled with about 2 ml of mineral oil. Ethanol fermentation was performed in an incubator shaker (model I2400, New Brunswick Scientific, Edison, NJ) at 30 °C for 72 h with continuous shaking at 150 rpm. The fermentation process was monitored by measuring the total weights of the fermentation flasks because the weight loss by CO2 evolution was proportional to the amount of ethanol produced during ethanol fermentation. Ethanol yield was defined as the ethanol concentration in fermentation broth. Fermentation efficiencies were calculated as a ratio of the experimentally determined ethanol yield to the theoretical ethanol yield. The total starch contents in the samples were used to calculate theoretical ethanol yields, assuming that 1.0 g of starch converts to 1.11 g of glucose and 1.0 g of glucose generates 0.511 g of ethanol. 2.6. Experimental design and optimization Ethanol production experiments were carried out with a sole nitrogen source (ammonium sulfate, urea, peptone, or yeast extract) at different concentrations in fermentation broth. All experiments were conducted in triplicate. A central composite design (CCD) with five coded levels (−1.41, −1, 0, +1, and +1.41) was used to investigate the possibility of partially replacing yeast extract with ammonium sulfate and urea. The concentration of yeast extract in the fermentation broth was controlled at 0.6%. The two independent variables used at five different levels were ammonium sulfate (X1 : 2, 10, 30, 50, and 58 mM) and urea (X2 : 4, 20, 60, 100, and 116 mM) concentrations. According to this design, the total number of treatment combinations was2k + 2k + no , where k is the number of independent variables and n0 is the number of repetitions of experiments at the center point. Experimental results of the CCD were used to fit with a second-order polynomial equation by a multiple regression technique (Eq. 1):
Y = βo +
k i=1
βi xi +
k
βii x2ii +
i=1
k
βi j xi x j
(1)
i< j
Reducing sugar contents of liquefied samples were determined by a Nelson–Somogyi method [29]. Glucose was used as a standard solution.
Where Y is the predicted response, β 0 is the offset term, β i is the ith linear coefficient, β ii is the ith quadratic coefficient, and β ij is the ijth interaction coefficient.
2.4. Determination of molecular weight distribution by gel permeation chromatography (GPC)
3. Results and discussion 3.1. Liquefaction of normal corn starch
The molecular weight distribution of the liquefied corn starch samples, after dissolving in dimethyl sulfoxide (DMSO), was determined by a GPC instrument (PL-GPC220, Polymer Laboratory, Amherst, MA) equipped with a refractive index detector and a ˚ 103 A, ˚ set of three Phenogel columns (300 × 7.8 mm, 10 μm, 105 A, ˚ Phenomenex, Torrance, CA) and eluted with 0.80 ml/min and 100 A, DMSO containing 5 mM NaNO3 . The oven and detector temperatures were controlled at 80 °C. Dextran standards were used to determine the relative molecular size. 2.5. Simultaneous saccharification and fermentation For the preparation of inoculums, active dry yeast (1 g) was dispersed in 19 ml of a preculture broth containing glucose
When the initial concentration of starch slurry was controlled at 340 g/l (w/v), the reducing sugar contents of corn starch hydrolysate increased gradually with liquefaction time. After the liquefaction of corn starch at 90 °C for 90 min, reducing sugar content reached approximately 12%. GPC analysis showed that the molecular weight distribution curve of starch hydrolysate had two peaks: one was located in the higher molecular weight region (about 16,0 0 0 g/mol), and the other was located in the lower molecular weight region (about 10 0 0 g/mol) (Fig. 1). Furthermore, the peak located in the low molecular weight region was much higher than that in the high molecular weight region (Fig. 1), suggesting that the liquefaction of corn starch solution could result in a very high proportion of maltodextrin with low molecular weight
Please cite this article as: Z. Li et al., Effects of nitrogen source on ethanol production in very high gravity fermentation of corn starch, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2016.10.055
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1.4
10
50 mM
40
100 mM
1 0.8 0.6 0.4 0.2
8
150 mM
35
250 mM
30
400 mM
6
25
Control
20 4
15 10
2
5
0 2
3
4
5
6
7
8
Fig. 1. Molecular weight distribution of starch hydrolysate obtained by the liquefaction of corn starch at 90 °C for 90 min.
35 25 mM
30
50 mM
12
24
36
48
60
72
Fermentation time (h) Fig. 3. Effects of urea concentration on ethanol yield and fermentation efficiency during the fermentation of starch hydrolysate with 12% reducing sugar content. Medium components: 340 g/l (w/v) normal corn starch hydrolysate, 50–400 mM urea, 0.1% (w/v) K2 HPO4 , and 0.02% (w/v) CaCl2 . The fermentation without urea supplementation was used as a control.
100 mM
6
25
200 mM 300 mM
20
400 mM
4
Control
15
10
2
Efficiency (%)
Ethanol yield (%, v/v)
0
0 0
log MW
8
Efficiency (%)
Ethanol yield (%, v/v)
1.2 dw/dlogM
3
5 0
0 0
12
24
36
48
60
S. cerevisiae cells associated with shortening of chronological life span in a concentration-dependent manner [32] and the utilization of ammonium sulfate, especially at high concentrations, caused a shift in the pH of culture medium to the more acidic side during the fermentation, resulting in inhibition of yeast growth. If the pH of culture medium could be controlled at 4.2 during the entire fermentation period, ethanol yield and fermentation efficiency might be improved.
72
Fermentation time (h) Fig. 2. Effects of ammonium sulfate concentration on ethanol yield and fermentation efficiency during the fermentation of starch hydrolysate with 12% reducing sugar content. Medium components: 340 g/l (w/v) normal corn starch hydrolysate, 25–400 mM ammonium sulfate, 0.1% (w/v) K2 HPO4 , and 0.02% (w/v) CaCl2 . The fermentation without ammonium sulfate supplementation was used as a control.
