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Original article Batch kinetics and modelling of ethanolic fermentation of whey

Salman Zafar,1 Mohammad Owais,2* Mohammed Saleemuddin2 & Sattar Husain1
1 Department of Chemical Engineering, Aligarh Muslim University, Aligarh 202 002, India 2 Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202 002, India (Received 1 April 2004; Accepted in revised form 10 September 2004)

Summary

The fermentation of whey by Kluyveromyces marxianus strain MTCC 1288 was studied using varying lactose concentrations at constant temperature and pH. The increase in substrate concentration up to a certain limit was accompanied by an increase in ethanol formation, for example, at a substrate concentration of 10 g L)1, the production of ethanol was 0.618 g L)1 whereas at 50 g L)1 it was 3.98 g L)1. However, an increase in lactose concentration to 100 g L)1 led to a drastic decrease in product formation and substrate utilization. The maximum ethanol yield was obtained with an initial lactose concentration of 50 g L)1. A method of batch kinetics was utilized to formulate a mathematical model using substrate and product inhibition constants. The model successfully simulated the batch kinetics observed at S0 10 and 50 g L)1 but failed in case of S0 100 g L)1 because of strong substrate inhibition.
Ethanol, inhibition kinetics, kinetic model, Kluyveromyces marxianus, lactose fermentation.

Keywords

Introduction

Because reserves of fossil fuels are dwindling, production of biofuel from microbial sources, using waste by-products as substrates, has acquired strategic signicance in recent years. Ethanol has emerged as one of the most viable options in the arena of non-conventional sources of energy. Ethanol has many applications in the chemical, pharmaceutical and food industries either as a raw material, solvent or fuel. The annual production of industrial ethanol is approximately 4 million tons, 80% of which is produced by fermentation. A global annual growth rate of ethanol consumption of around 5% has been predicted to occur over the next 20 years. Ethanol can be produced from chemical and biochemical processes. The raw material for the biochemical process primarily utilizes agroindustrial wastes like molasses, maize steep liquor, whey, etc.
*Correspondent: Fax: +91 571 2701081; e-mail: owais_lakhnawi@redimail.com doi:10.1111/j.1365-2621.2005.00957.x
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Whey is a major by-product of the dairy industry and its disposal without expensive sewage treatments represents a major source of water pollution. Cheese whey represents an important environmental problem because of the high volumes produced (to make 1 kg of cheese, 9 kg whey is generated) and its high organic matter content. Degradation of whey has a biological oxygen demand (BOD) ranging from 40 to 50 g L)1 and a chemical oxygen demand (COD) of 6080 g L)1 (von Stocker & Marison, 1993). Among various constituents of whey, lactose is the main component responsible for its high BOD and COD. Interestingly, it is a potential substrate for a variety of micro-organisms such as Candida pseudotropicalis, Kluyveromyces marxianus and K. lactis, etc. (Ruggeri, 1988; Porro et al., 1992; Dominguez et al., 1999, 2000). Therefore, the development of a highly productive process for fermenting lactose is of prime importance, as alcoholic fermentation can oer an alternative mode for bioremediation of cheese whey. Besides the basic sugar lactose, whey contains vitamins and minerals, which can improve the physiological

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activity of the yeast cells. To achieve a good utilization of lactose from whey it is especially important to choose a strain with suitable physiological characteristics and to model the kinetics of the process. The eect of initial substrate concentration on culture growth kinetics has not been studied extensively so far. It has also been reported that the rate of lactose fermentation is reduced by sugar concentrations above 2% and by the accumulation of more than 5% ethanol (Guimaraes, 1999). The maximum biotransformation of whey by K. marxianus has been reported by von Stocker & Marison (1993) to be 3035% when using immobilization techniques. In this research, a detailed set of batch kinetic data on fermentation of lactose by K. marxianus has been collected so as to acquire a better understanding of the environmental eects (the substrate availability, in particular) on nal ethanol accumulation during the fermentation process. An important aspect of this study included the development of a mathematical model that can predict suitable nutrient feeding strategies for selective ethanol production. These can then be experimentally implemented to minimize wasteful experimental trials for optimization of ethanol fermentation from lactose (or whey). The objectives of the present investigation were to study the eect of initial substrate concentration on the production of large amounts of biomass and ethanol by fermentation and to develop a mathematical model using the batch kinetic data.
Materials and methods

