Journal of Applied Microbiology 2001, 91, 900±906

Application of Doehlert design to determine the combined

effects of temperature, water activity and pH on conidial
germination of Penicillium chrysogenum

M. Sautour, A. Rouget, P. Dantigny, C. Divies and M. Bensoussan

Laboratoire de Microbiologie, UMR INRA 1082, ENS.BANA, Dijon, France

818/05/01: received 11 May 2001 and accepted 14 May 2001

M . S A U T O U R , A . R O U G E T , P . D A N T I G N Y , C . D I V I E S A N D M . B E N S O U S S A N . 2001.
Aims: The in¯uence 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°C,
0á86±0á98 water activity (aw) and pH 3á5±6á5. 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 coef®cients
close to 0á97.
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
signi®cant. The combined effect of temperature and water activity on T90 was also
demonstrated.
Signi®cance and Impact of the Study: Both the experimental set-up and the Doehlert
matrix were well suited to determine the in¯uence of environmental factors on mould
germination.

bacterial modelling (Gibson and Hocking 1997). The

INTRODUCTION
Under favourable environmental conditions, moulds can
grow on a large variety of substrates. Due to the appearance
of visible hyphae and the production of unpleasant odours,
mould growth causes economic losses (Bullerman 1984).
These drawbacks can be overcome by the addition of
preservatives such as sorbic acid to food products, although
this approach has come under criticism (Sofos and Busta
1981) as customers demand `more natural' and `fresher
foods'. Literature dealing with the prevention of mould
growth has emphasized the control of factors such as aw,
temperature (Ayerst 1969; MarõÂn et al. 1996; Cuppers et al.
1997; Ramos et al. 1998) and pH (Magan and Lacey 1984;
Skirdal and Eklund 1993; McQuilken et al. 1997). Due to
dif®culties in acquiring suf®cient, reproducible data, mod-
elling of mould growth has not developed as rapidly as
Correspondence to: P. Dantigny, Laboratoire de Microbiologie, UMR INRA
1082, ENS.BANA, 1 Esplanade Erasme, 21000 Dijon, France (email:
phdant@u-bourgogne.fr).
majority of the models describe the relationship between
environmental factors and fungal growth (Gibson et al.
1994; Cuppers et al. 1997). Few models (El Halouat and
Debevere 1997) concern the effect of environmental factors
on spore germination. However, it is of paramount import-
ance to assess the in¯uence of environmental factors on
spore germination as this is a primary step to mould
spoilage.
In this paper, the effects of temperature (T), water activity
(aw) and pH on the time to obtain 90% germination of
Penicillium chrysogenum conidia (T90) were determined.
Because of solute effects on fungal germination and growth
(Pitt and Hocking 1977), different humectants (e.g. glycerol
and sorbitol) were also examined. In order to reduce the
number of experiments, a Doehlert design was used.
Doehlert matrices (Doehlert 1970) present the advantage
of being easily expanded in both the variables space and the
experimental space (Quignon et al. 1997). The combined
effects were represented by means of a polynomial model
(Taragano and Pilosof 1999) for each humectant. A response

ã 2001 The Society for Applied Microbiology

 EFFECTS OF TEMPERATURE, WATER ACTIVITY AND pH ON GERMINATION OF P. CHRYSOGENUM 901

surface methodology was then applied to represent these was observed after inoculation of 104 spores (10 ll of a
effects. suspension of 106 spores ml±1) at the surface of YNB agar
media distributed into the small glass cylinders. After
inoculation, the glass cylinders could be removed from the
MATERIALS AND METHODS
lid. Without opening the devices, spores were examined
Mould twice daily for 48 h and then daily by microscopic observa-
tion (´100) or (´400) through the Petri dish lid. Spores were
Penicillium chrysogenum was isolated from a spoiled pastry
considered germinated when the length of the germ-tube was
product and identi®ed according to the descriptions of
equal to one half of the spore diameter (Paul et al. 1992).
Samson et al. (1995).

