- Email: [email protected]
Ethanol production from sweet sorghum juice using very high gravity technology: Effects of carbon and nitrogen supplementations
Bioresource Technology 100 (2009) 4176–4182
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Ethanol production from sweet sorghum juice using very high gravity technology: Effects of carbon and nitrogen supplementations Lakkana Laopaiboon a,b,*, Sunan Nuanpeng c, Penjit Srinophakun d, Preekamol Klanrit a, Pattana Laopaiboon a a
Department of Biotechnology, Faculty of Technology, Khon Kaen University, 123 Mittraparp Road, Khon Kaen 40002, Thailand Fermentation Research Center for Value Added Agricultural Products, Khon Kaen University, Khon Kaen 40002, Thailand c Graduate School, Khon Kaen University, Khon Kaen 40002, Thailand d Department of Chemical Engineering, Faculty of Engineering, Kasertsart University, Bangkok 10900, Thailand b
a r t i c l e
i n f o
Article history: Received 13 November 2008 Received in revised form 12 March 2009 Accepted 13 March 2009 Available online 17 April 2009 Keywords: S. cerevisiae Ethanol production VHG fermentation Sweet sorghum juice Sugarcane molasses
a b s t r a c t Ethanol production from sweet sorghum juice by Saccharomyces cerevisiae NP01 was investigated under very high gravity (VHG) fermentation and various carbon adjuncts and nitrogen sources. When sucrose was used as an adjunct, the sweet sorghum juice containing total sugar of 280 g l1, 3 g yeast extract l1 and 5 g peptone l1 gave the maximum ethanol production efficiency with concentration, productivity and yield of 120.68 ± 0.54 g l1, 2.01 ± 0.01 g l1 h1 and 0.51 ± 0.00 g g1, respectively. When sugarcane molasses was used as an adjunct, the juice under the same conditions gave the maximum ethanol concentration, productivity and yield with the values of 109.34 ± 0.78 g l1, 1.52 ± 0.01 g l1 h1 and 0.45 ± 0.01 g g1, respectively. In addition, ammonium sulphate was not suitable for use as a nitrogen supplement in the sweet sorghum juice for ethanol production since it caused the reduction in ethanol concentration and yield for approximately 14% when compared to those of the unsupplemented juices. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Ethanol production as an alternative to petroleum-based fuels can be produced from biomass, a plentiful renewable resource. To increase the productivity and cost effectiveness of ethanol production, many process improvements including very high gravity (VHG) technology have been studied. VHG fermentation technology is defined as the preparation and fermentation to completion of mashes containing 270 or more grams of dissolved solids per litre (Bayrock and Ingledew, 2001). It has several advantages for industrial applications such as the increase in both the ethanol concentration and the rate of fermentation, which reduce capital costs, energy costs per litre of alcohol and the risk of bacterial contamination (Thomas et al., 1996; Bvochora et al., 2000; Bayrock and Ingledew, 2001; Bai et al., 2004). Apart from sugarcane (in Brazil), corn grain (in USA), tapioca starch and sugarcane molasses (in Thailand), other agricultural raw materials rich in fermentable carbohydrates, including sweet sorghum, have been of particular interest for biological transformation into ethanol for use as fuel or fuel additive (Schaffert, 1995; Göksungur and Zorlu, 2001). Sweet sorghum has been promised as a large scale energy crop because its stalks contain
* Corresponding author. Address: Department of Biotechnology, Faculty of Technology, Khon Kaen University, 123 Mittraparp Road, Khon Kaen 40002, Thailand. Tel./fax: +66 43 362121. E-mail address: [email protected] (L. Laopaiboon). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.03.046
high fermentable sugar and it can be cultivated in nearly all temperatures and tropical climate areas (Sree et al., 1999). It is also one of the most drought resistant agricultural crops because of its capacity to remain dormant during the driest periods (Woods, 2000). It was reported that under appropriate environmental and nutritional conditions, Saccharomyces cerevisiae can produce and tolerate high ethanol concentrations (Thomas et al., 1996; Bafrncova et al., 1999). VHG fermentation process exploits the observation that the growth of S. cerevisiae is promoted and prolonged when very low but adequate levels of oxygen are present and assimilable nitrogen levels are not limiting (Casey and Ingledew, 1986). Several investigators have observed that yeast extract (Casey et al., 1984; Thomas and Ingledew, 1990; Jones et al., 1994; Bafrncova et al., 1999), ammonium (Jones et al., 1994), urea (Jones and Ingledew, 1994a), calcium and magnesium (Dombek and Ingram, 1986) have protective effects either on growth and fermentation or viability, which stimulate the fermentation rate and ethanol production. Our previous study showed that S. cerevisiae NP01 isolated from Long-pang (Chinese yeast cake) for Sato (Thai rice wine) making was a high-ethanol-producing strain under VHG condition (Laopaiboon et al., 2008). As total soluble solids in the sweet sorghum juice cv. KKU 40 has only 18°Bx (grams per 100 ml), ethanol fermentation under VHG supplemented with other carbon sources in order to raise the sugar content in the juice needs to be investigated. Characteristics of raw sweet sorghum juice cv. KKU 40 are shown in Table 1. The aim of this study was
L. Laopaiboon et al. / Bioresource Technology 100 (2009) 4176–4182 Table 1 Characteristics of raw sweet sorghum juice cv. KKU40 and sugarcane molasses from Mitr Phu Viang Sugar Mill, Nongrua, Khon Kaen, Thailand. Constituents
pH Total soluble solid (°Bx) Total sugar (g l-1) Glucosea (g l1) Fructosea (g l1) Sucrosea (g l1) b NHþ 4 –N (ppm) b NO 3 –N (ppm) Total Pc (ppm) Total Kd (ppm) Total Nad (ppm) Total Se (ppm) Total Caf (ppm) Total Mgf (ppm) Total Fef (ppm) Total Mnf (ppm) Total Cuf (ppm) Total Znf (ppm) a b c d e f
Contents Sweet sorghum juice
Molasses
4.9 18 173.02 20.85 16.80 124.05 21.4 4.4 20 1790 170 120 166 194 2 3 0.3 1.4
4.9 85 696.04 95.42 169.79 387.53 – – – – – – 1602 2985 150 56 2.8 7.4
By HPLC. MgO-Devarda alloy distillation method. Vanado-molybdate method. Flame photometry method. Turbidimetry method. Atomic absorption.
to compare the efficiency of ethanol production from sweet sorghum juice supplemented with sucrose or sugarcane molasses under VHG fermentation using S. cerevisiae NP01. The influences of yeast extract and peptone (YEP) or ammonium sulphate [(NH4)2SO4] as nitrogen sources for ethanol production were also studied. 2. Methods 2.1. Microorganism and inoculum preparation S. cerevisiae NP01 isolated from Long-pang (Chinese yeast cake) from Nakorn Pranom province, Thailand, was inoculated into a 250-ml Erlenmeyer flask containing 150 ml of yeast and malt extract (YM) medium. The medium contained (in g l1) yeast extract 3, peptone 5, malt extract 3 and glucose 10. The flask was incubated on a rotating shaker at 100 rpm, 30 °C for 15 h. To increase cell concentration, the yeast (approximately 3%) was transferred into a 500-ml Erlenmeyer flask with 360 ml of the YM medium containing 150 g l1 of glucose to give the initial cell concentration of 1 106 cells ml1. The flasks were further incubated under the conditions previous mentioned. After 15 h, the cells were harvested and used as an inoculum for ethanol production. 2.2. Raw materials Sweet sorghum juice cv. KKU40 modified from cv. Keller was obtained from Department of Agronomy, Faculty of Agriculture, Khon Kaen University, Thailand. After extraction, the juice was kept at 18 °C until use. Sugarcane molasses obtained from Mitr Phu Viang Sugar Mill, Nongrua, Khon Kaen, Thailand was kept at 4 °C until use. 2.3. Ethanol production medium Sweet sorghum juice (pH 4.9) containing total soluble solids of 18°Bx was adjusted with sucrose or molasses. The total soluble solids in the juice were adjusted to 24, 28 and 32°Bx by sucrose
4177
addition and to 28, 32 and 34°Bx by molasses addition, which corresponded to total sugar concentrations of approximately 240, 280 and 320 g l1, respectively. Then the juices were supplemented with 8 g l1 of YEP (3 g yeast extract and 5 g peptone) or 1.3 g l1 of (NH4)2SO4, and used as ethanol production (EP) medium without pH adjustment. The EP medium was transferred into a 500ml air-locked Erlenmeyer flask with a final working volume of 400 ml and autoclaved at 110 °C for 15 min. 2.4. Fermentation conditions The sterile EP medium containing various sugar and nitrogen supplements was inoculated to give the initial yeast cell concentration of 1 108 cells ml1. The fermentation was carried out in batch mode at 30 °C under static condition. The samples were withdrawn at time intervals for analysis. 2.5. Analytical methods The viable yeast cell numbers and total soluble solids of the fermentation broth were determined by direct counting method using haemacytometer and hand-held refractometer, respectively. The fermentation broth was centrifuged at 13,000 rpm for 10 min. The supernatant was then determined for residual total sugar in terms of total carbohydrate by phenol sulfuric acid method (Mecozzi, 2005). Ethanol concentration (P, g l1) was analyzed by gas chromatography (Shimadzu GC-14B, Japan, Solid phase: polyethylene glycol (PEG-20 M), carrier gas: nitrogen, 150 °C isothermal packed column, injection temperature 180 °C, flame ionization detector temperature 250 °C; C-R7 Ae plus Chromatopac Data Processor) and 2-propanol was used as an internal standard (modified from Laopaiboon et al., 2007). The ethanol yield (Y ps ;) was calculated as the actual ethanol produced and expressed as g ethanol per g total sugar utilized (g g1). The volumetric ethanol productivity (Q p ; g l1 h1) was calculated by ethanol concentration produced (P; g l1) divided by fermentation time giving the highest ethanol concentration. Fermentable nitrogen or formol nitrogen in the fermentation broth was analyzed by formol titration method (Zoecklein et al., 1995).
