 Original article
 Open Access
Multiplicative modelling of fourphase microbial growth
 María Jesús MunozLopez^{1, 2}Email author,
 Maureen P. Edwards^{3},
 Ulrike Schumann^{4, 5} and
 Robert S. Anderssen^{6}
https://doi.org/10.1186/s4073601500180
© MunozLopez et al. 2015
Received: 26 May 2015
Accepted: 14 September 2015
Published: 1 October 2015
Abstract
Microbial growth curves, recording the fourphases (lag, growth, stationary, decay) of the dynamics of the surviving microbes, are regularly used to support decisionmaking in a wide variety of health related activities including food safety and pharmaceutical manufacture. Often, the decisionmaking reduces to a simple comparison of some particular feature of the fourphases, such as the time at which the number of surviving microbes reaches a maximum. Consequently, in order to obtain accurate estimates of such features, the first step is the determination, from experimental measurements, of a quantitative characterization (model) of the fourphases of the growthdecay dynamics involved, which is then used to determine the values of the features. The multiplicative model proposed by Peleg and colleagues is ideal for such purposes as it only involves four parameters which can be interpreted biologically. For the determination of the four parameters in this multiplicative model from observational data, an iterative twostage linear least squares algorithm is proposed in this paper. Its robustness, which is essential to support successful comparative assessment, is assessed using synthetic data and validated using experimental data. In addition, for the multiplicative model, an analytic formula is derived for estimating the average lifetimes of the surviving microbes.
Keywords
Introduction
For microbial growth considerations in areas as diverse as food contamination and pharmaceutical manufacture, the key data are the fourphases (lag, growth, stationary, decay) of the growth dynamics of the surviving microbes [7, 8]. For the utilization of such data for comparative assessment, monitoring and predictive purposes, an appropriate model is required which accurately tracks the four phases [8]. Depending on the situation under consideration, such a model can be utilized in various ways. In food contamination situations, it can be used to compare different inactivation (heating) strategies in food processing or to comparatively assess the survival characteristics of different classes of microbes. In pharmaceutical situations, it can be used to predict the optimal time to harvest the surviving microbes, since it is only the surviving microbes that can be used to make the pharmaceutical. In the study of soil microbes, comparative assessment has been used to compare the chemical and physical factors which influence the relative levels of microbial carbon and nitrogen biomasses [10].
which models an initial growth (by having α>a) which is eventually dominated by the decay (by having b>β).
It is easier to describe the algorithm using this equation. Once the parameters a, b, α and β have been determined, one can then use the above relationships to determine m _{1}, m _{2}, t _{ cg } and t _{ cd }. These relationships are discussed from a biological interpretative perspective in subsections 2.3 and 2.4.
As explained in Edwards et al. [7], the importance of this model is that it is the solution of a nonautonomous ordinary differential equation which is able to track the fourphases. It therefore circumvents the shortcomings associated with models which are the solutions of autonomous ordinary differential equation, such as the Verhulst, since their solutions can only model the first, second and third phases, but not the fourth.
To determine the parameters in fitting the multiplicative model to observational survival data, Peleg and Corradini [8] suggest the use of mathematical software such as Mathematica. The challenge here is the need to find starting values for the parameters which are representative of the situation under consideration and to ensure that the solver used is stable with respect to measurement noise and limited data.
Here, it is shown how the special structure of the multiplicative model can be exploited to derive an iterative twostep procedure for the determination of the parameters. An assessment of its robustness, using synthetic data, is given. Validation is performed using microbial (fungal) survival measurements.
The paper has been organized in the following manner. The multiplicative model is discussed in Section 2 and an analytic average lifetime formula for it is derived. The algorithm is proposed in Section 3 and tested on synthetic data in Section 4. The application of the algorithm to real microbial survival data is the subject of Section 5 along with conclusions.
The model
The multiplicative model proposed by Peleg et al. (2009) [9] can be derived in various ways.
