http://www.fmfi-journal.org/paperInfo.aspx?ID=5354 Present study was carried out to obtain single cell oil using fermented apple pomace and banana peel. The variables selected for the final experiments were levels of pH (3.0, 4.0 and 5.0), fermentation time (24, 48 and 72 hrs) and inoculum level (0.8, 1.0 and 1.2 ml). Designed experiments were conducted randomly to reveal the effect of these variables on pH, sugar utilization and single cell oil yield. Fermentation of apple pomace (50%) with banana peel (50%) resulted in maximum single cell oil production (5.87%) by using R. minuta. Statistical analysis resulted in the optimum conditions (pH 3.0, fermentation time 56.4 hrs and inoculum level 1.12 ml) for maximum single cell oil production. The model F- value was found to be highly significant at 1% level of significance in case of pH, utilized sugar and single cell oil yield. Hence, second order model could be fitted to predict all the dependent parameters.
www.fmfi-journal.org Focusing on Modern Food Industry (FMFI) Volume 2 Issue 4, November 2013 170 Optimization of Pomace and Banana Peel Fermentation for Production of Single Cell Oil A. Kulkarni* 1 , A. Singh 2 , B.K Kumbhar 3 , M. Sahgal 4 Department of Post Harvest Process & Food Engineering, College of Technology, GB Pant University of Agri & Technology, Pantnagar 263 145 U.S Nagar, Uttarakhand, INDIA 2 corresponding author e-mail: [email protected]Abstract Present study was carried out to obtain single cell oil using fermented apple pomace and banana peel. The variables selected for the final experiments were levels of pH (3.0, 4.0 and 5.0), fermentation time (24, 48 and 72 hrs) and inoculum level (0.8, 1.0 and 1.2 ml). Designed experiments were conducted randomly to reveal the effect of these variables on pH, sugar utilization and single cell oil yield. Fermentation of apple pomace (50%) with banana peel (50%) resulted in maximum single cell oil production (5.87%) by using R. minuta. Statistical analysis resulted in the optimum conditions (pH 3.0, fermentation time 56.4 hrs and inoculum level 1.12 ml) for maximum single cell oil production. The model F- value was found to be highly significant at 1% level of significance in case of pH, utilized sugar and single cell oil yield. Hence, second order model could be fitted to predict all the dependent parameters. Key words Single Cell Oil; Fermentation; Pomace; Agro Waste Utilization Introduction The concept of single cell oil produced by lipid- producing microorganisms as the supplementary sources of conventional oils and fats has attracted attention since early 1980s. Hence, it is very important to develop new oil resources by using microbes, which offers many advantages compared with traditional methods using animal fat and plant oils. Single cell protein production technologies have arisen as a promising way to solve the problem of worldwide protein shortage. They evolved as bioconversion processes which turned low-value by-products, often wastes, into products with added nutritional and market value and since single cell protein belongs to one of the cheapest protein products in the market, its production is profitable. The ability of the organism to metabolize a wide variety of sugars indicates that other agricultural and food-processing wastes may be able to support its growth and oil production. Various juice industries produce huge quantity of apple pomace, which is notutilized for human consumption. The utilization of such wastes needs to be emphasized to save the resources, and protect the environment from pollution. Various microbiological transformations of apple pomace have been proposed to obtain valuable products i.e. single cell protein, enzymes, aroma compounds, ethanol etc. Apple pomace in combination with other substrates like molasses, orange peel, banana peel etc. can be the potential source to obtain single cell oil. Recycling of agricultural residue can be achieved naturally and artificially by microorganisms. Oleaginous yeasts are often considered for the production of single cell oil. Lipid yields from 0.08 to 0.11 g/g have been observed during batch and continuous cultures of oleaginous yeasts and molds on glycerol (Sajbidor et al., 1988; Chen and Chang, 1996; Papanikolaou and Aggelis, 2002) or on soluble starch (Chen and Chang, 1996; Chen and Liu, 1997) utilized as carbon substrate. Johnson et al. (2002) reported that maximum lipid production (66% w/w dry wt.) in Rhodotorula glutinis utilizing glucose in fed-batch fermentation under N- limiting conditions at 30°C, was at pH 4. The economics of these bioprocesses has become more favourable when zero or negative value waste substrates are utilized as carbon or nitrogen sources (Pan et al., 2009). Food processing wastes might have potential for recycling raw materials or for conversion into useful product of higher value (Rashad and Nooman, 2009). Solid state fermentation for the production of single cell oil using oil free rice bran waste and local isolate of Aspergillus niger as substrate was used. Sodium nitrate at C/N ratio of 1.387 was found to be an effective nitrogen-supplementing source, as it gave the higher biomass yield (Anupama et al. , 2001).