3. Results and discussion 3.1. VHG fermentation with sucrose as an adjunct and influences of various nitrogen sources to ethanol production Standard ethanol production medium (Melzoch et al., 1994) contains 3 g l1 of yeast extract and 5 g l1 of peptone which total fermentable nitrogen equals to 1129 mg l1. Therefore in this study yeast extract and peptone at those concentrations were supplemented in the sweet sorghum juice as nitrogen sources. To compare the effects of nitrogen source on ethanol production, the same amount of fermentable nitrogen in (NH4)2SO4 (1.3 g l1) was supplemented in the juice. A common nitrogen source, urea, was not studied in the research because it could react with ethanol yielding ethyl carbamate (urethane) as a product, resulting in lower ethanol concentration (Zoecklein et al., 1995). pH of the fermentation media was 4.9 which was in the optimum range for yeast growth and ethanol production (Narendranath and Power, 2005). Therefore, pH adjustment of the juice was not performed. However, profiles of pH during ethanol fermentation were monitored (data not shown). The pH of the broth at all conditions was slightly decreased to 4.5 after 12 h and relatively constant afterwards. Sugar consumption and ethanol production during batch fermentation of S. cerevisiae NP01 from the sweet sorghum juice sup-
300
Total sugar (g l -1)
120
A
100
250 80 200 60
150 100
40
50
20
0
0
350
120
B
Total sugar (g l -1)
300
100
250 80 200 60
150 100
40
50
20
0
0
350
Total sugar (g l -1)
120
C
300
100
250 80 200 60
150 100
40
50
20
0
Ethanol concentration (g l-1)
350
Ethanol concentration (g l-1)
L. Laopaiboon et al. / Bioresource Technology 100 (2009) 4176–4182
Ethanol concentration (g l-1)
4178
0 0
20
40
60
80
Time (h) Fig. 1. Sugar consumption and ethanol production during batch ethanol fermentation by S. cerevisiae NP01 from sweet sorghum juice supplemented with sucrose at various initial soluble solids and nitrogen sources: 24°Bx (s, d), 28°Bx (5, .) and 32°Bx (h, j), total sugar (open symbol) and ethanol (close symbol). (A) No extra nitrogen source, (B) supplemented with YEP and (C) supplemented with (NH4)2 SO4.
plemented with sucrose at the initial soluble solids of 24, 28 and 32°Bx and various nitrogen sources are shown in Fig. 1. The initial and final total sugar concentrations at the various conditions are shown in Table 2. The sugars were almost completely consumed
under high gravity (HG) fermentation (the initial soluble solids of 24°Bx). Under VHG conditions at the initial soluble solids of 28 and 32°Bx, stuck fermentation was observed with approximately 48–64 and 120–146 g l1 of total sugar remaining in the fermentation broth, respectively. The amount of sugar remaining was also dependent on supplemented nitrogen sources. Not all sugars in the media were completely utilized by the yeast. This might be due to thermal stress as described by Jones and Ingledew (1994a). They found that the amount of sugar that could be fermented decreased when fermentation temperature was above 25 °C. However, lower temperature might cause lower ethanol productivity. This was supported by Bai et al. (2008) who reviewed that the negative impact of high temperature on the ethanol fermentation performance was much worse under the VHG conditions than the regular fermentation. Table 2 also summarizes the important kinetic parameters (P; Q p and Y ps ) of the ethanol fermentation under various conditions. The results showed that initial total soluble solids or initial sugar concentration had significant effects on the main kinetic parameters. The juice containing initial total soluble solids of 28°Bx, corresponding to total sugar of approximately 280 g l1, and YEP gave the maximum ethanol concentration with the value of 120.68 ± 0.54 g l1. At the initial total soluble solids of 24 and 28°Bx, YEP enhanced the final ethanol concentrations approximately 3% compared to those of the control (no extra nitrogen supplement). Although the fermentation time was extended under the medium containing YEP (Table 2), this result might be explained that the yeast was able to produce ethanol at very high ethanol concentrations (more than 15% by volume), and thus, achieved a goal of VHG fermentation (Bai et al., 2008). The advantage of having higher ethanol concentration was that it could reduce energy consumption in distillation (Bai et al., 2004). Similar results were reported by Jones et al. (1994), who found that addition of yeast extract failed to enhance the ethanol productivity at 30 °C from sugarcane juice fortified with freeze-dried wheat hydrolysate or molasses adjuncts. However, because of lower ethanol productivity due to longer fermentation time in the presence of YEP, further studies will be carried out to improve the productivity. When (NH4)2SO4 at the same amount of fermentable nitrogen detected in YEP was used as a nitrogen supplement, P; Q p and Y ps obtained were significantly lower than those of the control and the juice supplemented with YEP, respectively. Negative effect of ammonium sulphate on final concentration might be due to more byproducts occurred. The amount of total sugar utilized was similar between the control media and the media supplemented with ammonium sulphate at all gravities, but Y ps of the latter was markedly lower (Table 2). There were some studies reported that excessive ammonium addition might cause the in-
Table 2 Kinetic parameters of ethanol production from sweet sorghum juice supplemented with sucrose at various initial total soluble solids and nitrogen sources by S. cerevisiae NP01. Extra nitrogen sources
None
YEP
(NH4)2SO4
a b c
°Bxa
24 28 32 24 28 32 24 28 32
Initial total sugar (g l1)
248.16 ± 1.39 285.10 ± 5.11 336.01 ± 2.38 236.06 ± 3.14 286.06 ± 4.71 336.89 ± 0.39 246.72 ± 1.21 287.17 ± 3.93 333.28 ± 4.71
Final total sugarb (g l1)
12.80 ± 3.05 63.76 ± 2.14 134.63 ± 2.34 5.96 ± 1.23 47.71 ± 2.67 120.30 ± 3.12 8.48 ± 0.59 59.98 ± 1.57 145.50 ± 3.18
Parameters (mean ± SD)c P (g l1)
Q p (g l1 h1)
Y ps (g g1)
t (h)
113.20 ± 0.81 117.28 ± 0.14 107.39 ± 1.07 116.71 ± 0.85 120.68 ± 0.54 108.23 ± 0.16 96.58 ± 0.73 101.48 ± 0.06 83.31 ± 0.06
2.83 ± 0.02 2.44 ± 0.00 2.24 ± 0.02 2.43 ± 0.02 2.01 ± 0.01 1.50 ± 0.00 2.01 ± 0.02 2.11 ± 0.00 1.74 ± 0.00
0.48 ± 0.03 0.53 ± 0.01 0.53 ± 0.00 0.51 ± 0.01 0.51 ± 0.00 0.50 ± 0.01 0.41 ± 0.00 0.45 ± 0.02 0.44 ± 0.02
40 48 48 48 60 72 48 48 48
Total soluble solids in °Bx. Total sugar at fermentation time. P; ethanol concentration; Q p , volumetric ethanol productivity; Y ps , ethanol yield and t, fermentation time. The experiments were performed in duplicate.
4179
L. Laopaiboon et al. / Bioresource Technology 100 (2009) 4176–4182
Table 3 Fermentable nitrogen utilized during ethanol production from sweet sorghum juice supplemented with sucrose at various initial total soluble solids and nitrogen sources by S. cerevisiae NP01. Extra nitrogen sources
°Bxa
Initial total sugar (g l1)
Final total sugarb (g l1)
None
24 28 32 24 28 32 24 28 32
248.16 ± 1.39 285.10 ± 5.11 336.01 ± 2.38 236.06 ± 3.14 286.06 ± 4.71 336.89 ± 0.39 246.72 ± 1.21 287.17 ± 3.93 333.28 ± 4.71
12.80 ± 3.05 63.76 ± 2.14 134.63 ± 2.34 5.96 ± 1.23 47.71 ± 2.67 120.30 ± 3.12 8.48 ± 0.59 59.98 ± 1.57 145.50 ± 3.18
YEP
(NH4)2SO4
Fermentable nitrogen (mg l1)
*
Initial
Utilized
657.28 ± 7.21 626.91 ± 0.00 594.96 ± 1.80 1138.96 ± 10.81 1149.15 ± 18.02 1098.19 ± 18.02 1114.40 ± 7.92 1094.80 ± 3.96 1061.20 ± 11.88
391.12 ± 1.80 294.29 ± 16.22 215.31 ± 19.82 420.40 ± 21.62 402.60 ± 50.45 287.90 ± 10.81 439.60 ± 3.96 380.80 ± 7.92 313.60 ± 7.92
a
See Table 1. See Table 1. The experiments were performed in duplicate and the results were expressed as mean ± SD.
b
9.0
A
8.5 8.0 7.5 7.0 6.5
production, and hence it would not be used for subsequent experiments. Bai et al. (2008) reported that assimilation nitrogen is the most important component in the fermentation medium. In this study, utilization of fermentable nitrogen in ethanol fermentation under various media is shown in Table 3. In the juice without nitrogen supplementation, the amount of fermentable nitrogen utilized decreased when sugar concentration in the juice increased. The results suggested that yeast nitrogen assimilation might be repressed under high sugar concentrations. Under HG conditions (24°Bx), the amount of nitrogen utilized in all media were similar. However, under VHG conditions at the same initial soluble solids, the yeast utilized fermentable nitrogen in the juice containing extra nitrogen sources more than the juice without nitrogen supplementation.