the Institute of Microbial Technology (MTCC), Chandigarh, India. The yeast was maintained on agar slants having the following composition (g L)1): lactose, 20; bactopeptone, 10; yeast extract, 5; agar, 20. A 24-h growth was preserved at a temperature of 4 C for further use. Inoculum preparation A loop of micro-organism from the slant was transferred into test tubes containing 10 mL sterile liquid medium, which had the same composition as was used for agar slants. The test tubes were incubated at 30 C for 72 h. Then the 200 mL of sterile liquid yeast medium was placed in 1000-mL Erlenmeyer asks and inoculated with 5% of the inocula and the asks were shaken on a rotary shaker at 3000 g at 30 C for 24 h. Subsequent transfer into the bioreactor was done when the biomass concentration in the inoculation asks was about 2.53.0 g L)1. Analytical techniques Biomass concentration was measured in terms of dry weight. Yeast cells were harvested by centrifugation at 200 g for 10 min at 4 C and then washed twice with distilled water and weighed after a 24-h period at 100 C. Lactose was determined by employing the dinitrosalicyclic acid (DNS) method for reducing sugars. 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. Fermentation The composition of the media for alcoholic fermentation was (in g L)1): lactose 10, 50 and 100; yeast extract, 3.0; malt extract, 1.5; (NH4)2HPO4, 2.0; (NH4)2SO4, 2.0; MgSO4, 0.5. Batch fermentation was in Erlenmeyer asks in an anaerobic shaker at initial concentrations of lactose of 10, 50 and 100 g L)1. The temperature was controlled at 34 C and pH was maintained at 4.5 by addition of sterile 6 n NaOH periodically. Samples were collected at an interval of 2 h. After recording the optical density at 580 nm, the remaining volume of the sample was centrifuged
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Chemicals Pure lactose was supplied by LobaChemie, Mumbai, India. Yeast extract, malt extract, bactopeptone, agar, diammonium phosphate, ammonium sulphate, magnesium sulphate were purchased from Hi Media Chemicals, Mumbai, India. All the chemicals were of analytical reagent grade. Micro-organism and maintenance For the alcoholic fermentation of whey, a strain of the yeast K. marxianus was used. Strain MTCC 1288 was procured from the culture collection of

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for 15 min. The supernatant was stored at 4 C for lactose and ethanol estimation.
50

Biomass (E) Lactose (E) Ethanol (E)

Biomass (S) Lactose (S) Ethanol (S)

Results and discussions

45

Effect of initial substrate concentration on the culture growth and product accumulation The batch kinetics of biomass and ethanol production from lactose was studied at dierent initial substrate concentrations (S0 10, 50 and 100 g L)1). Figure 1 shows the kinetics of batch fermentation of whey to ethanol by K. marxianus at S0 10 g L)1. Within 26 h, K. marxianus could metabolize most of the lactose to give biomass and ethanol concentrations of 9.53 and 0.618 g L)1 respectively. Upon increasing the concentration of lactose to 50 g L)1, ethanol yield was increased to 3.98 g L)1 at the end of fermentation while the biomass concentration increased insignicantly to 10.34 g L)1 as shown in Fig. 2. The low increase in biomass concentration can be attributed to the
Biomass (E) Lactose (E) Ethanol (E) 10 9 8 7 6 5 4 3 2 1 0 0 5 15 10 Time (h) 20 25 Biomass (S) Lactose (S) Ethanol (S)

40

Concentrations (g L1)

35 30 25 20 15 10 5 0 0 5 10 15 Time (h) 20 25

Figure 2 Comparison of the experimental (E) and simulated (S) kinetics of batch whey fermentation by Kluyveromyces marxianus at S0 50 g L)1, temperature 34 C and pH 4.5.