Experimental design
Media
An experimental domain was de®ned over 11±31°C, aw
The basal medium used for spore production was Malt
0á86±0á98 and pH 3á5±6á5. Two humectants were also
Extract Agar (bioMeÂrieux, Marcy l'Etoile, France). The
tested, glycerol and sorbitol. Each experiment was per-
germination media consisted of Yeast Nitrogen Base (YNB;
formed in duplicate.
6á7 g l±1; Difco) supplemented by glucose (20 g l±1) and
In contrast to a previous study (Sautour et al. 2001),
agar (15 g l±1), adjusted to different pH levels (3á5, 5á0 and
the experimental domain was suf®ciently wide to expect
6á5) and buffered (phosphate citrate). The aw in these media
an optimal response. Therefore, a second-order polyno-
was adjusted by substituting part of the water with an
mial relationship, which includes quadratic terms, was
identical weight of glycerol or sorbitol (w/w). The aw
required to surround this optimum. A response surface
measurements of the adjusted media were determined using
methodology was established to obtain a predictive model
an Aqualab CX2T (Decagon Devices, Pullman, WA, USA).
for the whole domain. Two types of experimental designs,
amongst the most widely used in response surface
Experimental set-up analysis, could be chosen for determining the model
coef®cients, central composite designs (CCD) and Doehl-
The device used in each experiment was made from a Petri
ert matrices. The location of the experiments in the
dish. The sterile Petri dish was opened within a laminar ¯ow
experimental domain depends on the design chosen.
cabinet. Three small glass cylinders (diameter 16 mm) were
Accordingly, the values of the coef®cients depend on
placed on the internal side of the lid and ®lled with the
the experimental design. However, whether coef®cients
appropriate sterile germination medium to about 1 mm
are signi®cant or not should not depend on the choice of
thickness. After solidi®cation, each surface was ready for
experimental design.
inoculation. In order to equilibrate the relative humidity
One advantage of the Doehlert design over the CCD is the
inside each device after inoculation, an appropriate water/
possibility of extended the domain by adding another factor
glycerol solution (15 ml) was poured into the Petri dish. The
or displacing the design towards a new experimental
aw of this solution was identical to that of the culture
domain.
medium on the lid. The devices sealed with Para®lmÒ
The Doehlert design allows the description of a region
constituted the closed incubation chambers (Fig. 1).
around an optimal response and contains k2 + k + 1 points
for k variables. For three variables, a set of 13 experiments is
Spore germination required and, in that case, one of the properties of the
Doehlert design is the uniform distribution of the experi-
Spores were collected by ¯ooding the surface of the plates
ments in a three-dimensional space (Fig. 2). Thus, 12
with sterile saline solution (8á5 g NaCl l±1 water) containing
experiments are equidistant from a central experiment
Tween 80 (0á1% v/v). Germination of Penicillium conidia

Lid

Parafilm
Fig. 1 Experimental set-up designed for
microscopic observation of the germination
process through the lid allowing constant aw
to be maintained

ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 91, 900±906
902 M . S A U T O U R ET AL.

temperature (X1), aw (X2) and pH (X3). Each experiment

could be located by its three coded values. The coded (Xi)
and experimental (natural) values of these three factors are
listed in Table 1. The natural values and the corresponding
coded values were used for setting up the experiments and
the model, respectively.

