Indian Journal of Biotechnology Vol 12, October 2013, pp 531-536
Fermentation variables for the fermentation of glucose and xylose using Saccharomyces cerevisiae Y-2034 and Pachysolan tannophilus Y-2460 G S Kocher* and Shivali Uppal Department of Microbiology, Punjab Agricultural University, Ludhiana 141 004, India Received 10 February 2012; revised 17 October 2012; accepted 10 January 2013 The fermentation variables (temperature, pH and agitation) were optimized by response surface methodology (RSM) alogorithm, Design Expert 7.1 and a response quadratic model was generated that revealed a correlation between all these parameters and also provided 23 solutions for process validation. Under the optimized conditions, the effect of inoculum size revealed 5.0 and 2.5% (v/v) of Saccharomyces cerevisiae Y-2034 and Pachysolan tannophilus Y-2460, respectively as optimum for sequential fermentation. The optimization of sequential fermentation led to improvement in total ethanol yield from 20.61 to 22.24 g L-1. Keywords: Ethanol, glucose, sequential fermentation, response surface methodology, xylose
Introduction Lignocellulosics, the potential substrate for biofuel production, are initially pretreated by stringent physicochemical processes to break open its crystalline structure. The process also releases free sugars or sugar complexes, consisting of some glucose and almost all xylose as hydrolysates. Although the amorphous cellulose is saccharified to produce ethanol, the hydrolysates produced remain unutilized as the mixture of glucose and xylose are not fermented by Saccharomyces cerevisiae or any other single fermenting yeast. It has been observed that the hydrolysate, if properly fermented, can decrease the overall cost of ethanol production from lignocellulosic biomass by 25%1,2. Approaches in both process engineering and strain engineering have been explored for enhancing ethanol yield. Process engineering by continuous culture3, the immobilization of one or two strains4, two stage fermentation by sequential culture in one bioreactor5 and separate fermentation in two bioreactors have been carried out3,6. Though ethanol production from xylose fermentation has been achieved using recombinant strains of S. cerevisiae with heterologous xylose reductase (XR) and xylitol dehydrogenase (XD) from Pichia stipitis along with overexpression of S. cerevisiae xylulokinase (XK)7, their xylose —————— *Author for correspondence: Tel; +91-161-2401960 ext. 330 E-mail:
[email protected]
consumption rates are not adequate enough for efficient bio-ethanol production from lignocellulosics. The strain construction strategies have not been successful and, therefore, there is an urgent need to explore other approaches, such as, developing an effective process for efficient conversion of lignocellulosic biomass to ethanol. Among process engineering attempts, cocultivation of two microorganisms in a single process for co-fermentation of glucose-xylose mixtures has been studied using glucose fermenting S. cerevisiae and pentose fermenting yeasts like P. stipitis, Pachysolan tannophilus and Candida shehatae; of which P. tannophilus has been studied extensively8. The present investigation was thus carried out to standardize sequential fermentation of glucose-xylose mixture by S. cerevisiae and P. tannophilus using response surface methodology (RSM) so that the process may be validated on a large scale. Material and Methods Lyophilized cultures of S. cerevisiae NRRL Y2034 and P. tannophilus NRRL Y-2460 were obtained from the Agricultural Research Culture Collection (NRRL), Peoria, IL, USA. The active cultures were maintained on a basic medium containing (g L-1): yeast extract 10, peptone 20 and agar 20 (pH 5.5), which was supplemented with either glucose (20 g L-1) or xylose (20 g L-1) as carbon source depending upon the treatment. All the cultures were subcultured every 15 d on the above medium.
