Natl. Acad. Sci. Lett. (November–December 2014) 37(6):529–533 DOI 10.1007/s40009-014-0273-0
RESEARCH ARTICLE
Optimization of Various Parameters for Utilization of Apple Pomace Amended with Molasses by Indigenous Yeast Isolates Govind Kumar • Manvika Sahgal • Mahesh Kumar Bharti Atindra K. Pandey • Anupama Singh
•
Received: 8 April 2013 / Revised: 4 December 2013 / Accepted: 15 December 2013 / Published online: 12 November 2014 Ó The National Academy of Sciences, India 2014
Abstract In present study, yeast strains YGM1 and YGM2 were evaluated and the selected strain YGM1 was evaluated for its ethanol producing efficiency using varying substrate combinations. Both strain YGM1 and YGM2 were able to grow in wide range of temperature from 25 to 40 °C. Based on cfu ml-1 and corresponding absorbance values of two, YGM1 was rapid multiplier and better performer at 37 °C. During fermentation, pH of the substrate first decreased and then increased. Alcohol producing ability of YGM1 in substrates; 100 % pomace, 75 % pomace, 25 % molasses, 25 % pomace: 75 % molasses and 100 % molasses was compared. Alcohol producing ability is directly related to sugar utilization maximum amount of
G. Kumar M. Sahgal Department of Microbiology, College of Basic Sciences and Humanities, College of Ag. and Tech, G. B. Pant University of Agriculture and Technology, U.S. Nagar, Pantnagar, Uttarakhand, India e-mail:
[email protected] M. K. Bharti (&) Department of Biochemistry, College of Basic Sciences and Humanities, College of Ag. and Tech, G. B. Pant University of Agriculture and Technology, U.S. Nagar, Pantnagar 263145, Uttarakhand, India e-mail:
[email protected] A. K. Pandey Molecular Biology and Biotechnology Division, National Bureau of Fish Genetic Resources, Lucknow 226002, UP, India A. Singh Department of Post Harvest and Food Processing, College of Ag. and Tech, G. B. Pant University of Agriculture and Technology, U.S. Nagar, Pantnagar, Uttarakhand, India
ethanol, 5.02 % was produced in pomace: molasses (75 %:25 %) and minimum 2.33 % in 100 % molasses. Maximum ethanol production, 5.02 % corresponded to maximum sugar utilization (11.31 %) in the 75 % pomace: 25 % molasses. Thus out of four combinations, 75 % pomace: 25 % molasses was better substrate for alcohol production. Keywords Apple pomace Fermentation Molasses Indigenous Invertase
Introduction India is the ninth largest producer of apple in the world contributing an annual production of 1.42 MT (million tonnes) from an area of 0.25 million hectares [1]. During processing for juice about 75 % of the fresh weight of fruit is recovered as juice and 25 % as pomace [2, 3]. Apple pomace has high chemical oxygen demand (C.O.D) and high biological oxygen demand that is why it’s biodegradable [4]. Production of ethanol when apple pomace is used as a substrate solely as it contains approximately 26.41 % dry matter, 17.35 % total sugar out of it 5–8 % is reducing sugar. It has been reported that production of ethanol increases at 16 % sugar concentration [5]. Several cellulosic substrates could be used to increase the sugar concentration of apple pomace. Molasses is one of them. It is a by-product of sugar industry and available in plenty at cheap price [6]. It’s reported that molasseses contain 51 % total reducing sugar out of which 45 % is fermentable and 5 % is non fermentable including hemicellulose, waxes and dextran [7]. Our main objective is optimizing various parameters for proper utilization of amended substrate by using effective yeast strain.
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Materials and Methods Two strains of Saccharomyces sp. (YGM1 and YGM2) obtained from culture collection at Department of Microbiology, G.B. P. U. A. & T., Pantnagar were used. The test strains were isolated from red delicious variety of apple and grown on Yeast peptone Dextrose agar (YPDA) media. For preparation of apple pomace, apple were washed, trimmed and cut in small pieces and then hydraulic press of Fred S. Carver Inc., USA, was used for removal of apple juice from apple pomace than solids material (apple pomace) was collected and molasses was obtained from Kichha sugar mill, Uttarakhand, India.
