Process Biochemistry 40 (2005) 801807

A mathematical model describing the effect of temperature variations on the kinetics of microbial growth in solid-state culture
Farah Diba H. Dalsenter a , Graciele Viccini a , Marcelo C. Barga a , David A. Mitchell a, , Nadia Krieger b
b

Departamento de Bioqu mica e Biologia Molecular, Universidade Federal do Paran, Cx. P. 19046 Centro Politcnico, Curitiba 81531-990 Paran, Brazil Departamento de Qu mica, Universidade Federal do Paran, Cx. P. 19081 Centro Politcnico, Curitiba 81531-990 Paran, Brazil Received 28 November 2003; accepted 7 February 2004

Abstract We present a model for the kinetics of microbial growth that incorporates the inuence of the temperature variations that typically occur during solid-state culture in large-scale bioreactors. The model proposes that the specic growth rate constant depends on the level of an essential component within the biomass, with the rate of the synthesis and denaturation reactions of this component depending on temperature according to the Arrhenius equation. Model predictions were compared to literature data for the growth of Rhizopus oligosporus in three different situations: (1) incubation of cultures at different but constant temperatures; (2) initial incubation of cultures at 37 C, followed by incubation at 50 C; and (3) tray culture. The model agreed reasonably with all three sets of experimental results with the use a single set of parameter values. This approach to modeling has good potential for application in models of solid-state culture bioreactors. 2004 Elsevier Ltd. All rights reserved.
Keywords: Solid-state fermentation; Mathematical modeling; Temperature variations; Fungal growth kinetics; Rhizopus oligosporus

1. Introduction Solid-state culture (SSC) involves the cultivation of microorganisms on moist solid substrates in situations where the continuous phase is a gas, with little or no liquid water in the inter-particle spaces. This culture technique has the potential to produce specic microbial products more efciently than submerged liquid culture. For example, in the production of fungal spores for use as biopesticides, greater spore yields are obtained in SSC and the spores produced in SSC are more robust and more virulent than those produced in submerged liquid culture [1,2]. Despite this potential, very few of the many SSC processes that are studied in the laboratory make the transition to production scale. This is largely due to the difculty in controlling the key process variables in large-scale bioreactors at the optimal values for growth and product formation.
Corresponding author. Tel.: +55-41-361-1658; fax: +55-41-266-2042. E-mail address: davidmitchell@ufpr.br (D.A. Mitchell). 0032-9592/$ see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2004.02.007

In the face of this difculty and the complexity of SSC systems, model-based strategies are required for the design and optimization of operation of large-scale bioreactors [3]. The models must describe not only the mass and energy balances in the bioreactor, which will determine the values of the key process variables, but also the kinetics of microbial growth as functions of these variables. One of the most important variables is the bed temperature, given that its control is one of the major difculties faced at large scale. Many SSC processes involve lamentous fungi, which can suffer damage from continuous mixing, so it is often necessary to operate the bioreactor in such a manner that the bed remains static for long periods. In this case, it is impossible to maintain the bed temperature at the optimum value for growth, and therefore, the microorganism will suffer variations in temperature during the process. For example, during times of peak heat production, the temperature can rise to values 10 C or more above the optimum temperature [4]. The kinetic equation should be capable of describing the effects of these temperature variations on growth.

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The approach that is currently used within models of SSC bioreactors to describe the effect of a varying temperature on growth is inadequate. In this approach, the so-called isothermal approach, various cultures of the microorganism are incubated at different temperatures, with each culture experiencing a constant temperature during the whole growth cycle (Fig. 1). The growth prole obtained at each temperature is analyzed by non-linear regression to extract the parameters of the kinetic equation. In this manner, a set of model parameters is generated for each temperature. Empirical equations are then tted by non-linear regression in order to express each parameter as a function of temperature [5]. Growth models developed using the isothermal approach predict that the growth rate at a particular instant depends solely on the temperature at that instant. However, in reality, the growth rate at a particular instant is inuenced by the temperature regime suffered by the organism before that instant [6]. Different modeling approaches have been developed in the area of food microbiology, in order to describe the effect of temperature variations below the optimum growth temperature. For example, Baranyi and Roberts [7] introduced an arbitrary factor to describe the physiological state of the cells. The value of this factor was expressed as a function of the environmental conditions and the specic growth rate constant within the growth kinetic equation was multiplied by this factor. The current work extends this idea to temperatures above the maximum, where processes for synthesis and degradation of an essential biomass component are expressed as functions of temperature. The model developed is suitable for incorporation into mathematical models describing SSC bioreactor operation.