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Fermentation Studies
The fermentation of glucose and xylose was carried out separately in 250 mL capacity Erlenmeyer flasks containing 185 mL of fermentation medium (about 2/3 of the total volume) comprising (g L-1): glucose 50, ammonium sulphate 1.0, potassium dihydrogen orthophosphate 1.0, yeast extract 1.0 and magnesium chloride 1.0 for glucose fermentation, and xylose 40, glucose 1.5, ammonium sulphate 1.0, potassium dihydrogen orthophosphate 1.0, yeast extract 1.0, magnesium chloride 1.0 (pH 5.5) for xylose fermentation. The flasks were autoclaved at 10 lb/in2 for 25 min, allowed to cool and inoculated with freshly prepared 7.5 and 1.5% (v/v) inocula of S. cerevisiae or P. tannophilus, respectively. The inoculated flasks were incubated at 30°C in a BOD incubator. The experiments with S. cerevisiae were performed as stationary flasks, while those with P. tannophilus were agitated at different speeds. In both types of fermentation, samples were collected aseptically at the designated intervals for determining number of cells by hemocytometer, centrifuged thereafter at 6000 rpm for 10 min and stored at –4°C. The stored samples were later analyzed for determining residual sugars9, ethanol10 and pH (pH meter, Make Hanna HI96107). Sequential Fermentation Studies
The sequential fermentation of glucose and xylose was studied by taking different ratios of glucose and xylose, i.e., 6:4 (30 g L-1 glucose & 20 g L-1 xylose), 7:3 (35 g L-1 glucose & 15 g L-1 xylose) and 8:2 (40 g L-1 glucose & 10 g L-1 xylose), in the fermentation medium. This medium was inoculated with 7.5% (v/v) S. cerevisiae prepared earlier and incubated at 28±1°C in a BOD incubator for 72 h till complete glucose utilization (checked qualitatively by paper chromatography and quantitatively by the method of Miller9). The ethanol produced as a result of glucose fermentation was distilled out in some of the treatments and the fermentation media was then supplemented with 1% yeast extract, pH adjusted to 5 and inoculated with 1.5% (v/v) P. tannophilus (prepared earlier) and incubated at 28±1°C on a rotary shaker (100 rpm) till complete xylose consumption. In fact, the experiments were planned in the form of four different treatments, i.e., aerobic distilled (I), aerobic undistilled (II), semiaerobic distilled (III) and semiaerobic undistilled (IV), of xylose fermentation. The samples collected periodically were centrifuged at 6000 rpm for
10 min and analyzed for residual sugars, pH and ethanol as above. Optimization of Sequential Fermentation Parameters by RSM
The optimization of fermentation parameters, viz., temperature (20-40ºC), agitation (50-150 rpm) and pH (4-6), for fermentation of glucose and xylose mixtures in sequential manner was carried out using a central composite design (CCD) developed using Design Expert 7.1 (Statease, MN, USA). A 3-factor and 2-level CCD consisting of 20 experimental runs for ethanol production as response was employed. The design consisted of a 23 CCD factorial design having 6 replicates at the central point and 6 axial points (α) to allow a better estimate of experimental error and to provide extra information about the activities within the design space. Experimental data from the CCD was analyzed using RSM algorithm, Design Expert 7.1 and fitted according to Eq. (1) as a second-order polynomial equation including main effects and interaction effects of each variable: y = βo +
∑
3
β
X i =1 i i
+
∑
3
β
X i =1 ii i
2
+
∑∑
3
β
X X j = i +1 ij i j
… (1) where, y=predicted response, bo=constant coefficient, bi=linear coefficient, bii=quadratic coefficient and bij=interaction coefficient. The analysis of variance (ANOVA) and surface plots were generated using Design Expert 7.1, and the optimized value of 3 independent variables for best response was determined using a numerical optimization package of the software. Effect of Inoculum Size
The effect of 5, 7.5 and 10% (v/v) inoculum size on glucose fermentation by S. cerevisiae Y-2034, and 1.5, 2.5, 5, 7.5 and 10% (v/v) on xylose fermentation by P. tannophilus Y-2460 was assessed by changing the initial inoculum levels in the co-substrate medium, and the fermentation was carried out (in triplicate) at 30±1°C temperature and pH 5.0 in a BOD incubator for glucose fermentation and on rotary shaker (100 rpm) for xylose fermentation. Residual sugars, pH and alcohol were estimated as described earlier. Results and Discussion The fermentation of glucose-xylose (3:2) mixture (total 50 g L-1) by S. cerevisiae Y-2034 and P. tannophilus Y-2460 under aerobic and semiaerobic conditions as a two-step fermentation
KOCHER & UPPAL: SEQUENTIAL FERMENTATION OF GLUCOSE AND XYLOSE
revealed that ethanol production in the glucose fermentation was significantly higher in the semi-aerobic conditions, while sugar consumption and change in pH were statistically non significant (Fig. 1). The ethanol yields in these fermentation treatments varied from 0.387 to 0.480 g g-1 substrate. In the second fermentation carried out by P. tannophilus Y-2460, the xylose consumption was complete between 120-144 h. The paper chromatogram of periodic sample also showed disappearance of glucose spots at 72 h and that of xylose at 144 h of fermentation (Fig. 2). However, the consumption results were significantly higher in both the distilled treatments, suggesting that ethanol present in the undistilled fermentation media
Fig. 1—Co-fermentation of glucose-xylose mixtures by S. cerevisiae Y-2034 and P. tannophilus Y-2460 under different conditions. [I, Aerobic distilled; II, Aerobic undistilled; III, Semiaerobic distilled; IV, Semi-aerobic undistilled]
Fig. 2—Paper chromatogram showing profile of glucose and xylose consumption during fermentation.