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parameters on dependent variables (pH change, ethanol production etc.). pH Change During Fermentation The change in pH was observed using digital pH meter (Microprocessor CP 931, Zimbabwe). Determination of Colony Forming Units Per ml of fermenting brew was determined. Estimation of Sugar
Temperature Tolerance of Yeast Strain by Measurement of Growth Pattern
Sugar was estimated using Dinitrosalicylic acid (DNSA) method of Miller [8].
For temperature tolerance YGM1 and YGM2 were inoculated in 250 ml yeast peptone dextrose broth (YPD) separately in 6 Erlenmeyer flasks and incubated at varying temperature levels, 25, 28, 31, 34, 37 and 40 °C in incubator shaker at 80 rpm (LT-W Adolf Kuhner AG, Switzerland) for 5 days. However colony forming unit’s ml-1 were observed and absorbance taken at 600 nm using UV visible spectrophotometer (Lamda 35, Perkin elmer, USA).
Ethanol Estimation by Colorimetric Method Alcohol was estimated colorimetrically according to method [9]. Fermentation Efficiency Fermentation efficiency calculated according to the method of Bhandari [10] using the formula:
Ethanol Tolerance of Yeast Strain Fermentation efficiency ¼ For ethanol tolerance of selected yeast strain YGM1 was carried out in YPD broth amended with varying concentrations of absolute alcohol (0, 3, 6, 9, 12, 15, and 18 %). The yeast culture was inoculated in 250 ml YPD broth and incubated in shaker at 80 rpm and 37 °C. The initial inoculum was kept at 104 cfu ml-1 liquid medium. The samples were withdrawn at an interval of 24 h till 144 h. Fermentation Experiment Log phase yeast culture, YGM1 was inoculated in 250 ml YPD broth then the substrates were used in four different combinations. Apple pomace mixed with molasseses in different ratios; pomace: 100 %, pomace: molasses = 75 %:25 %, pomace: molasses = 25 %:75 % and molasses: 100 %, as substrate for fermentation. Fermentation was carried out at fixed parameters, temperature (37 °C), agitation speed (40 rpm), dilution range of substrate (1:10), sample size (50–100 g), pH range (4.0–5.0) for 6 days and samples were withdrawn at regular intervals i.e., 0, 24, 48, 72, 96, 120, 144 h observation. Apple pomace was treated with enzyme, a-amylase at the rate of 0.3 g 600 ml-1 because it increases utilizable sugar in apple pomace whereas molasses was used untreated. Fermentation was carried out to see the effect of fixed
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Actual yield ð%Þ 100 Theoritical yield
Results Temperature Tolerance of Yeast Strain by Measurement of Growth Pattern Growth pattern of yeast strain, YGM1 and YGM2 at temperatures 25, 28, 31, 34, 37, and 40 °C was observed in YPD broth. As shown in (log cfu vs. time and absorbance vs. time), YGM 1 at 25, 28, 31, 34, 37 °C required 24 h to achieve log phase, showed 4.3, 5.2, 5.09, 4.5 and 4.9 log cfu ml-1 and showed absorbance 0.25, 1.2, 0.75, 0.6 and 0.62 at 600 nm respectively. Log phase lasted for 48 h at 28, 31, and 34 °C showed 5.1, 5.09, 5.0 respectively. At 25 and 37 °C it continued up 72 h showed 5.7 log cfu ml-1 at 37 °C. At 40 °C in terms of log cfu 0.08 ml-1, the growth was very slow. At 25 °C after 96 h the decrease in cell numbers was slow showed 1.3 log cfu ml-1 whereas, at 28, 31, 34 and 37 °C there was an abrupt decline showed 1.4, 1.42, 1.44, and 1.47 log cfu ml-1. For yeast strain YGM2, at 25 °C log phase was achieved at about 30 h observed 4.4 log cfu ml-1 and absorbance 0.25 at 600 nm, but lasted for about 84 h (2.3 log cfu ml-1). At 28, 31, 34, 37 and 40 °C required less than
Optimization of Various Parameters for Utilization
24 h to achieve log phase showed 5.5, 5.2, 4.8, 4.7, & 2.81 log cfu ml-1 and absorbance 1.0, 0.5, 0.6, 0.62, 0.25 at 600 nm which lasted till 50 h. At 28, 31, 34 and 37 °C stationary phase lasted from 50 to 96 h and then there was abrupt decline in cell numbers. At 25 °C stationary phase was very short. At 40 °C the decline in cell numbers (1.9 log cfu ml-1) started at 60 h and continued till 120 h.
