Cooling System Economy in Ethanol Production Using Thermotolerant Yeast Kluyveromyces Sp. IIPE453
1Sardar Swaran Singh National Institute of Renewable Energy, Kapurthala, India
2Biotechnology Area, Indian Institute of Petroleum, Dehradun, India
3Department of Chemical Engineering, Indian Institute of Technology, Roorkee, India
4SRM Research Institute, SRM University, Kattankulathur, Tamilnadu, India
5Department of Paper Technology, Indian Institute of Technology, Roorkee, Saharanpur Campus, India
The growth of thermotolerant/ thermophilic ethanol producing yeast and the fermentation processes using sugary substrates are exothermic processes. If the fermenters are heat insulated, the requirement of heat for maintaining the fermentation broth for ethanol production may be reduced considerably. The heat generated due to growth of thermotolerant yeast Kluyveromyces sp. IIPE453 was found to be 652kJ mol-1 at 50°C using glucose as a substrate. The heat generated due to ethanol formation by Kluyveromyces sp. IIPE453 was found to be 132.54kJ mol-1 of sugar consumed or 67.84kJ mol-1 of ethanol produced at 50°C using sugarcane molasses as substrate. This heat would be sufficient for maintaining the desired temperature, if insulated fermentation systems are used. Therefore, no additional heat would be required to maintain the temperature in fermentation process by thermotolerant yeast at 50°C.
At a glance: Figures
Keywords: metabolic heat, bioreactor, ethanol fermentation, thermotolerant yeast, cooling water
American Journal of Microbiological Research, 2013 1 (3),
Received March 11, 2013; Revised April 18, 2013; Accepted April 23, 2013Copyright © 2013 Science and Education Publishing. All Rights Reserved.
Cite this article:
- Kumar, Sachin, et al. "Cooling System Economy in Ethanol Production Using Thermotolerant Yeast Kluyveromyces Sp. IIPE453." American Journal of Microbiological Research 1.3 (2013): 39-44.
- Kumar, S. , Dheeran, P. , Singh, S. P. , Mishra, I. M. , & Adhikari, D. K. (2013). Cooling System Economy in Ethanol Production Using Thermotolerant Yeast Kluyveromyces Sp. IIPE453. American Journal of Microbiological Research, 1(3), 39-44.
- Kumar, Sachin, Pratibha Dheeran, Surendra P. Singh, Indra M. Mishra, and Dilip K. Adhikari. "Cooling System Economy in Ethanol Production Using Thermotolerant Yeast Kluyveromyces Sp. IIPE453." American Journal of Microbiological Research 1, no. 3 (2013): 39-44.
|Import into BibTeX||Import into EndNote||Import into RefMan||Import into RefWorks|
Most of the potential ethanologens that are in industrial use belong to mesophilic group (28-35ºC) [1, 2, 3, 4, 5]. However, bioethanol production by thermophiles has certain advantages over mesophiles and it needs to be investigated for ethanol production. Solvent tolerance, energy savings through reduced cooling costs, higher saccharification and fermentation rates, continuous ethanol removal and the reduced risk of contamination have stimulated a search for thermophilic or thermotolerant yeasts [6, 7, 8, 9, 10]. Less energy would be required for mixing of fermentation broth containing sugar, product and cell mass and product recovery in thermophilic fermentations because of lower viscosity, surface tension, higher vapor pressure and increased solubility of organic compounds . Ethanol production at high temperature is effective in tropical countries where average day-time temperatures (30-40°C) are usually high throughout the year [9, 12].
Where, ΔHr is heat generated due to biological reactions (kJ). The bacterium Zymomonas mobilis and yeast Saccharomyces cerevisiae are known to gain the energy required for growth and maintenance under anaerobic conditions by substrate phosphorylation processes through the Entner-Doudoroff pathway and glycolysis, respectively . Theerarattananoon et al.  reported overall metabolic heat generation 43kJ mol-1 glucose consumed or 32kJ mol-1 ethanol produced during fermentation using S. cerevisiae at 25ºC, when initial glucose concentration was kept at 300g l-1 glucose.
The heat loss to environment is negligible in the industrial scale bioreactors because these are operated nearly adiabatically due to much lower surface to volume ratio compared to laboratory-scale bioreactors , requiring huge quantity of cooling water to maintain optimum temperature. Approximately 60-70% of total chilling water load is used in bioreactors for ethanol production by using mesophiles . In tropical countries, the cooling water at 28°C is used to maintain the fermentation temperature at 32°C in bioreactor. Heat generation by exothermic sugar fermentation (Eq.(1)) by thermophilic/thermotolerant microorganisms has an impact on the net heat balance vis-à-vis cooling water requirement in such fermentation process.