Analysis and interpretation of the results

Y ˆ b0 ‡ b1 X1 ‡ b2 X2 ‡ b3 X3 ‡ b11 X12 ‡ b22 X22 ‡ b33 X32
Fig. 2 Cuboctahedron exhibiting the location of the 13 experimental
points for three different coded factors (e.g. X1, X2 and X3). Along
the X3 axis, in a medium slice, six experiments are located at the vertices
(2, 3, 5, 6, 8 and 11) and at a centre (1) of a hexagon and on both sides;
three experiments (4, 12 and 13) in an upper slice and three other
experiments (7, 9 and 10) in a lower slice are obtained
having the coded values (0, 0, 0) and are distributed on a
sphere with a radius of 1 (Fig. 2).
In this study, T90 was estimated, taking into account the
in¯uence of three environmental factors (i.e. variables),
Multiple regression analysis based on the least square
method was performed using Nemrod software (LPRAI,
Marseille, France). The analysis concerned the linear and
quadratic effects of the three factors and their interactions.
Thus, the equation giving T90 was a second-order polyno-
mial model with 10 coef®cients (b0, b1, b12¼b23):
‡ b12 X1 X2 ‡ b13 X1 X3 ‡ b23 X2 X3
where X1, X2 and X3 are coded factors studied.
The signi®cance of the coef®cients was evaluated by
multiple regression analysis based upon the F-test with
unequal variance (P < 0á05, P < 0á01 and P < 0á001).
RESULTS
Effects of temperature, water activity and pH
on germination time
The average T90 values obtained in the different conditions
are reported in Table 1 for both humectants. Prior to
assessing the in¯uence of the experimental conditions on

Table 1 Experimental matrix obtained by applying the Doehlert design methodology for three factors and experimental values for T90 (time (d) to
obtain 90% of the conidia of Penicillium chrysogenum germinated) for glycerol and sorbitol as humectants

Coded values Experimental values Mean T90 (d)

Experiment X1 X2 X3 T (°C) aw pH Glycerol Sorbitol

1 0 0 0 21 0á92 5 2á0 2á0

2 +1 0 0 31 0á92 5 1á5 2á0
3 + 0á5 + 0á866 0 26 0á98 5 0á5 0á5
4 + 0á5 + 0á289 0á816 26 0á94 6á5 1á0 1á0
5 )1 0 0 11 0á92 5 6á0 6á0
6 ) 0á5 ) 0á866 0 16 0á86 5 10á0 9á5
7 ) 0á5 ) 0á289 ) 0á816 16 0á90 3á5 4á0 4á5
8 + 0á5 ) 0á866 0 26 0á86 5 6á5 5á5
9 + 0á5 ) 0á289 ) 0á816 26 0á90 3á5 3á0 1á5
10 0 + 0á577 ) 0á816 21 0á96 3á5 1á0 1á5
11 ) 0á5 + 0á866 0 16 0á98 5 1á75 1á75
12 ) 0á5 + 0á289 + 0á816 16 0á94 6á5 2á0 2á0
13 0 ) 0á577 + 0á816 21 0á88 6á5 4á5 4á0

The experimental values (Ui) were calculated from the coded values (Xi) using the formula: Ui = Uoi + Xi.DUi, where Ui is the experimental value,
Uoi the centred value, Xi the coded value and DUi the range. For temperature, Uo1 = 21°C and DU1 = 10°C; for aw, Uo2 = 0á92 and DU2 = 0á07; for
pH, Uo3 = 5á0 and DU3 = 1á8.

ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 91, 900±906
 EFFECTS OF TEMPERATURE, WATER ACTIVITY AND pH ON GERMINATION OF P. CHRYSOGENUM 903

T90, the accuracy of the results should be examined. temperature. For glycerol and sorbitol, pH had no signi®-
Amongst a total of 26 different experiments (varying either cant in¯uence on T90. Figure 3 shows that the germination
the environmental conditions or the humectant), the results time of P. chrysogenum decreased with increasing aw from
in duplicate were identical in 15 cases. In the other cases, the 0á86 and with increasing temperature from 16°C. For both
difference between the assays (e.g. 0á5 d for T90 less than humectants, aw and temperature had a linear negative effect
equal to 2 d and otherwise 1 d) was due to the frequency in and a quadratic positive effect on T90 (see Table 2). This
readings. For example, in experiment no. 8 using glycerol as result suggests that, depending on the parameter values,
humectant, assuming less than 90% germination for both
the duplicates at 5 d and 100% and 80% germination at 6 d,
the T90 results would be 6 and 7 d, respectively, even if
all the spores had been well germinated shortly after 6 d,
leading to an average T90 value of 6á5 d. These results
exhibit reasonable reproducibility of the experiments real-
ized in duplicate.
For each experiment the differences between the results
obtained for glycerol and sorbitol (Table 1) could be
explained by the sampling methodology. Therefore, the
T90 values obtained for glycerol were not signi®cantly
different from those obtained for sorbitol. Furthermore, the
differences observed in T90 could not be explained only by
the variability in the experimental results. Accordingly, the
in¯uence of the environmental factors on T90 can be
examined.
The results of the multiple regression analysis which
provided the estimates of the model coef®cients are listed in
Table 2. The regression coef®cients, r2, were equal to 0á973
and 0á968 for glycerol and sorbitol, respectively; therefore,
C
about 97% of the fraction of the variation about the mean
could be explained by the models (Box and Draper 1987). It
can obviously be veri®ed that the response means coef®-
cients, b0, are the same as the experimental values of T90
obtained when all the factors are weighted with the coded
level 0, experiment 1. The greater the absolute value of the
linear coef®cients, b1±b3, the more important was the
variable in¯uencing the response. Therefore, in all cases,
the in¯uence of aw was greater than the in¯uence of