Figure 1 Comparison of the experimental (E) and simulated (S) kinetics of batch whey fermentation by Kluyveromyces marxianus at S0 10 g L)1, temperature 34 C and pH 4.5.

death of yeast cells at high ethanol concentrations (above 5%). Surprisingly, further enhancement of initial lactose concentration to 100 g L)1, resulted in a sharp decrease in rate of ethanol formation as well as in biomass production as shown in Fig. 3. The ethanol produced was less than 3.55 g L)1 and the amount of biomass produced was 8.54 g L)1, while the unconverted lactose at the end of fermentation was about 49 g L)1. The rate of formation of ethanol was slightly lower at S0 100 g L)1 as compared with S0 50 g L)1 although the lactose concentration was doubled. This abnormality might be due to the strong negative eect of high lactose concentration on the rate of fermentation. It is also evident from the growth kinetics at dierent substrate concentrations that an S0 of 50 g L)1 is likely to give the best possible ethanol yield in batch fermentation. An S0 lower than 50 g L)1 would result in reduced ethanol yield because of decreased substrate availability and S0 greater than 50 g L)1 would no longer increase ethanol production because of strong substrate

1 Concentrations (g L )

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Biomass (E) Lactose (E) Ethanol (E) 100 90 80

Biomass (S) Lactose (S) Ethanol (S)

Concentrations (g L1)

70 60 50 40 30 20 10 0 0 5 15 10 Time (h) 20 25

Figure 3 Comparison of the experimental (E) and simulated (S) kinetics of batch whey fermentation by Kluyveromyces marxianus at S0 100 g L)1, temperature 34 C and pH 4.5.

inhibition coupled with severe product inhibition of the enzymes responsible for converting lactose to ethanol. Model formulation The usual approach for mathematical modelling of bioreactors considers isothermal systems and is based on a single growth rate with variants of Monod kinetics. The models derived from Monod kinetics are simple in nature and easy to formulate. The second approach is that of structured models in which metabolic changes during cultivation are taken into account. This method results in detailed modelling of the regulating processes but it requires a larger number of measurements of variables. Another possibility is that of representing the system by using structured unsegregated cybernetic modelling, which is an intermediate approach between structured and unstructured models. The cybernetic modelling technique gives an enzyme-coupled model consisting of many

estimated parameters. Longhi et al. (2004) have suggested a novel approach of modelling strategy that evaluates the main metabolic routes for the variables of interest (lactose, oxygen, cell and ethanol) and yields a multi-route, unstructured kinetic model. The production or consumption rates for each route are derived but based on the experimental data. In most cases of inhibition kinetics, the equations are Monod-like relationships derived from theories on the inhibition of a single enzyme. The previous investigations in the modelling of ethanolic fermentation of cheese whey by Kluyveromyces species, based on simplied Monod kinetics, concentrated on two parameters the substrate limitation constant KS, and the yield coecient YX/S. Researchers have neglected the inuence of inhibition kinetics on cheese whey fermentation although it is well known that substrate and product inhibition play an important role for this particular type of fermentation (Guimaraes, 1999). The model proposed here takes into account two new parameters substrate inhibition constant KI,S and product inhibition constant KI,P which are specic for this particular type of fermentation. The inclusion of additional inhibition parameters in the model is based on the fact that the rate of lactose fermentation by K. marxianus is reduced by sugar concentrations above 2% and by the accumulation of more than 5% ethanol in the fermentation broth as shown by Guimaraes (1999). Therefore, we need to take into consideration the substrate inhibition, as well as the product inhibition, when formulating a model of fermentation of whey to ethanol by K. marxianus (Michel et al., 1987; Hill & Robinson, 1990). The batch kinetic data of K. marxianus strain MTCC 1288 at S0 50 g L)1, temperature 34 C and pH 4.5 were utilized for the formulation of the mathematical model. The following simplications were made while developing the mathematical model for batch fermentation of whey by K. marxianus: 1 The pH was known and controlled at a constant value throughout the modelling period. 2 The temperature remained constant during the fermentation process. 3 The substrate of importance was the carbon source (lactose) only.
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4 There was no process limitation by either nitrogen or phosphorus and growth factors (yeast extract, malt extract, etc.) and they are in excess in the fermentation medium. 5 The only product of importance was ethanol. All the other intermediates and by-products were neglected. 6 The conversion of lactose to ethanol took place in the absence of accumulation or consumption of any other intermediate metabolic product. The model developed here incorporates the eect of two inhibition constants substrate inhibition constant, KI,S and product inhibition constant, KI,P. The system of dierential equations summarized below, eqns 16, represents a general mathematical model capable of describing the batch kinetics of alcoholic fermentation of whey by K. marxianus:  llmax S KS S  1 1S=KI;S   KI;P ; KI;P P 1

