Energy & Fuels 2009, 23, 487–491
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Optimization of Fermentation Conditions for the Production of Ethanol from Stalk Juice of Sweet Sorghum by Immobilized Yeast Using Response Surface Methodology Xiaoyan Mei, Ronghou Liu,* Fei Shen, and Hanjing Wu Biomass Energy Engineering Research Centre, School of Agriculture and Biology, Shanghai Jiao Tong UniVersity, 800 Dongchuan Road, Minhang District, Shanghai 200240, People’s Republic of China ReceiVed June 4, 2008. ReVised Manuscript ReceiVed September 17, 2008
Optimization of three parameters, including initial total sugar concentration, supplement rate of (NH4)2SO4, and particles stuffing rate, was attempted using response surface methodology based on a Box-Behnken design for the optimal production of ethanol by immobilized yeast fermentation of stalk juice of Liaotian number 1 sweet sorghum cultivar in shaking flasks. The correlation analysis of the mathematical regression model indicated that the quadratic polynomial model could be employed to optimize ethanol production. The optimum conditions were found to be an initial total sugar concentration of 22.88%, supplement rate of (NH4)2SO4 of 0.244%, and particles stuffing rate of 25.15%. At the optimum conditions, the maximum predicted ethanol yield of 93.83% was obtained. The ethanol yield and fermentation time of verification experiments in the shaking flask were 92.37% and 14 h at the corresponding parameters, respectively, while they were 93.23% and 13 h, respectively, in a 5 L bioreactor, in which the predicted value of ethanol yield was very close to experimental values. In addition, the fermentation time of the stalk juice of sweet sorghum was about 3-4 times shorter with immobilized yeast than that of conventional fermentation technology. Thus, by immobilized yeast fermentation of the stalk juice of sweet sorghum, the Box-Behnken design was found to be the favorable strategy investigated with respect to the optimization of fermentation conditions for ethanol production.
Introduction With growing concerns for environmental pollution, energy security, and future oil supplies, the global community is seeking nonpetroleum-based alternative fuels, along with more advanced energy technologies, to increase energy use efficiency.1,2 Ethanol has excellent fuel properties for internal combustion engines. Because ethanol is less volatile than gasoline3 and has a low photochemical reactivity in the atmosphere, smog formation from evaporative emissions of pure ethanol can be less than for gasoline. Unlike fossil fuels, ethanol is a renewable energy source produced through fermentation of sugars. A dramatic increase in ethanol production using the current corn-starchbased technology may not be practical because corn production for ethanol will compete for the limited agricultural land needed for food and feed production.4 A potential source for low-cost ethanol production is to use energy crops, including sweet sorghum in China. Sweet sorghum [Sorghum bicolor (L.) Moench] is a high biomass- and sugar-yielding C4 plant. It has been noted for its potential as an energy crop because it can be cultivated in almost * To whom correspondence should be addressed. Telephone: 0086-2134205744. E-mail:
[email protected]. (1) Dai, X.; Wu, C.; Zhou, Z. Acta Energiae Solaris Sinnica 2001, 22, 124–130. (2) Prasad, S.; Singh, A.; Jain, N.; Joshi, H. C. Energy Fuels 2007, 21, 2415–2420. (3) Wyman, C. E. Production and UtilizationsApplied Energy Technology Series; Taylor and Francis: Oxford, U.K., 1996; pp 37-60. (4) Prasad, A.; Singh, H. C.; Jain, N. Resour. ConserV. Recycl. 2007, 50, 1–39.
