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Ethanol production from crude whey by Kluyveromyces marxianus
Biochemical Engineering Journal 27 (2006) 295–298
Short communication
Ethanol production from crude whey by Kluyveromyces marxianus Salman Zafar, Mohammad Owais ∗ Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India Received 25 January 2005; received in revised form 31 May 2005; accepted 31 May 2005
Abstract Kluyveromyces marxianus strain MTCC 1288 was employed to study the batch kinetics of ethanol and biomass production from crude whey. The yeast was able to metabolize most of the lactose within 22 h to give 2.10 g L−1 ethanol and 8.9 g L−1 biomass. The growth rate reached the peak value of 0.157 h−1 during the exponential phase but decreased significantly after the fermentation time of 12 h, presumably due to product inhibition. The specific ethanol formation rate attained the maximum value of 0.046 h−1 between 6 and 8 h of batch fermentation. The relationship between ethanol concentration and specific growth rate suggested a strong inhibitory effect of ethanol on the specific culture growth rate. © 2005 Published by Elsevier B.V. Keywords: Fermentation; Whey; Ethanol; Batch kinetics; Biomass
1. Introduction Cheese whey represents an important source of environmental pollution due to its enormous global production rate (to make 1 kg of cheese, 9 kg whey is generated) and high organic matter content, exhibiting the BOD and COD values of 50 and 80 g L−1 , respectively [9]. One of the most attractive options to check the proliferation of whey pollution is its bioconversion to ethanol employing yeast, especially Kluyveromyces species. The presence of lactose as the only fermentable carbohydrate in whey confines its use to selective fermentations involving microorganisms which are capable of breaking down lactose with the enzyme, -galactosidase [2–4]. Apart from lactose, whey also contains vitamins and minerals which may improve the physiological activity of the cells. Ethanol has tremendous applications in chemical, pharmaceutical and food industries in the form of raw material, solvent and fuel. The annual production of industrial ethanol is about four million tonnes, 80% of which is produced by fermentation. It is very important to choose a strain with suitable ∗
Corresponding author. Tel.: +91 571 2404025; fax: +91 571 2721776. E-mail address: owais [email protected] (M. Owais).
1369-703X/$ – see front matter © 2005 Published by Elsevier B.V. doi:10.1016/j.bej.2005.05.009
physiological characteristics to achieve a good utilization of lactose from whey. The main aspect of this investigation is to study the formation of ethanol from crude whey with Kluyveromyces marxianus leading to maximum lactose utilization and ethanol. Although a great deal of research has been accomplished with the cultures of the yeast strains, such as K. marxianus, Kluyveromyces lactis, Kluyveromyces fragilis, Candida pseudotropicalis, Candida intermedia and Torula cremoris on deproteinized whey [1,2,4,7,8], the use of crude whey as a culture media has not been explored in a comprehensive manner.
2. Materials and methods 2.1. Culture medium Crude (non-deproteinized, non-diluted and non-sterilized) whey was used as a culture medium. It contained 3.45% lactose and was fortified with 0.45% (NH4 )3 SO4 , 0.1% yeast extract, 0.1% malt extract and with a trace element solution containing: CuSO4 ·5H2 O, 0.3 mL L−1 ; MnSO4 ·H2 O, 0.8 mL L−1 ; MoO4 ·2H2 O, 0.4 mL L−1 ; ZnSO4 ·7H2 O,
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S. Zafar, M. Owais / Biochemical Engineering Journal 27 (2006) 295–298
3.0 mL L−1 and FeCl3 ·6H2 O, 4.0 mL L−1 made up with distilled water to 1 L. 2.2. Microorganism and maintenance K. marxianus strain MTCC 1288 was procured from the culture collection of the Institute of Microbial Technology (MTCC), Chandigarh (India). The strain was maintained on agar slant having the following composition: lactose, 20 g L−1 ; bactopeptone, 10 g L−1 ; yeast extract, 5 g L−1 ; agar, 20 g L−1 . A 24-h growth of the yeast was preserved at 4 ◦ C. 2.3. Analytical methods Biomass was measured in terms of dry weight. Yeast cells were harvested by centrifugation for 10 min at 10,000 rpm and then washed twice with distilled water and weighed after 24 h at 100 ◦ C. Lactose concentration was determined by employing the DNS method for reducing sugars [10]. Ethanol was estimated by the dichromate colorimetric method, which is based on the complete oxidation of ethanol by dichromate in the presence of sulphuric acid to form acetic acid [11]. 2.4. Fermentation Batch fermentation was performed in Erlenmeyer flasks in an anaerobic shaker at an agitation of 500 rpm. The temperature was controlled at 34 ◦ C and the pH was maintained at 4.5 by frequent addition of sterile 6N NaOH. Samples were collected at an interval of 2 h. After recording absorbance at 580 nm, the remaining volume of the sample was centrifuged at 4800 rpm for 15 min. The supernatant was stored at 4 ◦ C for lactose and ethanol estimation.