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interferes with sugar consumption11. Ethanol production in the xylose fermentation was also better in the previously distilled trials and the maximum total ethanol produced was higher (20.11 g L-1) in these treatments than the undistilled (18.07 g L-1) ones. After 144 h, ethanol content declined in all the treatments. This may be attributed to oxidation of the produced ethanol5. The total ethanol yield (0.407 g g-1) and combined fermentation efficiency (77.73%) were found to be the maximum when conditions were semi-aerobic and the fermentation medium was distilled after first fermentation. These observations suggest that if ethanol is harvested after first fermentation prior to initiation of the second, a better overall ethanol yield can be achieved from glucose-xylose mixtures. In case of 3 substrate ratios of glucose and xylose, viz., 6:4, 7:3 and 8:2 (total of 50 g L-1), ethanol yield was similar in all the three treatments for glucose fermentation (Fig. 3). However, in the second fermentation, a direct correlation between the xylose concentration and ethanol yield was observed, and total ethanol produced in the second fermentation was higher at 6:4 ratio (because of high initial xylose concentration in the treatment) but the ethanol yield of 0.371 g g-1 of xylose consumed was highest at 7:3 ratio. The total ethanol yield (from 0.437 to 0.413 g g-1) and combined fermentation efficiency (from 80.90% to 79.20%) declined gradually as the concentration of xylose increased from 10-20 g L-1. Similar observations have been made earlier and it has been attributed to the fact that as xylose concentration increases, ethanol production is delayed leading to low ethanol yield11.
Fig. 3—Co-fermentation of glucose-xylose mixtures (8:2, 7:3, 6:4) by S. cerevisiae Y-2034 and P. tannophilus Y-2460. [I, 8:2; II,7:3; III, 6:4)
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Optimization of Temperature, pH and Agitation by RSM
The ethanol production data fitted into the CCD was analysed using RSM algorithm Design Expert 7.1 and fitted in Eq. 1. Ethanol=+22.69+0.026*A+0.15*B–0.38*C+0.23* A*B+0.78*A*C–0.20*B*C–7.30*A2+0.50*B2–1.66*C2 … (2) The analysis has also provided a response quadratic model (Table 1). This ANOVA model revealed significant p-value for A2 (temperature) and C2 (agitation). The high value of R2 (0.9490) suggested a correlation between all the 3 parameters. The RSM model further provided 23 solutions, which can be used as temperature-pH-agitation combinations for optimized co-fermentation of glucose-xylose (7:3) by S. cerevisiae Y-2034 and P. tannophilus Y-2460 (Table 2). Besides finding 23 solutions, the RSM provided 3 design graphs, each by keeping one factor (out of 3) constant. The design of RSM revealed that on fixing agitation (at 100 rpm) and moving it within experimental range of pH between 4 and 6, the design graph predicts the maximum value of ethanol in the pH range of 5-5.5, though there is not much variability between pH 5 and 6. Also, at this constant agitation (100 rpm), maximum ethanol value was predicted in the range of 30-35ºC (Fig. 4). On fixing pH (at 5) and moving it within experimental range of temperature between 20 and 40ºC, the design graph predicts the maximum value of ethanol in the vicinity of 30ºC. Also, the maximum ethanol value was predicted near 100 rpm (Fig. 5). On fixing temperature (at 30ºC) and moving it within experimental range of pH between 4 and 6, the design graph predicted the maximum value of ethanol in the range of 5.5-6. Also, at this constant temperature