in total starch hydrolysates. These results suggest that the starch hydrolysate solution with high solids could be achieved for VHG ethanol production. 3.2. Effects of ammonium sulfate on ethanol production by S. cerevisiae In addition to the carbon source, the nitrogen source is one of the most important factors that affect the aerobic growth of S. cerevisiae, and thus the production of ethanol. S. cerevisiae is known to be capable of utilizing ammonium ions, which can be directly assimilated into a couple of amino acids, notably glutamate and glutamine [30,31]. We found that a certain amount of ethanol could be produced by S. cerevisiae using ammonium sulfate as the sole nitrogen source (Fig. 2). A positive relationship between ethanol yield and ammonium sulfate concentration occurred only during the first 15 h of fermentation (Fig. 2). However, as fermentation time increased, the low ammonium sulfate concentration yielded more ethanol than the higher concentration. The final fermentation results showed that 50 mM ammonium sulfate was most advantageous to the aerobic growth of S. cerevisiae, as indicated by the highest ethanol yield and fermentation efficiency. As a sole nitrogen source, even at the optimum concentration, final ethanol yield and fermentation efficiency were only 6.3% (w/v) and 26.5%, respectively (Table 1, Fig. 2). Thus, ammonium sulfate was not an ideal sole nitrogen source for ethanol production by S. cerevisiae in VHG fermentation, possibly because ammonium ions induced the death of aging
3.3. Effects of urea on ethanol production by S. cerevisiae Urea is an important and less expensive nitrogen source for yeast growth [33–35]. S. cerevisiae is able to degrade urea yielding ammonia, which is used to synthesize new complex nitrogenous molecules [36]. As a result, urea can be used as a sole nitrogen source for S. cerevisiae growth. The highest ethanol yield and fermentation efficiency were obtained when 150 mM urea was used (Table 1, Fig. 3). After 72 h of fermentation, ethanol yield and fermentation efficiency were 8.7% (v/v) and 36.5%, respectively (Table 1). If urea concentration was less than 100 mM, the nitrogen source might be not enough for the aerobic growth of S. cerevisiae. However, if urea concentration was too high, it would have a negative effect on the growth of S. cerevisiae. If urea concentration was higher than 200 mM, the ethanol yield was slightly lower than that at 150 mM urea. Although supplementation of 150 mM urea in growth media could result in higher ethanol yield (36.5%) than supplementation of 50 mM ammonium sulfate (26.5%), about 73.5% of corn starch hydrolysate remained unfermented. Thus, urea cannot be used as a sole nitrogen source for ethanol production in VHG fermentation. 3.4. Effects of peptone on ethanol production by S. cerevisiae Peptones are derived from animal milk, meat, or soy digested by proteolytic enzymes that contain small peptides, fats, metals, salts, vitamins, and many other biological compounds. Peptone is widely used as a key medium additive for growing fungi. A previous study showed that one of the best peptones for S. cerevisiae growth was from casein [37]. In the present study, casein peptone was supplemented into growth media as the sole carbon source in VHG fermentation. Ethanol yield increased as peptone concentration increased (Fig. 4). When peptone concentration was controlled at 2, 2.5, and 3%, after 72 h of fermentation, ethanol yield reached approximately 15.5, 17.1, and 17.9%, respectively, and fermentation efficiency reached 64.7, 71.3, or 74.6%, respectively
Please cite this article as: Z. Li et al., Effects of nitrogen source on ethanol production in very high gravity fermentation of corn starch, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2016.10.055
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Z. Li et al. / Journal of the Taiwan Institute of Chemical Engineers 000 (2016) 1–7 Table 1 Optimized ethanol yield and fermentation efficiency after 72 h of fermentation when using different nitrogen sources at optimum concentrations. Nitrogen source
Concentration
Assimilable N concentration (%)a
Ethanol yield (%, w/v)
Fermentation efficiency (%)
Ammonium sulfate Urea Peptone
50 mM 150 mM 2.5% 3.0% 2.0% 2.5%
0.14 0.42 0.09 0.11 0.10 0.13
6.3 ± 0.2a 8.7 ± 0.3b 17.1 ± 0.4c 17.9 ± 0.4d 20.5 ± 0.6e 21.0 ± 0.6e
26.5 ± 0.8a 36.5 ± 1.0b 71.3 ± 1.4c 74.6 ± 1.5d 85.7 ± 2.6e 87.7 ± 2.6e
Yeast extract
a It was calculated by multiplying concentration of nitrogen source by the content of assimilable nitrogen in the corresponding nitrogen source.
20
0.5%
24
80
60
2.5%
12
3% Control
40
8 20
4
100
0.3% 0.6%
20
80
0.8% 1%
16
1.5%
60
2%
12
2.5%
40
Control
8 20
4
0
0 0
12
24
36
48
60
72
Fermentation time (h) Fig. 4. Effects of peptone concentration on ethanol yield and fermentation efficiency during the fermentation of starch hydrolysate with 12% reducing sugar content. Medium components: 340 g/l (w/v) normal corn starch hydrolysate, 0.5–3% peptone, 0.1% (w/v) K2 HPO4 , and 0.02% (w/v) CaCl2 . The fermentation without peptone supplementation was used as a control.
Efficiency (%)
2%
Ethanol yield (%, v/v)
1.5%
16
Efficiency (%)
Ethanol yield (%, v/v)
1%
0
0 0
12
24
36
48
60
72
Fermentation time (h) Fig. 5. Effects of yeast extract concentration on ethanol yield and fermentation efficiency during the fermentation of starch hydrolysate with 12% reducing sugar content. Medium components: 340 g/l (w/v) normal corn starch hydrolysate, 0.3–2.5% yeast extract, 0.1% (w/v) K2 HPO4 , and 0.02% (w/v) CaCl2 . The fermentation without yeast extract supplementation was used as a control.
(Table 1, Fig. 4). When peptone concentration was controlled at above 3%, the ethanol concentration in the fermentation broth was only slightly higher than that at 3% peptone (data not shown). Peptone provides large numbers of amino acids and peptides, which are required for yeast growth. As a result, supplementation of peptone into growth media could result in much higher ethanol concentration than with ammonium sulfate or urea, suggesting that organic nitrogen sources might be more suitable for ethanol production by S. cerevisiae in VHG fermentation.
ter 72 h of fermentation, ethanol yield and fermentation efficiency were even slightly higher than those supplemented with 3% peptone, indicating that yeast extract was much more efficient than peptone for ethanol production by S. cerevisiae in VHG fermentation. This is probably due to the fact that the nutrients in yeast extract included more assimilable nitrogen, vitamins, and other growth factors, which could match more effectively the requirements of S. cerevisiae growth than those in peptone.