6.0
-1
Total sugar (g l )
B
8.5 8.0 7.5 7.0
120
A
300
100
250 80 200 60
150 100
40
6.0
50
20
5.5
0
0
6.5
5.0 9.0
350
C
8.5
-1
8.0 7.5 7.0 6.5 6.0 5.5
120
B
300
100
250 80 200 60
150 100
40
50
20
0
5.0
0 0
0
20
40
60
80
20
40
60
-1
350
5.0 9.0
Ethanol concentration (g l-1)
5.5
Total sugar (g l )
Log viable cell concentration (cells ml-1)
Log viable cell concentration (cells ml-1)
Log viable cell concentration (cells ml-1)
crease in higher alcohols (Beltran et al., 2005), acetic acid (Bely et al., 2003) or hydrogen sulphide (Wang et al., 2003). However, the byproducts were not monitored in our experiment. The results obtained indicate that (NH4)2SO4 seems not to be suitable for use as a nitrogen supplement in sweet sorghum juice for ethanol
Ethanol concentration (g l )
*
80
Time (h)
Time (h) Fig. 2. Yeast viability of S. cerevisiae NP01 during batch ethanol fermentation from sweet sorghum juice supplemented with sucrose at various initial total soluble solids and nitrogen sources: 24°Bx (d), 28°Bx (.) and 32°Bx (j). (A) No extra nitrogen source, (B) supplemented with YEP and (C) supplemented with (NH4)2 SO4.
Fig. 3. Sugar consumption and ethanol production during batch ethanol fermentation by S. cerevisiae NP01 from sweet sorghum juice supplemented with molasses at various initial total soluble solids: 28°Bx (s, d), 32°Bx (5, .) and 34°Bx (h, j), total sugar (open symbol) and ethanol (close symbol). (A) No extra nutrient, (B) supplemented with YEP.
4180
L. Laopaiboon et al. / Bioresource Technology 100 (2009) 4176–4182
Table 4 Kinetic parameters of ethanol production from sweet sorghum juice supplemented with molasses at various initial total soluble solids and nitrogen sources by S. cerevisiae NP01. Extra nitrogen sources
None
YEP
°Bxa
28 32 34 28 32 34
Initial total sugar (g l1)
242.56 ± 4.29 279.78 ± 2.36 315.89 ± 0.00 247.53 ± 4.08 277.02 ± 2.38 315.18 ± 5.56
Final total sugarb (g l1)
18.26 ± 2.12 63.29 ± 0.19 85.29 ± 1.60 23.94 ± 2.36 34.88 ± 1.98 63.57 ± 3.28
Parameters (mean ± SD)c P (g l1)
Q p (g l1 h1)
Y ps (g g1)
t (h)
100.54 ± 0.70 102.08 ± 2.63 98.29 ± 2.16 104.99 ± 1.51 109.34 ± 0.78 104.95 ± 2.18
2.51 ± 0.02 2.13 ± 0.05 2.05 ± 0.13 1.46 ± 0.02 1.52 ± 0.01 1.46 ± 0.03
0.45 ± 0.01 0.43 ± 0.00 0.43 ± 0.04 0.47 ± 0.04 0.45 ± 0.01 0.44 ± 0.02
40 48 48 72 72 72
The experiments were performed in duplicate. See Table 1. b See Table 1. c See Table 1. a
Sugar consumption and ethanol production during batch fermentation from the sweet sorghum juice supplemented with molasses under the presence and absence of YEP are shown in Fig. 3. The profiles of sugar and ethanol concentrations during fermentation were similar to those when using sucrose as the carbon adjunct (Fig. 1). Table 4 shows the initial and final total sugar concentrations at various conditions and summarizes the important kinetic parameters of the ethanol fermentation. P and Y ps of the broth containing YEP were higher than those of the control broth at all gravities. The amount of utilized sugar in the control medium and the medium supplemented with YEP at 28°Bx were similar, while the utilized sugar in the control media at 32 and 34°Bx were lower than those of the supplemented media at the same gravities (Table 4). However, the cell numbers at specific fermentation time throughout the experiments under various conditions were similar (Fig. 4). These results indicated that the supplemented YEP did not promote cell growth but it stimulated sugar utilization and ethanol
Log viable cell concentration (cells ml-1)
3.2. VHG fermentation using molasses as an adjunct and influences of various nitrogen sources to ethanol production
production under very high gravities. In contrast, the rate of ethanol fermentation (ethanol productivity) in the presence of YEP was significantly lower than that of the unsupplemented medium due to longer fermentation time (Table 4). These findings were consistent with those when using the juice supplemented with sucrose and YEP. The reasons of these results were previously described in Section 3.1. The juice supplemented with molasses and YEP at the initial total soluble solids of 32°Bx, corresponding to total sugar of approximately 280 g l1, gave the maximum ethanol concentration with the value of 109.34 ± 0.78 g l1. Under these conditions, the productivity and yield of ethanol were 1.52 ± 0.01 g l1 h1 and 0.45 ± 0.01 g g1, respectively. Y ps slightly decreased when sugar concentrations were increased (Table 4). Possible reasons were that some consumed sugar might be used for maintenance and/or converted to more byproducts. Ozmichi and Kargi (2007) reported that the ethanol yield decreased when the feed sugar content increased more than 200 g l1 due to high osmotic pressure and maintenance requirements at high sugar concentrations. Similar results were also observed by Jones et al. (1994) and Limtong et al. (2007).
9.0
Log viable cell concentration (cells ml-1)
In this study, fermentable nitrogen remaining in the control medium (without nitrogen supplementation) was 40–60% depending on the initial total soluble solids in the juice. This clearly indicated that nitrogen was not limited in the control medium, but the capability of nitrogen utilization or nitrogen requirement by yeast might depend on other factors such as yeast strain (Jiranek et al., 1995; Taillandier et al., 2007), sugar concentration, temperature, presence of oxygen (Valero et al., 2003) and initial fermentable nitrogen content (Taillandier et al., 2007). Table 3 also indicated that the amount of nitrogen consumption was not always related to ethanol production efficiency. Nitrogen utilized in the control medium was lower than that in the juice supplemented with (NH4)2SO4, but ethanol production from the control medium was higher. The amount of nitrogen consumption seemed to relate to type of nitrogen sources supplied (Table 3). Especially, in case of using ammonium salts to increase the nitrogen content, excessive ammonium addition might cause the increase in the byproducts (Beltran et al., 2005; Bely et al., 2003; Wang et al., 2003) as previously mentioned. Viability of the yeast during ethanol fermentation from sweet sorghum juice under various conditions is shown in Fig. 2. The results indicated that S. cerevisiae NP01 was a suitable microorganism for ethanol fermentation under VHG levels. It could survive and retain its metabolism under very high ethanol concentrations up to 120 g l1 (15% by volume) (Table 2) with high viable cells remaining in the fermentation broth (Fig. 2). Moreover, Nagashima (1990) reported that to achieve cell viability and ethanol production, appropriate amount of fermentable nitrogen must also be available. In our studies, fermentable nitrogen had still been available in the medium until the end of the fermentation.
9.0
A
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0
B
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 0
20
40
60
80
Time (h) Fig. 4. Yeast viability of S. cerevisiae NP01 during batch ethanol fermentation from sweet sorghum juice supplemented with molasses at various initial total soluble solids and nitrogen sources: 28°Bx (d), 32°Bx (.) and 34°Bx (j). (A) No extra nutrient, (B) supplemented with YEP.