In the present paper, the metabolic heat generation on sugar consumption or ethanol production by the thermotolerant yeast Kluyveromyces sp. IIPE453 is reported and the advantage on cooling water requirement over S. cerevisiae is compared.
2. Materials and Methods2.1. Microorganisms and Culture Conditions
A thermotolerant yeast, Kluyveromyces sp. IIPE453 (MTCC 5314)  was grown in Bioflow-110 bioreactor (working volume 5 liters) on medium SM containing g l-1, di-sodium hydrogen ortho phosphate, 0.15; potassium di-hydrogen ortho phosphate, 0.15; ammonium sulphate, 2.0; yeast extract, 1.0; glucose 20. The temperature and pH were controlled at 50°C and 5.0, respectively. The aeration rate in the bioreactor was provided at 1vvm and the agitation was maintained at 350rpm.2.2. Fermentation Conditions
The batch fermentation using the yeast Kluyveromyces sp. IIPE453 was carried out in a Bioflow-110 bioreactor (working volume 2 liters) using molasses as substrate, which contained the initial reducing sugar concentration of 300g l-1 . The components (in g l-1), di-sodium hydrogen ortho phosphate, 0.15; potassium di-hydrogen ortho phosphate, 0.15; ammonium sulphate, 1.0; yeast extract, 1.0 were mixed in the molasses solution and sterilized for 15min at 121°C. The temperature and pH were controlled at 50°C and 5.0, respectively. The agitation was maintained at 200 rpm. The initial dry cell weight (DCW) was kept at 4.5g l-1.2.3. Bioreactor
Bioflow-110 bioreactor of working volume 2 liters was used in the study. The bioreactor vessel was made of glass with internal diameter of 12.8cm, height of broth in the reactor of 17cm and the thickness of glass of 0.3cm, as shown in Figure 1.
3. Theory/Calculation3.1. Heat Generation
The ethanol fermentation process is an exothermic process. As the fermentation progresses (glucose fermentation to ethanol proceeds), the heat generated will raise the temperature of the fermentation broth. To maintain isothermal conditions, the excess heat generated is removed by using cooling coils within the reactor and/or jackets around the reactor with continuous flow of cooling medium (cold/chilled water). In a laboratory, with no fan/ventilator, the heat loss from the bioreactor surface to the surrounding air atmosphere shall take place by natural convection. If the reactor is used for cell growth, then aeration is done at a rate suitable for maintaining growth. In such a case, heat loss to the surrounding shall take place by (i) convective heat transport from the broth by bubbling air, (ii) natural convection from the reactor surface to the surrounding atmosphere, and (iii) heat loss by evaporation of water and ethanol.
The heat generation during growth or fermentation in a batch reactor can be given as:
Where, Q is the heat generated (kJ); m is the mass of fermentation/growth medium solution (kg); Cp is the specific heat (kJ kg-1 K-1); ΔT is the temperature difference of the fermentation broth over a time period (Δt) (K); and Qloss is the total heat loss from the reactor accounting for the heat loss due to aeration (during cell growth operation) and the heat loss from the reactor surface by natural convection. On differentiating Eq. (2) with respect to time, one gets
Where, is the heat generated per unit time (kJ h-1); Cp is considered to be insensitive to temperature change over a small temperature difference (ΔT ≈ 3 K); dT/dt is the slope of the curve between temperature and time, over a given time interval.
With inactive cells in the reactor, the temperature of the reactor will fall with time due to heat loss from the reactor surface to the surrounding.
Therefore, one gets,
The heat losses in a bioreactor during operation at high temperature may take place due to (i) convective heat transport from the broth by bubbling air, (ii) natural convection from the reactor surface to the surrounding atmosphere, and (iii) heat loss by evaporation of water and ethanol. The heat losses due to radiation, conduction and CO2 are assumed to be negligible.
The heat loss due to bubbling air and evaporation can be given as:
Where, is the heat loss due to bubbling air and evaporation (kJ h-1).