3.5. Effects of yeast extract on ethanol production by S. cerevisiae
3.6. Optimization using central composite design
Yeast extract is a water-soluble extract of selected autolyzed yeast cells. It can provide a wide range of amino acids, peptides, vitamins, inorganic salts, and carbon in growth media. As shown in Fig. 5, yeast extract had significant effects on ethanol yield and fermentation efficiency in VHG fermentation. When yeast extract concentration was controlled at 2%, after 72 h of fermentation, ethanol yield and fermentation efficiency reached approximately 20.3 and 84.5%, respectively (Table 1, Fig. 5), indicating that most of glucose was utilized by yeast to produce ethanol. If yeast extract concentration was controlled at 2.5% or above, the ethanol concentration in the fermentation broth was almost the same as that with 2.0% yeast extract (Table 1, Fig. 5). Furthermore, the ethanol fermentation with high yeast extract concentration (≥2%) took less time to reach the highest yield than those with low yeast extract concentration. The addition of a certain amount of yeast extract was highly advantageous to the aerobic growth of S. cerevisiae. Supplementing yeast extract into growth media could result in much higher ethanol concentration than the peptone alone at the same concentration. If yeast extract concentration was controlled at 0.8%, af-
Yeast extract was an ideal nitrogen source in growth media for ethanol production in VHG fermentation; however, the high cost of yeast extract (which is much more expensive than ammonium sulfate or urea) has a negative effect on the economics of industrialscale processes [22,38], so replacing yeast extract supplementation with less expensive nitrogen sources would increase profits. Although other nitrogen sources do not offer the same ethanol yields as yeast extract (Figs. 2–5), a combination of inexpensive urea and ammonium may be used to substitute for part of the yeast extract. The CCD was conducted in the vicinity of the optimum to locate the true optimum combination of urea (X1 ) and ammonium sulfate (X2 ) for ethanol yield (Y1 ) and fermentation efficiency (Y2 ). The levels of the variables for the CCD experiments were selected according to the results of the previous experiments. The design matrix and the corresponding experimental data are given in Table 2. Experimental results of the CCD fit well with the second-order polynomial equations:
Y1 (%, v/v) = 14.25 + 0.15X1 + 0.05X2 − 3.4 × 10−5 X1 X2 − 1.1 × 10−3 X1 2 − 9.0 × 10−4 X2 2
(2)
Please cite this article as: Z. Li et al., Effects of nitrogen source on ethanol production in very high gravity fermentation of corn starch, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2016.10.055
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5
a 20.1
Ethanol yield
18.975
17.85
16.725
15.6
50.00
100.00 80.00
40.00 30.00
X2: Ammonium sulfate
60.00
20.00
40.00
X1: Urea
20.00
10.00
b 84
Ethanol efficiency
79.25
74.5
69.75
65
50.00
100.00
40.00
80.00 30.00
X2: Ammonium sulfate
60.00 40.00
20.00
10.00
X1: Urea
20.00
Fig. 6. Response surfaces and contour lines of ethanol yield (A) and fermentation efficiency (B) showing interactions between urea and ammonium sulfate.
Y2 (% ) = 59.47 + 0.61X1 + 0.2X2 − 1.4 × 10 − 4.4 × 10−3 X1 2 − 3.8 × 10−3 X2 2
−4
X1 X2 (3)
The fit of the two models was assessed by the coefficient of determination R2 , which were both 0.956, indicating that only 4.4% of the variation was not explained by the models. The statistical significances of the response surface quadratic model equations were evaluated by an F-test ANOVA, which revealed
that the two regressions were statistically significant (P = 0.0 0 01) at the 99% confidence level. The contour plots described by the models Y1 and Y2 are represented in Fig. 6. The optimal concentrations for two inexpensive nitrogen sources obtained from the maximum point of the models were 69 mM for urea (X1 ) and 26 mM for ammonium sulfate (X2 ), respectively. The model predicted maximum responses of approximately 20.1% (v/v) ethanol concentration (Fig. 6A) and 83.8% fermentation efficiency (Fig. 6B).
Please cite this article as: Z. Li et al., Effects of nitrogen source on ethanol production in very high gravity fermentation of corn starch, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2016.10.055
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Z. Li et al. / Journal of the Taiwan Institute of Chemical Engineers 000 (2016) 1–7 Table 2 Design and responses of the central composite design (CCD). Tests
1 2 3 4 5 6 7 8 9 10 11 12 13
Coded and real values
Y1 Ethanol yield (%, w/v)
Y2 Fermentation efficiency (%)
X1 (Urea, mM)
X2 (Ammonium sulfate, mM)
Experimental
Predicted
Experimental
Predicted
−1 (20) 1 (100) −1 (20) 1 (100) −1.41 (4) 1.41 (116) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60)
−1 (10) −1 (10) 1 (50) 1 (50) 0 (30) 0 (30) −1.41 (2) 1.41 (58) 0 (30) 0 (30) 0 (30) 0 (30) 0 (30)
16.96 18.34 16.62 17.89 15.61 18.02 19.61 19.35 19.88 19.77 19.93 20.01 19.66
17.21 18.63 17.03 18.33 15.52 17.42 19.38 19.04 19.92 19.92 19.92 19.92 19.92
70.74 76.48 69.30 74.61 65.10 75.16 81.79 80.68 82.89 82.45 83.12 83.45 82.01
71.50 77.95 70.27 76.27 64.40 73.12 80.60 78.56 82.56 82.56 82.56 82.56 82.56
3.7. Validation of the optimized nitrogen source combination Experiments were done in triplicate using the optimized nitrogen source combination (0.6% yeast extract, 69 mM urea, and 26 mM ammonium sulfate) representing the maximum point of ethanol production to verify model results. The average ethanol yield and fermentation efficiency from validation tests were 20.2% (v/v) and 84.2%, respectively. The strong correlation between predicted and experimental values after optimization justified the validity of the response model and the existence of an optimum point. Supplementation of urea and ammonium sulfate could improve ethanol production by approximately 21% compared with using 0.6% yeast extract as the sole nitrogen source. Using the optimum combination of nitrogen sources could result in ethanol yield and fermentation efficiency in VHG fermentation comparable to those supplemented with 2% yeast extract, indicating that urea and ammonium sulfate synergistically enhanced ethanol production by S. cerevisiae in VHG fermentation with a substantial reduction of yeast extract. Furthermore, ethanol yield and fermentation efficiency in this study reach relatively high level. Commonly, VHG fermentation could achieve ethanol yield between 15 and 19 % (v/v) and fermentation efficiency of <80% [39,40]. 4. Conclusions Nitrogen sources were examined for ethanol production by S. cerevisiae in VHG fermentation of corn starch. Of the nitrogen sources examined, yeast extract was ideal for ethanol production, but its relative high prices made the partial substitution with urea and ammonium sulfate attractive to produce ethanol. The information presented in this work is a very good alternative to obtain >80% of fermentation efficiency from VHG ethanol fermentation. Besides yeast extract, the use of ammonium sulfate and urea yields a synergistic effect (observed as an increase more than the expected when used by separated) in ethanol yield and fermentation efficiency. Thus, ethanol could be produced with low-cost nitrogen sources in yeast growth media with high efficiency, especially in VHG ethanol fermentation. Acknowledgment This is contribution number 14-167-J from the Kansas Agricultural Experiment Station. References [1] Solomon BD. Biofuels and sustainability. Ann N Y Acad Sci 2010;1185:119–34.