L. Laopaiboon et al. / Bioresource Technology 100 (2009) 4176–4182
When the results of using sucrose and molasses were compared in terms of sugar utilization, the amount of the utilized total sugar under both carbon adjuncts were similar when compared at the same initial total sugar concentrations (Tables 2 and 4). These findings imply that molasses might contain some inhibitors (e.g., hydroxymethylfurfural, hexanol and heptanol) for yeast metabolism as previously reported (Glacet et al., 1985; Fattohi 1994). However, when the molasses was used as a carbon adjunct to raise sugar concentration to VHG levels, the inhibitors were diluted to a level which did not cause adverse effect on sugar utilization. Regarding to the important kinetic parameters for ethanol production; P, Q p and Y ps of the ethanol fermentation using molasses as an adjunct were significantly lower than those using sucrose as an adjunct (Tables 2 and 4). These results imply that some other nutrients in the molasses may promote the formation of byproducts of the ethanol fermentation. The advantage of using fermentable sugars (sweet sorghum juice, cane sugar or molasses) in this study especially under VHG fermentation is that they can reduce the overall fermentation time. Other forms of carbon such as starch or dextrins may be used to raise soluble solids to VHG levels as found in Jones and Ingledew (1994b). However, there were additional steps for pretreatment the raw materials before ethanol fermentation process such as double mashing and saccharification by glucoamylase, which was a drawback of using complex carbohydrate as the raw materials. 4. Conclusions This research achieves the goal of VHG fermentation technology that at least 15% (v/v) of ethanol is produced in the fermentation broth (Bai et al., 2008). The results obtained from this research have demonstrated that sucrose is a good adjunct to raise total soluble solids or sugar concentration of sweet sorghum juice to VHG levels for ethanol fermentations, while molasses causes the lower ethanol production efficiency. However, other methods for raising sugar content in sweet sorghum juice such as concentrating the juice by evaporation should be considered to reduce cost of the adjunct. Yeast extract and peptone promote ethanol fermentation under VHG levels. Other yeast products such as dried spent yeast may replace yeast extract and peptone as they are far less expensive. The effects of dried spent yeast on ethanol production will be investigated. According to the very high content of fermentable sugars in the sweet sorghum juice supplemented with cane sugar or molasses (Table 1), the optimum conditions in terms of processing parameters and/or fermentation processes to achieve complete sugar utilization under VHG levels need to be further studied. Acknowledgements This research was financially supported by the Thailand Research Fund (TRF) and Commission on Higher Education, Thailand. We would like to thank Assistant Prof. Dr. Paiboon Danviruthai, Faculty of Technology, Khon Kaen University (KKU) for providing the NP01 strain and Associate Prof. Dr. Prasit Jaisil, Faculty of Agriculture, KKU for providing sweet sorghum juice and Ms. Ratchanee Kortsree, Mitr Phu Viang sugar Mill, Khon Kaen, Thailand for providing molasses and Associate Prof. Dr. Aroonwadee Chanawong, Faculty of Associated Medical Sciences, KKU for her internal review of this paper and helpful suggestions. We also gratefully acknowledge the Royal Bangkok Sports Club (RBSC), Bangkok, Thailand and the Fermentation Research Center for Value Added Agricultural Products (FerVAAP) for financial support for Mr. Sunan Nuanpeng and Research Center for Environmental and Hazardous Substance Management, KKU and National Center of
4181
Excellence for Environmental and Hazardous Waste Management, KKU for some instrumentation supports. References Bafrncova, P., Smogrovicova, D., Slavikova, I., Patkova, J., Domeny, Z., 1999. Improvement of very high gravity ethanol fermentation by media supplementation using Saccharomyces cerevisiae. Biotechnol. Lett. 21, 337– 341. Bai, F.W., Chen, L.J., Zhang, Z., Anderson, W.A., Moo-Young, M., 2004. Continuous ethanol production and evaluation of yeast cell lysis and viability loss under very high gravity medium conditions. J. Biotechnol. 110, 287–293. Bai, F.W., Anderson, W.A., Moo-Young, M., 2008. Ethanol fermentation technologies from sugar and starch feedstocks. Biotechnol. Adv. 26, 89–105. Bayrock, D.P., Ingledew, W.M., 2001. Application of multistage continuous fermentation for production of fuel alcohol by very-high-gravity fermentation technology. J. Ind. Microbiol. Biotechnol. 27, 87–93. Beltran, G., Esteve-Zarzoso, B., Rozes, N., Mas, A., Guillamon, J.M., 2005. Influence of the timing of nitrogen additions during synthetic grape must fermentations on fermentation kinetics and nitrogen consumption. J. Agric. Food Chem. 53, 996– 1002. Bely, M., Rinaldi, A., Dubourdieu, D., 2003. Influence of assimilable nitrogen on volatile acidity production by Saccharomyces cerevisiae during high sugar fermentation. J. Biosci. Bioeng. 96, 507–512. Bvochora, J.M., Read, J.S., Zvauya, R., 2000. Application of very high gravity technology to the cofermentation of sweet stem sorghum juice and sorghum grain. Ind. Crop. Prod. 11, 11–17. Casey, G.P., Ingledew, W.M., 1986. Ethanol tolerance in yeasts. CRC Crit. Rev. Biotechnol. 13, 219–280. Casey, G.P., Magnus, C.A., Ingledew, W.M., 1984. High gravity brewing: effects of nutrition on yeast composition, fermentative ability, and alcohol production. Appl. Environ. Microbiol. 48, 639–646. Dombek, K.M., Ingram, L.O., 1986. Magnesium limitation and its role in apparent toxicity of ethanol during yeast fermentation. Appl. Environ. Microbiol. 52, 975– 981. Fattohi, N., 1994. Determination of molasses and yeast components by GC and HPLC. Zuckerind 119, 554–560. Glacet, A., Letoumeau, F., Leveque, P., Villa, P., 1985. Kinetics study of nitrate inhibition during alcoholic fermentation of beet molasses. Biotechnol. Lett. 7, 47–52. Göksungur, Y., Zorlu, N., 2001. Production of ethanol from beet molasses by Caalginate immobilized yeast cells in a packed-bed bioreactor. Turk. J. Biol. 25, 265–275. Jiranek, V., Langridge, P., Henscke, P.A., 1995. Amino acids and ammonium utilization by Saccharomyces wine yeasts from a chemically defined medium. Am. J. Enol. Vitic. 46, 75–83. Jones, A.M., Ingledew, W.M., 1994a. Fuel ethanol production: appraisal of nitrogenous yeast foods for very high gravity wheat mash fermentation. Process Biochem. 29, 483–488. Jones, A.M., Ingledew, W.M., 1994b. Fermentation of very high gravity wheat mash prepared using fresh yeast autolysate. Bioresour. Technol. 50, 97–101. Jones, A.M., Thomas, K.C., Ingledew, W.M., 1994. Ethanolic fermentation of blackstrap molasses and sugarcane juice using very high gravity technology. J. Agric. Food Chem. 42, 1242–1246. Laopaiboon, L., Thanonkeo, P., Jaisil, P., Laopaiboon, P., 2007. Ethanol production from sweet sorghum juice in batch and fed-batch fermentations by Saccharomyces cerevisiae. World J. Microbiol. Biotechnol. 23, 1497–1501. Laopaiboon, L., Nuanpeng, S., Srinophakun, P., Klanrit, P., Laopaiboon, P., 2008. Selection of Saccharomyces cerevisiae and investigation of its performance for very high gravity ethanol fermentation. Biotechnology 7, 493–498. Limtong, S., Sringiew, C., Yongmanichai, W., 2007. Production of fuel ethanol at high temperature from sugar cane juice by a newly isolated Kluyveromyces marxianus. Bioresour. Technol. 98, 3367–3374. Mecozzi, M., 2005. Estimation of total carbohydrate amount in environmental samples by the phenol–sulphuric acid method assisted by multivariate calibration. Chemom. Intell. Lab. Syst. 79, 84–90. Melzoch, K., Rychtera, M., Hábová, V., 1994. Effect of immobilization upon the properties and behaviour of Saccharomyces cerevisiae cells. J. Biotechnol. 32, 59– 65. Nagashima, M., 1990. Progress in ethanol production with yeasts. In: Hubert, V., Rene, De M. (Eds.), Yeast: Biotechnology and Biocatalysis. Marcel Dekker, New York. Narendranath, N.V., Power, R., 2005. Relationship between pH and medium dissolved solids in terms of growth and metabolism of Lactobacilli and Saccharomyces cerevisiae during ethanol production. Appl. Environ. Microbiol. 71, 2239–2243. Ozmichi, S., Kargi, F., 2007. Ethanol fermentation of cheese whey powder solution by repeated fed-batch operation. Enzyme Microb. Technol. 41, 169– 174. Schaffert, R.E., 1995. Sweet sorghum substrate for industrial alcohol. In: Dendy, D.A.V. (Ed.), Sorghum and Millets: Chemistry and Technology. American Association of Cereal Chemists, St. Paul, MN, USA, pp. 365–374. Sree, N.K., Sridhar, M., Rao, L.V., Pandey, A., 1999. Ethanol production in solid substrate fermentation using thermotolerant yeast. Process Biochem. 34, 115– 119.
4182
L. Laopaiboon et al. / Bioresource Technology 100 (2009) 4176–4182
Taillandier, P., Portugal, F.R., Fuster, A., Strehaiano, P., 2007. Effect of ammonium concentration on alcoholic fermentation kinetics by wine yeasts for high sugar content. Food Microbiol. 24, 95–100. Thomas, K.C., Ingledew, W.M., 1990. Fuel alcohol production: effects of free amino nitrogen on fermentation of very-high-gravity wheat mash. Appl. Environ. Microbiol. 56, 2046–2050. Thomas, K.C., Hynes, S.H., Ingledew, W.M., 1996. Practical and theoretical considerations in the production of high concentration of alcohol by fermentation. Process Biochem. 31, 321–331. Valero, J., Millan, C., Ortega, J.M., Mauricio, J.C., 2003. Concentration of amino acids in wine after the end of fermentation by Saccharomyces cerevisiae strains. J. Sci. Food Agric. 88, 830–835.