The heat loss due to natural convection can be given as :
Where, is the heat loss due to natural convection (kJ h-1); U is the overall heat transfer coefficient (kJ h-1 m-2 K-1); A is the surface area (m2) and ΔT is the temperature difference between the reactor surface and the surrounding atmosphere (K). The overall heat transfer coefficient can be given as:
Where, hb and ha are the convection heat transfer coefficients for broth and air, respectively (kJ h-1 m-2 K-1); Δx is the thickness of wall (m) and λ is the thermal conductivity of material (kJ h-1 m-1 K-1).
For the natural convection, the convection coefficient (ha) can be given as:
Where, Nu is the Nusselt number; λa is the thermal conductivity of the air (kJ h-1 m-1 K-1); and H is the height of the broth level in bioreactor (m). The Nusselt number can be evaluated as reported by Lienhard and Leinhard :
Where, Ra is the Rayleigh number; and Pr is the Prandtl number which can be given as:
Where, g is the gravity constant (m s-2); Tb and Ta are the temperatures of broth and surrounding atmosphere, respectively (K); νa and αa are the kinametic viscosity and thermal diffusivity of air (m2 s-1).
Surface area (A) of bioreactor can be given as:
Where, r and h are the radius and height of the level of broth in cylindrical section, respectively (m); rd and hd are the radius and height of spherical bottom, respectively (m). rd can be calculated as:
4. Results and Discussion
The temperature-time courses were recorded over a period of 20min in lag phase (0 hour) and log phase (12th hour) during the growth of yeast Kluyveromyces sp. IIPE453 at 50oC at 1 vvm aeration rate (Figure 2). The temperature was not controlled during recording of the temperature-time course. As shown in Figure 2, the temperature was decreased with time during lag phase because of high rate of heat loss to surrounding atmosphere. The lower rate of heat loss was observed during log phase than that of lag phase, shows the heat generation due to the growth of yeast. The heat generation due to growth of yeast was calculated using Eq. (6), which was found to be 26.1kJ h-1. The sugar consumption rate in 12th hour of the growth of yeast was found to be 0.04mol h-1. Therefore, the heat generation will be 652kJ mol-1 of sugar consumed during the growth of yeast Kluyveromyces sp. IIPE453 at 50°C.
The temperature-time courses were recorded over a period of 15min between zero and 24th hour during a batch fermentation using yeast Kluyveromyces sp. IIPE453 at 50°C. The temperature was not controlled during recording of the temperature-time course. As shown in Figure 3, the temperature was decreased with time because of high rate of heat loss to surrounding atmosphere at zero hour. The rate of heat loss was lower at 12th hour than that of zero hour, shows the heat generation due to the ethanol fermentation. The heat generation was calculated using Eq. (6), as given in Table 1. Figure 4 shows the heat generation in corresponding to sugar consumption rate and ethanol production rate. The heat generated in ethanol fermentation using Kluyveromyces sp. IIPE453 was found to be 132.54kJ mol-1 of sugar consumed or 67.84kJ mol-1 (2.46MJ l-1) of ethanol produced at 50°C. Theerarattananoon et al.  reported heat generation of 43-66kJ mol-1 of sugar consumed by S. cerevisiae at 25°C, which is almost half of the heat generated during ethanol fermentation using Kluyveromyces sp. IIPE453.
Table 1. Variation in rate of heat generation with rates of sugar consumption and ethanol production at different times during ethanol fermentation using Kluyveromyces sp. IIPE453 at 50ºC
The heat loss due to bubbling air (1 vvm aeration rate) and evaporation was calculated using Eq. (7), which was found to be 36kJ h-1. The surface area of the bioreactor vessel was calculated using Eq. (14) as 0.079m2. The heat loss due to natural convection was calculated using Eqs. (8)-(13) and found to be 187kJ h-1. The kinematic viscosity (νa), thermal diffusivity (αa) and thermal conductivity (λa) of air at surrounding temperature of 25oC are used as 1.595x10-5m2 s-1, 2.371x10-5m2 s-1 and 9.36kJ h-1 m-1 K-1, respectively. The convection heat transfer coefficient (ha), thermal conductivity of glass material (λ) and thickness of wall (Δx) are used as 108kJ h-1 m-2 K-1, 4.32kJ h-1 m-1 K-1 and 0.003m, respectively. In actual practice, the heat loss due to natural convection was calculated from Figure 2, which was found to be 58.7kJ h-1
In case of growth, the total heat loss will be the sum of heat loss due to bubbling air, natural convection and evaporation, which was found to be 94.7kJ h-1. For a growth of Kluyveromyces sp. IIPE453, a run of 24h , the total heat loss will be 2272.8kJ. The metabolic heat generation for 40g (0.222mol) sugar will be 145kJ. There is huge difference between heat loss and heat generation. The heat loss due to natural convection can be minimized using insulation and/or by decreasing S/V ratio. But heat loss due to bubbling air and evaporation cannot be controlled unless air at high temperature introduced in the reactor. The heat loss due to bubbling air and evaporation in 24h will be 864kJ. Therefore, during growth of the yeast the heat input will be required to maintain the desired temperature in the bioreactor.