Transcript
www.fmfi-journal.org Focusing on Modern Food Industry (FMFI) Volume 2 Issue 4, November 2013
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Optimization of Pomace and Banana Peel Fermentation for Production of Single Cell Oil A. Kulkarni*1, A. Singh2, B.K Kumbhar3, M. Sahgal4 Department of Post Harvest Process & Food Engineering, College of Technology, GB Pant University of Agri & Technology, Pantnagar 263 145 U.S Nagar, Uttarakhand, INDIA 2 corresponding author e-mail: [email protected] Abstract
Present study was carried out to obtain single cell oil using fermented apple pomace and banana peel. The variables selected for the final experiments were levels of pH (3.0, 4.0 and 5.0), fermentation time (24, 48 and 72 hrs) and inoculum level (0.8, 1.0 and 1.2 ml). Designed experiments were conducted randomly to reveal the effect of these variables on pH, sugar utilization and single cell oil yield. Fermentation of apple pomace (50%) with banana peel (50%) resulted in maximum single cell oil production (5.87%) by using R. minuta. Statistical analysis resulted in the optimum conditions (pH 3.0, fermentation time 56.4 hrs and inoculum level 1.12 ml) for maximum single cell oil production. The model F- value was found to be highly significant at 1% level of significance in case of pH, utilized sugar and single cell oil yield. Hence, second order model could be fitted to predict all the dependent parameters.
Key words
Single Cell Oil; Fermentation; Pomace; Agro Waste Utilization
Introduction
The concept of single cell oil produced by lipid-producing microorganisms as the supplementary sources of conventional oils and fats has attracted attention since early 1980s. Hence, it is very important to develop new oil resources by using microbes, which offers many advantages compared with traditional methods using animal fat and plant oils. Single cell protein production technologies have arisen as a promising way to solve the problem of worldwide protein shortage. They evolved as bioconversion processes which turned low-value by-products, often wastes, into products with added nutritional and market value and since single cell protein belongs to one of the cheapest protein products in the market, its production is profitable. The ability of the organism to metabolize a wide variety of sugars indicates that other agricultural and food-processing wastes may be able to support its growth and oil production.
Various juice industries produce huge quantity of apple pomace, which is notutilized for human consumption. The utilization of such wastes needs to be emphasized to save the resources, and protect the environment from pollution. Various microbiological transformations of apple pomace have been proposed to obtain valuable products i.e. single cell protein, enzymes, aroma compounds, ethanol etc. Apple pomace in combination with other substrates like molasses, orange peel, banana peel etc. can be the potential source to obtain single cell oil. Recycling of agricultural residue can be achieved naturally and artificially by microorganisms. Oleaginous yeasts are often considered for the production of single cell oil. Lipid yields from 0.08 to 0.11 g/g have been observed during batch and continuous cultures of oleaginous yeasts and molds on glycerol (Sajbidor et al., 1988; Chen and Chang, 1996; Papanikolaou and Aggelis, 2002) or on soluble starch (Chen and Chang, 1996; Chen and Liu, 1997) utilized as carbon substrate. Johnson et al. (2002) reported that maximum lipid production (66% w/w dry wt.) in Rhodotorula glutinis utilizing glucose in fed-batch fermentation under N-limiting conditions at 30°C, was at pH 4.
The economics of these bioprocesses has become more favourable when zero or negative value waste substrates are utilized as carbon or nitrogen sources (Pan et al., 2009). Food processing wastes might have potential for recycling raw materials or for conversion into useful product of higher value (Rashad and Nooman, 2009).