all temperate and tropical climate areas.5,6 The plant grows to a height from about 120 to above 400 cm, depending upon the varieties and growing conditions, and can be an annual or short perennial crop. More than 125 sweet sorghum germplasm resources have been registered in China. Most of the existing studies concern the breeding and harvesting of the sorghum plant and its conversion to ethanol using free cells of Saccharomyces cereVisiae in batch processes.7-9 Meanwhile, ethanol production from the juice of sweet sorghum using immobilized yeast fermentation has also received special attention recently because immobilized yeast fermentation has the advantage of rapid fermentation.10 In addition, nutrient substances in sufficient content of carbon, nitrogen, and certain minerals are needed for yeast to grow and reproduce. However, the content of inorganic salts existing in the stalk juice of sweet sorghum was not enough to meet the need of fermentation. The effects of the inorganic salts supplement dose of K2HPO4, (NH4)2SO4, and MgSO4 on ethanol fermentation by immobilized yeast using stalk juice of sweet sorghum has been researched recently. However, at present, the research on the optimization of fermentation conditions for production of ethanol from stalk (5) Giorgis, F.; Pirri, C. F.; Tresso, E.; Rava, P.; Paterson, A. M.; Dowling, G. R.; Chamberlain, D. A.; Billa, E.; Koullas, D. P.; Monties, B.; Koukios, E. G. Ind. Crop. Prod. 1997, 6, 297–302. (6) Mamma, D.; Koullas, D.; Fountoukidis, G.; Kekos, D.; Macris, B. J.; Koukios, E. Process Biochem. 1996, 31, 377–381. (7) Kimberley, B.; Ritter, C.; Lynne, M.; Ian, D.; David, G.; Jordan, R.; Scott, C. C. Euphytica 2007, 157, 161–176. (8) Bulawayo, B.; Bvochora, J. M.; Muzondo, M. I.; Zvauya, R. World J. Microbiol. Biotechnol. 1996, 12, 357–360. (9) Bvochora, J. M.; Read, J. S.; Zvauya, R. Ind. Crop. Prod. 2000, 11, 11–17. (10) Liu, R. H.; Shen, F. Bioresour. Technol. 2008, 99, 847–854.
10.1021/ef800429u CCC: $40.75 2009 American Chemical Society Published on Web 11/13/2008
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Table 1. Main Components of Stalk Juice of Liaotian Number 1 Sweet Sorghum Cultivar content of minerals (mg kg-1)
total sugar content (mg mL-1)
total nitrogen content (mg kg-1)
Cu2+
Fe3+
Mn2+
Ca2+
Zn2+
Mg2+
Na+
K+
water content (w/w, %)
ash content (w/w, %)
185.8
0.955
1.4
12.6
1.1
238.8
4.9
210.6
231.7
897.9
84.4
0.92
Table 2. Composition of Culture Media slant culture liquid medium medium
component glucose or sucrose (%) yeast extract (%) peptone (%) KH2PO4 (%) MgSO4 · 7H2O (%) agar (%) (NH4)2SO4 (%)
5.0 0.5 0.5 0.125 0.05 2.0
fermentation mash of stalk juice of sweet sorghum
5.0 0.5 0.5 0.125 0.05
0.125 0.05 according to experimental
design
juice of sweet sorghum by immobilized yeast using response surface methodology (RSM) is scarcely found in the literature. Thus, the objectives of this research were to develop a mathematical correlation between the initial total sugar concentration, supplement rate of (NH4)2SO4, and particles stuffing rate that is defined as a ratio of immobilized yeast particles weight to fermentation solution weight and ethanol yield by the application of the RSM using the Box-Behnken design11 and to optimize ethanol fermentation conditions of an initial total sugar concentration, supplement rate of (NH4)2SO4, and particles stuffing rate for maximum ethanol yield in a shaking flask. Materials and Methods Sweet Sorghum and Strain. Liaotian number 1 sweet sorghum cultivar was planted in an attached farm at the School of Agriculture and Biology of Shanghai Jiao Tong University, People’s Republic of China. Table 1 presents the main components of stalk juice of the sweet sorghum. The stalk of sweet sorghum whose leaves and leaf sheath were removed was extracted by a three-roller squeezer, then filtered by a 100 mesh screen, and finally, dehydrated to experimentally needed concentration by a vacuum rotary evaporator at 55-60 °C and 0.015 MPa. The strain of S. cereVisiae CICC 1308 was obtained from the Centre of Industrial Culture Collection of China. Culture Media and Microorganism Culture. The composition of culture media is shown in Table 2. The activated S. cereVisiae CICC 1308 was inoculated into a flesh slant culture medium in a test tube and cultivated in a thermotank at 30 °C for 1-3 days until the white lawn appeared on the slant culture and distributed evenly. Precultures were incubated for 20-24 h at 30 ( 0.5 °C, with orbital shaking at 150 rpm in a 25 mL test tube containing a loopful of yeast cells in 10 mL of liquid culture medium. When the yeast cells were up to 108 cells mL-1 observed by the microscope, the yeast cells in the liquid culture medium were mature. Fermentation culture was carried in a 500 mL flask with a working volume of 200-250 mL. The flask was inoculated with 20-25 mL of mature yeast cells of liquid culture medium and incubated aerobically at 30 °C, initial pH of 5.0, and agitation rate of 150 rpm for about 48 h. When the yeast cells were up to 108 cells mL-1, the yeast cells in fermentation culture medium were mature and could be used for the yeast cells immobilization. Yeast Culture and Immobilization. One loop of solid slant yeast was inoculated into a flesh slant culture medium in a 250 mL test tube and then cultivated in a thermotank at 32 °C with orbital shaking at 120 rpm for 20-40 h to obtain the liquid seed (cell number > 1.0 × 108 cells mL-1). The next step was to mix (11) Box, G. E. P.; Behnken, D. W. Technometrics 1960, 2, 455–475.