2. Model development 2.1. Kinetic model The model postulates that growth is controlled by the level within the cell of an essential component, denominated F. The level of this component in the cell is expressed in dimensionless terms, and therefore, varies between 0 and 1, where a value of 1 represents the normal level of the component within a fully healthy cell. The component not only plays a key role in determining the velocity of growth, but also is responsible for its own synthesis (Fig. 2). Further, it suffers thermal denaturation in a rst-order process. The rate of auto-synthesis of the component is controlled by the cell, according to the power-law version of the logistic equation, in an attempt to maintain the level at 1. Therefore, the equation for the level of the component in the cell is dF = kS F(1 F n ) kD F dt (1)

where F is the level of the key component, n the exponent in the power-law version of the logistic equation, t time, and kS and kD are the rate coefcients of the synthesis and denaturation reactions, respectively. These rate coefcients are expressed as functions of temperature according to the Arrhenius equation kS = AS exp kD = AD exp E S R(T + 273) ED R(T + 273) (2) (3)

25 C

30 C

35 C

40 C

45 C

50 C

where AS and AD are the frequency factors for the synthesis and denaturation reactions, respectively, ES and ED are the activation energies for the synthesis and denaturation reactions, respectively, R is the universal gas constant, and T is the temperature ( C). The organism grows according to the logistic equation X dX = FX 1 dt XM (4)

biomass

time adjust the kinetic equation to each curve to determine the value of for each temperature specific growth rate constant (h-1)

where X is biomass content of the solid substrate, XM the maximum possible biomass content and is the specic growth rate constant for a fully healthy cell at the optimum
Auto-synthesis in a regulated process Essential component Losses through thermal denaturation

25

30

35 40 45 50 temperature (C)

adjust an empirical equation to give =f(T)

cell Growth process (the velocity of which depends on the level of the essential component)
Fig. 2. Schematic representation of the kinetic model showing the central role of the essential component in affecting both growth and its own synthesis.

Fig. 1. The so-called isothermal approach to determining the effect of temperature on growth, which has been used to date in the development of kinetic equations for use within models for SSC bioreactors.

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temperature for growth. Therefore, changes in temperature do not change the specic growth rate constant for a perfectly healthy cell; that is, a cell with the normal concentration of the essential component. The effect of temperature on the growth rate of the cell is fully due to the effect of the temperature on the level of the essential component, F. 2.2. Simple model of a tray bioreactor A simple model of a tray bioreactor is used in the comparison of the model predictions with the work of Ikasari and Mitchell [8]. Given that there was little variation in the temperature across their tray, the whole bioreactor is represented by a single temperature. Heat removal through the sides of the tray is assumed to occur by convective cooling of the side walls. The dominant mechanism at the top and bottom of the tray is natural convection, which causes air to ow through the bed at a small velocity. An energy balance over the tray, therefore, gives dT = dt BYQ (dX/dt) + Fa Cpa (Ta T) hA(T TS ) +Fa ((Ha H) Cpv (Ha Ta HT)) Cpb B(1 + W)

In the denominator, Cpb is the overall heat capacity of the moist bed and W is the bed water content. The humidity of the air leaving the tray by natural convection (H) is calculated in two steps. First the vapor pressure in mmHg (Pvap ) is calculated according the Antoine equation [9] Pvap = exp 18.3036 3816.44 (T + 273.15) 46.13 (6)