Solid state fermentation for the production of single cell oil using oil free rice bran waste and local isolate of Aspergillus niger as substrate was used. Sodium nitrate at C/N ratio of 1.387 was found to be an effective nitrogen-supplementing source, as it gave the higher biomass yield (Anupama et al. , 2001).
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Keeping in mind the importance of food processing wastes and agro wastes, a study has been planned to work on production of single cell oil with the help of fermented apple pomace and banana peel with the specific objective i.e to study the effect of processing and operational parameters for enhanced recovery of single cell oil.
Materials and Methods
Apples (variety: red delicious) were purchased from the local market of Pantnagar, Uttarakhnad, India as per requirement and pomace was made using carver press (Model M-25). Banana peel in combination with apple pomace was also used for single cell oil production as banana peel contains lipid (2.2-10.9%) and rich in polyunsaturated fatty acids, particularly linoleic acid and γ-linolenic acid (Mohapatra, et al., 2010), addition of banana peel may help increase the oil recovery. Preliminary experiments were conducted to select dilution level (1:9, 1:10 and 1:15) of the different combinations of apple pomace and banana peel (25% banana peel and 75% apple pomace, 50% banana peel and 50% apple pomace).
Fermentation experiments were carried out at different fermenting conditions i.e. pH (3.0, 4.0, and 5.0), fermentation time (24, 48, and 72 h) and inoculum level (0.8, 1.0 and 1.2 ml). Fermenting liquor was inoculated with different concentrations of R. minuta, as this species is considered to be oleaginous and accumulates triacyl-glycerol from 20 to 70% of biomass under appropriate cultivation conditions. The samples were withdrawn at regular time intervals (0, 24, 48, 72 h) and analyzed for change in pH, amount of sugar utilized and single cell oil produced. Fermenting liquor was centrifuged at 5000 rpm for 10 min and biomass was dried in oven for 2 h at 70°C. The dried biomass was milled and then solvent extraction was done by using soxtec apparatus to extract single cell oil.
The final experiments were designed by considering the following constant, independent and dependent parameters as reported in Table 1 A and B.
TABLE 1 A CONSTANT PARAMETERS
S. No Parameter Value 1 Temperature of fermentation 30 0 C 2 Agitation 40 rpm 3 Pressing time 60 min 4 Crushing time 1 min. 5 Pressure applied 1-1.2 tonnes 6 Sample size 250 ml
7 Dilution Range 1:10
For apple pomace (50%) in combination with banana peel (50%)
TABLE 1 B INDEPENDENT AND DEPENDENT VARIABLES
Independent variables S. No Parameter Range Levels
Dependent variables 1 pH of each sample 2 Utilized sugar 3 Single cell oil
To find out all possible combinations of parameters, experiments were designed using Response Surface Methodology.
TABLE 2 PROCESS VARIABLES AND THEIR LEVELS
Independent variables Coded Levels
Name Code -1 0 1
Actual Levels
pH X1 3.0 4.0 5.0
Fermentation time (h)
X2 24 48 72
Inoculum level (ml)
X3 0.8 1.0 1.2
The Box Behenken design was used on three independent variables (pH, fermentation time (h) and inoculum level (ml)) at three levels. The experimental design is given in Table 3.
Full second order model was fitted in various responses and independent variables using least square regression analysis. Data analysis was done by using Design-Expert 7.0.0 software. The optimization was also carried out using Design-Expert 7.0.0. Effect of independent variables on the responses was
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interpreted using the models. Contour plots were drawn with the help of SURFER 9.0 to get the range of independent variables for product development.
Results and Discussions
The responses (pH, utilized sugar and single cell oil yield) were observed for different combinations of the experiments as mentioned in Table 3 and results of experiments with apple pomace in combination with banana peel have been reported in Table 4 along with the experiment number. A full second order mathematical model was fitted into each response.