[2] Falano T, Jeswani HK, Azapagic A. Assessing the environmental sustainability of ethanol from integrated biorefineries. Biotechnol J 2014;9(6):753–65. [3] Jeong SY, Lee JW. Optimization of pretreatment condition for ethanol production from oxalic acid pretreated biomass by response surface methodology. Ind Crop Prod 2016;79:1–6. [4] Tao X, Zheng D, Liu T, Wang P, Zhao W, Zhu M, et al. A novel strategy to construct yeast Saccharomyces cerevisiae strains for very high gravity fermentation. PlOS ONE 2012;7(2):e31235. [5] Liu CG, Wang N, Lin YH, Bai FW. Very high gravity ethanol fermentation by flocculating yeast under redox potential-controlled conditions. Biotechnol Biofuels 2012;5(1):61. [6] Puligundla P, Smogrovicova D, Obulam VS, Ko S. Very high gravity (VHG) ethanolic brewing and fermentation: a research update. J Ind Microbiol Biotechnol 2011;38(9):1133–44. [7] Wang FQ, Gao CJ, Yang CY, Xu P. Optimization of an ethanol production medium in very high gravity fermentation. Biotechnol Lett 2007;29(2):233–6. [8] Damore T, Stewart GG. Ethanol tolerance of yeast. Enzyme Microb Technol 1987;9(6):322–30. [9] Babrzadeh F, Jalili R, Wang C, Shokralla S, Pierce S, Robinson-Mosher A, et al. Whole-genome sequencing of the efficient industrial fuel-ethanol fermentative Saccharomyces cerevisiae strain CAT-1. Mol Genet Genomics 2012;287(6):485–94. [10] Lu J, Li X, Yang R, Yang L, Zhao J, Liu Y, et al. Fed-batch semi-simultaneous saccharification and fermentation of reed pretreated with liquid hot water for bio-ethanol production using Saccharomyces cerevisiae. Bioresour Technol 2013;144:539–47. [11] Ercan Y, Irfan T, Mustafa K. Optimization of ethanol production from carob pod extract using immobilized Saccharomyces cerevisiae cells in a stirred tank bioreactor. Bioresour Technol 2013;135:365–71. [12] Bialas W, Szymanowska D, Grajek W. Fuel ethanol production from granular corn starch using Saccharomyces cerevisiae in a long term repeated SSF process with full stillage recycling. Bioresour Technol 2010;101(9):3126–31. [13] Sarris D, Matsakas L, Aggelis G, Koutinas AA, Papanikolaou S. Aerated vs non-aerated conversions of molasses and olive mill wastewaters blends into bioethanol by Saccharomyces cerevisiae under non-aseptic conditions. Ind Crop Prod 2014;56:83–93. [14] Takaki M, Tan L, Murakami T, Tang YQ, Sun ZY, Morimura S, et al. Production of biofuels from sweet sorghum juice via ethanol–methane two-stage fermentation. Ind Crop Prod 2015;63:329–36. [15] Albers E, Larsson C, Liden G, Niklasson C, Gustafsson L. Influence of the nitrogen source on Saccharomyces cerevisiae anaerobic growth and product formation. Appl Environ Microbiol 1996;62(9):3187–95. [16] Watanabe T, Srichuwong S, Arakane M, Tamiya S, Yoshinaga M, Watanabe I, et al. Selection of stress-tolerant yeasts for simultaneous saccharification and fermentation (SSF) of very high gravity (VHG) potato mash to ethanol. Bioresour Technol 2010;101(24):9710–14. [17] Shen Y, Guo JS, Chen YP, Zhang HD, Zheng XX, Zhang XM, et al. Application of low-cost algal nitrogen source feeding in fuel ethanol production using high gravity sweet potato medium. J Biotechnol 2012;160(3–4):229–35. [18] Yue G, Yu J, Zhang X, Tan T. The influence of nitrogen sources on ethanol production by yeast from concentrated sweet sorghum juice. Biomass Bioenerg 2012;39(0):48–52. [19] Pereira FB, Guimaraes PM, Teixeira JA, Domingues L. Optimization of low-cost medium for very high gravity ethanol fermentations by Saccharomyces cerevisiae using statistical experimental designs. Bioresour Technol 2010;101(20):7856–63. [20] Laopaiboon L, Nuanpeng S, Srinophakun P, Klanrit P, Laopaiboon P. Ethanol production from sweet sorghum juice using very high gravity technology: effects of carbon and nitrogen supplementations. Bioresour Technol 20 09;10 0(18):4176–82. [21] Bach B, Sauvage F-X, Dequin S, Camarasa C. Role of γ -aminobutyric acid as a source of nitrogen and succinate in wine. Am J Enol Viticult 2009;60(4):508–16.
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Z. Li et al. / Journal of the Taiwan Institute of Chemical Engineers 000 (2016) 1–7 [22] Kawa-Rygielska J, Pietrzak W. Ethanol fermentation of very high gravity (VHG) maize mashes by Saccharomyces cerevisiae with spent brewer’s yeast supplementation. Biomass Bioenerg 2014;60:50–7. [23] Xiao DG, Wu SA, Zhu XD, Chen YF, Guo XW. Effects of soya fatty acids on cassava ethanol fermentation. Appl Biochem Biotechnol 2010;160(2):410–20. [24] Beltran G, Novo M, Rozes N, Mas A, Guillamon JM. Nitrogen catabolite repression in Saccharomyces cerevisiae during wine fermentations. FEMS Yeast Res 2004;4(6):625–32. [25] Arrizon J, Gschaedler A. Increasing fermentation efficiency at high sugar concentrations by supplementing an additional source of nitrogen during the exponential phase of the tequila fermentation process. Can J Microbiol 2002;48(11):965–70. [26] Huang LP, Dong T, Chen JW, Li N. Biotechnological production of lactic acid integrated with fishmeal wastewater treatment by Rhizopus oryzae. Bioprocess Biosyst Eng 2007;30(2):135–40. [27] Herpandi NH, Rosma A, Wan Nadiah WA. The tuna fishing industry: a new outlook on fish protein hydrolysates. Compr Rev Food Sci Food Saf 2011;10(4):195–207. [28] Yangcheng H, Jiang H, Blanco M, Jane JL. Characterization of normal and waxy corn starch for bioethanol production. J Agric Food Chem 2013;61(2):379–86. [29] Smogyi M. Notes on sugar determination. J Biol Chem 1952;195(1):19–23. [30] Nissen TL, Kielland-Brandt MC, Nielsen J, Villadsen J. Optimization of ethanol production in Saccharomyces cerevisiae by metabolic engineering of the ammonium assimilation. Metab Eng 20 0 0;2(1):69–77. [31] Mendes-Ferreira A, Mendes-Faia A, Leao C. Growth and fermentation patterns of Saccharomyces cerevisiae under different ammonium concentrations and its implications in winemaking industry. J Appl Microbiol 2004;97(3):540–5. [32] Santos J, Sousa MJ, Leao C. Ammonium is toxic for aging yeast cells, inducing death and shortening of the chronological lifespan. PlOS ONE 2012;7(5):e37090.