Wang, X.D., Bohlscheid, J.C., Edwards, C.G., 2003. Fermentative activity and production of volatile compounds by Saccharomyces grown in synthetic grape juice media deficient in assimilable nitrogen and/or pantothenic acid. J. Appl. Microbiol. 94, 349–359. Woods, J., 2000. Integrating Sweet sorghum and sugarcane for bioenergy: modelling the potential for electricity and ethanol production in SE Zimbabwe, Ph.D. Thesis, King’s College, London. Zoecklein, B.U., Fugelsang, K.C., Gump, B.H., Nury, F.S., 1995. Wine Analysis and Production. Chapman & Hall, New York.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Ethanol production from sweet sorghum juice using very high gravity technology: Effects of carbon and nitrogen supplementations Lakkana Laopaiboon a,b,*, Sunan Nuanpeng c, Penjit Srinophakun d, Preekamol Klanrit a, Pattana Laopaiboon a a
Department of Biotechnology, Faculty of Technology, Khon Kaen University, 123 Mittraparp Road, Khon Kaen 40002, Thailand Fermentation Research Center for Value Added Agricultural Products, Khon Kaen University, Khon Kaen 40002, Thailand c Graduate School, Khon Kaen University, Khon Kaen 40002, Thailand d Department of Chemical Engineering, Faculty of Engineering, Kasertsart University, Bangkok 10900, Thailand b
a r t i c l e
i n f o
Article history: Received 13 November 2008 Received in revised form 12 March 2009 Accepted 13 March 2009 Available online 17 April 2009 Keywords: S. cerevisiae Ethanol production VHG fermentation Sweet sorghum juice Sugarcane molasses
a b s t r a c t Ethanol production from sweet sorghum juice by Saccharomyces cerevisiae NP01 was investigated under very high gravity (VHG) fermentation and various carbon adjuncts and nitrogen sources. When sucrose was used as an adjunct, the sweet sorghum juice containing total sugar of 280 g l1, 3 g yeast extract l1 and 5 g peptone l1 gave the maximum ethanol production efficiency with concentration, productivity and yield of 120.68 ± 0.54 g l1, 2.01 ± 0.01 g l1 h1 and 0.51 ± 0.00 g g1, respectively. When sugarcane molasses was used as an adjunct, the juice under the same conditions gave the maximum ethanol concentration, productivity and yield with the values of 109.34 ± 0.78 g l1, 1.52 ± 0.01 g l1 h1 and 0.45 ± 0.01 g g1, respectively. In addition, ammonium sulphate was not suitable for use as a nitrogen supplement in the sweet sorghum juice for ethanol production since it caused the reduction in ethanol concentration and yield for approximately 14% when compared to those of the unsupplemented juices. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Ethanol production as an alternative to petroleum-based fuels can be produced from biomass, a plentiful renewable resource. To increase the productivity and cost effectiveness of ethanol production, many process improvements including very high gravity (VHG) technology have been studied. VHG fermentation technology is defined as the preparation and fermentation to completion of mashes containing 270 or more grams of dissolved solids per litre (Bayrock and Ingledew, 2001). It has several advantages for industrial applications such as the increase in both the ethanol concentration and the rate of fermentation, which reduce capital costs, energy costs per litre of alcohol and the risk of bacterial contamination (Thomas et al., 1996; Bvochora et al., 2000; Bayrock and Ingledew, 2001; Bai et al., 2004). Apart from sugarcane (in Brazil), corn grain (in USA), tapioca starch and sugarcane molasses (in Thailand), other agricultural raw materials rich in fermentable carbohydrates, including sweet sorghum, have been of particular interest for biological transformation into ethanol for use as fuel or fuel additive (Schaffert, 1995; Göksungur and Zorlu, 2001). Sweet sorghum has been promised as a large scale energy crop because its stalks contain
* Corresponding author. Address: Department of Biotechnology, Faculty of Technology, Khon Kaen University, 123 Mittraparp Road, Khon Kaen 40002, Thailand. Tel./fax: +66 43 362121. E-mail address: [email protected] (L. Laopaiboon). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.03.046
high fermentable sugar and it can be cultivated in nearly all temperatures and tropical climate areas (Sree et al., 1999). It is also one of the most drought resistant agricultural crops because of its capacity to remain dormant during the driest periods (Woods, 2000). It was reported that under appropriate environmental and nutritional conditions, Saccharomyces cerevisiae can produce and tolerate high ethanol concentrations (Thomas et al., 1996; Bafrncova et al., 1999). VHG fermentation process exploits the observation that the growth of S. cerevisiae is promoted and prolonged when very low but adequate levels of oxygen are present and assimilable nitrogen levels are not limiting (Casey and Ingledew, 1986). Several investigators have observed that yeast extract (Casey et al., 1984; Thomas and Ingledew, 1990; Jones et al., 1994; Bafrncova et al., 1999), ammonium (Jones et al., 1994), urea (Jones and Ingledew, 1994a), calcium and magnesium (Dombek and Ingram, 1986) have protective effects either on growth and fermentation or viability, which stimulate the fermentation rate and ethanol production. Our previous study showed that S. cerevisiae NP01 isolated from Long-pang (Chinese yeast cake) for Sato (Thai rice wine) making was a high-ethanol-producing strain under VHG condition (Laopaiboon et al., 2008). As total soluble solids in the sweet sorghum juice cv. KKU 40 has only 18°Bx (grams per 100 ml), ethanol fermentation under VHG supplemented with other carbon sources in order to raise the sugar content in the juice needs to be investigated. Characteristics of raw sweet sorghum juice cv. KKU 40 are shown in Table 1. The aim of this study was
L. Laopaiboon et al. / Bioresource Technology 100 (2009) 4176–4182 Table 1 Characteristics of raw sweet sorghum juice cv. KKU40 and sugarcane molasses from Mitr Phu Viang Sugar Mill, Nongrua, Khon Kaen, Thailand. Constituents
pH Total soluble solid (°Bx) Total sugar (g l-1) Glucosea (g l1) Fructosea (g l1) Sucrosea (g l1) b NHþ 4 –N (ppm) b NO 3 –N (ppm) Total Pc (ppm) Total Kd (ppm) Total Nad (ppm) Total Se (ppm) Total Caf (ppm) Total Mgf (ppm) Total Fef (ppm) Total Mnf (ppm) Total Cuf (ppm) Total Znf (ppm) a b c d e f
Contents Sweet sorghum juice
Molasses
4.9 18 173.02 20.85 16.80 124.05 21.4 4.4 20 1790 170 120 166 194 2 3 0.3 1.4
4.9 85 696.04 95.42 169.79 387.53 – – – – – – 1602 2985 150 56 2.8 7.4
By HPLC. MgO-Devarda alloy distillation method. Vanado-molybdate method. Flame photometry method. Turbidimetry method. Atomic absorption.
to compare the efficiency of ethanol production from sweet sorghum juice supplemented with sucrose or sugarcane molasses under VHG fermentation using S. cerevisiae NP01. The influences of yeast extract and peptone (YEP) or ammonium sulphate [(NH4)2SO4] as nitrogen sources for ethanol production were also studied. 2. Methods 2.1. Microorganism and inoculum preparation S. cerevisiae NP01 isolated from Long-pang (Chinese yeast cake) from Nakorn Pranom province, Thailand, was inoculated into a 250-ml Erlenmeyer flask containing 150 ml of yeast and malt extract (YM) medium. The medium contained (in g l1) yeast extract 3, peptone 5, malt extract 3 and glucose 10. The flask was incubated on a rotating shaker at 100 rpm, 30 °C for 15 h. To increase cell concentration, the yeast (approximately 3%) was transferred into a 500-ml Erlenmeyer flask with 360 ml of the YM medium containing 150 g l1 of glucose to give the initial cell concentration of 1 106 cells ml1. The flasks were further incubated under the conditions previous mentioned. After 15 h, the cells were harvested and used as an inoculum for ethanol production. 2.2. Raw materials Sweet sorghum juice cv. KKU40 modified from cv. Keller was obtained from Department of Agronomy, Faculty of Agriculture, Khon Kaen University, Thailand. After extraction, the juice was kept at 18 °C until use. Sugarcane molasses obtained from Mitr Phu Viang Sugar Mill, Nongrua, Khon Kaen, Thailand was kept at 4 °C until use. 2.3. Ethanol production medium Sweet sorghum juice (pH 4.9) containing total soluble solids of 18°Bx was adjusted with sucrose or molasses. The total soluble solids in the juice were adjusted to 24, 28 and 32°Bx by sucrose
4177
addition and to 28, 32 and 34°Bx by molasses addition, which corresponded to total sugar concentrations of approximately 240, 280 and 320 g l1, respectively. Then the juices were supplemented with 8 g l1 of YEP (3 g yeast extract and 5 g peptone) or 1.3 g l1 of (NH4)2SO4, and used as ethanol production (EP) medium without pH adjustment. The EP medium was transferred into a 500ml air-locked Erlenmeyer flask with a final working volume of 400 ml and autoclaved at 110 °C for 15 min. 2.4. Fermentation conditions The sterile EP medium containing various sugar and nitrogen supplements was inoculated to give the initial yeast cell concentration of 1 108 cells ml1. The fermentation was carried out in batch mode at 30 °C under static condition. The samples were withdrawn at time intervals for analysis. 2.5. Analytical methods The viable yeast cell numbers and total soluble solids of the fermentation broth were determined by direct counting method using haemacytometer and hand-held refractometer, respectively. The fermentation broth was centrifuged at 13,000 rpm for 10 min. The supernatant was then determined for residual total sugar in terms of total carbohydrate by phenol sulfuric acid method (Mecozzi, 2005). Ethanol concentration (P, g l1) was analyzed by gas chromatography (Shimadzu GC-14B, Japan, Solid phase: polyethylene glycol (PEG-20 M), carrier gas: nitrogen, 150 °C isothermal packed column, injection temperature 180 °C, flame ionization detector temperature 250 °C; C-R7 Ae plus Chromatopac Data Processor) and 2-propanol was used as an internal standard (modified from Laopaiboon et al., 2007). The ethanol yield (Y ps ;) was calculated as the actual ethanol produced and expressed as g ethanol per g total sugar utilized (g g1). The volumetric ethanol productivity (Q p ; g l1 h1) was calculated by ethanol concentration produced (P; g l1) divided by fermentation time giving the highest ethanol concentration. Fermentable nitrogen or formol nitrogen in the fermentation broth was analyzed by formol titration method (Zoecklein et al., 1995).
