Modelling the inhibitory effect of copper sulfate on the growth of Penicillium expansum and Botrytis cinerea.

AIMS: This study aimed to investigate the effect of copper sulfate (from 0 to 8 mmol kg(-1)) on radial growth rate and lag time of two moulds responsible for vine grapes spoilage: Penicillium expansum strain 25·03 and Botrytis cinerea, strains BC1 and BC2. METHODS AND RESULTS: A new model was developed to describe tailing and shoulders in the inhibition curves. Because of tailing, the minimum inhibitory concentration (MIC), was not defined as the concentration at which no growth was observed, but as the concentration at which the lag time was infinite. The concentrations at which µ = µ(opt)/2, (Cu50), were in the range of 2·2-2·6 mmol kg(-1). Radial growth rate of P. expansum and the reciprocal of the lag time were linearly correlated (r = 0·84). In contrast, in the range 0-4 mmol kg(-1), an inhibition of growth of B. cinerea was observed whereas germination remained unaffected (i.e. the lag time was constant). In the range 4-8 mmol kg(-1) , the radial growth rate of B. cinerea was almost constant (c. 1 mm day(-1)), but germination was inhibited (i.e. the lag time was increased). CONCLUSIONS: The MIC values were 4·7 mmol kg(-1) for P. expansum, 8·2 and 7·3 mmol kg(-1) for B. cinerea strain BC1 and BC2, respectively, demonstrating that some isolates of these moulds are resistant to copper. SIGNIFICANCE AND IMPACT OF THE STUDY: Copper concentrations at 4 mmol kg(-1) would be sufficient to control the development of these isolates, but the toxicity of copper should be extended to other isolates and evaluated in vineyards.

Use of the Weibull model to describe inactivation of dry-harvested conidia of different Penicillium species by ethanol vapours.

AIMS: This study aimed at modelling the effect of ethanol vapours, in the range 0.7-7.5 kPa, on the inactivation of dry-harvested conidia of Penicillium chrysogenum, Penicillium digitatum and Penicillium italicum. METHODS AND RESULTS: Survival curves were modelled by a Weibull model: log (N/N(0)) = -1/2.303 (t/alpha)(beta). The shape parameter beta was different from one in all cases, indicating that the classical first-order kinetics approach is the exception rather than the rule. Survival curves exhibited upward concavity (beta < 1) with the notable exception of P. chrysogenum at ethanol vapour pressures 0.7 and 1.5 kPa. The scale parameter alpha (h) varied greatly depending on the ethanol vapour pressure and on the species. CONCLUSIONS: For safety reasons, it is recommended not to exceed an ethanol vapour pressure of 3.3 kPa. At 2.8 kPa, more than 4 log(10) reductions in viable conidia were achieved for all the species after 24-h exposure. SIGNIFICANCE AND IMPACT OF THE STUDY: Ethanol has GRAS status in the USA and represents an interesting alternative to fungicides. The effectiveness of ethanol vapours to inactivate dry-harvested conidia of some Penicillium was demonstrated in this study.

Strategies for improving production and purification of a recombinant protein: rP30 of Toxoplasma gondii expressed in the yeast Schizosaccharomyces pombe.

Many problems concerned with the production and the purification of recombinant proteins must be addressed prior to launching an industrial production process. Among these problems, attention is focused on low-level expression that complicates the purification step and can jeopardise the process. The expression of a membrane protein, rP30, of Toxoplasma gondii in the yeast Schizosaccharomyces pombe led to a secretion of only 0.5 microg ml(-1). In order to obtain a sufficient quantity for biochemical characterization and evaluation in vitro diagnostic test development, strategies for both production and purification had to be optimized. First, the influence of four nitrogen sources (three peptones and yeast extract) on the growth rate, but also on the separation between the protein and the components of the fermentation broth was assessed. Second, batch and fed-batch fermentations were compared in terms of final biomass and rP30 concentrations. Third, three different protocols that included fixed and expanded bed ion exchange chromatography were compared for processing a large volume of feedstock. By using the most appropriate strategies, i.e. fed-batch fermentation, capture on EBA cation exchanger and affinity chromatography polishing, a purification factor of 1778 and a yield of 49% were achieved. These performances allowed a 12.5-fold increase for the overall rP30 process productivity.

Influence of inoculum preparation on the growth of Penicillium chrysogenum.

AIMS: The influence of the spore preparation on subsequent fungal growth of Penicillium chrysogenum was assessed. METHODS AND RESULTS: The influence of four factors [the nature of the diluting solution (physiological water and physiological water added with Tween-80), the age of the sporulating culture (4, 8 and 12 days), the strain (737, 738 and 740) and the inoculum size (102, 103, 104 and 105 spores ml(-1)] on two responses (i.e. the radial growth rate, mu, and the lag time, lambda) was studied using an experimental screening methodology. CONCLUSIONS: The main conclusion was the strong effect of the inoculum size on lambda. In contrast, the diluting solution had no effect on both the experimental responses. In order to obtain the highest growth rates, it is recommended to use 4-day-old sporulating cultures with an inoculum size of 102 spores ml(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: There is a need for standardizing spore preparation in predictive mycology. The screening methodology is a powerful tool to determine the influence of qualitative and quantitative factors on various biological responses and can be applied widely in microbiology.

