Food Microbiology, 1993, 10, 43-59

A non-autonomous differential equation to model bacterial growth

J. Baranyi, T. A. Roberts and P. McClure*
A F R C Institute o f Food Research, Reading, Laboratory, Earley Gate, W h i t e k n i g h t s Road, R e a d i n g R G 6 2EF, U K Received 26 A p r i l 1992 In order to describe the dynamics of growing bacterial cultures a non-autonomous differential equation is applied. The model describes the lag phase as an adjustment period and for the lag-parameter a new definition is introduced. Some mathematical aspects of the model are described and, on the basis of more than 500 growth curves, its statistical properties are compared with the Gompertz-approach commonly used in food microbiology.

Introduction
Several approaches to modelling bacterial growth can be found in the microbiological literature. Following the classification of Roels and Kossen (1978) we deal here with unstructured, nonsegregated growth models. This means that we suppose that (1) the biomass is homogenous; (2) the mass concentration is the only dependent variable of the system; and (3) environmental parameters like temperature, chemicals, etc. are not involved in the model (the possible dependency on them is expressed through the dependency on the mass concentration). In food microbiology several basic sigmoid functions (logistic, Gompertz, etc.; see, for example France and Thornley 1984), as empirical models, have become widely used. A comparative study about them was published by Zwietering et al. (1990). However, with the use of more and more exact methods in microbiology, the demand for less empirical growth
*Present address: Unilever Research, Colworth Laboratory, Colworth House, Sharnbrook, Bedford MK44 1LQ, UK. 0740-0020/93/010043 + 17 $08.00/0

models is increasing (Whiting 1992). The commonly used simple growth model of population theory is a first order ordinary differential equation where the growth rate does not depend on time directly (autonomous, non-segregated, unstructured models). As an example one can consider the model of Turner et al. (1976), which is general enough to include the logistic, Gompertz, Richards, Bertalanffy etc. growth functions. The typical representation of a bacterial batch culture is to plot the logarithm of the cell concentration against time and, in most cases, the result is a sigmoid curve. A possible empirical solution is to fit a basic sigmoid function, augmented with an additive term, to these data. This way was followed by many authors in recent years in food microbiology (among them Gibson et al. 1988, Buchanan and Cygnarowicz 1990, Zwietering et al. 1990). We should keep in mind that in this case these models should not be referred to as the logistic, Gompertz etc. growth models which are meant to apply to the cell concentration and not to its logarithm. In this paper we show a new way to apply fundamental growth models
1993 Academic Press Limited

44

J. Baranyi et al.
al. (1967). Multiply the right-hand side of this differential equation by a smooth function (adjustment function) having the property that it is closer and closer to 1 as the experimental time passes. This operation expresses our wish to describe the gradually diminishing effect of the previous environment. The result is a non-autonomous, separable, first order ordinary differential equation. In this way the lag phase (adjustment) is formally separated from the exponential and stationary phase which can be regarded as parts of the potential growth defined by the autonomous model. It is especially important in food safety investigations to investigate the duration of the lag phase. The classical definition of the lag (Pirt 1975) assumes that the logarithm of the cell concentration forms a sigmoid curve against time and the intercept of the tangent at the inflexion with the lower asymptote is considered as the turning point indicating the end of the lag phase. By means of the adjustment function we will give a less geometrical definition for the lag. Our lag parameter, as well as the maximum specific growth rate of the new model, will be compared with other definitions of the respective parameters. In many respects the new model is a generalization of other models already used in the literature.

generally accepted in population theory. A mathematically formalized approach is shown which assumes that a given environment determines the potential g r o w t h rate of the culture that is higher than the actual growth rate if the time is close to the inoculation. The ratio of the actual and the potential growth rate characterizes the process of adjustment of the cells to the new environment. The most important mathematical theorems on the model have been shown in Baranyi et al. (1992) and are collected in the Appendix. Before starting an experiment to study bacterial growth in a given environment the cells are normally grown in a favourable, more or less optimal environment, in the so-called subculture, to a get a sufficient amount of cells for inoculation. Denote the pre-inoculation environment (the subculture) by El and the actual (post-inoculation) environment by E2. Suppose that E1 is sig-nificantly different (in this case more favourable) from the actual environment, E 2. Our aim is to create a model which describes the lag as the process of adjustment to the new environment. Note that this approach already suggests a non-autonomous model, because it takes a sudden external effect on the system into account. The mathematical formalization of the above concept can be outlined as follows. We can consider the time of inoculation as zero time. Suppose that if the effect of E1 is neglected (or, which is the same, E1 = E2) then some well-known autonomous differential equation of the form
yc = i-t(x)x (0 < t < ~; 0 < x)

Theory
In most papers on unstructured, nonsegregated models of population dynamics the starting point is the assumption that the growth of a population in a given environment is described by the first order ordinary differential equation
ic=tz(x)x

describes the bacterial growth in E2, where x denotes the cell concentration, /z(x) denotes the specific growth rate. In this case the growth rate does not depend on time directly, only through the cell concentration. This basic assumption is an implication of the fifth hypothesis of the framework of Frederickson et

(0_<t <o% 0 < x )

(la)

with the initial value

x(O)=xo

(0 <Xo < xm~)