Then the humidity of the air, which is assumed to be saturated, is calculated as Pvap H = 0.6246 (7) P Pvap where P is the total pressure of the system and the factor 0.6246 is the ratio of the molecular weight of water (18 g mol1 ) to the average molecular weight of dry air (28.82 g mol1 ). In the case of the air entering the bottom of the bed, T is replaced with Ta , and the vapor pressure (Pvapa ) and humidity (Ha ) of the entering air are calculated with the same equations. The quantity of dry solids in the circular tray is calculated as
1 2 B = b L( 2 )

(5)

where T is the temperature of the bed. In the rst term of the numerator, which describes metabolic heat production, B is the total mass of solids in the bed and YQ is the metabolic heat yield. In the second term, which describes heat removal by the dry air, Fa , Cpa , and Ta are the ow rate, heat capacity, and temperature of dry air, respectively. In the third term, which describes heat removal by convection to the surroundings, h is the overall heat transfer coefcient at the tray wall, A is the area of contact between the bed and the surroundings and TS is the temperature of the surroundings. In the fourth term, which describes heat removal by the water vapor, is the enthalpy of evaporation of water, Ha and H are the humidities of the air entering and leaving the bed, respectively, and Cpv is the heat capacity of water vapor.
Table 1 Symbols used in the kinetic model and their values Symbol AD AS ED ES n F kS kD R t X XM Value and units

(8)

where b is the density of the substrate bed, and L and are the height and diameter of the tray, respectively. The area for conductive heat transfer (A) is calculated as the area of the side walls A = L 2.3. Solution of the models The parameter values used in the growth kinetic model are given in Table 1 [810]. The determination of these parameters is described in Section 3. The parameter values for the energy balance calculations are given in Table 2 [8,9,1114]. (9)

Source Fitting of Figs. 3 and Fitting of Figs. 3 and Fitting of Figs. 3 and Fitting of Figs. 3 and Fitting of Figs. 3 and Calculated by Eq. (1) Calculated by Eq. (2) Calculated by Eq. (3) Independent variable [8] [10] [8] [10] Fitting of data, see text 4 4 4 4 4

7.56 1061 h1 10.23077 h1 386800.5 J mol1 14289.27 J mol1 10 (dimensionless) Fo = 1 (dimensionless) h1 h1 8.314 J mol1 C1 to = 0 h Xo = 1.97 mg-biomass cm2 (Fig. 3) Xo = 0.001 kg-biomass kg-substrate1 (Figs. 4 and 5) XM = 5.06 mg-biomass cm2 (Fig. 3) XM = 0.125 kg-biomass kg-substrate1 (Figs. 4 and 5) 0.0988 or 0.236 h1

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Table 2 Symbols used in the energy balance calculations and their values Symbol A B Cpa Cpb Cpv Fa h H Ha L P Pvap Pvapa T Ta Ts W YQ b Value and units m2 kg 1007 J kg-dry-air1 C1 2500 J kg-solid1 C1 1867 J kg-vapor1 C1 4.835 105 kg-dry-air h1 36000 J h1 m2 C1 kg-vapor kg-dry-air1 kg-vapor kg-dry-air1 0.1 m 760 mmHg mmHg mmHg To = 30 C 26 C 37 C 1 kg-water kg-IDS1 8.366 106 J kg-biomass1 0.3 m 2500900 J kg-vapor1 178.5 kg-dry-solids m3 Source Eq. (9) Eq. (8) [9] [11] [9] Chosen to simulate natural convection [12] Eq. (7) Eq. (7) [8] Chosen as 1 atm Eq. (6) Eq. (6) Eq. (5) [8] [8] [8] [13] [8] [9] [14]

Biomass (mg cm -2)

0 0 10 20 30 Time (h) 40

Fig. 3. Comparison between the predictions of the model and the experimental data of Ikasari et al. [6] for an experiment in which various temperature proles were imposed on a culture of Rhizopus oligosporus during the growth cycle. Note that prior to zero time on this graph, all cultures had been incubated at 37 C for 20 h. () Experimental data and ( - ) model predictions for a culture incubated at 37 C throughout the period 040 h (case 1); ( ) experimental data and (- - - -) model predictions for a culture incubated at 50 C throughout the period 040 h (case 2); () experimental data () and model predictions for a culture incubated at 50 C during the period 010 h and then returned to 37 C (case 3).