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[33] Navarathna DH, Das A, Morschhauser J, Nickerson KW, Roberts DD. Dur3 is the major urea transporter in Candida albicans and is co-regulated with the urea amidolyase Dur1,2. Microbiology 2011;157(Pt 1):270–9. [34] Pereira FB, Guimaraes PM, Teixeira JA, Domingues L. Optimization of low-cost medium for very high gravity ethanol fermentations by Saccharomyces cerevisiae using statistical experimental designs. Bioresour Technol 2010;101(20):7856–63. [35] Mukhtar K, Asgher M, Afghan S, Hussain K, Zia-Ul-Hussnain S. Comparative study on two commercial strains of Saccharomyces cerevisiae for optimum ethanol production on industrial scale. J Biomed Biotechnol 2010;2010:419586. [36] Coulon J, Husnik JI, Inglis DL, van der Merwe GK, Lonvaud A, Erasmus DJ, et al. Metabolic engineering of Saccharomyces cerevisiae to minimize the production of ethyl carbamate in wine. Am J Enol Viticult 2006;57(2):113–24. [37] Dufosse L, De la Broise D, Guerard F. Evaluation of nitrogenous substrates such as peptones from fish: a new method based on Gompertz modeling of microbial growth. Curr Microbiol 2001;42(1):32–8. [38] Nancib A, Nancib N, Meziane-Cherif D, Boubendir A, Fick M, Boudrant J. Joint effect of nitrogen sources and B vitamin supplementation of date juice on lactic acid production by Lactobacillus casei subsp. rhamnosus. Bioresour Technol 2005;96(1):63–7. [39] Baeyens J, Kang Q, Appels L, Dewil R, Lv Y, Tan T. Challenges and opportunities in improving the production of bio-ethanol. Prog Energ Combust 2015;47:60–88. [40] Chu-Ky S, Pham T-H, Bui K-LT, Nguyen T-T, Pham K-D, Nguyen H-DT, et al. Simultaneous liquefaction, saccharification and fermentation at very high gravity of rice at pilot scale for potable ethanol production and distillers dried grains composition. Food Bioprod Process 2016;98:79–85.
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Journal of the Taiwan Institute of Chemical Engineers 0 0 0 (2016) 1–7
Contents lists available at ScienceDirect
Journal of the Taiwan Institute of Chemical Engineers journal homepage: www.elsevier.com/locate/jtice
Effects of nitrogen source on ethanol production in very high gravity fermentation of corn starch Zhaofeng Li a,b, Donghai Wang c, Yong-Cheng Shi a,∗ a
Department of Grain Science and Industry, Kansas State University, Manhattan, KS 66506, USA School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, PR China c Department of Biological and Agricultural Engineering, Kansas State University, Manhattan, KS 66506, USA b
a r t i c l e
i n f o
Article history: Received 3 July 2016 Revised 25 October 2016 Accepted 27 October 2016 Available online xxx Keywords: Nitrogen source Starch Ethanol Saccharomyces cerevisiae Very high gravity fermentation
a b s t r a c t Nitrogen sources, the critical media component, were optimized to enhance ethanol production by Saccharomyces cerevisiae in very high gravity (VHG) fermentation of corn starch (340 g/l). Screening experiments revealed yeast extract as an ideal nitrogen source for ethanol production. When yeast extract concentration was controlled at 2%, ethanol yield and fermentation efficiency reached approximately 20.3% and 84.5%, respectively, after 72 h of fermentation. To reduce ethanol production cost, yeast extract supplementation was partially replaced with less expensive nitrogen sources, namely urea and ammonium sulfate. Combined effects of the three nitrogen sources on ethanol production were determined through central composite design. The optimum combination of nitrogen sources (0.6% yeast extract, 69 mM urea, and 26 mM ammonium sulfate) enabled ethanol yield and fermentation efficiency comparable to those supplemented with 2% yeast extract, indicating that urea and ammonium sulfate synergistically enhanced ethanol production by S. cerevisiae in VHG fermentation. © 2016 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction Fuel ethanol is a sustainable energy source that is intended to provide a more environmentally friendly alternative to fossil fuels such as diesel and gasoline [1–3]. World ethanol production for transport fuel has increased more than four-fold in the past decade and reached about 93 billion liters in 2014. In conventional alcoholic fermentation, a substrate containing 180– 220 g/l total sugars is used to achieve ethanol concentration of 10–14% (v/v) [4]. Very high gravity (VHG) fermentation technology can considerably increase both fermentation productivity and ethanol concentration while consuming less water and energy [5,6]. VHG is defined as ‘‘the preparation and fermentation to completion of mashes containing 27 g or more dissolved solids per 100 g mash’’ [7]. An important consideration for VHG fermentation is that a high final ethanol concentration subjects yeast to ethanol stress, which decreases its growth and cell viability [8]. Saccharomyces cerevisiae (S. cerevisiae), the budding yeast, is used universally for industrial production of fuel ethanol because of its ability to produce high ethanol concentrations [9–14]. Yeast
∗
Corresponding author. Fax: +1 7855327010. E-mail address: [email protected] (Y.-C. Shi).
requires an adequate supply of nutrients to grow. In addition to fermentable sugars and inorganic sources, the nitrogen source is an essential component of yeast growth media [15]. Lack of nitrogen source leads to a significant reduction in ethanol yield, and such negative effect cannot be omitted particularly in VHG fermentation [16,17]. S. cerevisiae is able to use a wide variety of nitrogen sources for growth, such as organic nitrogen, inorganic nitrogen, or a combination of both. Previous studies have shown that ammonium ion [18], urea [18,19], peptone [20], yeast extract [20], corn steep liquor [19], free amino acid [21], spent brewer’s yeast [22], and other nitrogen sources [23], could improve the growth of yeast cells and increase ethanol production; however, not all nitrogen sources contribute to yeast growth equally well [24,25]. In addition, the nitrogen source is usually the most expensive component of microbial growth media [26,27]. Because the nitrogen source is one of the main contributors in the total material cost, there is great need to replace costly nitrogen sources with less expensive ones for ethanol production. In this study, we investigated VHG fermentation of corn starch (340 g/l, w/v) and compared the effects of the readily available and often-used nitrogen sources (ammonium sulphate, urea, peptone, and yeast extract) on ethanol production by S. cerevisiae. Furthermore, a central composite design (CCD) was used to investigate the possibility of replacing high-cost nitrogen sources with more economical ones.