(30ºC), the maximum ethanol value was predicted in the range of 100-120 rpm (Fig. 6). Among the 23 solutions, the effect of inoculum size on ethanol production was studied by taking solution number 13 that also validated the RSM Table 2—RSM solution for temperature, pH and agitation for co-fermentation of glucose-xylose mixture by S. cerevisiae and P. tannophilus No. 1
Temperature (°C) 30.65
pH 5.93
Agitation (rpm) 90.24
Ethanol Desirability (gL-1) 23.2904 1.000
2
30.12
5.98
105.53
23.2418
1.000
3
30.34
5.87
94.22
23.2436
1.000
4
31.10
5.93
92.79
23.2322
1.000
5
30.35
5.97
92.11
23.3563
1.000
6
29.88
5.96
92.39
23.3425
1.000
7
30.06
5.97
77.90
23.2375
1.000
8
29.75
5.90
97.56
23.2411
1.000
9
29.34
5.97
86.73
23.3066
1.000
10
30.59
5.97
78.79
23.2253
1.000
11
29.96
5.92
83.39
23.2558
1.000
12
29.78
5.94
96.66
23.3003
1.000
13
29.48
5.97
100.35
23.2692
1.000
14
30.30
5.86
95.46
23.2302
1.000
15
30.29
5.91
90.93
23.2853
1.000
16
29.94
5.96
85.81
23.3218
1.000
17
30.47
5.86
89.40
23.2222
1.000
18
31.11
5.98
81.74
23.212
1.000
19
30.95
5.93
94.91
23.2567
1.000
20
29.31
5.95
92.70
23.2884
1.000
21
29.84
4.00
97.15
23.0506
0.994
22
29.85
4.00
96.45
23.0502
0.994
23
29.87
4.00
98.83
23.0486
0.993
Table 1—ANOVA for response quadratic model: Analysis of variance table (partial sum of squares Type III) Source
Sum of df Mean F value p-value squares square Prob > F Model 813.84 9 90.43 20.66 F values less than 0.0500 are significant; R2 = 0.9490.
Fig. 4—Design graph showing effect of temperature and pH at constant agitation.
KOCHER & UPPAL: SEQUENTIAL FERMENTATION OF GLUCOSE AND XYLOSE
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Fig. 5—Design graph showing effect of temperature and agitation at constant pH.
Fig. 7—Effect of inoculum size (v/v) on co-fermentation of glucose-xylose mixtures by (a) S. cerevisiae Y-2034 (I, 5%; II, 7.5%; & III, 10%); & (b) P. tannophilus Y-2460 (I, 1.5%; II, 2.5%; III, 5%; IV, 7.5%; V, 10%).
Fig. 6—Design graph showing effect of pH and agitation at constant temperature.
(Table 2). It revealed statistically similar results of the three parameters with different inoculum sizes suggesting that 5% (v/v) S. cerevisiae Y-2034 inoculum having initial cell load of 1.62× 106 cells/mL was sufficient for first (glucose) fermentation (Fig. 7a). The results presented for the second (xylose) fermentation (in the mixture of glucose and xylose) revealed a direct relation with xylose consumption and inoculum size (Fig. 7b). The ethanol production was highest with 2.5% inoculum at 144 h, which was significantly better than the control (1.5% v/v). Thus, the total ethanol yield and combined fermentation efficiency improved to 0.448 g g-1 and 87.59%, respectively. Similar observations have been made where it was found that a 10 times increase in the inoculum size from 3.6× 105 cells/100 mL to 3.6×106 cells/mL decreased
the ethanol yield12. However, the increase in ethanol production from 1.5 to 2.5% (v/v) inoculum size has been due to the fact that increasing the inoculum size decreases the severity of ethanol inhibition, which leads to a better ethanol production13. Therefore, in the present study, total ethanol yield improved from 20.61 g L-1 to 22.24 g L-1. Thus, by optimization of fermentation parameters through RSM, the combined ethanol yield of glucose-xylose mixture (7:3) increased to 0.448 g g-1 with a fermentation efficiency of 87.59%, which is higher than control (0.407 g g-1 and 77.73%). The data recorded in the present study was found at par with the earlier study with recombinant Saccharomyces 1400 (pLNH33)14, where the maximum ethanol yield of 0.46 g g-1 from glucose-xylose mixture at 90% fermentation efficiency was reported. While Tain et al15 reported a fermentation efficiency of only 67.14% from glucose-xylose mixture (3:2) by S. cerevisiae YS58. The present optimized conditions are being employed for fermentation of lignocellulosic hydrolysates in the laboratory.