YP/S denotes yield coecient for product on substrate (kgP kgS)1) and mS denotes maintenance coecient for cells (kgS kgX)1 h)1). Eqn 1 describes the rate of biomass formation featuring Monod-type substrate limitation, the substrate inhibition model of Andrews and the product inhibition model of Jerusalimsky (Moser, 1989; Rehm & Reed, 1995). Eqn 3 represents the rate of substrate consumption which describes the substrate utilized for growth as well as for maintenance. Eqn 5 describes the rate of ethanol formation featuring the relationship proposed by Gaden and co-workers (Aiba et al., 1968; Luong, 1985; Moser, 1989; Blanch & Clark, 1997). The system of dierential equations comprising eqns 2, 4 and 6 describing the batch kinetics of whey fermentation by K. marxianus, was solved using RungeKutta method of the fourth order.
Evaluation of model parameters

dX S lmax dt KS S



1 1S=KI;S



KI;P KI;P P

 X; 2

  1 qS l mS ; YX=S

  h 1  l S  dS 1 KI;P max X dt YX=S KS S 1 S=KI;S KI;P P i mS X ; 4  qP  YP=S l; YX=S

    dP YP=S lmax S 1 KI;P X; 6 dt YX=S KS S 1S=KI;S KI;P P where S is substrate concentration (g L)1), X is biomass concentration (g L)1), P is product concentration (g L)1), l denotes specic growth rate (h)1), lmax denotes maximum specic growth rate (h)1), qS denotes specic substrate utilization rate (h)1), qP is specic product formation rate (h)1), KS is substrate limitation constant (g L)1), KI,S is substrate inhibition constant (g L)1), KI,P is product inhibition constant (g L)1), YX/S is yield coecient for cells on substrate (kgX kgS)1),
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In order to be able to use the general mathematical model developed in the preceding section for computer simulation studies of the bioreactor performance and to verify its capability to describe the fermentation dynamics, numerical values of model parameters used in the model equations have to be determined. The model identication procedure is based on minimizing the deviations between the model predictions and the actual experimental data. A non-linear regression technique was used for this purpose in combination with a set of computer programs, including MathCAD (Mathsoft Engineering and Education Inc., Cambridge, MA, USA) and Polymath (Cache Corporation, Austin, TX, USA). The model equations described previously were simulated on a computer using optimum values of model parameters (Table 1). Figure 2 compares the experimental and simulated batch kinetics of ethanol fermentation at S0 50 g L)1. The model was further tested to simulate the experimental kinetics at S0 10 g L)1 using the model parameters for S0 50 g L)1 (Fig. 1). The experimental data and model simulation shown in Figs 1 and 2 demonstrated the validity of the model. The model was unable to describe the batch kinetics at S0 100 g L)1 because of strong substrate inhibition (Fig. 3).

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Table 1 Model parameters and parametric sensitivity values for batch fermentation (S0 50 g L)1) Model parameters KS (g L)1) KI,S (g L)1) KI,P (g L)1) lmax (h)1) YX/S (g g)1) mS (g g)1 h)1) YP/S (g L)1) Value 16.068 16.096 5.201 0.401 0.219 0.158 0.127 APS 0.009 0.053 )0.157 )1.773 )0.877 3.923 )0.917 RPS 0.124 0.121 0.095 0.215 0.409 0.071 0.101

the overall model formulation (Table 1). Therefore, this parameter should be estimated with extreme care and accuracy. It was necessary to evaluate the results of model identication from the standpoint of data analysis. The F-test was used with a degree of signicance, a 0.05, to check the validity of the model from a statistical point of view. The results of the F-distribution test showed that the model is acceptable under the given set of conditions.
Conclusions

APS, absolute parametric sensitivity; RPS, relative parametric sensitivity.