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observed till 24 h only after which, there was gradual decline. In the initial stages of fermentation the increase in cell count indicated the cell multiplication, but as the anaerobic condition was created by evolution of carbon dioxide, the growth decreases gradually. Total viable cell decreased which was due to exhaustion of nutrients and increase in amount of ethanol at the later stages of fermentation.
Ethanol Tolerance of Yeast Strain Sugar Utilization/Residual Sugar Thus, tolerance of yeast strain was observed at 0, 3, 6, 9, 12, 15 and 18 % ethanol concentration. Yeast strain YGM1 at 0 % ethanol achieved log phase at 24 h which lasted till 96 h. In this case stationary phase continued till 120 h. At 3, 6, 9, 12 and 15 % log phase is achieved within 24 h but growth represented as number of cells decreased with increase in ethanol concentration. At 3 %, log phase lasted till 72 h, after which there was steady decline. At 6 %, log phase lasted till 96 h and then there was sharp decline (data not shown). Solid State Fermentation Fermentation studied in different substrate combinations was performed using strain YGM1 at 37 °C and 40 rpm for 6 days. The progress of fermentation was monitored by measuring change in pH, recording amount of sugar utilized, residual sugar and amount of ethanol produced. Culture growth during fermentation was observed by measuring optical density at 600 nm and counting corresponding values of colony forming units (cfu) ml-1 of fermentation brew. pH The change in pH during growth is represented in Table 1. Growth of YGM1 in different substrate combinations resulted in gradual decrease in pH. From initial pH of 4.0 it declined to pH 3.13 at 72 h in 100 % pomace. In 25 % pomace: 75 % molasses, pH decreased from 4.8 to 3.78 till 72 h. It decreased from initial pH of 4.3–3.71 at 72 h in 75 % pomace: 25 % molasses (Table 1). Cell Count Initial cell count was 5 9 104 cfu ml-1 in 100 % pomace, 9 9 104 cfu ml-1 in 75 % pomace: 25 % molasses, 7 9 104 cfu ml-1 in 25 % pomace : 75 % molasses and 7 9 103 ml-1 in 100 % molasses. At 72 h colony forming units increased to 3.12 9 107 cfu ml-1 in 100 % pomace, to 4.3 9 106 in 75 % pomace and 25 % molasses. In 25 % pomace and 75 % molasses cfu was observed till 48 h whereas in 100 % molasses increase in colony forming was
Amount of sugar utilized and the corresponding values of residual sugar, in different substrate combinations, are presented in table. In 100 % pomace the initial sugar concentration was 12.73 % which decreased to 3.16 % at 120 h. In 75 % pomace: 25 % molasses initial sugar was 15.08 % and decrease to 3.70 %, in 25 % pomace: 75 % molasses it decreased from 18.18 to 13.05 % and from 21.50 to 18.74 % in case of 100 % molasses At 72 h of fermentation the maximum sugar utilization of 11.31 % was observed in 75 % pomace: 25 % molasses (Table 1). At 72 h the minimum utilization of sugar, 2.73 %, was observed in 100 % molasses. Better sugar utilization was observed in 100 % pomace and pomace in combination of molasseses in the ratio of (3:1). Hence, both could be used as substrates for alcoholic fermentation but latter is better of the two. Sugar utilization was affected by initial sugar concentration and pH both. Ethanol Production In 100 % pomace maximum amount of ethanol, 4.8 % was produced at 72 h, after which it decreases to 3.98 % at 