In case of fermentation, the total heat loss will be due to natural convection, which is found to be 58.7kJ h-1. For a run of ethanol fermentation on molasses in first 32h, the total sugar consumption was 334g or 1.86mol . The total heat loss in 32h will be 1878.4kJ. The metabolic heat generation for 1.86mol sugar will be 212kJ. There is also a huge difference between heat loss and heat generation. The heat loss due to natural convection can be minimized using insulation and/or by decreasing S/V ratio. Therefore, no additional heat would be required to maintain the desired temperature for fermentation in the bioreactor.
In a case study of Indian distillery, fermentation of 400 m3 wort using S. cerevisiae completes in 32h with 8% (v/v) ethanol concentration, which requires cooling water at 25ºC at the rate of 550m3 h-1. Therefore, 550m3 cooling water per m3 of ethanol is required to maintain temperature at 32ºC in a bioreactor. The power requirement for cooling tower and circulating to the bioreactor is ~70Kw h-1 or ~0.07kW l-1 of ethanol produced. For a fermentation run, the cooling water is required for 20h, which will consume ~5.04MJ l-1 of ethanol produced. The energy required for fermentation and distillation is 12.63MJ l-1 of ethanol . Therefore, about 40% of energy utilization can be minimized by eliminating the cooling during ethanol fermentation. The metabolic heat may be sufficient to maintain the temperature of bioreactor at 50ºC.
The metabolic heat generated due to ethanol fermentation by thermotolerant yeast Kluyveromyces sp. IIPE453 was higher than reported for mesophilic yeast S. cerevisiae. Ethanol fermentation using thermotolerant yeast has the advantage over mesophiles in terms of minimization of the cooling water requirement. Therefore, no additional heat may be required to maintain the temperature in fermentation process by thermotolerant yeast at 50ºC. In addition, using thermotolerant yeast for ethanol fermentation power requirement for maintaining temperature in a bioreactor can be minimized, which accounts ~40% of total power requirement in fermentation and distillation using mesophiles. The current study is a lab-scale study and future study on a large-scale is required to validate the results.
We thank Dr M.O. Garg, Director IIP, Dehradun for his valuable suggestion and encouragement to carry out this research work. One of the authors (Sachin Kumar) gratefully acknowledges Senior Research Fellowship awarded by Council of Scientific and Industrial Research (CSIR), India.
|||Kosaric, N, “Ethanol- potential source of energy and chemical products,” In: Rehm HJ, Reed G (eds) Biotechnology, Vol 6, Products of Primary Metabolism, 2nd edn. VCH Publishers Inc, Germany, 121-203, 1996.|
|||Abdel-Fattah, W.R., Fadil, M., Nigam, P. and Banat, I.M, “Isolation of thermotolerant ethanlogenic yeasts and use of selected strains in industrial scale fermentation in an Egyptian distillery,” Biotechnology and Bioengineering, 68 (5), 531-535, Jun.2000.|
|||Lin, Y. and Tanaka, S, “Ethanol fermentation from biomass resources: current state and prospects,” Applied Microbiology and Biotechnology, 69(6), 627-642, Feb.2006.|
|In article||CrossRef PubMed|
|||Phisalaphong, M., Srirattana, N. and Tanthapanichakoon, W, “Mathematical modeling to investigate temperature effect on kinetic parameters of ethanol fermentation,” Biochemical Engineering Journal, 28(1), 36-43, Feb.2006.|
|||Kumar, S., Singh, S.P., Mishra, I.M. and Adhikari, D.K, “Recent advances in production of bioethanol from lignocellulosic biomass,” Chemical Engineering and Technology, 32(4), 517-726, Apr.2009.|
