Biochemical Engineering Journal 40 (2008) 465–472

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Biochemical Engineering Journal

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Modelling the stress inducing biphasic growth and pediocin production by

Pediococcus acidilactici NRRL B-5627 in re-alkalized fed-batch cultures
Nelson P. Guerra ∗ , Paula Fajardo Bernárdez, Lorenzo Pastrana Castro
Departamento de Quı́mica Analı́tica y Alimentaria, Facultade de Ciencias de Ourense, Universidade de Vigo, As Lagoas, 32004 Ourense, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Five re-alkalized fed-batch cultures of Pediococcus acidilactici NRRL B-5627 were carried out in culture
Received 8 May 2006 media prepared with mussel processing wastes (MPWs) and whey under different fermentation condi-
Received in revised form 16 January 2008 tions. The shift from homolactic to mixed acid product formation and the biphasic kinetics observed for
Accepted 8 February 2008
cell growth, nitrogen consumption and pediocin production were the most noteworthy observations of
these cultures. For a better understanding of the culture dynamics and for a suitable description of the
Keywords:
bacteriocin production system, an unstructured mathematical model based on the two phases of nitro-
Fed-batch culture
gen consumption was developed. The model was expressed in terms of biomass, product accumulation
Fermentation
Glucose
and substrate utilization. Excellent agreements between model predictions and experimental data were
Growth kinetics achieved in the five re-alkalized fed-batch cultures and a reasonable description for each parameter in
Lactose each growth phase was provided by the model. Using published experimental data by other researchers,
Modelling model agreement was also found for the growth of Ped. acidilactici NRRL B-5627 in a repeatedly re-alkalized
culture in a synthetic culture medium. The developed model appears to be useful for the design, scale-up,
control and optimization of the production of potentially probiotic cultures and bacteriocins.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction bacteriocins have a wide inhibitory spectrum of activity, which

In recent years, lactic acid bacteria (LAB) have received consid-
erable attention due to their probiotic nature. Probiotics are viable
microorganisms that once ingested by humans and animals in suf-
ficient amounts, produce beneficial physiology effects by assisting
in the establishment of an intestinal population which is beneficial
to the host and antagonistic to harmful bacteria. Many LAB (Pedio-
coccus, Lactobacillus, Lactococcus, Bifidobacterium and Streptococcus
species) have been used as probiotics because they fulfil many of
the probiotic criteria that include: (i) safety (non-pathogenicity and
antibiotic resistance characteristics), (ii) survival and persistence in
the stressful environment of the stomach (acidic pH and bile) and in
the intestinal ecosystem of the host, (iii) adherence to host epithe-
lial tissue, (iv) good technological properties, (v) ability for pro-
ducing high amounts of antimicrobial substances antagonistic to
pathogen growth, and (vi) beneficial to the host in some way [1–3].
Although nisin has been the most studied bacteriocin [4,5], the
pediocin family has gained increasing interest due to their poten-
tial applications in food, veterinary and pharmaceutical industries
[6–8]. Pediocins are generally produced by Pediococcus strains,
which are organisms generally recognized as safe (GRAS). These
∗ Corresponding author. Tel.: +34 988 387062; fax: +34 988 387001.
E-mail address: nelsonpg@uvigo.es (N.P. Guerra).
includes both spoilage and pathogenic organisms, such as Liste-
ria monocytogenes, Enterococcus faecalis, Staphylococcus aureus and
Clostridium perfringens [7,8].
A fed-batch cultivation technique with successive re-
alkalizations of the culture medium has been successfully used
to produce large amounts of probiotic biomass and bacteriocins
in an economic way using culture media from food wastes [9]. In
these cultures, two well-differentiated phases of fermentation (a
homolactic fermentation phase and a mixed acid fermentation
phase) were observed. In addition, cells displayed a diauxic (bipha-
sic) growth pattern, which could be probably related with two
distinct phases of nitrogen utilization [9]. The biphasic nitrogen
metabolism had been observed before during diauxic growth
of Lactococcus lactis [10] and Streptococcus thermophilus [11] in
milk, with qualitative differences in the amino acids that were
taken up and utilized during each growth phase. However, the
use of mathematical models describing the effect of the nitrogen
concentration on the growth of these LAB are scarce. On the other
hand, the sigmoidal equations (such as the logistic and the Gom-
pertz equations) have found to be unsuitable for the simulation
of fed-batch fermentation processes [12]. For this reason, there
is a growing interest in using models with a more mechanistic
approach to elucidate the effect of single environmental factors,
such as the limiting substrate or product concentration on the
specific growth rate [13].