http://dx.doi.org/10.1016/j.jtice.2016.10.055 1876-1070/© 2016 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Z. Li et al., Effects of nitrogen source on ethanol production in very high gravity fermentation of corn starch, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2016.10.055
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2. Materials and methods 2.1. Materials Normal corn starch was obtained from National Starch Food Innovation, now Ingredion Inc. (Bridgewater, NJ). Active dry yeast (S. cerevisiae, Red Star Ethanol Red) was obtained from Lesaffre Yeast Corp. (Milwaukee, WI). The yeast contained > 20 × 109 viable cells/g [22,28]. Thermostable α -amylase (Liquozyme SC DS) and glucoamylase (Spirizyme SC DS) were obtained from Novozymes (Franklinton, NC). Enzyme activity of Liquozyme SC DS was 240 kilo Novo units (KNU)/g, and one KNU was defined as the amount of enzyme that hydrolyzed 5.26 g of starch (soluble starch) per hour under Novo Nordisk’s standard conditions for α -amylase determination (37 ± 0.05 °C, 0.3 mM Ca2+ , and pH 5.6). Enzyme activity of Spirizyme SC DS was 750 AGU/g, and one AGU is defined as the amount of enzyme that hydrolyzed 1 μM maltose/min under standard conditions (37 ± 0.05 °C, pH 4.3, 23.2 mM maltose, and reaction time of 5 min). Yeast extract (5.1% of assimilable nitrogen), the water-soluble portion of autolyzed fresh yeast, and peptone (3.7% of assimilable nitrogen), a mixture of peptides and free amino acids from pancreatic digest of casein, were purchased from Thermo Fisher Scientific (Santa Clara, CA). Ammonium sulfate (21.0% of assimilable nitrogen), urea (46.5% of assimilable nitrogen), and other general chemicals were also purchased from Thermo Fisher Scientific. 2.2. Liquefaction of normal corn starch A pressure reactor from Parr Instrument Company (Moline, IL) was used for the liquefaction of normal corn starch. Corn starch was mixed with distilled water to obtain starch slurry at 340 g/l (w/v). Corn starch slurry was adjusted to about pH 5.8 with 0.1 N HCl and thermostable α -amylase (Liquozyme SC DS, 9 KNU/100 g dry starches) was then added to the corn starch slurry. The mixture of corn starch and enzyme was placed in a beaker in the pressure reactor. The temperature of the mixture was ramped from 25 to 90 ± 2 °C with continuous agitation at 200 rpm in about 30 min. After the temperature reached 90 °C, the mixture was kept at this temperature and agitating rate of 200 rpm for 90 min for starch liquefaction. The liquefied solution was then used for ethanol fermentation. 2.3. Reducing sugar analysis
(20 g/l), peptone (5 g/l), yeast extract (3 g/l), KH2 PO4 (1 g/l), and MgSO4 ·7H2 O (0.5 g/l) and incubated at 38 °C for 30 min in an incubator shaking at 200 rpm. The fermentation broth containing the liquefied sample (100 g), activated yeast culture (1 ml), glucoamylase (Spirizyme SC DS, 2 U/g dry starches), K2 HPO4 (1 g/l), CaCl2 (0.2 g/l), and a nitrogen source were adjusted to pH 4.2 with 2 M HCl and added to each flask, which was subsequently sealed with an S-shaped airlock filled with about 2 ml of mineral oil. Ethanol fermentation was performed in an incubator shaker (model I2400, New Brunswick Scientific, Edison, NJ) at 30 °C for 72 h with continuous shaking at 150 rpm. The fermentation process was monitored by measuring the total weights of the fermentation flasks because the weight loss by CO2 evolution was proportional to the amount of ethanol produced during ethanol fermentation. Ethanol yield was defined as the ethanol concentration in fermentation broth. Fermentation efficiencies were calculated as a ratio of the experimentally determined ethanol yield to the theoretical ethanol yield. The total starch contents in the samples were used to calculate theoretical ethanol yields, assuming that 1.0 g of starch converts to 1.11 g of glucose and 1.0 g of glucose generates 0.511 g of ethanol. 2.6. Experimental design and optimization Ethanol production experiments were carried out with a sole nitrogen source (ammonium sulfate, urea, peptone, or yeast extract) at different concentrations in fermentation broth. All experiments were conducted in triplicate. A central composite design (CCD) with five coded levels (−1.41, −1, 0, +1, and +1.41) was used to investigate the possibility of partially replacing yeast extract with ammonium sulfate and urea. The concentration of yeast extract in the fermentation broth was controlled at 0.6%. The two independent variables used at five different levels were ammonium sulfate (X1 : 2, 10, 30, 50, and 58 mM) and urea (X2 : 4, 20, 60, 100, and 116 mM) concentrations. According to this design, the total number of treatment combinations was2k + 2k + no , where k is the number of independent variables and n0 is the number of repetitions of experiments at the center point. Experimental results of the CCD were used to fit with a second-order polynomial equation by a multiple regression technique (Eq. 1):
Y = βo +
k i=1
βi xi +
k
βii x2ii +
i=1
k
βi j xi x j
(1)
i< j
Reducing sugar contents of liquefied samples were determined by a Nelson–Somogyi method [29]. Glucose was used as a standard solution.
Where Y is the predicted response, β 0 is the offset term, β i is the ith linear coefficient, β ii is the ith quadratic coefficient, and β ij is the ijth interaction coefficient.
2.4. Determination of molecular weight distribution by gel permeation chromatography (GPC)
3. Results and discussion 3.1. Liquefaction of normal corn starch
The molecular weight distribution of the liquefied corn starch samples, after dissolving in dimethyl sulfoxide (DMSO), was determined by a GPC instrument (PL-GPC220, Polymer Laboratory, Amherst, MA) equipped with a refractive index detector and a ˚ 103 A, ˚ set of three Phenogel columns (300 × 7.8 mm, 10 μm, 105 A, ˚ Phenomenex, Torrance, CA) and eluted with 0.80 ml/min and 100 A, DMSO containing 5 mM NaNO3 . The oven and detector temperatures were controlled at 80 °C. Dextran standards were used to determine the relative molecular size. 2.5. Simultaneous saccharification and fermentation For the preparation of inoculums, active dry yeast (1 g) was dispersed in 19 ml of a preculture broth containing glucose
When the initial concentration of starch slurry was controlled at 340 g/l (w/v), the reducing sugar contents of corn starch hydrolysate increased gradually with liquefaction time. After the liquefaction of corn starch at 90 °C for 90 min, reducing sugar content reached approximately 12%. GPC analysis showed that the molecular weight distribution curve of starch hydrolysate had two peaks: one was located in the higher molecular weight region (about 16,0 0 0 g/mol), and the other was located in the lower molecular weight region (about 10 0 0 g/mol) (Fig. 1). Furthermore, the peak located in the low molecular weight region was much higher than that in the high molecular weight region (Fig. 1), suggesting that the liquefaction of corn starch solution could result in a very high proportion of maltodextrin with low molecular weight
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1.4
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1 0.8 0.6 0.4 0.2
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Fig. 1. Molecular weight distribution of starch hydrolysate obtained by the liquefaction of corn starch at 90 °C for 90 min.
35 25 mM
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Fermentation time (h) Fig. 3. Effects of urea concentration on ethanol yield and fermentation efficiency during the fermentation of starch hydrolysate with 12% reducing sugar content. Medium components: 340 g/l (w/v) normal corn starch hydrolysate, 50–400 mM urea, 0.1% (w/v) K2 HPO4 , and 0.02% (w/v) CaCl2 . The fermentation without urea supplementation was used as a control.
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0 0
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S. cerevisiae cells associated with shortening of chronological life span in a concentration-dependent manner [32] and the utilization of ammonium sulfate, especially at high concentrations, caused a shift in the pH of culture medium to the more acidic side during the fermentation, resulting in inhibition of yeast growth. If the pH of culture medium could be controlled at 4.2 during the entire fermentation period, ethanol yield and fermentation efficiency might be improved.