(lb)

where x is the number of individuals per

Bacterial growth model

unit (concentration). The quantity ix(x) is called the specific growth rate and for the classical growth functions it is assumed that (a)/x(x) is defined for x0 < x < xm~ (x,~ is fixed) (b) t~(Xo) > 0 and tX(Xm~) = 0 (C) /X(X) is continuously differentiable in its domain and d l x / d x is strictly negative there. Under these conditions the so-called initial value problem defined above (differential equation together with its initial value) has a unique solution, denoted here as fit). This solution is monotone increasing and converges to xm~ as t ~ ~ (Vance 1990). Equation (la) is a so-called autonomous differential equation because its right-hand side depends on the unknown dependent variable itself and it does not depend on time directly. Its general solution takes the form x(t) = f(t-T), where T is a constant characterizing a shift parallel to the t axis (it can be considered as a delay in growth). The value of T can be uniquely defined by fixing an initial value for x(0). Depending on the choice of/~(x), different sigmoid functions can be obtained which satisfy (la). Note that the condition (c) corresponds to the assumption that the larger the population density the lower is its specific growth rate. As can be shown mathematically, this means that the time-derivative of the logarithm of the wanted growth function is strictly monotone decreasing. Consider the experimental time (i.e. the time elapsed from inoculation) as the independent variable, t. Fix the time of the inoculation as t = 0. Our model will be defined for non-negative values of t in the following way. We postulate that after inoculation the cell concentration of the culture is described by the initial value problem

45

yc = a(t)tL(x)x (0 < _ _ t < ~; 0 < x) (2a) x(O) = Xo (0 < xo < x.,,=) (2b)

where: (a)/~(x) is independent of E1 and satisfies the conditions assumed under (la) and (lb). (b) a(t) depends on E1 and E2 and 0<a(t)<l (0<t<~). (3) Furthermore if a(t) ~ 1 monotone increasingly as t --> ~ then we say that a(t) is an a d j u s t m e n t function from E1 to E2. We say that (la), (lb) define the potential growth, and (2a), (2b) define the actual growth in the environment E2. The interpretation of these definitions is as follows: The given, actual environment E2 and the inoculum level, Xo, uniquely determine the potential growth curve, according to which the population would be able to grow if the previous environment had been the same as the present environment (E~ = E2: no need to adjust, that is a(t) -= 1). The potential growth of the population is described by the autonomous equation (la). The actual growth, however, is described by (2a) and (2b) which means that after inoculation (a sudden change in the environment from El to Ee), the cells' actual specific growth rate is heavily influenced by the fact that the time is close to zero. Later, however, the effect of the previous environment diminishes, until some time after the inoculation it has little or no effect and the cells grow essentially at their potential growth rate, /x(x), defined by the new environment, E2. Therefore the ratio of the actual and the potential growth rate, i.e. the adjustment function, is expected to increase from zero (no growth because of lagging) to 1 (total adjustment). Theorem 1 in the Appendix gives the solution g(t) for the actual growth curve by means of the functions f and a. Its form is: g(t) = f(A(t)), (4)

46 J. Baranyi et al.
where A(t) is the integral function of a(t). A class of adjustment functions of the form tn an(t) = - - , (5) A" + t n where A and n are positive numbers, proved to be generally very effective when fitting our viable counts data. This adjustment function can be derived in the following way: Suppose that the growth is controlled by a critical substrate, or product, say P, which is vital to ensure growth in the new environment and it was present in a negligibly small amount in the previous environment. Furthermore suppose that the dependence of growth on this product follows the well-known Michaelis-Menten rule:
P(t) = Kp + P(t) ~ (x)x,

(6)

is built up around t = A (the larger is n, the faster is the accumulation of P) where A is the time point were P = Kp. Substitute the latter expression in (7) and the adjustment function of the form (5) can be obtained. The interpretation above explains the next definitions. We call the parameter of the adjustment function of the form (5) the lag p a r a m e t e r . In what follows we refer to an(t) as a d j u s t m e n t function of order n. Note that Theorem 2 in the Appendix gives an estimation of what happens if the adjustment function is derived in a different way. Roughly it can be said that if two adjustment functions are close to each o t h e r (this 'closeness' is measured by the integral of their difference), then the respective actual growth curves will also be close to each other. Some properties of the adjustment function (5) are:
- a,(O) = 0; an(A) = 1/2 - an(t) is strictly monotone increasing an(t) ---> 1 (t ~ ~) - if n > 1 then a~ d a n / d t = O a t t = O b/ an(t) has an inflexion point at
-

where Kp is the so-called saturation constant. This gives the adjustment function the form:
P(t) a(t) = Kp + P(t) .

(7)

After rearrangement:
P(t)

T n = ~/(n - 1 ) / ( n + 1) A.

(10)

~(t) =

go
1+P(t)

(8)

Generally it simplifies the calculation of the An(t) integral function if the next relation is used:
An(t)=

go
The quantity P ( t ) / K s is dimensionless. Suppose that atter inoculation the production of the critical product depends on E2 and is proportional to the n-th power of time:
P(t)Kp= ( t ) n

fs
t

An + s n ds = A

-Bn

(II)

where,
I Bn(t) =
o

1 --ds.
l +s n

(12)

(9)

Theoretically the integral function

Bn(t) can be expressed by elementary

Here n characterizes the rate at which P

functions for a fixed positive integer, n, but for larger values of n the expression

Bacterial growth model

is more and more complicated. For bacteriological data representing a broad range of growth conditions for a variety of organisms, an adjustment function of order 4 proved to be satisfactory to characterize the transition from the lag to the exponential phase. In this case the expression for B4(t) is: 1 B4(t) =
__ In

47

t2 + X/2t + 1
_ + ~/(t)

2V2

t2 - ~ / 2 t + 1

(13) where, V2t arctan - 1 -t ~

(t < 1) (t = 1) (14)

y(t)=

7r/2 arctan X/2t

1 -

t2

+Tr ( t > l )