Table 2 Model coef®cients obtained for glycerol and sorbitol as

humectants

Coef®cient Glycerol Sorbitol

Response means b0 2á00*** 2á00***

T b1 )1á91*** )2á28***
aw b2 )3á99*** )3á45***
pH b3 )0á10ns )0á10ns
T2 b11 1á75*** 1á63***
aw2 b22 2á83*** 2á38***
pH2 b33 )0á27ns )0á34ns
T.aw b12 1á59*** 1á88*** C
T.pH b13 )0á56ns 0á56ns
aw.pH b23 0á03ns )0á44ns Fig. 3 Contour plots of the in¯uence of water activity and tempera-
ture on the time (d) to obtain 90% of the conidia of Penicillium
*** Signi®cant (P < 0á001). chrysogenum germinated on YNB medium at pH 5 for (a) glycerol and
ns
Not signi®cant. (b) sorbitol as humectants

ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 91, 900±906
904 M . S A U T O U R ET AL.
optimum values for aw in the range 0á86±0á98 and tempera-
ture in the range 16±31°C capable of minimizing T90 could
be obtained. For glycerol and sorbitol, a signi®cant inter-
action between aw and temperature was demonstrated. The
positive values for b12 suggested a synergistic in¯uence of
both variables on the spore germination process.
Model ®tting
In order to determine the optimum conditions for germi-
nation, surface response contour plots were drawn. One
factor was ®xed arbitrarily at the centre of the matrix, while
the two other factors varied. By ®xing the pH at 5, the effect
of temperature and aw on T90 is shown in Fig. 3 for the
different humectants. The optimum conditions are sche-
matized as an ellipse. The centre of the ellipse (e.g. glycerol,
23á4°C, 0á963 and sorbitol, 23á6°C, 0á966) represents the
optimum conditions in terms of temperature and aw at pH 5.
In these optimum conditions, the model predicts germina-
tion times of about 0á5 d for both glycerol and sorbitol. In
order to prevent food spoilage due to P. chrysogenum it is,
therefore, recommended to avoid these optimum conditions.
Figure 3 shows that the conditions which allowed the
slowest germination times were 16°C and aw 0á860 and 16°C
and aw 0á865 for glycerol and sorbitol, respectively. In these
conditions, the model prediction for glycerol (T90 about
9á5 d) was a little greater than for sorbitol (T90 about 8á5 d).
DISCUSSION
An experimental set-up to determine germination time has
been described. The control of humidity (by means of a
solution of the same aw as the medium and hermetic closure
of the experimental set-up) was required in order to
maintain a constant aw. It appeared that the main interest
in the proposed method was the observation of the spores
through the Petri dish lid without opening the dishes. A
similar system has been described by Magan and Lacey
(1984). In contrast, MarõÂn et al. (1996) opened the dishes to
aseptically remove three discs at each sampling, thus leading
to many manipulations.
The spores were considered germinated when the length
of the germ-tube was equal to one half of the spore diameter
and a test was considered positive when 90% of the
inoculated conidia were germinated. Another protocol (i.e.
spore considered germinated when the germ-tube length
was equal to or greater than the diameter of a spore and a
test considered positive when 10% of the spores had
germinated) has been suggested (Magan and Lacey 1984;
MarõÂn et al. 1996; McQuilken et al. 1997). Obviously, the
protocol does affect the germination time. Magan and Lacey
(1984) recommended the ®gure of 10% in preference to
larger percentages to obtain a better estimate of the
ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 91, 900±906
minimum aw allowing germination. However, this was not
the purpose of the present study. The in¯uence of
environmental factors on T10 (time to obtain 10% of the
germinated conidia) has been assessed. Although the