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Fermentation time (h) Fig. 2. Effects of ammonium sulfate concentration on ethanol yield and fermentation efficiency during the fermentation of starch hydrolysate with 12% reducing sugar content. Medium components: 340 g/l (w/v) normal corn starch hydrolysate, 25–400 mM ammonium sulfate, 0.1% (w/v) K2 HPO4 , and 0.02% (w/v) CaCl2 . The fermentation without ammonium sulfate supplementation was used as a control.
in total starch hydrolysates. These results suggest that the starch hydrolysate solution with high solids could be achieved for VHG ethanol production. 3.2. Effects of ammonium sulfate on ethanol production by S. cerevisiae In addition to the carbon source, the nitrogen source is one of the most important factors that affect the aerobic growth of S. cerevisiae, and thus the production of ethanol. S. cerevisiae is known to be capable of utilizing ammonium ions, which can be directly assimilated into a couple of amino acids, notably glutamate and glutamine [30,31]. We found that a certain amount of ethanol could be produced by S. cerevisiae using ammonium sulfate as the sole nitrogen source (Fig. 2). A positive relationship between ethanol yield and ammonium sulfate concentration occurred only during the first 15 h of fermentation (Fig. 2). However, as fermentation time increased, the low ammonium sulfate concentration yielded more ethanol than the higher concentration. The final fermentation results showed that 50 mM ammonium sulfate was most advantageous to the aerobic growth of S. cerevisiae, as indicated by the highest ethanol yield and fermentation efficiency. As a sole nitrogen source, even at the optimum concentration, final ethanol yield and fermentation efficiency were only 6.3% (w/v) and 26.5%, respectively (Table 1, Fig. 2). Thus, ammonium sulfate was not an ideal sole nitrogen source for ethanol production by S. cerevisiae in VHG fermentation, possibly because ammonium ions induced the death of aging
3.3. Effects of urea on ethanol production by S. cerevisiae Urea is an important and less expensive nitrogen source for yeast growth [33–35]. S. cerevisiae is able to degrade urea yielding ammonia, which is used to synthesize new complex nitrogenous molecules [36]. As a result, urea can be used as a sole nitrogen source for S. cerevisiae growth. The highest ethanol yield and fermentation efficiency were obtained when 150 mM urea was used (Table 1, Fig. 3). After 72 h of fermentation, ethanol yield and fermentation efficiency were 8.7% (v/v) and 36.5%, respectively (Table 1). If urea concentration was less than 100 mM, the nitrogen source might be not enough for the aerobic growth of S. cerevisiae. However, if urea concentration was too high, it would have a negative effect on the growth of S. cerevisiae. If urea concentration was higher than 200 mM, the ethanol yield was slightly lower than that at 150 mM urea. Although supplementation of 150 mM urea in growth media could result in higher ethanol yield (36.5%) than supplementation of 50 mM ammonium sulfate (26.5%), about 73.5% of corn starch hydrolysate remained unfermented. Thus, urea cannot be used as a sole nitrogen source for ethanol production in VHG fermentation. 3.4. Effects of peptone on ethanol production by S. cerevisiae Peptones are derived from animal milk, meat, or soy digested by proteolytic enzymes that contain small peptides, fats, metals, salts, vitamins, and many other biological compounds. Peptone is widely used as a key medium additive for growing fungi. A previous study showed that one of the best peptones for S. cerevisiae growth was from casein [37]. In the present study, casein peptone was supplemented into growth media as the sole carbon source in VHG fermentation. Ethanol yield increased as peptone concentration increased (Fig. 4). When peptone concentration was controlled at 2, 2.5, and 3%, after 72 h of fermentation, ethanol yield reached approximately 15.5, 17.1, and 17.9%, respectively, and fermentation efficiency reached 64.7, 71.3, or 74.6%, respectively
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Z. Li et al. / Journal of the Taiwan Institute of Chemical Engineers 000 (2016) 1–7 Table 1 Optimized ethanol yield and fermentation efficiency after 72 h of fermentation when using different nitrogen sources at optimum concentrations. Nitrogen source
Concentration
Assimilable N concentration (%)a
Ethanol yield (%, w/v)
Fermentation efficiency (%)
Ammonium sulfate Urea Peptone
50 mM 150 mM 2.5% 3.0% 2.0% 2.5%
0.14 0.42 0.09 0.11 0.10 0.13
6.3 ± 0.2a 8.7 ± 0.3b 17.1 ± 0.4c 17.9 ± 0.4d 20.5 ± 0.6e 21.0 ± 0.6e
26.5 ± 0.8a 36.5 ± 1.0b 71.3 ± 1.4c 74.6 ± 1.5d 85.7 ± 2.6e 87.7 ± 2.6e
Yeast extract
a It was calculated by multiplying concentration of nitrogen source by the content of assimilable nitrogen in the corresponding nitrogen source.
20
0.5%
24
80
60
2.5%
12
3% Control
40
8 20
4
100
0.3% 0.6%
20
80
0.8% 1%
16
1.5%
60
2%
12
2.5%
40
Control
8 20
4
0
0 0
12
24
36
48
60
72
Fermentation time (h) Fig. 4. Effects of peptone concentration on ethanol yield and fermentation efficiency during the fermentation of starch hydrolysate with 12% reducing sugar content. Medium components: 340 g/l (w/v) normal corn starch hydrolysate, 0.5–3% peptone, 0.1% (w/v) K2 HPO4 , and 0.02% (w/v) CaCl2 . The fermentation without peptone supplementation was used as a control.
Efficiency (%)
2%
Ethanol yield (%, v/v)
1.5%
16
Efficiency (%)
Ethanol yield (%, v/v)
1%
0
0 0
12
24
36
48
60
72
Fermentation time (h) Fig. 5. Effects of yeast extract concentration on ethanol yield and fermentation efficiency during the fermentation of starch hydrolysate with 12% reducing sugar content. Medium components: 340 g/l (w/v) normal corn starch hydrolysate, 0.3–2.5% yeast extract, 0.1% (w/v) K2 HPO4 , and 0.02% (w/v) CaCl2 . The fermentation without yeast extract supplementation was used as a control.
(Table 1, Fig. 4). When peptone concentration was controlled at above 3%, the ethanol concentration in the fermentation broth was only slightly higher than that at 3% peptone (data not shown). Peptone provides large numbers of amino acids and peptides, which are required for yeast growth. As a result, supplementation of peptone into growth media could result in much higher ethanol concentration than with ammonium sulfate or urea, suggesting that organic nitrogen sources might be more suitable for ethanol production by S. cerevisiae in VHG fermentation.
ter 72 h of fermentation, ethanol yield and fermentation efficiency were even slightly higher than those supplemented with 3% peptone, indicating that yeast extract was much more efficient than peptone for ethanol production by S. cerevisiae in VHG fermentation. This is probably due to the fact that the nutrients in yeast extract included more assimilable nitrogen, vitamins, and other growth factors, which could match more effectively the requirements of S. cerevisiae growth than those in peptone.