A low value was used for the heat transfer coefcient and the air ow rate due to the absence of forced convection currents at the tray surface [12]. All models were solved in FORTRAN using the subroutine DRKGS to perform the numerical integration [15].

3. Results 3.1. Estimation of the parameters of the kinetic model The values of the parameters of the kinetic model were determined on the basis of two different experimental studies from the literature, which were carried out with the same strain of Rhizopus oligosporus: Mitchell [16] incubated R. oligosporus at various temperatures, according to the isothermal approach, and plotted the linear extension from the inoculation point after 24 h against temperature, while Ikasari et al. [6] obtained dry weight proles for cultures of R. oligosporus that were shifted between 37 and 50 C during the growth cycle. The model in this case consisted of Eqs. (1)(4), with the experimentally-imposed temperature used as input rather than being calculated. Values of AS , AD , ES , ED , and n were selected that gave a reasonable t to the data in both Figs. 3 and 4 (see Table 1). In the experiment of Ikasari et al. [6], all cultures were incubated at 37 C for 20 h before different temperature regimes were imposed. One culture was maintained at 37 C for a further 40 h (Fig. 3, case 1). Non-linear regression of the logistic equation against this growth prole gave a value of of 0.0988 h1 , and this value was used for the specic growth rate constant in the model. In this case, the growth

rate decelerated only when the biomass approached XM . Two cultures were shifted to 50 C, which caused a signicant decrease in the growth rate (Fig. 3, cases 2 and 3). When a culture was incubated at 50 C throughout (Fig. 3, case 2), the growth rate fell to zero. When a culture was returned to 37 C after 10 h at 50 C (Fig. 3, case 3), the growth rate only began to accelerate about 10 h after the return to 37 C. The model predictions agreed reasonably well with both these experimental curves.

1.0

Relative growth

0.8 0.6 0.4 0.2 0.0 40 42 44 46 48 Temperature (oC) 50

Fig. 4. Comparison between the predictions of the model and the results of Mitchell [16] for an experiment carried out using the isothermal approach with Rhizopus oligosporus, in which the amount of growth was determined after 24 h. () Amount of biomass produced by 24 h predicted by the model, where a relative growth of 1 is equal to 8.03 mg-biomass g-solid1 ; () linear extension measured by Mitchell [16], where a relative growth of 1 is equal to 10.75 mm.

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In modeling the experiment of Mitchell [16], the value of of 0.0988 h1 calculated from the data of Ikasari et al. [6] was used, since Mitchell [16] used a similar agar medium. The amount of growth predicted by the model after 24 h was plotted against the temperature of incubation (Fig. 4). Note that the model predicts the results in terms of biomass produced, whereas the experimental data was measured in terms of linear extension. Therefore, for comparative purposes both the predictions and experimental results are plotted in dimensionless terms as fractions of the corresponding value obtained at 40.65 C, the temperature at which Mitchell [16] noted the highest growth rate. Experimentally, the amount of biomass obtained after 24 h decreased most rapidly for incubation temperatures from 41 to 46 C [16], while the model predicted that the interval of rapid decrease would be from 42 to 49 C. The model suggested that a small amount of growth would be observed at 50 C. Mitchell [16] did not test this temperature, however, the predictions are supported by the results of Ikasari et al. [6] who obtained a small amount of growth for a culture incubated at 50 C throughout the growth cycle. 3.2. Use of the model to predict the performance of a tray bioreactor Given that the values selected for AS , AD , ES , ED , and n allowed a reasonable description of the effect of temperature on growth in two separate experiments, these values were used for the simulation of the growth of R. oligosporus in a tray bioreactor [8], using Eqs. (1)(9). The value used for the specic growth rate constant was 0.236 h1 , obtained by non-linear regression of the logistic equation against data for growth of this organism in SSC [10]. Ikasari and Mitchell [8] did not measure biomass concentrations, only the overall O2 uptake rate and the temperature. In the case of the temperature prole, there was very good agreement of the model predictions with the experimental results, with a rapid increase in the temperature to approximately 50 C in approximately 15 h, and then a slow decrease in the temperature, which still remained above 43 C after 72 h (Fig. 5a). The experimental O2 uptake prole of Ikasari and Mitchell [8] was compared with the growth rate as calculated by Eq. (4). Given the fact that the model and experimental results use different units, the O2 uptake rate prole of Ikasari and Mitchell [8] was scaled to give the same maximum value as obtained for the growth rate in the model, and then all data points were divided by this maximum value, such that the two curves were normalized to a range of 01. There is remarkably good agreement between the two proles (Fig. 5b). This agreement is largely due to the contribution of the factor F in Eq. (4). The effect of this factor can be understood by noting that the logistic kinetics for the growth of R. oligosporus were based on growth in a ask culture maintained at 37 C [10]. This logistic growth curve was normalized in the same manner as described for