Fig. 1. Experimental kinetics of batch culture of crude whey by Kluyveromyces marxianus at temperature 34 ◦ C and pH 4.5.
the uptake of the substrate. qS exhibited an increase between the fermentation time of 18 and 20 h apparently due to the increased metabolic activity of the cells before their death. qP attained the peak value of 0.046 h−1 during the batch fermentation time of 6 and 8 h (Fig. 4), i.e., during the exponential phase of the process. The increased lactose consumption by the yeast cells during this period may have been responsible for the early upsurge in the production of ethanol.
3. Results and discussions The batch kinetics of ethanol production from crude whey was studied in detail. Fig. 1 shows the batch kinetics of bioconversion of whey to ethanol by K. marxianus. Most of the initial lactose (35 g L−1 ) was metabolized by the yeast within 22 h resulting in the formation of 2.10 g L−1 ethanol. The biomass produced was about 8.9 g L−1 . The growth rate reached a maximum value of 0.157 h−1 during the exponential phase (Fig. 2) but it decreased considerably after the fermentation time of 12 h. This may be attributed to the accumulation of ethanol in the broth leading to product inhibition. The peak specific substrate consumption rate was observed within the fermentation time of 10 h, which is an indication of maximum substrate utilization during the exponential phase (Fig. 3). The decrease in lactose consumption at 6 and 8 h of fermentation may have been the consequence of accumulation of lactose into the cell, thereby reducing
Fig. 2. Variation of specific growth rate with time for batch culture of crude whey by Kluyveromyces marxianus.
S. Zafar, M. Owais / Biochemical Engineering Journal 27 (2006) 295–298
297
Fig. 5. Relationship between specific growth rate and ethanol formation for batch fermentation of whey by Kluyveromyces marxianus. Fig. 3. Variation of specific substrate consumption rate with time for batch culture of crude whey by Kluyveromyces marxianus.
Fig. 5 shows the relationship between P and µ which reflects a strong inhibitory effect of ethanol on the specific growth rate [6]. But it is important to realize that the plotted relationship is based on an actual observed behaviour and does not represent the effect of ethanol alone (organic compounds like glycerol, lactic acid, etc. are also produced during fermentation of whey). The relationship between qS and S has been shown in Fig. 6. The overall lactose consumption represents a combination of sugar consumed for different cellular functions including growth and for the production of ethanol and other
Fig. 6. Relationship between substrate utilization rate and lactose consumption for batch fermentation of whey by Kluyveromyces marxianus.
products. The scatter of the points in Fig. 6 suggest that the lactose consumption relationship can be mathematically described by a combination of rate equation for linear relationship and hyperbolic relationship between sub-process rates and substrate concentration as described by the following equation. dS S = k1 SX + k2 X dt KS + S
(1)
4. Conclusions Fig. 4. Variation of specific product formation rate with time for batch culture of whey by Kluyveromyces marxianus.
The relationship between P and µ as measured after the lag phase in batch cultures suggested a strong inhibitory effect
S. Zafar, M. Owais / Biochemical Engineering Journal 27 (2006) 295–298
298
of ethanol on the specific growth rate of yeast cells. Further improvement in ethanol yield may be obtained by employing suitable nutrient feeding strategy and studying the influence of initial lactose concentration on selective ethanol production. The cellular growth rate and product formation rate reached a maximum value of 0.157 and 0.046 h−1 , respectively, during the exponential phase which is an indication of maximum substrate consumption within this period. The results obtained in this investigation have a great importance in processes based on batch mode of operation since the rate of lactose fermentation by K. marxianus is reduced by sugar concentrations above 2% and by the accumulation of ethanol in the fermentation broth [5]. This raises new perspectives for alcoholic fermentation of whey by Kluyveromyces sp. since financially viable strategy for disposal of whey continues to be a big challenge for the cheese industry.
Acknowledgements We wish to express thanks to Prof. M. Idrees, Chairman, Department of Chemical Engineering (A.M.U., Aligarh) and Prof. M. Saleemuddin, Coordinator, Interdisciplinary Biotechnology Unit (A.M.U., Aligarh) for the material and financial help for the completion of this study.