Effect of pH, Fermentation Time and Inoculum Level on the Process
1) pH
During the fermentation of apple pomace in combination with banana peel, the range was found to be 2.86 (Expt. no.14) to 5.02 (Expt. no. 1). During initial stage of fermentation, there could be accumulation of acidic intermediates which were formed during the growth phase of yeast cells. But during the later stage, increase in pH was observed because these intermediates formed during the fermentation were soon utilized and hence pH increased.
2) Sugar Utilization
Observations presented in Table 4 were recorded for the utilized sugar for several combinations of the experiments performed (Expt. 1-17), as mentioned in Table 3. In case of fermentation of apple pomace in combination with banana peel, the utilized sugar ranged from 1.27 to 7.52%. The maximum sugar (7.52%) was utilized in the case of experiment with initial pH 4.0, fermentation time 72 h and inoculum level of 1.2 ml. Minimum sugar (1.27%) was utilized when the sample was kept for 24 h of fermentation at the initial pH of 4.0 and inoculums level 0.8 ml. The reason for this might be the fact that within 24 h, yeast isolates entered lag phase and hence the cell multiplication was observed to be slow as they took some time (24 h) to acclimatize themselves. But after 48 h of fermentation period, the yeast isolates entered the log phase (exponential phase) cell, started multiplying faster and therefore rate of sugar utilization was observed to be faster (up to 72 h of fermentation).
3) Single Cell Oil Yield
The results of yield of single cell oil during fermentation of apple pomace in combination with banana peel are reported in Table 4. Yield of single cell oil varied from 4.14% (Expt. no. 6) to 5.87% (Expt. no. 2). Experiment no. 6 indicated the conditions where sample was treated with 0.8 ml of inoculum, pH level was kept as 4.0 and fermentation time was 24 h, whereas experiment no. 2 implied the fermenting conditions having 1.0 ml of
inoculum, pH level of 5.0 and fermentation time of 72 h. This clearly indicated that an increase in the initial pH and the fermentation time leads to enhanced single cell oil production.
The effect of three independent parameters viz. pH, fermentation time and inoculum level can be observed from the Table 4. The maximum single cell oil yield in the case of fermentation of apple pomace in combination of banana peel was 5.87% when sample was treated with 1.0 ml of inoculum at pH level of 5.0 and after 72 h of fermentation. Similarly, in the case of fermentation time i.e. 72 h, maximum conversion of sugar (7.52%) was observed for the time interval of 48-72 h of fermentation during experiments with apple pomace in combination with banana peel. Sugar utilization during the later stages of fermentation (i.e. 72 h) was found to be maximum because the yeast cells in the log phase of their growth cycle convert the maximum amount of substrate into product.
Development of Second Order Model
A second order response function for three independent variables has the following general form:
Where, β0, βii, βij and Xi, Xj are constants and variables (coded). The experimental data were then analyzed employing multiple regression techniques to develop response functions and variable parameters optimized for best outputs. The regression coefficients of complete second order model and their significance are reported Table 5.
Effect of pH, Fermentation Time and Inoculum Level on pH During Fermentation of Apple Pomace and Banana Peel
In the experiments of fermentation of apple pomace and banana peel, the maximum change (0.16) was obtained in the experiment where the initial pH was 3.0 and the sample was kept for 48 h fermentation with inoculation of 1.2 ml while the corresponding minimum value (0.02) was obtained when the sample was inoculated with 1.0 ml strain of R. minuta at pH 5.0 and the sample was kept for 24 h for fermentation.
Full second order model, Eqn. 1 was fitted to the responses observed for levels of pH and various experimental conditions using multiple regression analysis. Results obtained are given in Table 5. The coefficient of determination (R2) for the regression
∑ ∑ ∑ ∑ = + = = =
+ + + = 3
1 i
2 i ii
3
1 i j j i ij
3
1 i
2
1 i i i 0 X β X X β X β β Y
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model for this parameter was 99.28%, implying that the model could account for 99.28% data. Model was highly significant (p <0.01) with F-value of 106.79. Therefore, second order model was found to be adequate in describing change in pH content.
TABLE 4 EXPERIMENTAL DATA ON SINGLE CELL OIL RECOVERY FROM APPLE POMACE IN COMBINATION WITH BANANA PEEL
Where, X1 is pH, X2 is fermentation time, h and X3 is inoculum level, ml.