agitate mixtures of 2% sodium alginate and distilled water, then heat and sterilize mixtures after they were completely dissolved, and subsequently, add the liquid seed of yeast to Na-alginate colloid solution, making them uniformly blended. Afterward, 2% CaCl2 solution, which had been sterilized and cooled down, was gradually instilled to produce the immobilized yeast particles of about 3 mm in diameter, and then particles were immobilized for 24 h in the CaCl2 solution. After all, immobilized particles after washing 3 times by aseptic water were inoculated into multiplication culture medium, 100 mL of culture medium with 50 g of particulate, and then cultivated for 12-24 h with orbital shaking at 32 °C and 120 rpm. When yeast cell were up to 1.0 × 108 cells mL-1, it was suitable to be applied into ethanol fermentation. Equipments. The main equipment used in this research included an atomic absorption spectrophotometer (AA-6800, Shimadzu, Japan), a gas chromatograph (GC-2010, Shimadzu, Japan), and a 5 L bioreactor (GUJS-5C, East Biotech. Co., Zhenjiang, China). Analytical Methods. Sugars were determined using the 3,5dinitrosalicylic acid (DNS) method.12 Nitrogen content was determined using the Kjeldahl method.13 Trace element concentrations were measured by atomic absorption spectrometry (AAS).14 pH was determined by a glass-electrode pH meter. Ethanol content in fermentation mash was measured by gas chromatography (GC) with a headspace, flame ionization detector (FID).15 The exterior standard method was applied. The chromatogram column and samples injection were a stainless-steel column and auto-headspace samples injection. Ethanol (GR) was used for the standard curve. The column temperature was controlled at 200 °C. N2 was used as a carrier gas (40 mL min-1). The flow rates of H2 and O2 were 40 and 500 mL min-1, respectively. Samples of 0.3 µL were injected directly into the column. All determinations were performed by means of standard curves, and results were the mean of two repetitions. To determinate the end of ethanol fermentation, the CO2 weight loss rate of every hour from the beginning of ethanol fermentation in the shaking flask was calculated. When the CO2 weight loss rate is less than 0.1 g h-1, the fermentation is finished. While as for the bioreactor, releasing no CO2 is regarded as the symbol of the end of fermentation. Ethanol yield (Y) is calculated by eq 1
Y ) Yi/(0.511So)
(1)
where Y is the ethanol yield (%), Yi is actual measured ethanol content (%), So is initial total sugar concentration (%), and 0.511 is the theoretical conversion rate from glucose to ethanol. All of the data are statistically analyzed by the data packet of Design-Expert 7.0 Trial from Stat-Ease. Experimental Design and Optimization. On the basis of previous studies,10,16 three independent variables, including initial total sugar concentration, supplement rate of (NH4)2SO4, and particles stuffing rate, were chosen for the further optimization studies using a RSM. A Box-Behnken design11 with three variables at three levels and a total of 17 runs was used for the study. The three levels of each variable were coded as -1, 0, and +1, which corresponded to the lower, middle, and higher (12) Hu, M. F. Food Analysis; Southwest China Normal University Press: Chong Qing, People’s Republic of China, 1993; pp 173-174. (13) Hao, Y. Y.; Liu, R. H. J. Anhui Agri. Sci. 2006, 34, 3429–3431. (14) Zhang, S. H. Food Analysis; China Light Industry Press: Beijing, People’s Republic of China, 2004; pp 250-254. (15) Mu, J. L.; Wang, J.; Chen, Z. H. Liq. Making 2005, 32, 77–78. (16) Liu, R. H.; Li, J. X.; Shen, F. Renewable Energy 2008, 33, 1130– 1135.