Appendix A. Nomenclature
k1 k2 KS P
constant constant saturation constant ethanol concentration
qP qS S X
specific product formation rate specific substrate consumption rate lactose concentration biomass concentration
Greek letter µ specific growth rate
References [1] D. Barba, F. Beolchini, G.D. Re, G.D. Giacomo, F. Veglio, Kinetic analysis of Kluyveromyces lactis fermentation of whey: batch and fed-batch operations, Process Biochem. 36 (2001) 531–536. [2] C. Compagno, B.M. Ranzi, A. Tura, E. Martegani, Bioconversion of lactose/whey to fructose diphosphate with recombinant Saccharomyces cerevisiae cells, Biotechnol. Bioeng. 42 (1993) 393–400. [3] L. Dominguez, L. Nelson, J.A. Teixiera, Alcohol production from cheese whey permeate using genetically modified flocculent yeast cells, Biotechnol. Bioeng. 72 (2000) 507–514. [4] S. Grba, T. Vesna, D. Stanzer, Selection of yeast strain Kluyveromyces marxianus for alcohol and biomass production on whey, J. Chem. Biochem. Eng. 45 (1998) 24–32. [5] W.V. Guimaraes, Fermentation of sweet whey by ethanologenic Escherichia coli, Biotechnol. Bioeng. 40 (1999) 41–45. [6] G.H.T. Luong, Kinetic of ethanol inhibition in alcohol fermentation, Biotechnol. Bioeng. 27 (1985) 280–285. [7] A. Michel, F. Jacob, J. Perrier, S. Poncet, Yeast production from crude sweet whey, Biotechnol. Bioeng. 30 (1987) 780–783. [8] D. Porro, E. Martegani, B.M. Ranzi, L. Alberghina, Lactose/whey utilization and ethanol production by transformed S. cerevisiae cells, Biotechnol. Bioeng. 39 (1992) 799–805. [9] U. von Stocker, I.W. Marison, Unconventional utilization of whey in Switzerland, in: T.K. Ghose (Ed.), Bioprocess Engineering, IRS Press, Oxford, United Kingdom, 1993, pp. 330–364. [10] K. Tasun, P. Ghose, K. Ghen, Sugar determination of DNS method, Biotechnol. Bioeng. 12 (1970) 921. [11] M.B. William, D. Reese, Colorimetric determination of ethyl alcohol, Anal. Chem. 22 (1950) 1556.
Short communication
Ethanol production from crude whey by Kluyveromyces marxianus Salman Zafar, Mohammad Owais ∗ Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India Received 25 January 2005; received in revised form 31 May 2005; accepted 31 May 2005
Abstract Kluyveromyces marxianus strain MTCC 1288 was employed to study the batch kinetics of ethanol and biomass production from crude whey. The yeast was able to metabolize most of the lactose within 22 h to give 2.10 g L−1 ethanol and 8.9 g L−1 biomass. The growth rate reached the peak value of 0.157 h−1 during the exponential phase but decreased significantly after the fermentation time of 12 h, presumably due to product inhibition. The specific ethanol formation rate attained the maximum value of 0.046 h−1 between 6 and 8 h of batch fermentation. The relationship between ethanol concentration and specific growth rate suggested a strong inhibitory effect of ethanol on the specific culture growth rate. © 2005 Published by Elsevier B.V. Keywords: Fermentation; Whey; Ethanol; Batch kinetics; Biomass
1. Introduction Cheese whey represents an important source of environmental pollution due to its enormous global production rate (to make 1 kg of cheese, 9 kg whey is generated) and high organic matter content, exhibiting the BOD and COD values of 50 and 80 g L−1 , respectively [9]. One of the most attractive options to check the proliferation of whey pollution is its bioconversion to ethanol employing yeast, especially Kluyveromyces species. The presence of lactose as the only fermentable carbohydrate in whey confines its use to selective fermentations involving microorganisms which are capable of breaking down lactose with the enzyme, -galactosidase [2–4]. Apart from lactose, whey also contains vitamins and minerals which may improve the physiological activity of the cells. Ethanol has tremendous applications in chemical, pharmaceutical and food industries in the form of raw material, solvent and fuel. The annual production of industrial ethanol is about four million tonnes, 80% of which is produced by fermentation. It is very important to choose a strain with suitable ∗
Corresponding author. Tel.: +91 571 2404025; fax: +91 571 2721776. E-mail address: owais [email protected] (M. Owais).