(See, for example, Korn and Korn 1973). Some theoretical aspects of this adj u s t m e n t function are summarized in Theorems 4-5 in the Appendix. Since the adjustment function, an(t), is the derivative of An(t), and an(t) converges to 1 as the time elapses, An(t) plays the role of delayed time; i.e. A,(t) is closer and closer to the function t-A and the actual growth function is closer and closer to a delayed potential growth function, fit-A). Therefore, this delay, A, can be considered as a good approximation of the length of the lag phase in the actual growth. Now we examine two important classes of autonomous growth as potential growth. (a) Pure exponential g r o w t h as potential growth When collecting data from experiments, sometimes, for one reason or other, there are no data to indicate the stationary phase of the growth curve. In these cases a computer program fitted a sig-

moid curve to the data can easily fail because of the lack of information to estimate the p a r a m e t e r characterizing the stationary phase. Still it is desirable not to waste the results of these experiments. The solution can be either to fix the p a r a m e t e r characterizing the stationary phase or to have a growth function which models only the lag and the exponential phase. The new approach introduced in this paper is suitable to get a growth function of the latter type. For this purpose consider the pure exponential growth as potential growth and combine it with our adjustment function given in (5). (Although in this case the condition (c) under (la) and (lb) is not satisfied, according to the Theorems in the Appendix the connection between the potential and actual growth remains the same as above). The respective equations are: Potential growth curve (solution of (la)-(lb)): fit) = xoexp (l~m~t). Actual growth (2a)-(2b)): curve (solution of

g(t) = xoexp (/~mo~A,(t)). where A,(t) is defined by (11), (12). In the study of batch cultures in food microbiology it is more common to consider the logarithm of the cell concentration as the dependent variable. If this is the natural logarithm: y(t) = In x(t), then the slope of the tangent of the y(t) curve will be the specific growth rate. Transform the actual growth curve into the y,t plane: y(t) =Yo + I~:,An(t) where Yo = In xo. By means of the obtained model (15) (15)

48

J. Baranyi et al.
growth curve at the transition between the lag and the exponential phase (Fig. 1). If n is large t hen the transition is practically a breakpoint, j u s t as in the model of Kono (1968).

we can d emo n s tr at e the role of the par a m e t e r s ~ and n also in the following way: (i) Second-derivative-concept. Recently, B u c h a n a n and Cygnarowicz (1990) defined the end of the lag phase as the time point where the second derivative of the logarithm of the actual growth curve has its maximum, i.e. where the third derivative of A , ( t ) is zero. Since an(t) is the first derivative of A,(t), the point in question is the inflexion point T, of the an(t) a d j u s t m e n t function. It can be seen from Eqn (10) t h a t in our a d j u stmen t function, T, is very close to (for example, for n = 4:T4 ~- 0-9 ~). In fact from (10) it follows t h a t T, ~ A monotone increasingly as n ~ oo. This demonstrates again why we can call out A p a r a m e t e r the lag parameter. (Note t h a t the classical lag definition by means of the t a n g e n t drawn to the inflexion of the growth curve could not be applied here because the growth curve itself has no inflexion?) (ii) Step-function-concept. It can be checked t h a t a,(t) --~ a~(t) as n -o ~, where a=(t) is the step function 0 a=(t) = 1/2 1 (t < A) (t = A) (t > A).

(b) Logistic g r o w t h as p o t e n t i a l g r o w t h
For a n o t h e r example consider the wellknown logistic growth model as potential growth. In this case /x(x) =/~m~ 1

(x)
-

(18)

Xmax

where/xm~ is the limit of/z(x) as x -~ 0. The respective equations are: Potential growth curve (solution of (la)-(lb)):

]~t) = 1+ .

Xm~
xmax

Xo
Actual growth (2a)-(2b)):

1 ) e-t'm't
(solution

(19) of

curve
Xrna.x

f'(t)

1+

1)e -~m~A,(t) ( 20 )
Xo

(16)

where A , ( t ) is defined by (11), (12).

This function was implicitly used by Kono (1968) to model the lag phase. There the a u t h o r used a constant factor which was 0 for t < t 1 and 1 for t > t~, where t, was a 'suitable' time (see also Barford et al. 1982). In our model A plays the role of t~. For a=(t) the definite integral function is A=(t), where 0 A=(t) = t - ,\ (t < ~) (t >_~) (17)

,J"
6
i

n"

'~

I~| mG hi" "

f"

t I'J

f
"

.."'"

...'"'

8 4

9 -

I(

..'"'"
-.ii-- PO0) ........ A2(t)

2 :l.l.l-I:/~ru~

I - - r~('l - , - ~Lo(,I I
0
1 5 I I0 I 15 1 20 I 25

(Theorem 4 in the Appendix). Therefore the actual growth curve of our model converges to the function /~,~A~(t) as n --* ~. The p a r a m e t e r n characterizes the c u r v a t u r e of the

Time ( h )

Fig. 1. Influence of the parameter n on the growth curve simulated by the new model. P0(t), Potential growth curve. Ai(t), Actual growth curve at n = i (i = 2, 4, 10).

Bacterial growth model

As with (15), we transform the obtained model (20) to the y,t plane, where y(t) = In x(t). We get:
y(t) = ym~-ln (1 + (e-'Y~-Yo-1)e-t~=An(t)), y(t) = Yo + I~m~,An(t) +

49

lln(1 m

+e""~A"(t)-II
em{ymax--Yo} ]

(22b)

(21a) where y , ~ = In x,~. As can be checked, the formula above can be written in another form:

Results
Our concept will be demonstrated with the fourth order adjustment function as(t) combined with the logistic growth model above as potential growth curve. Choosing n = 4 for the order of the adjustment function has mainly computational advantages. By means of the Eqns (11), (14) it is possible to substitute an explicit expression for the actual growth curve in a curvefitting procedure. We found that, generally, higher values of n (usually between 6 and 10) give better fit, but either fixing the curvature parameter n at an integer value larger than 4, or fitting its value to a parameter estimating procedure, needs some computational tricks and it is advisable to adjust some well-known codes (for example Press et al. 1990) to solve these problems. These computational aspects of our approach are being summarized presently in a separate paper. From several points of view Zwietering et al. (1990) found the Gompertz curve the best from a range of growth models. Also Gibson et al. (1988) reported that the Gompertz function was the best fitting curve when analysing their data. This is why we chose the Gompertz function to compare it with one of our family of growth models: the logistic growth combined with fourth order adjustment function. In this case the formula for the logarithm of the cell concentration of a growing culture is given by (21) and (11). Gibson et al. (1988) published the estimates of the maximum specific growth rate and the lag obtained by fitting four parameter Gompertz curves to a number of S a l m o n e l l a - d a t a . For a numeric

y(t) =yo + P-m~An(t) + In 1 +

egmo~.(t))_~ ~ ]
g

(21b) This other form of the same function highlights the connection with the case, when the pure exponential growth was used for potential growth (see Eqn (15)). It is well-known that i f h << 1 then ln(1 + h) ~ h therefore the formula (21b) shows that close to the inoculation, where the fraction in (21b) is small, practically there is no difference between (15) and (21a) - that is, the maximum population does not yet influence the growth. In the Discussion we return to the computational consequences of this connection. Without going into detail, we mention the possibility of choosing the more general Richards-curve (see for example, France and Thornley 1984) as potential growth. It differs from the logistic curve in the sense that it has an extra (positive) parameter, denoted by m here, which characterizes the curvature before the stationary phase. The choice m = 1 is equivalent to the choice of the logistic curve. The formulae, respective to (21a) and (21b) can be written as:
y(t ) = y , ~ - - ~

1 In (1+ (e~"~-y~
(22a)

50

J. Baranyi et al.
T a b l e 1. T h e d a t a p o i n t s o f t h e Salmonellae g r o w t h c u r v e w i t h c o d e = 1 i n G i b s o n e t al. (1988).

d e m o n s t r a t i o n we selected t h e first 40 curves of t h a t dataset. O u r c u r v e f i t t i n g p r o c e d u r e was r u n on t h e s a m e datapoints. Program 1 fitted four p a r a m e t e r G o m p e r t z curves, as s u g g e s t e d by Gibson et ai. (1988), to t h e m e a s u r e d loglo counts by a s t a n d a r d least s q u a r e s method using Marquardt's algorithm w i t h single precision a r i t h m e t i c (Press et al. 1990). T h e m a x i m u m specific g r o w t h r a t e was calculated as the slope of the t a n g e n t at the inflexion of the yl(t) curve w h e r e yl(t) is a fitted G o m p e r t z function, a u g m e n t e d by a n additive par a m e t e r (see Gibson et al. 1988). T h e lag was calculated as the i n t e r c e p t of this m a x i m u m slope with the lower a s y m p tote of t h e curve. In w h a t follows,/~1 a n d ~ will d e n o t e the v a l u e s of t h e s e g r o w t h p a r a m e t e r s as e s t i m a t e d by Program 1. Program 2 fitted o u r f o u r - p a r a m e t e r model, a logistic p o t e n t i a l g r o w t h with f o u r t h o r d e r a d j u s t m e n t function, u s i n g the s a m e M a r q u a r d t - m e t h o d . T h e estim a t e d g r o w t h p a r a m e t e r s (/~m~, maxim u m p o t e n t i a l g r o w t h rate; A, lag-par a m e t e r of the a d j u s t m e n t function) will be d e n o t e d by p~ a n d A2 respectively. In Table 1 we list the v a l u e s which were the d a t a p o i n t s for the first curve, with code = 1, in Gibson et al. (1988). Fig. 2 shows the curves fitted by Prog r a m 1 a n d Program 2. T h e m a i n differences can be s u m m a rized as follows: (1) At t h e t i m e t = 0 the slope, p r o d u c e d by the new model, is exactly zero (only n o n - a u t o n o m o u s models can h a v e t h a t property). This is a consequence of the fact t h a t the v a l u e of o u r a d j u s t m e n t function is zero at t h e origin; (2) T h e new model gave a practically s t r a i g h t line ( l o g a r i t h m of the logistic growth) in t h e e x p o n e n t i a l phase. This is a r e s u l t of the construction: in this p h a s e t h e p o t e n t i a l g r o w t h is d o m i n a n t ;

No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Time (h) 0.00 1.17 2.00 2.92 3.92 4.96 5-96 8-08 10.2 13.1 19.8 21.3 22.8 23.8 24.7 26.7 27.7 28.8 29.7 31.3 32-8 49.8

Log counts (loglo ml-b 3.39 3-39 3-47 3.46 3.57 3.70 3.98 5.41 4.96 5-74 7.45 7.79 8.10 8.24 8.46 8.69 8.66 8.67 9.16 8.69 8.76 8.78

(3) T h e c u r v a t u r e before the s t a t i o n a r y p h a s e is m o r e p r o n o u n c e d in the new model. This, again, shows the difference

g 8
o

//

~ Profrom2 ]
:: ...... ~ o g r o m /
I 20
Time ( h )

I I0

I
40

Fig. 2. Comparison of the fits given by Program 1 and Program 2 on viable count data (see Table 1). A1 = 3.78 and/~1 = 0.69 (estimate of Program 1); A2 = 2.58 and/~2 = 0.56 (estimate of Program 2).