Relationship between spore germination kinetics and lag time during growth of Mucor racemosus.

AIMS: Spore germination requires microscopic observation whereas fungal growth results in a macroscopic examination. This paper aims at establishing a relationship between the percentage of germinated spores and parameters easily available from visible development. METHODS AND RESULTS: About 225 spores of Mucor racemosus were inoculated on PDA medium and incubated at 15 degrees and 25 degrees C. Germination kinetics were modelled by a logistic function. Fungal development provided two parameters, a growth rate, micro, and a lag period, lambda, defined as the slope of the straight line of the graph radius (mm) vs time (h) and the intercept of this line with the X-axis, respectively. CONCLUSIONS: It was found that the lag period coincided with the completion of the germination process, although the number of spores inoculated should be controlled carefully. SIGNIFICANCE AND IMPACT OF THE STUDY: Providing that this result can be generalized, this procedure would constitute a significant breakthrough for predicting food spoilage by moulds.

Comparison of the effects of temperature and water activity on growth rate of food spoilage moulds.

The influence of temperature (T) and water activity (aw) on the growth rate (mu) of seven moulds (Alternaria alternata, Aspergillus flavus, Cladosporium cladosporioides, Mucor racemosus, Penicillium chrysogenum, Rhizopus oryzae and Trichoderma harzianum) was assessed in suboptimal conditions. Firstly, the dependence of fungal growth on temperature, at aw 0.99, was modelled through an approach described previously for bacteria. A dimensionless growth rate variable: mu(dimalpha)=mu/mu (optalpha) depended on the following normalised temperature: T(dim)=(T-T(min))/(T(opt)- T(min)) according to a power function: mu(dimalpha)=[T(dim)]alpha, where alpha was an exponent to be estimated. Secondly, the same approach was used to describe the influence of aw on fungal growth, at the respective optimum temperatures for each mould. Similarly, mu(dimbeta)=mu/mu(optbeta) depended on the following normalised water activity: a(wdim)=(aw-a(wmin))/(a(wopt)-a(wmin)) according to a power function: mu(dimbeta)=[a(wdim)](beta). Results show: (i) for each mould, the alpha-value is significantly less than the beta-value, confirming that water activity has a greater influence than temperature on fungal development; (ii) the alpha-values and the beta-values depend on the mould; (iii) the alpha-value is less than 1 for the mesophilic mould A. flavus, whereas the other moulds are characterised by higher alpha-values ranging from 1.10 to 1.54; (iv) the mesophilic A. flavus exhibits a low beta-value, 1.50, compared to the hydrophilic T. harzianum, beta=2.44, while beta-values are within the range (1.71-2.37) for the other moulds.

Application of Doehlert design to determine the combined effects of temperature, water activity and pH on conidial germination of Penicillium chrysogenum.

AIMS: The influence of temperature, water activity and pH on the time necessary for germination of 90% of Penicillium chrysogenum conidia inoculated (T90) was determined. METHODS AND RESULTS: A new experimental device was developed for easy monitoring of the germination process. Experiments were carried out according to a Doehlert matrix at 11-31 degrees C, 0.86-0.98 water activity (a(w)) and pH 3.5-6.5. In these conditions, a second order polynomial relationship between T90 and the environmental factors was established for the different humectants used throughout this study (e.g. glycerol and sorbitol) with regression coefficients close to 0.97. CONCLUSIONS: For both humectants, the major effect of temperature and water activity on T90 was highlighted, whereas the effect of pH on T90 in these experimental conditions was not significant. The combined effect of temperature and water activity on T90 was also demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: Both the experimental set-up and the Doehlert matrix were well suited to determine the influence of environmental factors on mould germination.

A temperature-type model for describing the relationship between fungal growth and water activity.

Growth of Penicillium chrysogenum, Aspergillus flavus, Cladosporium cladosporioides and Alternaria alternata at their respective optimum temperatures was studied in Potato Dextrose Agar (PDA) medium at different water activities (a(w)) adjusted with glycerol. The growth rate (mu) was expressed as the increase in colony radius per unit of time. This paper extends the model that showed the relationship between temperature and bacterial growth rate developed by Rosso et al. [J. Theor. Biol. 162 (1993) 447] to describe the influence of a(w) on fungal development. An excellent correlation between the experimental data and the model predictions was obtained, the regression coefficients (r2) were greater than 0.990, with the exception of that for A. flavus (r2 = 0.982). In addition, the use of such a model allows predictions of the cardinal water activities: a(wmin), a(wopt) and a(wmax). The estimation of the minimum water activity (a(wmin)) was in accordance with data literature for all the moulds considered here, but seemed to be slightly underestimated for P. chrysogenum and A. flavus when compared to our experimental values. The estimations of the optimal water activity (a(wopt)) and the optimal growth rate (muopt) were in excellent agreement to the experimental results for the four moulds. Through this example, it is suggested that the same approach for modelling can be used for various microorganisms (e.g. bacteria and moulds), and different environmental parameters (e.g. temperature and water activity).