2.1 From first principles
where t _{ cg } denotes the characteristic growth time and m _{1} characterizes the rate of growth.
where t _{ cd } denotes the characteristic decay time and m _{2} characterizes the rate of decay.
Justification for this being a realistic model of a fourphase growthdecay process is given in Edwards et al. [7], where it is shown that such a structure corresponds to the solution of a nonautonomous ordinary differential equation model of a quite general growthdecay process.
2.2 Solution of the nonautonomous von Bertalanffy equation
which becomes, when θ(t) is a constant because β=b=1, the standard exponential growthdecay equation.
For the von Bertalanffy Eq. (5), Edwards and Anderssen [6] have performed a Lie point symmetry analysis to identify the regularity that \(\bar {\alpha }\), \(\bar {\beta }\), \(\bar {a}\), \(\bar {b}\) and ψ(t) must satisfy in order for (5) to have interesting classes of analytic solutions (often referred to technically as nontrivial symmetries). Such symmetries can then be utilized to explore for new closed form solutions.
2.3 Biological interpretation of the parameters α, β, a and b
The relevance of the above two derivations for Eqs. (1) and (2) is that they shed light on how to interpret the parameters α, β, a and b biologically and study their interactive interdependence. The starting point is Eq. (2) rewritten in its equivalent form (6).
For the standard decay Eq. (6), when θ(t) is a constant θ _{0} and the population corresponds to a discrete ensemble of members, as holds for microbial growthdecay, the characteristic time of the exponential decay 1/θ _{0} corresponds algebraically to the “mean lifetime”, ℓ _{0}, of the members in the ensemble. (The algebraic details are contained in the Appendix.) In addition, if all the individual lifetimes are measured with respect to the same initial reference state, then 1/θ _{0} corresponds to the arithmetic mean of these individual times.
As highlighted in the Appendix, the mean lifetime concept can be extended to any fourphase microbial growthdecay situation which decays to zero. This generalized mean lifetime will be denoted by ℓ _{ θ }. Its importance relates to the fact that it measures a key biologically relevant feature, the average life time of the microbes in a fourphase growthdecay situation. From a food safety perspective, ℓ _{ θ } can be used to identify strategies that allow inactivation to be performed effectively, whereas, from a pharmaceutical perspective, an understanding of the value of ℓ _{ θ } is required to guarantee the time optimal harvesting of live microbes.
In particular, with respect to given growthdecay data, the algorithm is used to determine the values of the parameters α, β, a and b which are then substituted into Eq. (7) which can be evaluated using Matlab.
As is clear from the form of the right hand side of Eq. (7), the value of ℓ _{ α,β,a,b } can be used to compare different scenarios for the parameters α, β, a and b. For example, since together α and β identify how a particular microbial population grows, the values of α and β could be fixed and the values of a and b varied to find the minimum value of the average lifetimes ℓ _{ α,β,a,b } as a characterization for an optimal strategy for performing inactivation.
Comparative values for ℓ _{ α,β,a,b } for various growthdecay dynamics are discussed in subsection 5.
2.4 Biological interpretation of the parameters t _{ cg }, t _{ cd }, m _{1} and m _{2}
If m _{1}=m _{2}=1, the parameters t _{ cg } and t _{ cd } correspond, respectively, to the characteristic times of the growth and decay. In particular, they characterize how quickly the growth and decay of the microbes within a population occur, with the rate of growth (decay) being inversely proportional to the value of t _{ cg } (t _{ cd }). Consequently, the values of t _{ cg } and t _{ cd } give an immediate indicative illustration of the relative strengths of the growth and decay dynamics.
Consequently, multiple choice of α and β (a and b) will generate the same value for t _{ cg } (t _{ cd }). Such ambiguities are resolved by determining α and β (a and b) from the experimental data of the growthdecay dynamics under consideration. The linear least squares procedures, as outlined in section 3.1, achieve this by first estimating the values of α and β, and then the values of a and b, separately in an iterative manner.