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Acknowledgement The authors are greatful to NRRL, USA for providing the yeast cultures used in the present study. The help received from Dr H S Oberoi, Principal Scientist, Central Institute of Post Harvest Engineering and Technology, Ludhiana in statistical planning is also acknowledged. References 1
2 3
4
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6
Jeffries T W & Kurtzman C P, Taxonomy, genetics, and strain selection of xylose-fermenting yeasts, Enzyme Microb Technol, 16 (1994) 922-932. Saha B C, Hemicellulose bioconversion, J Ind Microbiol Biotechnol, 30 (2003) 279-291. Grootjen D R J, Jansen M L, Van der Lans R G J M & Luyben K Ch A M, Reactors in series for the complete conversion of glucose/xylose mixtures by Pichia stipitis and Saccharomyces cerevisiae, Enzyme Microb Technol, 13 (1991) 828-833. Lebeau T, Jouenne T & Junter G A, Simultaneous fermentation of glucose and xylose by pure and mixed cultures of Saccharomyces and Candida shehatae immobilized in a two-chambered bioreactor, Enzyme Microb Technol, 21(1997) 265-272. Fu N & Peiris P, Co-fermentation of a mixture of glucose and xylose to ethanol by Zymomonas mobilis and Pachysolen tannophilus, World J Microbiol Biotechnol, 24 (2007) 1091-1097. Taniguchi M, Itaya T, Tohma T & Fujii M, Ethanol production from a mixture of glucose and xylose by coculture of Pichia stipitis and a respiratory-deficient mutant of Saccharomyces cerevisiae, J Ferment Bioeng, 83 (1997) 364-370.
7
8
9
10
11
12
13
14
15
Katahira S, Ito M, Takema H, Fujita Y, Tanino T et al, Improvement of ethanol productivity during xylose and glucose co-fermentation by xylose assimilating S. cerevisiae via expression of glucose transporter Sut1, Enzyme Microb Technol, 43 (2008) 115-119. Fu N, Peiris P, Markham J & Bavor J, A novel co-culture process with Zymomonas mobilis and Pichia stipitis for efficient ethanol production on glucose/xylose mixtures, Enzyme Microb Technol, 45 (2009) 210-217. Miller G L, Use of dinitrosalicyclic acid reagent for determination of reducing sugars, Anal Chem, 31 (1959) 426-428. Caputi A Jr & Wright D, Collaborative study of the determination of ethanol in wine by chemical oxidation, J Assoc Offic Anal Chem, 52 (1969) 85-89. Zhao L, Yu L, Zhang X & Tan T, The ethanol tolerance of Pachysolen tannophilus in fermentation on xylose, Appl Biochem Biotechnol, 160 (2008) 378-85. El-Diwany A I, El-Abyad M S, El-Refai A H, Sallam L & Allam R F, Some physiological parameters for ethanol production from beet molasses by Saccharomyces cerevisiae Y-7, Bioresour Technol, 42 (1992) 191-195. Vega J L, Navarro A R, Clansen T C & Gaddy J L, Effect of inoculum size on ethanol inhibition modelling and other fermentation parameters, Biotechnol Bioeng, 29 (1987) 633-638. Krishnan M S, Ho N W Y & Tsao G T, Fermentation kinetics of ethanol production from glucose and xylose by recombinant Saccharomyces 1400 (pLNH33), Appl Biochem Biotechnol, 77-79 (1999) 373-388. Tian S, Zang J, Pan Y, Liu J, Yuan Z et al, Construction of a recombinant yeast strain converting xylose and glucose to ethanol, Fron Biol China, 3 (2008) 165-169.