Bacterial growth model

between the curvature of the logarithm of the logistic curve and the curvature of the Gompertz curve before the stationary phase. Before comparing Program 1 and Program 2 on a larger dataset we show an example for the use of the pure exponential growth as potential growth: Leave out those points from the dataset in Table 1 where the loglo counts are larger t h a n 4 (let only the first seven points remain). Fig. 3 shows a comparison between the results produced by Program 1 and Program 3 where Program 3 is based on pure exponential growth as potential growth combined with fourth order adjustment function. Here the estimated values, suggested by the Gompertz-fit for the m a x i m u m specific growth rate and the lag time, are unrealistically high. The reason is t h a t the inflexion point lies outside the region of the seven experimental data. The new model gives much better estimates of both parameters. Note t h a t considering the m a x i m u m slope inside the experimental region as m a x i m u m specific growth rate could im-

51

prove the results of Program 1, but even this cannot overcome the difficulty t h a t it indicates an ill-conditioned problem. The more general comparison of Program 1 and Program 2 below will be based on the following estimates; the subscripts 1 and 2 indicating the Gompertz-curve (Program 1) and the new model (Program 2), respectively. length of the lag phase (AI, A2) m a x i m u m specific growth rate (/~1,/~2) residual mean squares (rms l, rms2) standard error of lag (s(A~), s(A2)) standard error of/~ (s(t~1), s(~), respectively) (see Table 2). Comparing the outputs, the goodnessof-fit of the new model is generally at least as good as the goodness-of-fit of the Gompertz-curve. The residual mean squares were lower in the case of the new model in 35 cases. The lag and m a x i m u m specific growth rate estimated by the new model were slightly lower t h a n in the Gompertz case. One reason for this is t h a t in the exponential phase the Gompertz curve shows a curvature and our model is practically a straight line in this phase (a consequence of the fact the Gompertz curve should be used for the cell concentration r a t h e r t h a n for its logarithm!). The estimated standard error of the lag was on average a little lower, and t h a t of the m a x i m u m specific growth rate was generally much lower in the output of the new model. A demonstrative way to represent the results of similar comparisons on a large number of growth curves is shown below. We ran the two programs mentioned above on more t h a n 500 sets of growth data obtained from batch cultures of various kinds of micro-organisms (Listeria, Salmonella, Yersinia, Escherichia coli). The order of magnitude of the measured cell populations

...-" :'Y

....... P r o g r o m

51

i 5

IO Time ( h )

15

Fig. 3. Comparison of the fits given by Program I and Program 2 on the first seven datapoints of Table 1. A1 = 6.08 and/~1 = 1.45 (estimate of Program 1); A2= 4-44 and/12 = 0.81 (estimate of Program 2).

52

J. Baranyi et al.

Table 2. C o m p a r i s o n o f t h e G o m p e r t z - f u n c t i o n ( P r o g r a m 1) a n d t h e n e w m o d e l ( P r o g r a m 2 ) u s i n g t h e first f o r t y g r o w t h c u r v e s o f t h e s ~ l m o n e H a e d a t a o f G i b s o n et al. (1988). code

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
rms 1 rms 2 ~t1 ~2 s(/tl)

s(~2)
0.82 1.06 0.84 12.3 0.52 0.74 0-46 0.54 0.55 0.49 0.49 0.42 0-25 6.02 1.05 1.12 11.3 2-67 0.48 0.98 2.64 1.74 2.72 2.81 11.1 8.26 0.84 1.44 1-74 6.25 1.23 22.5 1.93 1.54 5-92 0.47 28.7 4-87 1.35 1.19

~tl
0.69 0.27 1.18 0.04 0.58 0.57 0.60 0.59 0.58 0.58 0.58 0.57 1.45 0-15 0.74 0.36 0.08 0.26 1.17 0-97 0.20 0.69 0.70 0.84 0-04 0.23 0.57 1.17 2.29 0.27 1.02 0.04 0.67 2.34 0.27 1.20 0.11 0.26 0-74 1.15

P~2
0.56 0.25 1.02 0.04 0.49 0.49 0.51 0.50 0.49 0.49 0.50 0.49 1.20 0.15 0.66 0.32 0.08 0.22 0.95 0.81 0.18 0.62 0.65 0.69 0.03 0.20 0.47 0.91 1.70 0.22 0.79 0.03 0.52 2.05 0.22 0.96 0.08 0.21 0.60 0.97

s(~l)
0.054 0.031 0.157 0.005 0.043 0.057 0.030 0.034 0.037 0-040 0.040 0.040 0-135 0.031 0.120 0.036 0.008 0.040 0-127 0.218 0.033 0.158 0.189 0.215 0-006 0.055 0.057 0.183 0.816 0.056 0.124 0.009 0.122 0.809 0.048 0-092 0.039 0.043 0.124 0.158

s(p~2)
0.026 0.007 0.116 0.003 0.013 0.019 0.013 0.015 0.013 0-013 0.014 0.011 0.043 0-018 0.051 0.015 0.010 0.013 0.052 0.064 0.008 0.052 0.104 0.116 0.001 0.022 0.015 0.115 0.387 0.019 0.069 0.002 0.045 0.506 0.017 0.041 0.016 0.016 0.035 0.104

0.24 0.12 0.26 0.19 0.13 0.18 0.09 0.10 0.11 0.12 0.12 0.12 0.17 0.37 0.26 0.16 0.33 0.28 0.19 0.39 0.17 0.42 0.55 0.57 0.20 0.58 0.19 0-31 0.772 0.49 0.26 0.33 0.33 0.69 0.47 0.15 0-63 0.37 0-34 0.28

0.20 0.10 0.24 0-19 0.09 0.13 0.08 0.10 0.10 0.09 0.08 0.08 0-10 0.35 0.22 0.14 0.30 0.28 0.16 0.32 0-16 0.30 0.43 0.62 0.13 0.49 0.12 0.32 0-64 0.40 0.26 0.31 0-25 0.53 0.42 0-10 0.60 0.32 0.22 0.28