2.5 Properties of the multiplicative model
Sufficient conditions, in terms of the parameters in the more compact form (2) for the multiplicative model, which guarantee a fourphase structure, are given by α>a (which guarantees initial growth) and b>β (which guarantees subsequent decay). Alternatively, in terms of the general form of Eq. (6) with an arbitrary θ, fourphase dynamics is guaranteed if θ(t) is initially positive, which guarantees initial growth, and \(\int _{0}^{\infty } \theta (\tau)d\tau =\infty \), which guarantees that subsequent decay occurs and goes to zero.
which will play a key role in the formulation of the algorithm.
which yields a connection back to the multiplicative model being the solution of a particular form of the von Bertalanffy equation.
The algorithm
The essence of the current situation is the fitting of the multiplicative model (2) to given experimental data, which reduces to the determination of estimates for the four parameters α, β, a, b. However, the multiplicative model is nonlinear and the amount of experimental data available is usually quite limited. The standard procedure proposed by various authors is to use some nonlinear regression software package such as is available in Matlab. The limitation here is the need to find starting values for the parameters which are representative of the situation under consideration. In comparative assessment situations, it is important that the estimates of the values of the parameters α, β, a, b correctly characterize the situations being compared. For example, if the value of the parameter b was used to assess the effectiveness of different inactivation strategies, then the estimates of b, utilized for the comparative assessment, must correctly represent the actual decay occurring so that no incorrect action or advice was implemented.
As explained below, because of the way in which the estimation is performed, the determination of the parameters α, β, a, b is essentially unique in that the estimation is performed, iteratively, as two separate steps involving first the growth phase, to determine α and β, and then the decay phase, to determine a and b.
In a sense, compared with nonlinear least squares methods, the proposed algorithm is an example of “let the data decide”. The rationale is that if one just uses a nonlinear solver to do the parameter identification, then no specific structure is exploited within the data which relates to subsets of the parameters. In the algorithm proposed here, this is possible as, in the multiplicative model, the model is separable into a growth component, involving only α and β, and a decay component, involving only a and b.
3.1 Estimating the parameters
In the past, different methods have been proposed and used to model microbial growth and decay dynamics.
For instance, in order to assess the nature of the initial lagphase of growthdecay dynamics, Baranyi et al. [3] proposed the use of detection times. However, this requires that the detection times be limited to the initial exponential growth in order to avoid underestimating the rate of growth of the lagphase.
into the nonautonomous Eq. (11). Various choices for ϕ(t) have been proposed and analysed by Baranyi and colleagues. However, they have not chosen a form for ϕ(t) that corresponds to that for the nonautonomous equation which generates the multiplicative model (2). In particular, their emphasis is on modelling the growth of the total population.
Peleg and Corradini [8], for determination of the parameters in the multiplicative model (1), suggest nonlinear least squares. The difficulty is that representative starting values for the parameters must be chosen for the implementation of such methods, which the proposed algorithm avoids.
The algorithm proposed and implemented here, which explicitly exploits the properties of logarithms, is based on the iterative use of two linear least squares approximations applied to different phases of a growth curve. Its advantage is that it can be iterated to obtain successively better approximations for the parameters α, β, a, b. This type of algorithm does not appear to have been published in the microbial growth modelling literature, though it has been used to determine the parameters of the stretched exponential (Kohlrausch) function in rheological and biological applications [1].
The fourth, fifth, ⋯ steps in the implementation now iterate, respectively, between the second and third steps.
3.2 Algorithm implementation
Because the implementation of the algorithm involves the evaluation of logarithms, the choice of the scale for the times becomes an important issue. In the situations studied here, the basic time scale is days.
However, for measurements made at fractions of a day, the logarithms will be negative. Consequently, to avoid this potential difficulty, it is best to work with a time scale (hours, minutes or seconds) such that all the times, at which measurements were made, are greater than one.
The validation of the algorithm using synthetic data

the accuracy of the recovery of the parameters, and

the quality of the reconstructions of the fourphase growthdecay dynamics curves compared with the actual N(t).