The degree of signicance of individual parameters was evaluated by the method of parametric sensitivity analysis. The absolute parametric sensitivity (APS), which characterizes the direction in which a selected parameter is acting, was calculated as follows (Goswami & Srivastava, 2000): APS @f Df % ; @ki ki 7

where f is the optimized function and ki is the parameter. The APS characterizes the direction in which the parameter under consideration is acting. Its positive value leads to increased dierence between the model and the experimental data, and vice versa. For a mutual comparison of the calculated parametric sensitivities and the indication of the signicance of individual parameters, the relative parametric sensitivities (RPS) is expressed, dened as follows (Goswami & Srivastava, 2000):     ki @f ki Df  % : 8 RPS  f@ki  fDki  YX/S was found to be the most sensitive parameter indicating the maximum eect of the parameter on

The model was found to be capable of reecting all batch culture phases to a certain degree of accuracy. The parameters of the proposed model were found to be in agreement with values found in the literature. This was particularly true for parameters YX/S and KS. The estimated value of KS was slightly higher than those predicted by Moresi et al. (1990) and Barba et al. (2001). In addition, the estimated value for lmax was lower than that estimated by Moresi et al. (1990), Barba et al. (2001) and Longhi et al. (2004). This disagreement might be attributed not only to a dierent species of Kluyveromyces but also to either dierent operating procedures or dierent modelling strategies (Table 2). It can be concluded that the kinetic model for batch ethanolic fermentation of whey by K. marxianus using substrate and product inhibition constants simulates the microbial growth, substrate consumption and product formation to a good degree of accuracy. The model considers the inhibition of fermentation by lactose as well as ethanol, which have been largely neglected in previous studies. These results will be very useful in the phase of process design of cheese whey
Table 2 Comparative account of kinetic parameters of the proposed model and earlier reports

Parameters lmax 0.401 0.318 0.93 0.43 n.a. 0.55 n.a. n.a KS 16.07 n.a 1.2 9.1 n.a. n.a. n.a. n.a KI,S 16.10 n.a. n.a. n.a. n.a. n.a. n.a. n.a KI,P 5.20 n.a n.a. n.a. n.a. n.a. n.a. n.a YX/S 0.22 0.07 n.a. 0.36 0.52 0.31 0.12 0.45 Micro-organism K. K. K. K. K. K. K. K. marxianus fragilis fragilis lactis fragilis marxianus fragilis fragilis Reference This work von Stocker & Marison (1993) Moresi et al. (1990) Barba et al. (2001) Bernstein et al. (1977) Longhi et al. (2004) Beausejour et al. (1981) Harju et al. (1976)

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utilization, where the main operating conditions have to be established for an ecient fermentation process. The model predictions in the computer process simulation could be used to direct further experimental work on bioreactor performance and optimization in dierent unconventional operating modes. The proposals for future work include studying the inuence of pH on the model parameters, optimization of the operating conditions, generalization of the model to other fermentation processes and implementation of model-based control technologies. It is clear that an alternative experimental strategy needs to be implemented to overcome the problems of limited success of fermentation of lactose in whey permeate directly into ethanol, as the yields and alcohol tolerances of the organisms capable of directly fermenting lactose are low.
Acknowledgments

The authors express their gratitude to Prof. M. Idrees, Chairman, Department of Chemical Engineering (AMU, Aligarh) for providing the necessary facilities to complete this study. This work was supported by FIST-DST programme.
Nomenclature
KI,P KI,S KS mS P qP qS S t X YP/S YX/S l lmax Product inhibition constant (g L)1) Substrate inhibition constant (g L)1) Saturation constant (g L)1) Maintenance coefcient for cells on oxygen substrate (kgS kgX)1 h)1) Product concentration (g L)1) Specic ethanol production rate (h)1) Specic substrate utilization rate (h)1) Substrate concentration (g L)1) Time (h) Biomass concentration (g L)1) Yield coefcient for product on substrate (kgP kgS)1) Yield coefcient for cells on substrate (kgX kgS)1) Specic growth rate of cells (h)1) Maximum specic growth rate of cells (h)1)

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