(a)
50

Temperature (oC)

40

30

20

(b)
Relative growth rate
3

0 0 20 40 60 Time (h) 80

Fig. 5. Comparison of the predictions of the model with the experimental data of Ikasari and Mitchell [8] for an experiment in which Rhizopus oligosporus was cultivated in a tray bioreactor. (a) Temperature predictions: () temperature prole predicted by the model; () experimentally-measured temperatures. (b) Relative growth rate predictions, calculated by scaling the maximum growth rate calculated on the basis of the experimentally-determined O2 uptake rate results of Ikasari and Mitchell [8] to the same value as the maximum growth rate predicted by the model and then dividing by the maximum predicted growth rate. () Relative growth rate predicted by the model; () relative growth rate calculated from the O2 uptake rate results of Ikasari and Mitchell [8]; (- - -) relative growth rate, for logistic growth kinetics with F = 1 throughout the entire growth cycle, that is, for growth at 37 C.

the other two curves and plotted in Fig. 5b. Since much higher growth rates are obtained, the normalized growth rate exceeds 1, reaching values of over 3. In other words, the maximum growth rate obtained at 37 C is over three times greater than the maximum growth rate obtained in the tray culture, in which the temperature rises to values as high as 50 C. The importance of the F factor becomes apparent when it is realized that the two curves in Fig. 5b are both plotted using Eq. (4), the only difference being that for the dashed curve (growth at 37 C) F remains equal to 1 whereas for the solid curve (the model predictions) F varies according to the kinetic model.

4. Discussion The growth kinetic model represented by Eqs. (1)(4) has managed, with a single set of values for AD , AS , ED , ES , and n, to describe reasonably well the effect of temperature on the growth of R. oligosporus in three different types of

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growth experiments involving temperature effects: one in which cultures were maintained at a constant temperature throughout the whole growth cycle, one in which cultures were shifted between 37 and 50 C and one in which the temperature changed continuously within a bioreactor. The agreement of the shape of the predicted growth rate prole with the experimental O2 uptake rate results of Ikasari and Mitchell [8] is particularly interesting since the O2 uptake rate gives a reasonable estimate for the growth rate. The results of the present work give insights into observations made in the literature. Saucedo-Castaneda et al. [13], in their mathematical model of heat transfer within a packed-bed bioreactor, expressed the specic growth rate constant as a function of temperature according to experimental results obtained using the isothermal approach. However, the bed temperatures predicted by their model were signicantly lower than the measured values and they were only able to predict the temperatures measured experimentally when the growth rate was assumed to remain constant as the temperature increased. Stuart et al. [17] obtained temperatures as high as 48 C during cultivations in a rotating drum bioreactor, whereas, in experiments done in agar plates according to the isothermal approach, signicant growth was not observed at temperatures above 40 C [16]. Our model can explain growth at temperatures higher than those at which growth is noted in isothermal experiments: when growth is initiated at the optimal temperature, it is quite possible for the growth rate to remain high for a time at deleterious temperatures, only decelerating as the level of the essential component within the cell decreases. Note that the ability of the model to predict this behavior does not prove that there is a single, heat-labile, essential component on which growth rate and the synthesis of that component depends. Without conrmation of biochemical mechanisms, the model must be considered as being only empirical. However, it is interesting to speculate as to whether there is a cellular component that might represent the essential component, F. The deleterious effects of high temperature may in part be due to denaturation of ribosomes and enzymes and problems with the uidity of membranes [18]. Ribosomes are good candidates for the essential component, since they participate both in the production of cell material and their own synthesis. One of the only studies of the effect of temperature on ribosome levels, undertaken with Escherichia coli, showed that an upshift in temperature caused a transient period during which both the number of functioning ribosomes and the total number of ribosomes decreased, with effects on growth only becoming apparent 2 h after the upshift [19]. The approach taken in formulating the model is different from that which has previously been used in models that describe growth in SSC processes. In the majority of cases, the specic growth rate has been expressed solely as a function of the current temperature [5]. In the work of Baranyi and Roberts [7], both the specic growth rate constant and the