Though the comparison of the reconstructions of the growthdecay dynamics is indicatively important, the key issue is the robustness, accuracy and reliability of the recovery of the parameters, as it is those that will be used for subsequent decisionmaking and comparative assessments.
4.1 The synthetic data analysis
The exact synthetic data used to test the algorithm was generated using the discrete multiplicative model data {N(t _{ i })} of Eq. (20) with the parameter values α=6, β=1.5, a=4, b=2 and N _{0}=100.
4.1.1 Exact synthetic data inversion
For the synthetic data situation without noise, only 7 data points are needed to perform the parameter estimation using the algorithm, which returns the correct values α=6, β=1.5, a=4, b=2.
4.1.2 Simulation studies of nonexact synthetic data inversion
It is known that, in carefully performed measurements of microbial growthdecay dynamics, the measurement error does not depend on the size of the population as it evolves. Consequently, it was only necessary to test the robustness of the algorithm with respect to Gaussian error perturbations.
This difference represents a direct illustration of how fundamental β and b are to determining the growth and decay, respectively, in order to accurately recover a fourphase structure. It implies that a good fit to a fourphase structure cannot be achieved by simply varying α and a unless good estimates of β and b have been determined. This interpretation is implicit in the proposed algorithm, as illustrated in Eqs. (19) and (17), which highlight that β and b are the slopes of the straight lines that are fitted to the logarithmic data. This relates to the fact that, in terms of the linearity of the algebra of Eqs. (19) and (17), the constants ln(α) and ln(a) do not influence the actual slopes β and b of the straight line fits to the logarithmic data.
Furthermore, the algorithm estimates β and b separately using, respectively, a growth component and a decay component of the fourphase structure. Consequently, this illustrates the uniqueness in the determination of the parameters α, β, a, b and, hence, the values of t _{ cg } and t _{ cd } of Eq. (8).
Application of the algorithm to microbial survival data and conclusions
5.1 Recovery of the parameters α, β, a, b
In order to illustrate the practicality of the algorithm for real data, it was applied to the measurements from a study of the growthdecay dynamics for the filametus fungus Fusarium oxysporum.
Fusarium oxysporum is a plant pathogenic fungus with a wide host range causing a variety of diseases contributing to crop losses all over the globe. To obtain microbial growth data in a closed environment we monitored the growth of the fungus Fusarium oxysporum in minimal media. A primary potato dextrose broth culture was inoculated with Conidiospores from a −80 °C frozen stock and grown at 28 °C, shaking at 200 rpm for 2 days. Cells were collected by centrifugation, suspended in water, the optical density at 260 nm was measured and the cell concentration determined by comparison with a standard curve. A fresh secondary minimal medium culture was inoculated with 1.0E6 cells/ml and grown as above. In a temporal fashion, 1000 μ l samples were removed from the culture, the cells collected by centrifugation and suspended in water (between 100 μ l and 500 μ l) adjusting the suspension volume as the culture became denser. Care was taken that cells were well suspended at all times by vigorous vortexing. Cells were then stained with Propidium Iodide for 5 minutes. Microscopic images were taken using three independent 5 μ l subsamples imaging at least 7 independent regions of each sample. Bright field and fluorescence images were taken and the total number of cells counted using the bright field image. Dead cell counts were obtained from fluorescent images as propidium iodide permeates the membranes of dead cells staining these red. The average number of total and dead cells was determined and, as the cell suspension was more concentrated than the culture, the suspension volume was taken into account to determine the proportional number of total and dead cells in the culture.
The measurements represent a situation where the data is sparse and has only been measured for part of the decay phase. Nevertheless, it contains sufficient data to allow the algorithm to recover useful estimates of the parameters α, β, a, b, which can be used to evaluate ℓ _{ θ } of Eq. (7).