120 h. Similarly in pomace : molasses (3:1) maximum ethanol production was 5.02 % at 72 h which then gradually decreased to 4.58 % at 120 h. In pomace: molasses (1:3) maximum ethanol 2.8 % was attained at 48 h; there was gradual decrease till 96 h and then abrupt decline was observed as given in table. In 100 % molasses maximum ethanol, 2.33 % was achieved at 24 h; there was gradual decline till 72 h and then sharp decline was observed. Ethanol production in relation to residual sugar was studied. The decrease in residual sugar resulted in increased production of ethanol (Table 1). Pomace (100 %) and pomace: molasses (3:1) were better substrates for ethanol production as is evident by amount of ethanol produced and decreased residual sugar levels, latter is better of the two. The results obtained are in confirmation with earlier reports. Determination of Fermentation Efficiency Table shows fermentation efficiency of yeast strain in different substrate containing 100 % Pomace, 75 %
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Table 1 Fermentation efficiency of yeast strain in different substrate combination Substrate 100 % pomace
75 % P: 25 % M
25 % P: 75 % M
100 % molasses
Time (h)
Residual sugar (%)
SD
0
12.73
±0.02
24
8.17
48
5.32
72
Utilized sugar (%)
SD
Ethanol (%)
SD
pH change
SD
Fermentation efficiency (%)
SD
0.00
±0.00
0.04
±0.01
4.00
±0.10
00.61
±0.01
±0.21
4.56
±0.01
1.98
±0.02
3.20
±0.02
30.43
±0.01
±0.30
7.41
±0.03
2.30
±0.02
3.15
±0.01
35.35
±0.01
3.67
±0.13
9.06
±0.02
4.80
±0.01
3.13
±0.01
73.77
±0.01
96
3.64
±0.02
9.09
±0.01
4.00
±0.10
3.48
±0.01
61.49
±0.01
120
3.61
±0.08
9.12
±0.03
3.98
±0.05
3.67
±0.02
61.17
±0.01
144 0
3.59 15.08
±0.20 ±0.05
9.14 0.00
±0.06 ±0.00
3.96 0.80
±0.01 ±0.03
3.80 4.30
±0.02 ±0.01
60.86 10.38
±0.02 ±0.01
24
10.03
±0.09
5.05
±0.02
3.13
±0.02
4.06
±0.02
40.61
±0.01
48
6.21
±0.05
8.87
±0.03
4.39
±0.02
3.86
±0.01
56.95
±0.01
72
3.77
±0.14
11.31
±0.03
5.02
±0.01
3.71
±0.02
65.13
±0.01
96
3.72
±0.20
11.36
±0.02
4.60
±0.10
3.98
±0.02
59.69
±0.02
120
3.70
±0.10
11.38
±0.01
4.58
±0.01
3.69
±0.01
59.42
±0.02
144
3.69
±0.12
11.39
±0.02
4.11
±0.01
4.36
±0.02
53.32
±0.01
0
18.18
±0.25
0.00
±0.00
0.04
±0.01
4.80
±0.01
00.22
±0.01
24
14.44
±0.03
3.74
±0.02
1.78
±0.01
4.74
±0.01
19.16
±0.01
48
13.49
±0.11
4.69
±0.10
2.80
±0.06
4.16
±0.01
30.13
±0.01
72
13.16
±0.05
5.02
±0.02
1.98
±0.01
3.83
±0.02
21.30
±0.01
96
13.08
±0.05
5.10
±0.02
1.80
±0.05
3.84
±0.01
19.37
±0.02
120
13.05
±0.04
5.13
±0.02
0.99
±0.01
3.80
±0.01
10.65
±0.02
144
13.05
±0.08
5.15
±0.02
0.79
±0.01
3.78
±0.01
8.50
±0.03
0 24
21.50 18.82
±0.05 ±0.14
0.00 2.68
±0.00 ±0.02
0.08 2.33
±0.01 ±0.02
5.00 4.89
±0.02 ±0.02
00.73 21.20
±0.01 ±0.01
48
18.78
±0.09
2.72
±0.02
2.07
±0.01
4.52
±0.01
18.83
±0.02
72
18.77
±0.09
2.73
±0.02
1.89
±0.03
3.98
±0.01
17.20
±0.01
96
18.75
±0.03
2.75
±0.02
0.73
±0.02
4.02
±0.01
6.64
±0.01
120
18.75
±0.01
2.75
±0.01
0.69
±0.01
4.00
±0.07
6.28
±0.02
144
18.74
±0.01
2.77
±0.02
0.65
±0.01
4.00
±0.06
5.915
±0.00
Boldface data represents highest values of different parameters
Pomace: 25 % molasses, 25 % Pomace: 75 % molasses and 100 % molasses at different time interval was observed. The maximum fermentation efficiency was observed 73.77 % in case of 100 % Pomace at 72 h. In case 75 % Pomace: 25 % molasses of 65.13 % fermentation efficiency was observed at 72 h. The minimum 21.20 % fermentation efficiency was observed in case of 100 % molasses.