rate of change in the level of the physiological factor were expressed as functions of temperature. In the present work, the specic growth rate constant does not depend on temperature, the sole effect of temperature being to determine the ability of the organism to grow at its intrinsic specic growth rate, which in turn depends on the timetemperature treatment that the organism has previously undergone. The effect of this is to delay responses to shifts in temperature. As the temperature rises, it takes time for the essential component F to denature. On the other hand, when the temperature falls from deleterious values to values at which the organism is capable of growing in perfect health, it takes time for the normal levels of the essential component F to be reestablished. This raises the question as to whether the specic growth rate constant is actually a function of temperature. According to the Arrhenius equation, the rate of chemical reactions increases with temperature, and therefore, it might be expected that the maximum specic growth rate that the organism is capable of would increase with temperature. In fact, this is probably the dominant effect below the optimum temperature for growth. However, growth is not a simple chemical reaction, but rather requires the coordination of many chemical reactions, in a system overlaid with control schemes, some of which, such as the heatshock system, respond directly to increases in temperature. Possibly this coordination is adversely affected at temperatures above the optimum temperature for growth. These metabolic processes are so complex that any model attempting to describe them mechanistically would be too complex to be useful within a mathematical model of growth in SSC bioreactors. The model proposed in the current work aims to capture this effect while maintaining simplicity. Some models of growth in SSC include death kinetics [5]. The model presented in the present work would give similar results if all the viable biomass were considered to be fully healthy and F were used to denote the fraction of the total biomass that was viable, with X denoting the total biomass, both dead and viable. Of course, Eq. (1) would then need to be expressed in terms of the growth and death rates. However, the predictions would not be identical. In the current model, F can increase more rapidly than the organism grows. In fact, it can increase in the absence of growth: when X = XM there is no growth, but if F is less than one it can increase. In the case of a death-kinetic model, it would only make sense to allow the fraction F to increase through the growth of living biomass, and not through the conversion of dead biomass to viable biomass, and therefore, the fraction F could only increase if X also increased.

5. Conclusion The ability of the model to describe several sets of data in which R. oligosporus was subjected to different temperature

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regimes suggests that, in developing mathematical models for SSC bioreactors, it is worthwhile to use kinetic models of the type developed in the current work, rather than to use kinetic models derived from data obtained by the so-called isothermal approach.

Acknowledgements This work was done under a grant given by the Brazilian-Argentinean Biotechnology Committee (CBAB, Comit e Brasileiro-Argentino de Biotechnologia), using funds provided by the Brazilian Ministry of Science and Technology (MCT, Ministrio de Ci encia e Tecnologia) and administered by the Brazilian National Council for Scientic and Technological Development (CNPq, Conselho Nacional de Desenvolvimento Cient co e Tecnolgico). Farah Diba Dalsenter, Graciele Viccini, Marcelo Barga, Nadia Krieger and David Mitchell thank CNPq for research scholarships.

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