Effect of independent variables on pH at linear, quadratic and interactive level was revealed using ANOVA. The results showed that the effect was highly significant (p < 0.01) at linear level. The model F-value was also found to be highly significant at 1% level of significance.
Effect of pH, Fermentation Time and Inoculum Level on the Utilized Sugar During Fermentation of Apple Pomace and Banana Peel
The utilized sugar after fermentation ranged from 1.27 to 7.52%. The maximum sugar (7.52%) utilized indicated the conditions where samples of apple pomace and banana peel was treated with the combination of 1.2 ml of inoculum, pH level was kept at 4.0 and the fermentation period of 72 h while the corresponding process conditions for the minimum utilization (1.27%) were at 0.8 ml inoculated sample kept for 24 h at the initial pH of 4.0.
Full second order model, Eqn. 2 was fitted to the responses for utilized sugar and experimental conditions using multiple regression analysis. The results obtained are reported in Table 6.
The coefficient of determination (R2) for the regression model for utilized sugar was 92.72%, which implied that the model could account for 92.72% data. Model was highly significant (p < 0.01) with F-value as 9.90. Lack of fit was insignificant; therefore, second order model was adequate in describing utilized sugar content.
Where, X1 is pH, X2 is fermentation time, h and X3 is inoculum level, ml.
Effect of independent variables on sugar utilized at linear, quadratic and interactive level was determined. Results showed that the effect was highly significant (p < 0.01) at linear level and (p < 0.05) at quadratic level. The model F-value was also found to be highly significant at 1% level of significance.
Total effect of individual parameter on sugar utilized was calculated using the sequential sum of squares and it was observed that only fermentation time affected the utilization of sugar at 1% level of
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significance.
Effect of pH, Fermentation Time and Inoculum Level on Single Cell Oil Production During Fermentation of Apple Pomace and Banana Peel
Full second order model, Eqn. 3 was fitted to the responses for single cell oil yield and experimental conditions using multiple regression analysis. The results obtained are reported in Table 5. The coefficient of determination (R2) for the regression model for ethanol yield was 83.61%, which implied that the model could account for 83.61% data. Lack of fit was insignificant; therefore, second order model was adequate in describing utilized sugar content.
Where, X1 is pH, X2 is fermentation time, h and X3 is inoculum level, ml.
The single cell oil yield varied from 4.14% to 5.87%. The maximum single cell oil yield of 5.87% indicated the conditions when the sample was inoculated with 1.0 ml of inoculum, pH level was kept at 5.0 and the period of fermentation was 72 h. While the minimum yield (4.14%) implied the conditions of inoculation with 0.8 ml of inoculum, pH level of 4.0 and the fermentation period was 24 h.
Total effect of individual parameter on single cell oil yield was calculated using the sequential sum of squares. There was no significant effect of pH, fermentation time and inoculum level on the parameter.
Optimization of Process Parameters for Fermentation of Apple Pomace and Banana Peel
Optimization of processing conditions was carried out to minimize the pH change from the fermentation using software design expert 7.0.0. The minimum change in pH at the optimum conditions was found to be 0.02. Optimum values of different parameters thus obtained are given in Table 6.
TABLE 6 OPTIMUM VALUE OF PARAMETERS DURING FERMENTATION OF APPLE POMACE AND BANANA PEEL
Value pH, (X1)
Fermentation time, h
(X2)
Inoculum level, ml (X3)
Coded -1 0.35 -0.6 Actual 3 56.4 1.12
In order to show the effect of variables and to determine the operating range for best result, contour
plots were drawn. The contour equations were developed by keeping other two variables either at centre point or optimum value and reported in Table 7 and 8, respectively.
TABLE 7 CONTOUR EQUATION FOR PH AT CENTRE POINT DURING FERMENTATION OF APPLE POMACE AND BANANA PEEL
The contours are shown in Fig. 1 for various combinations of interactive terms either at optimum value or centre point. From the Fig. 1 A1 and 1B1, it was observed that pH value was independent of fermentation time. Fig. 1A2 and 1B2 showed that the pH was independent of inoculum level. From Fig. 1A3 and 1B3 it is clear that as the inoculum level and fermentation time increased, the change in pH was gradually insignificant.