1369-703X/$ – see front matter © 2005 Published by Elsevier B.V. doi:10.1016/j.bej.2005.05.009
physiological characteristics to achieve a good utilization of lactose from whey. The main aspect of this investigation is to study the formation of ethanol from crude whey with Kluyveromyces marxianus leading to maximum lactose utilization and ethanol. Although a great deal of research has been accomplished with the cultures of the yeast strains, such as K. marxianus, Kluyveromyces lactis, Kluyveromyces fragilis, Candida pseudotropicalis, Candida intermedia and Torula cremoris on deproteinized whey [1,2,4,7,8], the use of crude whey as a culture media has not been explored in a comprehensive manner.
2. Materials and methods 2.1. Culture medium Crude (non-deproteinized, non-diluted and non-sterilized) whey was used as a culture medium. It contained 3.45% lactose and was fortified with 0.45% (NH4 )3 SO4 , 0.1% yeast extract, 0.1% malt extract and with a trace element solution containing: CuSO4 ·5H2 O, 0.3 mL L−1 ; MnSO4 ·H2 O, 0.8 mL L−1 ; MoO4 ·2H2 O, 0.4 mL L−1 ; ZnSO4 ·7H2 O,
296
S. Zafar, M. Owais / Biochemical Engineering Journal 27 (2006) 295–298
3.0 mL L−1 and FeCl3 ·6H2 O, 4.0 mL L−1 made up with distilled water to 1 L. 2.2. Microorganism and maintenance K. marxianus strain MTCC 1288 was procured from the culture collection of the Institute of Microbial Technology (MTCC), Chandigarh (India). The strain was maintained on agar slant having the following composition: lactose, 20 g L−1 ; bactopeptone, 10 g L−1 ; yeast extract, 5 g L−1 ; agar, 20 g L−1 . A 24-h growth of the yeast was preserved at 4 ◦ C. 2.3. Analytical methods Biomass was measured in terms of dry weight. Yeast cells were harvested by centrifugation for 10 min at 10,000 rpm and then washed twice with distilled water and weighed after 24 h at 100 ◦ C. Lactose concentration was determined by employing the DNS method for reducing sugars [10]. Ethanol was estimated by the dichromate colorimetric method, which is based on the complete oxidation of ethanol by dichromate in the presence of sulphuric acid to form acetic acid [11]. 2.4. Fermentation Batch fermentation was performed in Erlenmeyer flasks in an anaerobic shaker at an agitation of 500 rpm. The temperature was controlled at 34 ◦ C and the pH was maintained at 4.5 by frequent addition of sterile 6N NaOH. Samples were collected at an interval of 2 h. After recording absorbance at 580 nm, the remaining volume of the sample was centrifuged at 4800 rpm for 15 min. The supernatant was stored at 4 ◦ C for lactose and ethanol estimation.
Fig. 1. Experimental kinetics of batch culture of crude whey by Kluyveromyces marxianus at temperature 34 ◦ C and pH 4.5.
the uptake of the substrate. qS exhibited an increase between the fermentation time of 18 and 20 h apparently due to the increased metabolic activity of the cells before their death. qP attained the peak value of 0.046 h−1 during the batch fermentation time of 6 and 8 h (Fig. 4), i.e., during the exponential phase of the process. The increased lactose consumption by the yeast cells during this period may have been responsible for the early upsurge in the production of ethanol.
3. Results and discussions The batch kinetics of ethanol production from crude whey was studied in detail. Fig. 1 shows the batch kinetics of bioconversion of whey to ethanol by K. marxianus. Most of the initial lactose (35 g L−1 ) was metabolized by the yeast within 22 h resulting in the formation of 2.10 g L−1 ethanol. The biomass produced was about 8.9 g L−1 . The growth rate reached a maximum value of 0.157 h−1 during the exponential phase (Fig. 2) but it decreased considerably after the fermentation time of 12 h. This may be attributed to the accumulation of ethanol in the broth leading to product inhibition. The peak specific substrate consumption rate was observed within the fermentation time of 10 h, which is an indication of maximum substrate utilization during the exponential phase (Fig. 3). The decrease in lactose consumption at 6 and 8 h of fermentation may have been the consequence of accumulation of lactose into the cell, thereby reducing
Fig. 2. Variation of specific growth rate with time for batch culture of crude whey by Kluyveromyces marxianus.
S. Zafar, M. Owais / Biochemical Engineering Journal 27 (2006) 295–298
297
Fig. 5. Relationship between specific growth rate and ethanol formation for batch fermentation of whey by Kluyveromyces marxianus. Fig. 3. Variation of specific substrate consumption rate with time for batch culture of crude whey by Kluyveromyces marxianus.