3.8 14. 4-9 75. 7.4 7-3 7.6 7.4 7.0 7.2 7.8 6.8 2.8 38. 7.0 17. 84. 8.7 2.5 2.0 5.7 10. 13. 6.3 45. 24. 7.2 5-0 5.6 26. 5.1 43. 8.9 5.9 23. 5-7 71. 22. 8.8 4.7

2.6 12. 4.0 60. 5.5 5-6 5.7 5.5 4.9 5-3 6.0 5.0 1.9 366.0 14. 81. 6.2 1.3 1.3 5.8 8.5 12. 4.3 4018. 4.4 3.4 3.9 17. 3.0 29. 5.6 5-0 15. 4.1 34. 14. 5.8 3.4

1.28 1.49 0.69 12.8 0.76 1.05 0.49 0.55 0.67 0.68 0.65 0.72 0.41 5.32 1.10 1.01 9.00 3.90 0.63 1.69 5-18 2.26 2.41 2.46 29.2 8.73 1.29 1.07 1.18 6.05 0-97 38.7 2.01 1.09 5.66 0-50 23.7 4.76 1.69 0.97

w e r e b e t w e e n 2 a n d 10 in t e r m s of 10based logarithm. During the generation of t h e s e d a t a , t h e s u b c u l t u r e c o n d i t i o n s (El) w e r e d e l i b e r a t e l y close to t h e optimum pH value and temperature for g r o w t h w i t h no a d d e d s o d i u m c h l o r i d e to r e d u c e t h e w a t e r a c t i v -

ity. C o n d i t i o n s u n d e r w h i c h t h e o r g a n isms subsequently multiplied (E~) g e n e r a l l y d i f f e r e d w i t h r e s p e c t to a t l e a s t one of t h o s e f a c t o r s c o n t r o l l i n g growth. I n Fig. 4 t h e y c o o r d i n a t e is t h e difference b e t w e e n t h e r m s v a l u e s :

Bacterial growth model

y(Fig. 4) =
rmsz - rmse

53

To be able to c o m p a r e t h e e s t i m a t e d s t a n d a r d e r r o r s of the different lag a n d specific g r o w t h r a t e values, in Figs 5 a n d 6 we took the difference b e t w e e n t h e r e l a t i v e e r r o r s of the r e s p e c t i v e estim a t e s as t h e y coordinate:
r l i = S(Ai)/Ai rmi = s(lxi)/lxi

(i = 1,2 r e s p e c t i v e l y to y(Fig. 5) = y(Fig. 6) =

rll - rl2 rml - rm2 = s(lzl)//~l

Program

i)

= s(Ai)/Ai - s(Ae)/A2
- s(lz~)/lx2

In all t h e s e plots (Figs 4, 5, 6) the e s t i m a t e d v a l u e s of/z~ are t a k e n for the x-coordinate. T h e s e plots d e m o n s t r a t e t h a t for v e r y slow curves, w h e n the g r o w t h r a t e is less t h a n a b o u t 0.1 h -z, t h e r e is no significant difference b e t w e e n the two

fits, b u t at h i g h e r g r o w t h r a t e s t h e new model is g e n e r a l l y b e t t e r in t e r m s of goodness-of-fit. As far as the s t a n d a r d e r r o r s of t h e e s t i m a t e d g r o w t h p a r a m e t e r s are conc e r n e d the p r o p e r t i e s are d i f f e r e n t a t g r o w t h r a t e s less t h a n a b o u t 0.3 h -z a n d at h i g h e r g r o w t h rates. F o r slower g r o w t h c u r v e s t h e s t a n d a r d e r r o r of ~t is less if we use t h e G o m p e r t z curve, b u t at f a s t e r g r o w t h c u r v e s the new model is m o r e a d v a n t a g e o u s . H o w e v e r , a t slower g r o w t h r a t e s t h e s t a n d a r d e r r o r of the lag p a r a m e t e r is m u c h b e t t e r if we use the n e w model a n d a t h i g h e r r a t e s t h e r e is no significant difference b e t w e e n t h e two models in this respect.

Discussion
Apart from certain statistical and

0.10[

.,
me4lg o l , ..,"

B'"
i I B ,ll i II . " l.

n.O0 I ~ ' ' :-'~'." ~ _ ,,.. ~ "1~'.' "'"'.I"

","

.-

~t

t'o

~,'.

I 0

-0.10 0.00

/~l of P r o n t o

1.00
I

Fig. 4. Comparison of the goodness-of-fit of the Oompertz function (Program 1) and the new model (Program 2) on more than 500 growth curves. The comparison is characterized by the

difference in the residual mean square: r m s z - r m s 2. The points above the horizontal axis represent those cases when the fit of the new model was better.

54

J. Baranyi et al.
1.00

=="
.-

.,.
|

I=
",="

"=
.

..?-... [.
I I I Ii ~

."., . : ....,......
i i
~

...
I ~ ~
~ i
.

"~,~ 0"00 ~,..~ :. .-...~.

,_

,.

,....

. . . . .