constant values were different, the main effects of tempera-
ture and aw and the combined effects of temperature and aw
on the conidial germination of P. chrysogenum were also
demonstrated (results not shown).
The main effects of aw and temperature have also been
reported in the literature for fungal growth (Holmquist et al.
1983) and conidial germination of Fusarium spp. (MarõÂn
et al. 1996). The in¯uence of temperature on growth was
second after aw in order of signi®cance (Holmquist et al.
1983). The major in¯uence of aw on the germination process
is in accordance with the data of Ayerst (1969) and Seiler
(1976) on moulds growing on minimally processed foods.
In contrast, in this study no signi®cant in¯uence of pH, in
the range 3á5±6á5, was highlighted. These results are in
accordance with those of McQuilken et al. (1997) who
reported that maximum germination occurred between pH
4á5 and 6á2. In addition, the optimum temperature and aw
have been determined through the model equations at pH 5,
which is optimum for most moulds (Lacey 1989). The
combined effect of temperature and aw has been reported for
fungal growth (Ayerst 1969; Horner and Anagnostopoulos
1973; Pitt 1993) and germination (Ayerst 1969; MarõÂn et al.
1996). The present results were in accordance with this
literature.
It has been reported that the effect of the changes in aw,
temperature and pH on fungal growth was broadly the
same for media adjusted by either glycerol or sucrose
(Horner and Anagnostopoulos 1973). A similar observation
was made by Pitt and Hocking (1977) for germination
time. Eventually, these authors concluded (Hocking and
Pitt 1979) that, with the notable exception of NaCl, the
differences in effects caused by the various solutes were
slight. Accordingly, they recommended the use of glycerol
for controlling aw.
Experiment designs according to Doelhert matrices were
carried out to determine the optimal conditions for conidial
germination. According to Henika (1982), this methodolo-
gical approach is well suited to work on predictive
microbiology in the agro-food area. Previously, other
authors have used polynomial models to describe Aspergillus
development (Fang et al. 1994). The multifactorial analysis
allowed the development of a model with a good quality of
®t and taking account of possible interaction effects between
the environmental factors. However, the prediction given by
the model was valid only for the strain/medium and
within the particular experimental domain (Delignette-
Muller 1997). Any extrapolation to other species or growth
substrates, and outside the limits of the considered factors,
would be hazardous; consequently, it is important to specify
 EFFECTS OF TEMPERATURE, WATER ACTIVITY AND pH ON GERMINATION OF P. CHRYSOGENUM
the areas in which the model cannot be used (Baranyi et al.
1996).
As the experiments were mostly performed on optimal
culture medium, the fungal development in the laboratory
appeared faster than in food; thus, some caution is needed in
use of the model. Predictive mycology studies the behaviour
of moulds under different physico-chemical conditions. It
can help in the identi®cation of critical points in the
production and distribution process and the optimization of
production and distribution chains (Zwietering et al. 1990).
The modelling approach introduced in this paper is in
accordance with these objectives and could contribute to
improving the microbiological safety and shelf-life of food
products.
ACKNOWLEDGEMENTS
This research was partially supported by ACTIA (Paris).
The authors would like to thank V. Huchet and D. Thuault
from ADRIA, V. Stahl from AERIAL M. Sergent and M.C.
Guilhem from LPRAI (Marseille).
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