Fig. 5 shows the relationship between P and µ which reflects a strong inhibitory effect of ethanol on the specific growth rate [6]. But it is important to realize that the plotted relationship is based on an actual observed behaviour and does not represent the effect of ethanol alone (organic compounds like glycerol, lactic acid, etc. are also produced during fermentation of whey). The relationship between qS and S has been shown in Fig. 6. The overall lactose consumption represents a combination of sugar consumed for different cellular functions including growth and for the production of ethanol and other
Fig. 6. Relationship between substrate utilization rate and lactose consumption for batch fermentation of whey by Kluyveromyces marxianus.
products. The scatter of the points in Fig. 6 suggest that the lactose consumption relationship can be mathematically described by a combination of rate equation for linear relationship and hyperbolic relationship between sub-process rates and substrate concentration as described by the following equation. dS S = k1 SX + k2 X dt KS + S
(1)
4. Conclusions Fig. 4. Variation of specific product formation rate with time for batch culture of whey by Kluyveromyces marxianus.
The relationship between P and µ as measured after the lag phase in batch cultures suggested a strong inhibitory effect
S. Zafar, M. Owais / Biochemical Engineering Journal 27 (2006) 295–298
298
of ethanol on the specific growth rate of yeast cells. Further improvement in ethanol yield may be obtained by employing suitable nutrient feeding strategy and studying the influence of initial lactose concentration on selective ethanol production. The cellular growth rate and product formation rate reached a maximum value of 0.157 and 0.046 h−1 , respectively, during the exponential phase which is an indication of maximum substrate consumption within this period. The results obtained in this investigation have a great importance in processes based on batch mode of operation since the rate of lactose fermentation by K. marxianus is reduced by sugar concentrations above 2% and by the accumulation of ethanol in the fermentation broth [5]. This raises new perspectives for alcoholic fermentation of whey by Kluyveromyces sp. since financially viable strategy for disposal of whey continues to be a big challenge for the cheese industry.
Acknowledgements We wish to express thanks to Prof. M. Idrees, Chairman, Department of Chemical Engineering (A.M.U., Aligarh) and Prof. M. Saleemuddin, Coordinator, Interdisciplinary Biotechnology Unit (A.M.U., Aligarh) for the material and financial help for the completion of this study.
Appendix A. Nomenclature
k1 k2 KS P
constant constant saturation constant ethanol concentration
qP qS S X
specific product formation rate specific substrate consumption rate lactose concentration biomass concentration
Greek letter µ specific growth rate
References [1] D. Barba, F. Beolchini, G.D. Re, G.D. Giacomo, F. Veglio, Kinetic analysis of Kluyveromyces lactis fermentation of whey: batch and fed-batch operations, Process Biochem. 36 (2001) 531–536. [2] C. Compagno, B.M. Ranzi, A. Tura, E. Martegani, Bioconversion of lactose/whey to fructose diphosphate with recombinant Saccharomyces cerevisiae cells, Biotechnol. Bioeng. 42 (1993) 393–400. [3] L. Dominguez, L. Nelson, J.A. Teixiera, Alcohol production from cheese whey permeate using genetically modified flocculent yeast cells, Biotechnol. Bioeng. 72 (2000) 507–514. [4] S. Grba, T. Vesna, D. Stanzer, Selection of yeast strain Kluyveromyces marxianus for alcohol and biomass production on whey, J. Chem. Biochem. Eng. 45 (1998) 24–32. [5] W.V. Guimaraes, Fermentation of sweet whey by ethanologenic Escherichia coli, Biotechnol. Bioeng. 40 (1999) 41–45. [6] G.H.T. Luong, Kinetic of ethanol inhibition in alcohol fermentation, Biotechnol. Bioeng. 27 (1985) 280–285. [7] A. Michel, F. Jacob, J. Perrier, S. Poncet, Yeast production from crude sweet whey, Biotechnol. Bioeng. 30 (1987) 780–783. [8] D. Porro, E. Martegani, B.M. Ranzi, L. Alberghina, Lactose/whey utilization and ethanol production by transformed S. cerevisiae cells, Biotechnol. Bioeng. 39 (1992) 799–805. [9] U. von Stocker, I.W. Marison, Unconventional utilization of whey in Switzerland, in: T.K. Ghose (Ed.), Bioprocess Engineering, IRS Press, Oxford, United Kingdom, 1993, pp. 330–364. [10] K. Tasun, P. Ghose, K. Ghen, Sugar determination of DNS method, Biotechnol. Bioeng. 12 (1970) 921. [11] M.B. William, D. Reese, Colorimetric determination of ethyl alcohol, Anal. Chem. 22 (1950) 1556.