-1-00 0.00 ~1 of Pro~rom I

t'O0

Fig. 5. Comparison of the estimated relative standard errors of the lag when fitting the Gompertz function (Program 1) and the new model (Program 2) to more than 500 growth curves. The points above the horizontal axis represent the cases when the relative standard error of the lag estimated by the new model was smaller. numerical considerations like linearity and stability it is not incorrect to use any mathematical formula to describe a system empirically; practice will prove or disprove its usefulness. Still, it is a reasonable aim to choose models which can be connected to generally accepted mathematical descriptions of nature. The well-known growth models of population dynamics can be derived from autonomous differential equations. A property of these models is t h a t their solution has zero slope only if the solution is constant. Consequently a growth curve which shows zero derivative at the beginning (at t = 0), m u s t be described by non-autonomous differential equation. Following this concept we introduced the term adjustment function and potential growth by formalized definitions and we constructed a family of growth models, advantageous properties of which were demonstrated. Two features of our concept are t h a t the definition of lag is independent of the shape of the growth curve and the effect of the previous environment is separated from t h a t of the present environment. Only the adjustment function is influenced by the previous environment, the potential growth is not. In this way the model separates from the effects of the present and the pre-inoculation environment. In the introduction we mentioned t h a t in our experiments the subculture, used for getting sufficient a m o u n t of bacteria for inoculation, plays the role of the previous environment, El. this means t h a t if the conditions in the sub-

Bacterial growth model

0.20

55

| J

",,

"

::.,.
F

0.00

~.

,:,"
...

-,.

..

...
n i i n I

"%

.~:
.;',.'"
, ,w Ik '1,

".~."'"

,.
%

-.

..

:,.....
.

,,m
B

- 0 - 2 0 0.00

"

I.O0 ,u.q o f

Pro{lrom I

Fig. 6. Comparison of the estimated relative standard errors of the maximum specific growth rate when fitting the Gompertz function (Program 1) and the new model (Program 2) to more than 500 growth curves. The points above the horizontal axis represent the cases when the relative standard error of the maximum specific growth rate estimated by the new model was smaller. c u l t u r e w e r e v e r y close to the conditions in the a c t u a l c u l t u r e t h e n the population should c a r r y on growing w i t h o u t a lag period a n d in this case we should use a n a d j u s t m e n t function identical to i (a(t) -= 1). In Table 3 we show such a d a t a s e t . T h e r e the o r g a n i s m (C. jejuni) was g r o w n in a f a v o u r a b l e e n v i r o n m e n t before the inoculation and t h e condition in the a c t u a l c u l t u r e was v i r t u a l l y identical to t h a t of the subculture. ( G r o w t h curves p r o d u c e d u n d e r such conditions were not included in t h e set of g r o w t h d a t a c o n s i d e r e d in t h e Results). I f t h e r e is no difference b e t w e e n the s u b c u l t u r e a n d the actual e n v i r o n m e n t t h e n the r e l e v a n t model should h a v e a n adjustm e n t function of a(t) -= 1. This e x p r e s s e s t h e fact t h a t t h e e x p e r i m e n t does not contain i n f o r m a t i o n on t h e lag which, in our concept, would h a v e b e e n a n adjustm e n t period to the new e n v i r o n m e n t . We fitted two curves to the d a t a of Table 3. T h e first curve was Richards' model with a c u r v a t u r e p a r a m e t e r m = 0.25 [see E q n s (22a) a n d (22b)] a n d with a n a d j u s t m e n t function of a(t) =- 1 (i.e. An(t) =- t) to be r e l e v a n t to this experim e n t . Also we fitted the d a t a by Prog r a m 1 as described above. T h e r e s u l t s are s h o w n in Fig. 7. As can be seen, t h e r e is no problem with the goodnessof-fit in e i t h e r case, b u t the e x t r a p o l a tion to the t i m e before the inoculation is different. This plot suggests t h a t the m a x i m u m specific g r o w t h r a t e m i g h t h a v e b e e n r e a c h e d a r o u n d or before the inoculation. This is also a n e x a m p l e of

56

J. Baranyi et al.
ate an a d j u s t m e n t function with two parameters: A, which can be considered as l a g - p a r a m e t e r and n which characterizes the c u r v a t u r e after the lag phase. Based on our experience and for computational convenience we do not suggest the fitting of n but choosing its value at n = 4. Even in this case one m u s t be careful at program m i ng the formula (21a) or (21b), because, when fitting data, overflow error can occur even if the final results are ordinary numbers of usual magnitude. This situation can be avoided if for small values of An(t) (especially at t = 0 where An(t) = 0), the (21b) form of the model is used instead of (21a) and t hen the logarithm t e r m can be omitted, as it is discussed u n d e r (21b). For large values of A,(t), however, the form (21a) is suggested, where, again, the logarithm t e r m can be omitted and the value of the dependent variable is practically Ym~. Here the definition of the terms 'small' and 'large' depends on the required accuracy and

T a b l e 3. V i a b l e c o u n t d a t a o n C. jejuni w h e n t h e e n v i r o n m e n t in t h e subc u l t u r e d i d n o t differ f r o m t h e a c t u a l environment.

No. 1 2 3 4 5 6 7 8 9 10 11 12

Time (h) 0.00 2-98 5.08 7.00 8.87 15.53 19-10 21.82 41.28 45.10 66.72 93-O2

Log counts (loglo m1-1) 3.08 3.64 4.36 4.43 4.63 5.00 5-63 5-81 7.08 7.35 7.78 8.47

an instance when the Gompertz-fit gives a negative value for the lag. As a simplest case with explicit formulae we suggested the logistic growth for the potential growth and a Michaelis-Menten type relation to cre9.00

/
6.00

g 8

_o

J
3.00

- ........

RO(t} Progrom I

0.00

-25

.:,""/ 0
I

I 25

Time

(h)

1 50

I 75

I00

Fig. 7. Applying the Gompertz function (Program 1) and Richards' growth function as potential growth without adjustment function (RO(t)) to the datapoints of Table 3.