Ethanol from Stalk Juice of Sweet Sorghum
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Table 3. Independent Variables and Their Levels Employed in the Box-Behnken Design coded level variables
-1
0
1
initial total sugar concentration (X1, %) supplement rate of (NH4)2SO4 (X2, %) particles stuffing rate (X3, %)
18 0.1 20
22 0.2 25
26 0.3 30
Table 4. Box-Behnken Design Conditions and Ethanol Yield run number
X1
X2
X3
experimental ethanol yield (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
1 1 -1 0 -1 1 0 0 -1 0 0 -1 0 0 1 0 0
-1 0 -1 0 0 0 0 1 0 -1 0 1 0 1 1 0 -1
0 1 0 0 -1 -1 0 -1 1 1 0 0 0 1 0 0 -1
85.05 86.56 78.28 93.40 80.45 88.82 93.40 88.95 82.63 84.50 92.51 85.89 92.51 90.73 88.82 92.51 82.73
values, respectively. For individual parameters these were initial total sugar concentration (X1, %), supplement rate of (NH4)2SO4 (X2, %), and particles stuffing rate (X3, %). The fermentation experiments for optimization were conducted in 250 mL flasks, with a working value of 100 mL. The other operating conditions were maintained at a temperature of 34 ( 1 °C, rotational speed of shaking flasks of 140 ( 5 rpm, and pH 4.5. The flask and bioreactor were maintained under anaerobic conditions. The software Design-Expert (version 7.0, Stat-Ease, Inc., Minneapolis, MN) was used for experimental design, data analysis, and quadratic model building. The response surface graphs were obtained using the software to understand the effect of variables individually and in combination and to determine their optimum levels. The experimental setup of RSM is shown in Table 3. Five replicates at the center of the design were used to allow for estimation of a pure error sum of squares. Experiments were randomized to maximize the effects of unexplained variability in the observed responses because of extraneous factors. A full quadratic equation, shown as follows, was used for this model: k
Y ) β0 +
∑ j)1
k
βjXj +
∑β X +∑ ∑β XX 2
jj j
j)1
jj i j
(2)
i F value of the model was 0.0500 indicate that the model terms are insignificant. The particles stuffing rate (X3) and interaction between the supplement rate of (NH4)2SO4 (X2) and particles stuffing rate (X3) are insignificant because of p > 0.05. A lackof-fit value of 3.712 implies that the lack of fit is not significant relative to the pure error when p ) 0.1188 > 0.05 also supports the fitness of the model. Because of no anomalous point in the data, it is not necessary to bring in a higher degree term. Therefore, with an effective term of X1, X2, X1 X2, X1X3, X12, X22, and X32 in eq 3, the diminished form of eq 3 can be expressed as eq 4 Y ) 92.87 + 2.75X1 + 2.98X2 - 0.96X1X2 - 1.11X1X3 5.24X12 - 3.12X22 - 3.02X32 (4) The simplified response surface model developed in this study for predicting the ethanol yield was adequate. Joglekar and May17 suggested that, for a good fit of a model, R2 should be at least 0.80. The R2 value for this response variable was higher than 0.80, indicating that the regression model explained the fermentation well. The R2 value was 0.9772 for the ethanol yield. The fit degree of the model was high enough to explain 97.72% of ethanol yield. Thus, this model can be applied to predict ethanol yield (Y) within the experimental setting range. In addition, the validity of this model can also be demonstrated by the fact that the predicted value of ethanol yield in eq 4 was very close to the experimental value in Table 6. The result is Table 5. ANOVA for the Response Surface Quadratic Model3
source
sum of squares (SS)
degree of freedom (df)
mean squares (MS)
model X1 X2 X3 X1X2 X1X3 X2X3 X12 X22 X32 residual lack of fit pure error total
357.050 60.471 70.966 1.509 3.700 4.912 0.000 115.460 41.016 38.315 3.593 2.644 0.949 360.643
9 1 1 1 1 1 1 1 1 1 7 3 4 16
39.672 60.471 70.966 1.509 3.700 4.912 0.000 115.460 41.016 38.315 0.513 0.881 0.237
F value
p value Prob > F
77.288 117.808 138.254 2.940 7.208 9.569 0.000 224.937 79.907 74.644