|||Anderson, P.J., McNeil, K. and Watson, K, “High-efficiency carbohydrate fermentation to ethanol at temperatures above 40°C by Kluyveromyces marxianus var. marxianus isolated from sugar mills,” Applied and Environmental Microbiology, 51(6), 1314-1320, Jun.1986.|
|||Lynd, L.R, “Production of ethanol from lignocellulosic materials using thermophilic bacteria: Critical evaluation of potential and review,” In: Fiechter A (ed) Advances in Biochemical Engineering/Biotechnology, Vol 38, Lignocellulosic Materials, Springer, New York, 1-52, 1989.|
|||Banat, I.M., Nigam, P., Singh, D., Marchant, R. and McHale, A.P, “Review: Ethanol production at elevated temperatures and alcohol concentrations: Part I - Yeasts in general,” World Journal Microbiology and Biotechnology, 14(6), 809-821, Nov.1998,|
|||Sree, N.K., Sridhar, M., Rao, L.V. and Pandey, A, “Ethanol production in solid substrate fermentation using thermotolerant yeast,” Process Biochemistry, 34(2), 115-119, Feb.1999.|
|||Koskinen, P.E.P., Beck, S.R., Örlygsson, J. and Puhakka, J.A, “Ethanol and hydrogen production by two thermophilic, anaerobic bacteria isolated from icelandic geothermal areas,” Biotechnology and Bioengineering, 101(4), 679-690, Nov.2008.|
|In article||CrossRef PubMed|
|||Lee, J, “Biological conversion of lignocellulosic biomass to ethanol,” Journal of Biotechnology, 56, 1-24(1), Jul.1997.|
|||Limtong, S., Sringiew, C. and Yongmanitchai, W, “Production of fuel ethanol at high temperature from sugar cane juice by a newly isolated Kluyveromyces marxianus,” Bioresource Technology, 98(17), 3367-3374, May.2007.|
|||Plesser, T., Müller, S.C., Hess, B., Lamprecht, I. and Schaarschmidt, B, “Periodic heat production by oscillating glycolysis in a cytoplasmic medium extracted from yeast,” FEBS Letters, 189(1), 42-44, Sep.1985.|
|||von Stockar, U., Duboc, P., Menoud, L. and Marison, I.W, “On-line calorimetry as a technique for process monitoring and control in biotechnology,” Thermochimca Acta, 300 (1-2), 225-236, Oct.1997.|
|||Meier-Schneiders, M. and Schäfer, F, “Quantification of small enthalpic differences in anaerobic microbial metabolism a calorimetry-supported approach,” Thermochimca Acta, 275(1), 1-16, Apr.1996.|
|||Theerarattananoon, K., Lin, Y.H. and Peng, D.Y, “Metabolic heat evolution of Saccharomyces cerevisiae grown under very-high-gravity conditions,” Process Biochemistry, 43(11), 1253-1258, Nov.2008.|
|||Türker, M, “Development of biocalorimetry as a technique for process monitoring and control in technical scale fermentations,” Thermochimca Acta, 419(1-2), 73-81, Sep.2004.|
|||Chongvatana, P, “Cooling system in ethanol plant with starch base feedstock,” ASHARE Thailand Chapter Journal, 1, 30-33, 2007-2008.|
|||Kumar, S., Singh, S.P., Mishra, I.M. and Adhikari, D.K, “Ethanol and xylitol production from glucose and xylose at high temperature by Kluyveromyces sp. IIPE453,” Journal of Industrial Microbiology and Biotechnology, 36(12), 1483-1489, Dec.2009.|
|In article||CrossRef PubMed|
|||Kumar, S., Singh, S.P., Mishra, I.M. and Adhikari, D.K, “Feasibility of ethanol production with enhanced sugar concentration in bagasse hydrolysate at high temperature using Kluyveromyces sp. IIPE453,” Biofuels, 1(5), 697-704, Sep.2010.|
|||Colombié, S., Malherbe, S. and Sablayrolles, J.M, “Modeling of heat transfer in tanks during wine-making fermentation,” Food Control, 18(8), 953-960, Aug.2007.|
|||Lienhard, J.H. and Leinhard, J.H, A heat transfer textbook, Phlogiston Press Cambridge, Massachusetts, USA, 2004.|
|||Khatiwada, D. and Silveira, S, “Net energy balance of molasses based ethanol: The case of Nepal,” Renewable and Sustainable Energy Reviews, 13(9), 2515-2524, Dec.2009.|