Bacterial growth model

on the limit of n u m b e r - r e p r e s e n t a t i o n of the given computer. A more complicated version of the new model can be obtained by (22a) and (22b), which contains two c u r v a t u r e par a m eter s , n and m. However, the fitting of these c u r v a t u r e p a r a m e t e r s is not easy and can cause computational problems. This is why we fixed their value as n = 4 and m = 1 which seemed to be a good compromise between the goodnessof-fit and convenience. This simplest version of our model contains the following parameters: logarithm of the inoculum, lag-parameter, potential m a x i m u m growth rate, Yo; A; ~m~; logarithm of the m a x i m u m population density,

57

y~.

which r e p r e s e n t the m ai n characteristics of a sigmoid curve. Exam i nat i on of non-autonomous growth models is a developing field in population theory (see for example, Vance 1990). They seem especially useful when modelling the a d j u s t m e n t of the population to changing (or new) e n v i r o n m e n t (Coleman 1978). In this paper we showed a possible way to apply this t heory in food microbiology.

Acknowledgement
The authors t h a n k the referees for several helpful suggestions.

References
Baranyi, J., Roberts, T. A. and McClure, P. (1992). Theorems on a non-autonomous growth model describing the adjustment of the bacterial population to a new environment. Sixth IMA Conference on the Mathematical Theory of the Dynamics of Biological Systems, Oxford, 1-3 July, 1992. Barford, J. P., Pamment, N. B. and Hall, R. J. (1982). Lag phases and transients. In Mi, robial population dynamics. (Ed Bazin, M.). CRC Press, pp. 55-91. Boca Raton, FL. Buchanan, R. L. and Cygnarowicz, M. L. (1990) A mathematical approach toward defining the duration of the lag phase. Food Microbiol. 7, 237-240. Coleman, B. D. (1978) Nonautonomous logistic equations as models of the adjustment of populations to environmental change. Math. Biosci. 45, 159-173. France, J. and Thornley, J. H. M. (1984) Mathematical models in agriculture. Butterworth. Oxford. Frederickson, A. G., Ramkrishna, D. and Tsuhiya, H. M. (1967). Statistics and dynamics of procaryotic cell populations. Math. Biosci. 1,327-374. Gibson, A. M., Bratchell, N. and Roberts, T. A. (1988). Predicting microbial growth: growth responses of salmonellae in a laboratory medium as affected by pH, sodium chloride and storage temperature. Int. J. Food Microbiol. 6, 155-178. Kono, T. (1968) Kinetics of microbial cell growth. Biotech. Bioeng. 10, 105-131. Korn, G. and Korn, T. (1973). Mathematical handbook for scientists and engineers. New York. McGraw-Hill. MacDonald, N. (1978). Time lags in biological models. Berlin. Springer. Pirt, S. J. (1975). Principles of microbe and cell cultivation. London. Blackwell. Press, W. H., Flannery, B. P., Teukolsky, S. A. and Vetterling, W. T. (1990) Numerical recipes. Cambridge. Cambridge University Press. Roels, J. A. and Kossen, N. W. F. (1978). On the modelling of microbial metabolism. In Progress in industrial microbiology, vol. 14. (Ed. Bull, M. J.), pp. 95-203. Amsterdam. Elsevier. Turner, M. E., Bradley, E. L., Kirk, K. A. and Pruitt, K. M. (1976). A theory of growth. Math. Biosci. 29, 367-373. Vance, R. R. (1990) Population growth in a time-varying environment. J. Theor. Biol. 3'/, 438-454.

58

J. Baranyi et al.

Vance, R. R. and Coddington, E. A. (1989) A nonautonomous model of population growth. J. Math. Biol., 27, 491-506. Whiting, C. (1992) Letter to the editor. Food Microbiol. 9(2), 173-174. Zwietering, M. H., Jongenburger, I., Rombouts, F. M. and van't Riet, K. (1990) Modelling of the bacterial growth curve. Appl. Environ. Microbiol. 56, 1875-1881.

APPENDIX Below we s u m m a r i z e some m a t h e m a t i c a l t h e o r e m s on t h e new model. Thf~se theor e m s w e r e p r e s e n t e d in B a r a n y i et al. (1992). C o n s i d e r the initial v a l u e p r o b l e m (la), (lb). T h e t h e o r e m s below are valid also if we consider the case of limited g r o w t h rate, so i n s t e a d of condition (c) u n d e r (la), (lb) we suppose the m u c h w e a k e r c o n s t r a i n t :

dtL/dx is limited

(0 < x < xm~).

Theorem 1 L e t a(t) be a n a d j u s t m e n t function as defined u n d e r (2a), (2b). T h e n t h e solution

of the initial v a l u e problem (2a) a n d (2b) is

g(t) = f(A(t)),
where
t

A(t) = f a(s)ds o

Theorem 2 Let a(t) a n d fl(t) a d j u s t m e n t functions a n d let g~(t), go(t) d e n o t e the respective
solutions of (2a), (2b). If t

I f (~(s) - fl (s))dsr <

0

then Ig~(t) -go(t)l < ell~(x)xl,~ < et~(Xo)Xm~. L e t the a d j u s t m e n t function h a v e the form tn

an(t) =
for some A,n > O. In this ease

An + t n

An(t) = ~j ~
0

-ff

where

Bn(t)=

fl
t 0

l + snds.

Bacterial growth model

Theorem 3
If n > 1 then the improprius integral B , = l i m Bn(t)
t--~

59

exists. M o r e o v e r if n - o ~ t h e n Bn ~ 1.

Theorem 4
I f n ~oo t h e n B~(t) ~ B~(t) u n i f o r m l y on (0,~) w h e r e

B~(t) =

t 1

(t < 1) (t > 1)"

C o n s e q u e n c e : if n ~oo t h e n A,(t) -o A~(t) u n i f o r m l y on (0,~), w h e r e

A~(t) =

0 t-k

(t < n) (t >_h)"

Theorem 5
U s i n g t h e n o t a t i o n s above: if n - - ~ t h e n

IgA~(t)) ~ f(A~(t)) u n i f o r m l y on (0,~).