A) At Centre Point A1)
Single cell oil = 5.03+0.055X1+0.48X2+0.098X3+0.021X1X2+0.027X1X3+0.033X2X3+0.24X12+0.23X22-0.30X32 (3)
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A2)
A3)
B) At Optimum Point
B1)
B2)
B3)
FIG. 1 CONTOUR PLOTS FOR PH DURING FERMENTATION OF APPLE POMACE AND BANANA PEEL
Optimization of Process Parameters for Utilized Sugar during Fermentation of Apple Pomace and Banana Peel
Optimization of processing conditions was carried out to maximize the sugar utilization from the fermentation process. The maximum utilized sugar at the optimum conditions was found to be 7.52%. Optimum values of different parameters thus obtained are given in Table 8.
In order to show the effect of variables and to determine the operating range for best result, contour plots were drawn. The contour equations were developed by keeping other two variables either at centre point or optimum value. The contours are shown in Fig. 2 for various combinations of interactive terms either at optimum value or centre point.
A) At Centre Point A1)
A2)
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A3)
B) At Optimum Point
B1)
B2)
B3)
FIG. 2 CONTOUR PLOTS FOR UTILIZED SUGAR DURING
FERMENTATION OF APPLE POMACE AND BANANA PEEL
Optimization of Process Parameters for Single Cell Oil During Fermentation of Apple Pomace and Banana Peel
The maximum single cell oil yield at the optimum conditions was found to be 5.87%. Optimum values of different parameters thus obtained are given in Table 9.
In order to show the effect of variables and to determine the operating range for best result, contour plots were drawn (Fig 3).
A) At Centre Point A1)
A2)
A3)
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B) At Optimum Point B1)
B2)
B3)
FIG. 3 CONTOUR PLOTS FOR SINGLE CELL OIL DURING
FERMENTATION OF APPLE POMACE AND BANANA PEEL
The contours are shown in Fig. 3 for various combinations of interactive terms either at optimum value or centre point. From the Fig. 3 A1 and 3 B1 it is clear that as fermentation time increased, there was increase in single cell oil production. Fig. 3A2 and 3 B2 showed that there was maximum single cell oil production at Saddle point at the centre. From Fig. 3 A3 and 3 B3 it is clear that as the inoculum level and fermentation time was increased, single cell oil production was also increased.
The optimum levels of variables in coded and actual form for fermentation of apple pomace in combination with banana peel have been reported in Table 9 and 10 respectively.
TABLE 9 CONSTRAINTS FOR OPTIMIZATION FOR FERMENTATION PROCESS OF APPLE POMACE AND BANANA PEEL
Name Goal Lower limit
Upper limit
pH Is in range -1 +1 Fermentation time minimize -1 +1
Inoculum level minimize -1 +1
pH minimize 2.86 5.02
Utilized sugar maximum 1.27 7.52 Single cell oil maximum 4.14 5.87
TABLE 10 OPTIMUM LEVELS OF VARIABLES FOR FERMENTATION PROCESS OF APPLE POMACE AND BANANA PEEL
Independent variables Coded levels Actual levels
pH (X1) -1 3
Fermentation time (X2) 0.35 56.40 h Inoculum level (X3) -0.6 1.12 ml
From the above results, it is clear that single cell oil production increased in the case where apple pomace (50%) in combination with the banana peel (50%) was fermented when optimum value pH of sample was kept as 3.0, fermentation time 56.40 h and inoculum level as 1.12 ml.
Conclusions
Through the experimental studies conducted under already described conditions, it could be concluded that fermentation of apple pomace and banana peel resulted in enhanced single cell oil recovery with 5.87% yield when pH was 5.0 and fermentation period was 72 h. Optimum values of parameters from the optimization process for production of single cell oil using apple pomace and banana peel were found to be 3.0 pH, fermentation time 56.4 h and inoculum level 1.12 ml. Initial pH and inoculum level both affected the process of single cell oil production but effect of fermentation time was found to be more significant.
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