3. Results and discussion 3.1. VHG fermentation with sucrose as an adjunct and influences of various nitrogen sources to ethanol production Standard ethanol production medium (Melzoch et al., 1994) contains 3 g l1 of yeast extract and 5 g l1 of peptone which total fermentable nitrogen equals to 1129 mg l1. Therefore in this study yeast extract and peptone at those concentrations were supplemented in the sweet sorghum juice as nitrogen sources. To compare the effects of nitrogen source on ethanol production, the same amount of fermentable nitrogen in (NH4)2SO4 (1.3 g l1) was supplemented in the juice. A common nitrogen source, urea, was not studied in the research because it could react with ethanol yielding ethyl carbamate (urethane) as a product, resulting in lower ethanol concentration (Zoecklein et al., 1995). pH of the fermentation media was 4.9 which was in the optimum range for yeast growth and ethanol production (Narendranath and Power, 2005). Therefore, pH adjustment of the juice was not performed. However, profiles of pH during ethanol fermentation were monitored (data not shown). The pH of the broth at all conditions was slightly decreased to 4.5 after 12 h and relatively constant afterwards. Sugar consumption and ethanol production during batch fermentation of S. cerevisiae NP01 from the sweet sorghum juice sup-
300
Total sugar (g l -1)
120
A
100
250 80 200 60
150 100
40
50
20
0
0
350
120
B
Total sugar (g l -1)
300
100
250 80 200 60
150 100
40
50
20
0
0
350
Total sugar (g l -1)
120
C
300
100
250 80 200 60
150 100
40
50
20
0
Ethanol concentration (g l-1)
350
Ethanol concentration (g l-1)
L. Laopaiboon et al. / Bioresource Technology 100 (2009) 4176–4182
Ethanol concentration (g l-1)
4178
0 0
20
40
60
80
Time (h) Fig. 1. Sugar consumption and ethanol production during batch ethanol fermentation by S. cerevisiae NP01 from sweet sorghum juice supplemented with sucrose at various initial soluble solids and nitrogen sources: 24°Bx (s, d), 28°Bx (5, .) and 32°Bx (h, j), total sugar (open symbol) and ethanol (close symbol). (A) No extra nitrogen source, (B) supplemented with YEP and (C) supplemented with (NH4)2 SO4.
plemented with sucrose at the initial soluble solids of 24, 28 and 32°Bx and various nitrogen sources are shown in Fig. 1. The initial and final total sugar concentrations at the various conditions are shown in Table 2. The sugars were almost completely consumed
under high gravity (HG) fermentation (the initial soluble solids of 24°Bx). Under VHG conditions at the initial soluble solids of 28 and 32°Bx, stuck fermentation was observed with approximately 48–64 and 120–146 g l1 of total sugar remaining in the fermentation broth, respectively. The amount of sugar remaining was also dependent on supplemented nitrogen sources. Not all sugars in the media were completely utilized by the yeast. This might be due to thermal stress as described by Jones and Ingledew (1994a). They found that the amount of sugar that could be fermented decreased when fermentation temperature was above 25 °C. However, lower temperature might cause lower ethanol productivity. This was supported by Bai et al. (2008) who reviewed that the negative impact of high temperature on the ethanol fermentation performance was much worse under the VHG conditions than the regular fermentation. Table 2 also summarizes the important kinetic parameters (P; Q p and Y ps ) of the ethanol fermentation under various conditions. The results showed that initial total soluble solids or initial sugar concentration had significant effects on the main kinetic parameters. The juice containing initial total soluble solids of 28°Bx, corresponding to total sugar of approximately 280 g l1, and YEP gave the maximum ethanol concentration with the value of 120.68 ± 0.54 g l1. At the initial total soluble solids of 24 and 28°Bx, YEP enhanced the final ethanol concentrations approximately 3% compared to those of the control (no extra nitrogen supplement). Although the fermentation time was extended under the medium containing YEP (Table 2), this result might be explained that the yeast was able to produce ethanol at very high ethanol concentrations (more than 15% by volume), and thus, achieved a goal of VHG fermentation (Bai et al., 2008). The advantage of having higher ethanol concentration was that it could reduce energy consumption in distillation (Bai et al., 2004). Similar results were reported by Jones et al. (1994), who found that addition of yeast extract failed to enhance the ethanol productivity at 30 °C from sugarcane juice fortified with freeze-dried wheat hydrolysate or molasses adjuncts. However, because of lower ethanol productivity due to longer fermentation time in the presence of YEP, further studies will be carried out to improve the productivity. When (NH4)2SO4 at the same amount of fermentable nitrogen detected in YEP was used as a nitrogen supplement, P; Q p and Y ps obtained were significantly lower than those of the control and the juice supplemented with YEP, respectively. Negative effect of ammonium sulphate on final concentration might be due to more byproducts occurred. The amount of total sugar utilized was similar between the control media and the media supplemented with ammonium sulphate at all gravities, but Y ps of the latter was markedly lower (Table 2). There were some studies reported that excessive ammonium addition might cause the in-
Table 2 Kinetic parameters of ethanol production from sweet sorghum juice supplemented with sucrose at various initial total soluble solids and nitrogen sources by S. cerevisiae NP01. Extra nitrogen sources
None
YEP
(NH4)2SO4
a b c
°Bxa
24 28 32 24 28 32 24 28 32
Initial total sugar (g l1)
248.16 ± 1.39 285.10 ± 5.11 336.01 ± 2.38 236.06 ± 3.14 286.06 ± 4.71 336.89 ± 0.39 246.72 ± 1.21 287.17 ± 3.93 333.28 ± 4.71
Final total sugarb (g l1)
12.80 ± 3.05 63.76 ± 2.14 134.63 ± 2.34 5.96 ± 1.23 47.71 ± 2.67 120.30 ± 3.12 8.48 ± 0.59 59.98 ± 1.57 145.50 ± 3.18
Parameters (mean ± SD)c P (g l1)
Q p (g l1 h1)
Y ps (g g1)
t (h)
113.20 ± 0.81 117.28 ± 0.14 107.39 ± 1.07 116.71 ± 0.85 120.68 ± 0.54 108.23 ± 0.16 96.58 ± 0.73 101.48 ± 0.06 83.31 ± 0.06
2.83 ± 0.02 2.44 ± 0.00 2.24 ± 0.02 2.43 ± 0.02 2.01 ± 0.01 1.50 ± 0.00 2.01 ± 0.02 2.11 ± 0.00 1.74 ± 0.00
0.48 ± 0.03 0.53 ± 0.01 0.53 ± 0.00 0.51 ± 0.01 0.51 ± 0.00 0.50 ± 0.01 0.41 ± 0.00 0.45 ± 0.02 0.44 ± 0.02
40 48 48 48 60 72 48 48 48
Total soluble solids in °Bx. Total sugar at fermentation time. P; ethanol concentration; Q p , volumetric ethanol productivity; Y ps , ethanol yield and t, fermentation time. The experiments were performed in duplicate.
4179
L. Laopaiboon et al. / Bioresource Technology 100 (2009) 4176–4182
Table 3 Fermentable nitrogen utilized during ethanol production from sweet sorghum juice supplemented with sucrose at various initial total soluble solids and nitrogen sources by S. cerevisiae NP01. Extra nitrogen sources
°Bxa
Initial total sugar (g l1)
Final total sugarb (g l1)
None
24 28 32 24 28 32 24 28 32
248.16 ± 1.39 285.10 ± 5.11 336.01 ± 2.38 236.06 ± 3.14 286.06 ± 4.71 336.89 ± 0.39 246.72 ± 1.21 287.17 ± 3.93 333.28 ± 4.71
12.80 ± 3.05 63.76 ± 2.14 134.63 ± 2.34 5.96 ± 1.23 47.71 ± 2.67 120.30 ± 3.12 8.48 ± 0.59 59.98 ± 1.57 145.50 ± 3.18
YEP
(NH4)2SO4
Fermentable nitrogen (mg l1)
*
Initial
Utilized
657.28 ± 7.21 626.91 ± 0.00 594.96 ± 1.80 1138.96 ± 10.81 1149.15 ± 18.02 1098.19 ± 18.02 1114.40 ± 7.92 1094.80 ± 3.96 1061.20 ± 11.88
391.12 ± 1.80 294.29 ± 16.22 215.31 ± 19.82 420.40 ± 21.62 402.60 ± 50.45 287.90 ± 10.81 439.60 ± 3.96 380.80 ± 7.92 313.60 ± 7.92
a
See Table 1. See Table 1. The experiments were performed in duplicate and the results were expressed as mean ± SD.
b
9.0
A
8.5 8.0 7.5 7.0 6.5
production, and hence it would not be used for subsequent experiments. Bai et al. (2008) reported that assimilation nitrogen is the most important component in the fermentation medium. In this study, utilization of fermentable nitrogen in ethanol fermentation under various media is shown in Table 3. In the juice without nitrogen supplementation, the amount of fermentable nitrogen utilized decreased when sugar concentration in the juice increased. The results suggested that yeast nitrogen assimilation might be repressed under high sugar concentrations. Under HG conditions (24°Bx), the amount of nitrogen utilized in all media were similar. However, under VHG conditions at the same initial soluble solids, the yeast utilized fermentable nitrogen in the juice containing extra nitrogen sources more than the juice without nitrogen supplementation.