3.5. Effects of yeast extract on ethanol production by S. cerevisiae
3.6. Optimization using central composite design
Yeast extract is a water-soluble extract of selected autolyzed yeast cells. It can provide a wide range of amino acids, peptides, vitamins, inorganic salts, and carbon in growth media. As shown in Fig. 5, yeast extract had significant effects on ethanol yield and fermentation efficiency in VHG fermentation. When yeast extract concentration was controlled at 2%, after 72 h of fermentation, ethanol yield and fermentation efficiency reached approximately 20.3 and 84.5%, respectively (Table 1, Fig. 5), indicating that most of glucose was utilized by yeast to produce ethanol. If yeast extract concentration was controlled at 2.5% or above, the ethanol concentration in the fermentation broth was almost the same as that with 2.0% yeast extract (Table 1, Fig. 5). Furthermore, the ethanol fermentation with high yeast extract concentration (≥2%) took less time to reach the highest yield than those with low yeast extract concentration. The addition of a certain amount of yeast extract was highly advantageous to the aerobic growth of S. cerevisiae. Supplementing yeast extract into growth media could result in much higher ethanol concentration than the peptone alone at the same concentration. If yeast extract concentration was controlled at 0.8%, af-
Yeast extract was an ideal nitrogen source in growth media for ethanol production in VHG fermentation; however, the high cost of yeast extract (which is much more expensive than ammonium sulfate or urea) has a negative effect on the economics of industrialscale processes [22,38], so replacing yeast extract supplementation with less expensive nitrogen sources would increase profits. Although other nitrogen sources do not offer the same ethanol yields as yeast extract (Figs. 2–5), a combination of inexpensive urea and ammonium may be used to substitute for part of the yeast extract. The CCD was conducted in the vicinity of the optimum to locate the true optimum combination of urea (X1 ) and ammonium sulfate (X2 ) for ethanol yield (Y1 ) and fermentation efficiency (Y2 ). The levels of the variables for the CCD experiments were selected according to the results of the previous experiments. The design matrix and the corresponding experimental data are given in Table 2. Experimental results of the CCD fit well with the second-order polynomial equations:
Y1 (%, v/v) = 14.25 + 0.15X1 + 0.05X2 − 3.4 × 10−5 X1 X2 − 1.1 × 10−3 X1 2 − 9.0 × 10−4 X2 2
(2)
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Z. Li et al. / Journal of the Taiwan Institute of Chemical Engineers 000 (2016) 1–7
5
a 20.1
Ethanol yield
18.975
17.85
16.725
15.6
50.00
100.00 80.00
40.00 30.00
X2: Ammonium sulfate
60.00
20.00
40.00
X1: Urea
20.00
10.00
b 84
Ethanol efficiency
79.25
74.5
69.75
65
50.00
100.00
40.00
80.00 30.00
X2: Ammonium sulfate
60.00 40.00
20.00
10.00
X1: Urea
20.00
Fig. 6. Response surfaces and contour lines of ethanol yield (A) and fermentation efficiency (B) showing interactions between urea and ammonium sulfate.
Y2 (% ) = 59.47 + 0.61X1 + 0.2X2 − 1.4 × 10 − 4.4 × 10−3 X1 2 − 3.8 × 10−3 X2 2
−4
X1 X2 (3)
The fit of the two models was assessed by the coefficient of determination R2 , which were both 0.956, indicating that only 4.4% of the variation was not explained by the models. The statistical significances of the response surface quadratic model equations were evaluated by an F-test ANOVA, which revealed
that the two regressions were statistically significant (P = 0.0 0 01) at the 99% confidence level. The contour plots described by the models Y1 and Y2 are represented in Fig. 6. The optimal concentrations for two inexpensive nitrogen sources obtained from the maximum point of the models were 69 mM for urea (X1 ) and 26 mM for ammonium sulfate (X2 ), respectively. The model predicted maximum responses of approximately 20.1% (v/v) ethanol concentration (Fig. 6A) and 83.8% fermentation efficiency (Fig. 6B).
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Z. Li et al. / Journal of the Taiwan Institute of Chemical Engineers 000 (2016) 1–7 Table 2 Design and responses of the central composite design (CCD). Tests
1 2 3 4 5 6 7 8 9 10 11 12 13
Coded and real values
Y1 Ethanol yield (%, w/v)
Y2 Fermentation efficiency (%)
X1 (Urea, mM)
X2 (Ammonium sulfate, mM)
Experimental
Predicted
Experimental
Predicted
−1 (20) 1 (100) −1 (20) 1 (100) −1.41 (4) 1.41 (116) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60) 0 (60)
−1 (10) −1 (10) 1 (50) 1 (50) 0 (30) 0 (30) −1.41 (2) 1.41 (58) 0 (30) 0 (30) 0 (30) 0 (30) 0 (30)
16.96 18.34 16.62 17.89 15.61 18.02 19.61 19.35 19.88 19.77 19.93 20.01 19.66
17.21 18.63 17.03 18.33 15.52 17.42 19.38 19.04 19.92 19.92 19.92 19.92 19.92
70.74 76.48 69.30 74.61 65.10 75.16 81.79 80.68 82.89 82.45 83.12 83.45 82.01
71.50 77.95 70.27 76.27 64.40 73.12 80.60 78.56 82.56 82.56 82.56 82.56 82.56
3.7. Validation of the optimized nitrogen source combination Experiments were done in triplicate using the optimized nitrogen source combination (0.6% yeast extract, 69 mM urea, and 26 mM ammonium sulfate) representing the maximum point of ethanol production to verify model results. The average ethanol yield and fermentation efficiency from validation tests were 20.2% (v/v) and 84.2%, respectively. The strong correlation between predicted and experimental values after optimization justified the validity of the response model and the existence of an optimum point. Supplementation of urea and ammonium sulfate could improve ethanol production by approximately 21% compared with using 0.6% yeast extract as the sole nitrogen source. Using the optimum combination of nitrogen sources could result in ethanol yield and fermentation efficiency in VHG fermentation comparable to those supplemented with 2% yeast extract, indicating that urea and ammonium sulfate synergistically enhanced ethanol production by S. cerevisiae in VHG fermentation with a substantial reduction of yeast extract. Furthermore, ethanol yield and fermentation efficiency in this study reach relatively high level. Commonly, VHG fermentation could achieve ethanol yield between 15 and 19 % (v/v) and fermentation efficiency of <80% [39,40]. 4. Conclusions Nitrogen sources were examined for ethanol production by S. cerevisiae in VHG fermentation of corn starch. Of the nitrogen sources examined, yeast extract was ideal for ethanol production, but its relative high prices made the partial substitution with urea and ammonium sulfate attractive to produce ethanol. The information presented in this work is a very good alternative to obtain >80% of fermentation efficiency from VHG ethanol fermentation. Besides yeast extract, the use of ammonium sulfate and urea yields a synergistic effect (observed as an increase more than the expected when used by separated) in ethanol yield and fermentation efficiency. Thus, ethanol could be produced with low-cost nitrogen sources in yeast growth media with high efficiency, especially in VHG ethanol fermentation. Acknowledgment This is contribution number 14-167-J from the Kansas Agricultural Experiment Station. References [1] Solomon BD. Biofuels and sustainability. Ann N Y Acad Sci 2010;1185:119–34.
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Please cite this article as: Z. Li et al., Effects of nitrogen source on ethanol production in very high gravity fermentation of corn starch, Journal of the Taiwan Institute of Chemical Engineers (2016), http://dx.doi.org/10.1016/j.jtice.2016.10.055