Prediction of conidial germination of Penicillium chrysogenum as influenced by temperature, water activity and pH.

AIMS: Conidial germination of Penicillium chrysogenum was carried out under operating conditions compatible with a pastries manufacturing process. METHODS AND RESULTS: A range, limited by two experimental values, was defined for each environmental factor tested: temperature (15 or 25 degrees C), water activity (0.75 or 0.85) and pH (3.5 or 5.5). A closed device was made, which maintained an equilibrium between water activity of the culture medium and atmospheric relative humidity during 25 days, to follow spore germination. The combined effects of temperature, water activity and pH on spore germination were studied by applying factorial design methodology. CONCLUSIONS: Higher rates of spore germination were associated with a high level of water activity. The incubation temperature also had a positive effect. A significant positive interaction between water activity and temperature was observed. Under these specific experimental conditions, pH did not have a significant effect on conidial germination. SIGNIFICANCE AND IMPACT OF THE STUDY: A model describing the behaviour of fungal conidia is proposed.

Influence of the growth rate calculation on the relationship between growth rate and temperature.

Three calculations of the growth rate (e.g. slope of a plot of the log10 of cfu ml-1 vs time, mum of the Gompertz equation and the reciprocal of time to obtain 108 cfu ml-1) were compared for Escherichia coli TG1 growing in tryptone soy broth medium at temperatures ranging from 14 to 39 degrees C. Up to now, the influence of using such different definitions on the relationship between microbial growth rate and temperature has never been investigated. In order to compare these calculation procedures, a dimensionless analysis based on the following normalized variables, mudim = mu/muopt and Tdim = [T-Tmin]/[Topt-Tmin], was used (Dantigny 1998). The influence of suboptimal temperatures on the growth rate was represented by means of a Belehràdek-type model based on a power function law: [mudim] = [Tdim]alpha. The influence of the different growth rate calculations on the model constants was assessed. Despite the great dependence of the raw growth rate values on the calculation procedure, the dimensionless analysis demonstrated that the alpha-value is independent of the growth rate definition. This result suggests that any definition for the growth rate can be utilized in studies aimed at determining the influence of temperature on microbial growth and highlights the interest of using dimensionless variables to overcome differences in the order of magnitude of the growth rate data and to avoid confusion between definitions.

Transition rate kinetics from ethanol oxidation to glucose utilisation within a structured model of baker's yeast.

The transition rate kinetics from ethanol oxidation to glucose utilisation, within a structured model of baker's yeast, described previously, were experimentally identified. The shift in metabolism has been assessed through glucose pulses during batch growth on ethanol. The influence of glucose concentration (between 0.25 g l-1 and 0.90 g l-1) and initial biomass concentration (between 0.61 g l-1 and 1.44 g l-1) on the transition rate was determined. The transition rate can not be described by a first-order saturation-type kinetics with respect to glucose only. A corrective term, which takes into account biomass concentration should be included.

Modeling of the aerobic growth of Saccharomyces cerevisiae on mixtures of glucose and ethanol in continuous culture.

A structured model dedicated to fed-batch growth of baker's yeast is detailed in steady-state conditions. The simulated results for aerobic growth on mixtures of glucose and ethanol are provided. The model differentiates and identifies glucose utilisation (either oxido-reductive or by oxidation), state 1 and ethanol oxidation, state 2. Ethanol can be oxidised when glucose concentration is below a certain value, s(crit), only; ethanol is excreted when glucose concentration exceeds s(crit). The amount of ethanol co-consumed with glucose is controlled by s(crit) through the transition rate from X1 to X2. Two major novelties are introduced for modeling glucose metabolism. (1) The specific growth rate on glucose is constant, equal to D(crit), at low glucose concentrations, but follows Monod kinetics at high glucose concentrations. (2) Non-constant yields (i.e., Yx/s and Ye/s) are determined by means of dimensionless groups when the specific growth rate on glucose exceeds D(crit). The model is developed on experimentally easily accessible parameters found in the literature for Saccharomyces cerevisiae H1022. A remarkable prediction of the experimental data obtained by Rieger et al. (1983) (J. Gen. Microbiol. 129, 653-661) is highlighted. The simulations suggest that the specific growth rate on ethanol may be underestimated in limited respiratory capacity based models.

Simulation of L/A control in fed-batch culture of baker's yeast.

L/A controllers have extended their use from continuous to fed-batch fermentation where the control is applied from the start of an initial batch phase. As opposed to proportional integral derivative (PID) controllers where even a startup procedure is recommended prior to fed-batch, the L/A controller is not upset by an early connection. It is easily retuned continuously by means of ethanol measurements and can cope with a large range of output conditions. The performance of an L/A algorithm, which uses biomass concentration as the controlled variable, is assessed through simulation. The self-contained algorithm is relatively simple with no greater intrinsic complexity than modern PID stand alone controllers.