6.0
-1
Total sugar (g l )
B
8.5 8.0 7.5 7.0
120
A
300
100
250 80 200 60
150 100
40
6.0
50
20
5.5
0
0
6.5
5.0 9.0
350
C
8.5
-1
8.0 7.5 7.0 6.5 6.0 5.5
120
B
300
100
250 80 200 60
150 100
40
50
20
0
5.0
0 0
0
20
40
60
80
20
40
60
-1
350
5.0 9.0
Ethanol concentration (g l-1)
5.5
Total sugar (g l )
Log viable cell concentration (cells ml-1)
Log viable cell concentration (cells ml-1)
Log viable cell concentration (cells ml-1)
crease in higher alcohols (Beltran et al., 2005), acetic acid (Bely et al., 2003) or hydrogen sulphide (Wang et al., 2003). However, the byproducts were not monitored in our experiment. The results obtained indicate that (NH4)2SO4 seems not to be suitable for use as a nitrogen supplement in sweet sorghum juice for ethanol
Ethanol concentration (g l )
*
80
Time (h)
Time (h) Fig. 2. Yeast viability of S. cerevisiae NP01 during batch ethanol fermentation from sweet sorghum juice supplemented with sucrose at various initial total soluble solids and nitrogen sources: 24°Bx (d), 28°Bx (.) and 32°Bx (j). (A) No extra nitrogen source, (B) supplemented with YEP and (C) supplemented with (NH4)2 SO4.
Fig. 3. Sugar consumption and ethanol production during batch ethanol fermentation by S. cerevisiae NP01 from sweet sorghum juice supplemented with molasses at various initial total soluble solids: 28°Bx (s, d), 32°Bx (5, .) and 34°Bx (h, j), total sugar (open symbol) and ethanol (close symbol). (A) No extra nutrient, (B) supplemented with YEP.
4180
L. Laopaiboon et al. / Bioresource Technology 100 (2009) 4176–4182
Table 4 Kinetic parameters of ethanol production from sweet sorghum juice supplemented with molasses at various initial total soluble solids and nitrogen sources by S. cerevisiae NP01. Extra nitrogen sources
None
YEP
°Bxa
28 32 34 28 32 34
Initial total sugar (g l1)
242.56 ± 4.29 279.78 ± 2.36 315.89 ± 0.00 247.53 ± 4.08 277.02 ± 2.38 315.18 ± 5.56
Final total sugarb (g l1)
18.26 ± 2.12 63.29 ± 0.19 85.29 ± 1.60 23.94 ± 2.36 34.88 ± 1.98 63.57 ± 3.28
Parameters (mean ± SD)c P (g l1)
Q p (g l1 h1)
Y ps (g g1)
t (h)
100.54 ± 0.70 102.08 ± 2.63 98.29 ± 2.16 104.99 ± 1.51 109.34 ± 0.78 104.95 ± 2.18
2.51 ± 0.02 2.13 ± 0.05 2.05 ± 0.13 1.46 ± 0.02 1.52 ± 0.01 1.46 ± 0.03
0.45 ± 0.01 0.43 ± 0.00 0.43 ± 0.04 0.47 ± 0.04 0.45 ± 0.01 0.44 ± 0.02
40 48 48 72 72 72
The experiments were performed in duplicate. See Table 1. b See Table 1. c See Table 1. a
Sugar consumption and ethanol production during batch fermentation from the sweet sorghum juice supplemented with molasses under the presence and absence of YEP are shown in Fig. 3. The profiles of sugar and ethanol concentrations during fermentation were similar to those when using sucrose as the carbon adjunct (Fig. 1). Table 4 shows the initial and final total sugar concentrations at various conditions and summarizes the important kinetic parameters of the ethanol fermentation. P and Y ps of the broth containing YEP were higher than those of the control broth at all gravities. The amount of utilized sugar in the control medium and the medium supplemented with YEP at 28°Bx were similar, while the utilized sugar in the control media at 32 and 34°Bx were lower than those of the supplemented media at the same gravities (Table 4). However, the cell numbers at specific fermentation time throughout the experiments under various conditions were similar (Fig. 4). These results indicated that the supplemented YEP did not promote cell growth but it stimulated sugar utilization and ethanol
Log viable cell concentration (cells ml-1)
3.2. VHG fermentation using molasses as an adjunct and influences of various nitrogen sources to ethanol production
production under very high gravities. In contrast, the rate of ethanol fermentation (ethanol productivity) in the presence of YEP was significantly lower than that of the unsupplemented medium due to longer fermentation time (Table 4). These findings were consistent with those when using the juice supplemented with sucrose and YEP. The reasons of these results were previously described in Section 3.1. The juice supplemented with molasses and YEP at the initial total soluble solids of 32°Bx, corresponding to total sugar of approximately 280 g l1, gave the maximum ethanol concentration with the value of 109.34 ± 0.78 g l1. Under these conditions, the productivity and yield of ethanol were 1.52 ± 0.01 g l1 h1 and 0.45 ± 0.01 g g1, respectively. Y ps slightly decreased when sugar concentrations were increased (Table 4). Possible reasons were that some consumed sugar might be used for maintenance and/or converted to more byproducts. Ozmichi and Kargi (2007) reported that the ethanol yield decreased when the feed sugar content increased more than 200 g l1 due to high osmotic pressure and maintenance requirements at high sugar concentrations. Similar results were also observed by Jones et al. (1994) and Limtong et al. (2007).
9.0
Log viable cell concentration (cells ml-1)
In this study, fermentable nitrogen remaining in the control medium (without nitrogen supplementation) was 40–60% depending on the initial total soluble solids in the juice. This clearly indicated that nitrogen was not limited in the control medium, but the capability of nitrogen utilization or nitrogen requirement by yeast might depend on other factors such as yeast strain (Jiranek et al., 1995; Taillandier et al., 2007), sugar concentration, temperature, presence of oxygen (Valero et al., 2003) and initial fermentable nitrogen content (Taillandier et al., 2007). Table 3 also indicated that the amount of nitrogen consumption was not always related to ethanol production efficiency. Nitrogen utilized in the control medium was lower than that in the juice supplemented with (NH4)2SO4, but ethanol production from the control medium was higher. The amount of nitrogen consumption seemed to relate to type of nitrogen sources supplied (Table 3). Especially, in case of using ammonium salts to increase the nitrogen content, excessive ammonium addition might cause the increase in the byproducts (Beltran et al., 2005; Bely et al., 2003; Wang et al., 2003) as previously mentioned. Viability of the yeast during ethanol fermentation from sweet sorghum juice under various conditions is shown in Fig. 2. The results indicated that S. cerevisiae NP01 was a suitable microorganism for ethanol fermentation under VHG levels. It could survive and retain its metabolism under very high ethanol concentrations up to 120 g l1 (15% by volume) (Table 2) with high viable cells remaining in the fermentation broth (Fig. 2). Moreover, Nagashima (1990) reported that to achieve cell viability and ethanol production, appropriate amount of fermentable nitrogen must also be available. In our studies, fermentable nitrogen had still been available in the medium until the end of the fermentation.
9.0
A
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0
B
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 0
20
40
60
80
Time (h) Fig. 4. Yeast viability of S. cerevisiae NP01 during batch ethanol fermentation from sweet sorghum juice supplemented with molasses at various initial total soluble solids and nitrogen sources: 28°Bx (d), 32°Bx (.) and 34°Bx (j). (A) No extra nutrient, (B) supplemented with YEP.