5.2 Evaluation of average lifetimes ℓ _{ α,β,a,b }
5.3 Conclusions
For the determination of the four parameters α,β,a,b in the multiplicative model (2), a simple, easily implementable, iterative twostage linear least squares algorithm has been proposed. Its robustness has been confirmed by testing it on synthetic data. Its practicality has been demostrated by applying it to measured growthdecay for the fungus Fusarium oxysporum.
In addition, for the multiplicative model, an analytic formula has been derived for estimating the average lifetimes ℓ _{ α,β,a,b } of the surviving microbes, which has been applied to the synthetic and measured data.
Overall, it appears that the numerical performance of the algorithm and the average liftime estimate will be useful in the support of decisionmaking related to health issues such as food safety and pharmaceutical manufacture.
Appendix
Mean lifetime for microbial growthdecay for the multiplicative model
The standard decay model
⇓
⇓ Solve:
⇓
⇓ Transform N(t) to an exponential probability distribution:
⇓
⇓ The mean of the exponential distribution is λ:
The generalized decay model
⇓
⇓ Solve:
⇓
⇓ Regularity: θ(0)>0 and \(\int _{0}^{\infty } \theta (\tau)d\tau =\infty \)
⇓
⇓ Transform N _{ θ }(t) to a probability distribution:
⇓
⇓ Compute the mean of \({\mathcal P}(N_{\theta }(t))\):
Declarations
Acknowledgements
The authors thank the reviewer whose comments helped improved the clarity of the paper. The third author wishes to acknowledge the support of Prof. Thomas Preiss (Department of Genome Science, School of Medical Sciences (JCSMR), Australian National University, Garran Road, ACT 2601, Australia) for his support to carry out this research.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
Authors’ Affiliations
References
 Anderssen, R.S., Helliwell, C.A.: Information recovery in molecular biology: causal modelling of regulated promoter switching experiments. J. Math. Biol. 67, 105–122 (2013).MathSciNetView ArticleMATHGoogle Scholar
 Anderssen, R.S., Husain, S., Loy, R.J.: The Kohlrausch function: properties and applications. ANZIAM J (E). 45, C800—C816 (2004).MathSciNetMATHGoogle Scholar
 Baranyi, J., Pin, C.: Estimating bacterial growth parameters by means of detection times. Appl. Enviro. Microbiol. 65(2), 732–736 (1999).Google Scholar
 Baranyi, J., Roberts, T.A., McClure, P.: A nonautonomous differentialequation to model bacterialgrowth. Food Microbio. 10(1), 43–59 (1993).View ArticleMATHGoogle Scholar
 Baranyi, J., Roberts, T.A., McClure, P.: Some properties of a nonautonomous determistic growthmodel describing the adjustment of the bacterial population to a new environment. IMA. J. Math. Appl. Med. Bio. 10(4), 293–299 (1993).View ArticleMATHGoogle Scholar
 Edwards, M.P., Anderssen, R.S.: Symmetries and solutions of the nonautonomous von bertalanffy equation. Commun. Nonlinear Sci. Numer. Simulat. 22, 1062–1067 (2015).MathSciNetView ArticleMATHGoogle Scholar
 Edwards, M.P., Schumann, U., Anderssen, R.S.: Modelling microbial growth in a closed environment. J. MathforIndustry. 5, 33–40 (2013).MathSciNetMATHGoogle Scholar
 Peleg, M., Corradini, M.G.: Microbial growth curves: what the models tell us and what they cannot. Crit. Rev. Food Sci. Nutr. 51(10), 917–945 (2011).View ArticleGoogle Scholar
 Peleg, M., Corradini, M.G., Normand, M.D.: Isothermal and Nonisothermal Kinetic Models of Chemical Processes in Foods Governed by Competing Mechanisms. J. Agric. Food Chem. 57(16), 7377–7386 (2009).View ArticleGoogle Scholar
 Wardle, D.A.: A comparativeassessment of factors which influence microbial biomass carbon and nitrogen levels in soil. Biol. Rev. Camb. Philos. Soc. 67, 321–358 (1992).View ArticleGoogle Scholar