Discussion Commercial alcoholic fermentation takes place at high temperature so yeast strains suitable for fermentation should be able to grow at high temperature. They were screened for their tolerance to temperature. The strain YGM 1 was less sensitive to temperature as is evident from results. The strain YGM1, at 28, 31, 34 and 37 °C,
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achieved log phase early, 24 h, which continued for longer period, till 72 h. Better growth results in increased biomass which is directly related to ethanol producing efficiency of strains [11]. Thus strain YGM1 was selected for further studies. Ethanol tolerance is an important characteristic for the selection of strain for ethanol production along with strain stability during fermentation. Ethanol tolerance was represented as ethanol concentration which completely suppressed the growth of culture [12]. The results show that growth of yeast strain YGM1 was good in YPD broth supplemented with 3 and 6 % absolute alcohol as compared to higher concentration. Ethanol inhibits the growth and causes inactivation of enzymes such as hexokinase [13], and dehydrogenase [14]. Inhibition of growth leads to less biomass of yeast which is directly related to ethanol production. Enzymes, hexokinase and dehydrogenase play an important role in glycolysis. Based on these observations it is concluded that strains YGM1 could tolerate 6 %
Optimization of Various Parameters for Utilization
absolute alcohol. Just before fermentation starts, pomace contained malic acid which affects the pH and fermentation rate [15]. High pH value favours the formation of glycerol instead of alcohol [16]. During fermentation, variations in acidity and pH might be due to differential utilization of malic acid and production of varying proportion of volatile acid due to metabolic activity of YGM1. During fermentation pH of substrate first decreased and then increased. The initial fall in the pH is due to accumulation of acidic intermediates which were formed during growth phase of yeast cells. These intermediates were soon utilized and converted to ethanol hence, pH increased. Similar observations have been reported earlier also. Best ethanol yields are obtained at initial pH range of 4.5–4.7 as the buffering capacity of the medium is relatively high [17] whereas, at higher pH values production of glycerol and organic acids are favoured. The reported range for maximum alcohol production is pH 3.2–3.6 by wine yeasts [18]. Better growth of yeast results in increased biomass which is directly related to ethanol producing efficiency of strains [11]. The change in the viable count of yeast cells during fermentation is represented in table. Higher ethanol yields are obtained when sugar concentration is in the range of 9-15 %. Higher initial sugar level could be limiting for growth of yeast cell [5].
Conclusion YGM1 was tolerant towards higher temperature as compared to YGM2. Among various substrate combinations sugar utilization was maximum (11.38 %) and pH decline (4.3-3.71) was favourable for ethanol production in 75 % Pomace:25 % Molasses (5.02 %) and fermentation efficiency were 65.13 % at 72 h of intervals. Acknowledgments
This work was finantially supported by AICRP.
Conflict of interest The author declare that there are no conflicts of interest.
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