L. Laopaiboon et al. / Bioresource Technology 100 (2009) 4176–4182
When the results of using sucrose and molasses were compared in terms of sugar utilization, the amount of the utilized total sugar under both carbon adjuncts were similar when compared at the same initial total sugar concentrations (Tables 2 and 4). These findings imply that molasses might contain some inhibitors (e.g., hydroxymethylfurfural, hexanol and heptanol) for yeast metabolism as previously reported (Glacet et al., 1985; Fattohi 1994). However, when the molasses was used as a carbon adjunct to raise sugar concentration to VHG levels, the inhibitors were diluted to a level which did not cause adverse effect on sugar utilization. Regarding to the important kinetic parameters for ethanol production; P, Q p and Y ps of the ethanol fermentation using molasses as an adjunct were significantly lower than those using sucrose as an adjunct (Tables 2 and 4). These results imply that some other nutrients in the molasses may promote the formation of byproducts of the ethanol fermentation. The advantage of using fermentable sugars (sweet sorghum juice, cane sugar or molasses) in this study especially under VHG fermentation is that they can reduce the overall fermentation time. Other forms of carbon such as starch or dextrins may be used to raise soluble solids to VHG levels as found in Jones and Ingledew (1994b). However, there were additional steps for pretreatment the raw materials before ethanol fermentation process such as double mashing and saccharification by glucoamylase, which was a drawback of using complex carbohydrate as the raw materials. 4. Conclusions This research achieves the goal of VHG fermentation technology that at least 15% (v/v) of ethanol is produced in the fermentation broth (Bai et al., 2008). The results obtained from this research have demonstrated that sucrose is a good adjunct to raise total soluble solids or sugar concentration of sweet sorghum juice to VHG levels for ethanol fermentations, while molasses causes the lower ethanol production efficiency. However, other methods for raising sugar content in sweet sorghum juice such as concentrating the juice by evaporation should be considered to reduce cost of the adjunct. Yeast extract and peptone promote ethanol fermentation under VHG levels. Other yeast products such as dried spent yeast may replace yeast extract and peptone as they are far less expensive. The effects of dried spent yeast on ethanol production will be investigated. According to the very high content of fermentable sugars in the sweet sorghum juice supplemented with cane sugar or molasses (Table 1), the optimum conditions in terms of processing parameters and/or fermentation processes to achieve complete sugar utilization under VHG levels need to be further studied. Acknowledgements This research was financially supported by the Thailand Research Fund (TRF) and Commission on Higher Education, Thailand. We would like to thank Assistant Prof. Dr. Paiboon Danviruthai, Faculty of Technology, Khon Kaen University (KKU) for providing the NP01 strain and Associate Prof. Dr. Prasit Jaisil, Faculty of Agriculture, KKU for providing sweet sorghum juice and Ms. Ratchanee Kortsree, Mitr Phu Viang sugar Mill, Khon Kaen, Thailand for providing molasses and Associate Prof. Dr. Aroonwadee Chanawong, Faculty of Associated Medical Sciences, KKU for her internal review of this paper and helpful suggestions. We also gratefully acknowledge the Royal Bangkok Sports Club (RBSC), Bangkok, Thailand and the Fermentation Research Center for Value Added Agricultural Products (FerVAAP) for financial support for Mr. Sunan Nuanpeng and Research Center for Environmental and Hazardous Substance Management, KKU and National Center of
4181
Excellence for Environmental and Hazardous Waste Management, KKU for some instrumentation supports. References Bafrncova, P., Smogrovicova, D., Slavikova, I., Patkova, J., Domeny, Z., 1999. Improvement of very high gravity ethanol fermentation by media supplementation using Saccharomyces cerevisiae. Biotechnol. Lett. 21, 337– 341. Bai, F.W., Chen, L.J., Zhang, Z., Anderson, W.A., Moo-Young, M., 2004. Continuous ethanol production and evaluation of yeast cell lysis and viability loss under very high gravity medium conditions. J. Biotechnol. 110, 287–293. Bai, F.W., Anderson, W.A., Moo-Young, M., 2008. Ethanol fermentation technologies from sugar and starch feedstocks. Biotechnol. Adv. 26, 89–105. Bayrock, D.P., Ingledew, W.M., 2001. Application of multistage continuous fermentation for production of fuel alcohol by very-high-gravity fermentation technology. J. Ind. Microbiol. Biotechnol. 27, 87–93. Beltran, G., Esteve-Zarzoso, B., Rozes, N., Mas, A., Guillamon, J.M., 2005. Influence of the timing of nitrogen additions during synthetic grape must fermentations on fermentation kinetics and nitrogen consumption. J. Agric. Food Chem. 53, 996– 1002. Bely, M., Rinaldi, A., Dubourdieu, D., 2003. Influence of assimilable nitrogen on volatile acidity production by Saccharomyces cerevisiae during high sugar fermentation. J. Biosci. Bioeng. 96, 507–512. Bvochora, J.M., Read, J.S., Zvauya, R., 2000. Application of very high gravity technology to the cofermentation of sweet stem sorghum juice and sorghum grain. Ind. Crop. Prod. 11, 11–17. Casey, G.P., Ingledew, W.M., 1986. Ethanol tolerance in yeasts. CRC Crit. Rev. Biotechnol. 13, 219–280. Casey, G.P., Magnus, C.A., Ingledew, W.M., 1984. High gravity brewing: effects of nutrition on yeast composition, fermentative ability, and alcohol production. Appl. Environ. Microbiol. 48, 639–646. Dombek, K.M., Ingram, L.O., 1986. Magnesium limitation and its role in apparent toxicity of ethanol during yeast fermentation. Appl. Environ. Microbiol. 52, 975– 981. Fattohi, N., 1994. Determination of molasses and yeast components by GC and HPLC. Zuckerind 119, 554–560. Glacet, A., Letoumeau, F., Leveque, P., Villa, P., 1985. Kinetics study of nitrate inhibition during alcoholic fermentation of beet molasses. Biotechnol. Lett. 7, 47–52. Göksungur, Y., Zorlu, N., 2001. Production of ethanol from beet molasses by Caalginate immobilized yeast cells in a packed-bed bioreactor. Turk. J. Biol. 25, 265–275. Jiranek, V., Langridge, P., Henscke, P.A., 1995. Amino acids and ammonium utilization by Saccharomyces wine yeasts from a chemically defined medium. Am. J. Enol. Vitic. 46, 75–83. Jones, A.M., Ingledew, W.M., 1994a. Fuel ethanol production: appraisal of nitrogenous yeast foods for very high gravity wheat mash fermentation. Process Biochem. 29, 483–488. Jones, A.M., Ingledew, W.M., 1994b. Fermentation of very high gravity wheat mash prepared using fresh yeast autolysate. Bioresour. Technol. 50, 97–101. Jones, A.M., Thomas, K.C., Ingledew, W.M., 1994. Ethanolic fermentation of blackstrap molasses and sugarcane juice using very high gravity technology. J. Agric. Food Chem. 42, 1242–1246. Laopaiboon, L., Thanonkeo, P., Jaisil, P., Laopaiboon, P., 2007. Ethanol production from sweet sorghum juice in batch and fed-batch fermentations by Saccharomyces cerevisiae. World J. Microbiol. Biotechnol. 23, 1497–1501. Laopaiboon, L., Nuanpeng, S., Srinophakun, P., Klanrit, P., Laopaiboon, P., 2008. Selection of Saccharomyces cerevisiae and investigation of its performance for very high gravity ethanol fermentation. Biotechnology 7, 493–498. Limtong, S., Sringiew, C., Yongmanichai, W., 2007. Production of fuel ethanol at high temperature from sugar cane juice by a newly isolated Kluyveromyces marxianus. Bioresour. Technol. 98, 3367–3374. Mecozzi, M., 2005. Estimation of total carbohydrate amount in environmental samples by the phenol–sulphuric acid method assisted by multivariate calibration. Chemom. Intell. Lab. Syst. 79, 84–90. Melzoch, K., Rychtera, M., Hábová, V., 1994. Effect of immobilization upon the properties and behaviour of Saccharomyces cerevisiae cells. J. Biotechnol. 32, 59– 65. Nagashima, M., 1990. Progress in ethanol production with yeasts. In: Hubert, V., Rene, De M. (Eds.), Yeast: Biotechnology and Biocatalysis. Marcel Dekker, New York. Narendranath, N.V., Power, R., 2005. Relationship between pH and medium dissolved solids in terms of growth and metabolism of Lactobacilli and Saccharomyces cerevisiae during ethanol production. Appl. Environ. Microbiol. 71, 2239–2243. Ozmichi, S., Kargi, F., 2007. Ethanol fermentation of cheese whey powder solution by repeated fed-batch operation. Enzyme Microb. Technol. 41, 169– 174. Schaffert, R.E., 1995. Sweet sorghum substrate for industrial alcohol. In: Dendy, D.A.V. (Ed.), Sorghum and Millets: Chemistry and Technology. American Association of Cereal Chemists, St. Paul, MN, USA, pp. 365–374. Sree, N.K., Sridhar, M., Rao, L.V., Pandey, A., 1999. Ethanol production in solid substrate fermentation using thermotolerant yeast. Process Biochem. 34, 115– 119.
4182
L. Laopaiboon et al. / Bioresource Technology 100 (2009) 4176–4182
Taillandier, P., Portugal, F.R., Fuster, A., Strehaiano, P., 2007. Effect of ammonium concentration on alcoholic fermentation kinetics by wine yeasts for high sugar content. Food Microbiol. 24, 95–100. Thomas, K.C., Ingledew, W.M., 1990. Fuel alcohol production: effects of free amino nitrogen on fermentation of very-high-gravity wheat mash. Appl. Environ. Microbiol. 56, 2046–2050. Thomas, K.C., Hynes, S.H., Ingledew, W.M., 1996. Practical and theoretical considerations in the production of high concentration of alcohol by fermentation. Process Biochem. 31, 321–331. Valero, J., Millan, C., Ortega, J.M., Mauricio, J.C., 2003. Concentration of amino acids in wine after the end of fermentation by Saccharomyces cerevisiae strains. J. Sci. Food Agric. 88, 830–835.
Wang, X.D., Bohlscheid, J.C., Edwards, C.G., 2003. Fermentative activity and production of volatile compounds by Saccharomyces grown in synthetic grape juice media deficient in assimilable nitrogen and/or pantothenic acid. J. Appl. Microbiol. 94, 349–359. Woods, J., 2000. Integrating Sweet sorghum and sugarcane for bioenergy: modelling the potential for electricity and ethanol production in SE Zimbabwe, Ph.D. Thesis, King’s College, London. Zoecklein, B.U., Fugelsang, K.C., Gump, B.H., Nury, F.S., 1995. Wine Analysis and Production. Chapman & Hall, New York.