1369-703X/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2008.02.001
466 N.P. Guerra et al. / Biochemical Engineering Journal 40 (2008) 465–472
In this study, the growth of Ped. acidilactici NRRL B-5627
was followed in five re-alkalized fed-batch fermentations in cul-
ture media prepared with mussel processing wastes and whey
by using different nutrient feeding strategies and cycles of re-
alkalization and feeding. From the experimental data obtained,
a mathematical model for pediocin fermentation was developed.
The growth-associated pediocin production, which has previously
been described as a function of pH [14,15], has been remodelled
according to the mechanistic growth model developed in this study.
Published experimental data obtained by other researchers [16]
from a repeatedly re-alkalized culture of Ped. acidilactici NRRL B-
5627 in a synthetic culture medium were also fitted with the
proposed model.
2. Materials and methods
2.1. Microorganisms and media
Pediocin-producing strain Ped. acidilactici NRRL B-5627 was
obtained from the Northern Regional Research Laboratory (NRRL,
Peoria, IL, USA). Carnobacterium piscicola CECT 4020, the indicator
strain in the bacteriocin assay, was obtained from the Spanish Type
Culture Collection (CECT). Stock cultures were maintained at 4 ◦ C
on MRS agar slants. Working cultures were grown in MRS broth
at 30 ◦ C and 200 rpm. The mean composition of the culture media
used for the different re-alkalized fed-batch cultures is shown in
Table 1.
2.2. Fermentation conditions
Seed culture was prepared in 250 ml Erlenmeyer flask contain-
ing 50 ml of deproteinized whey supplemented with a 2% yeast
extract (DWYE2 medium) or 50 ml of mussel processing wastes
(MPWs) medium agitated in shaker rotating at 200 rpm and 30 ◦ C
for 18 h.
To obtain kinetic data for the development of a mathematical
model capable of describing the pediocin production system by
Ped. acidilactici, five re-alkalized fed-batch cultures were carried
out in MPW medium (fermentations 1 and 2) and DWYE2 medium
(fermentations 3–5). These fermentations were carried out at a
controlled temperature of 30 ◦ C in a 6 l bench top fermentor (New
Brunswick Scientific, New Jersey) equipped with pH, temperature
and dissolved oxygen controllers. The working volume was 4 l. The
fermentor was sterilized at 121 ◦ C for 25 min, cooled and then inoc-
ulated with 2% (v/v) inoculum. An aeration flow rate of 0.5 l/h and
an agitation speed of 200 rpm were maintained throughout the
incubation period in all the cultures.
Fed-batch fermentations were carried out as a batch fermen-
tation without pH-control during the first 8 h (for fermentations
in MPW medium) or 12 h (for fermentations in DWYE2 medium),
when the lower steady pH was reached in the cultures [14,15].
Then, a sample of 100 ml was taken from the fermentation medium
to perform analytical determinations. After determining the total
sugars concentration in the sample withdrawn, the medium was
Table 1
Mean composition (g/l) of the culture media prepared with whey (CW and DW) and
mussel processing wastes (MPWs and CMPW)
CW DW MPW
Total sugars 48.11 20.54 5.33
Proteins 5.02 2.04 1.82
Total nitrogen 1.05 0.45 0.65
Total phosphorous 0.43 0.25 0.14
Ashes 5.10 3.00 2.43
Solid residue 62.70 26.60 32.10
re-alkalized up to a set pH of 6.3 (for fermentations in MPW
medium) or 7.0 (for fermentations in DWYE2 medium) with 5 mol/l
NaOH. The volume of fermentation medium in the fed-batch fer-
mentations was maintained constant (dVF /dt = 0) by matching the
volumes added to the fermentor (volume of 5 mol/l NaOH plus the
volume of feeding substrates) with the sampling volumes (100 ml).
From the difference between the sampling volume (100 ml) and
the volume of alkali added for re-alkalizing the medium, the neces-
sary volumes of feeding substrates to restore the initial total sugars
concentration in the fermentation media were calculated. The vol-
umes of each feeding substrate were calculated by applying mass
balance equations for the total sugars around the fermentor. The
volumes of feeding media were completed with distilled sterile
water, when this was necessary. These sampling, feeding and re-
alkalization strategies were repeated every 8 h (in MPW cultures)
and 12 h (in DWYE2 cultures), respectively, until the producer strain
was unable to bring about the decrease of pH.
The fermentation and feeding media used in each culture are
shown in Table 2. The preparation of these culture media was pre-
viously described [14,15].
2.3. Analytical determinations
The concentrations of biomass, total phosphorous, nitrogen,
protein and sugars, pediocin, lactic acid, acetic acid, ethanol and
butane-2,3-diol were determined by methods described in previ-
ous works [9,14,15].
3. Results and discussion
3.1. Re-alkalized fed-batch fermentations in MPW medium using
different feeding substrates (fermentations 1 and 2)
The growth kinetic of Ped. acidilactici NRRL B-5624 in the first
re-alkalized fed-batch fermentation in MPW medium (fermenta-
tion 1) is shown in Fig. 1. A 240 g/l concentrated glucose was
used to fed the growing culture. As it can be observed, biphasic
kinetics of growth and pediocin production were obtained dur-
ing the incubation period. Each growth phase was characterized
by an exponential and a stationary growth phase. In addition, the
nitrogen source seemed to be consumed in two steps. This obser-
vation suggests that in the first step, the pediocin-producing strain
consumed the nitrogen fraction easier to assimilate, and in the
second step, the strain consumed the nitrogen fraction more dif-
ficult to metabolize [9,10,17,18]. Assuming the existence of these
two nitrogen consumption phases, the existence of a critical nitro-
gen concentration which indicates the end of the first phase and
the beginning of the second phase of nitrogen assimilation could
be postulated.
Table 2
Culture media used for the different re-alkalized fed-batch fermentations
Fermentation no. Fermentation Feeding media
medium
Fermentation 1 MPW A 240 g/l concentrated glucose
Fermentation 2 MPW CMPW
Fermentation 3 DWYE2 A 400 g/l concentrated glucose
CMPW
Fermentation 4 DWYE2 CWYE2 and a 400 g/l concentrated glucose
101.33 Fermentation 5 DWYE2 CWYE4 and a 400 g/l concentrated glucose
3.47
MPW: mussel processing wastes medium; CMPW: concentrated mussel processing
0.84
wastes medium; DWYE2 diluted whey medium supplemented with 2% (w/v) yeast
0.26
extract; CWYE2 concentrated whey medium supplemented with 2% (w/v) yeast
3.42
extract; CWYE4 concentrated whey medium supplemented with 4% (w/v) yeast
42.12
extract.
 N.P. Guerra et al. / Biochemical Engineering Journal 40 (2008) 465–472
Fig. 1. Fed-batch fermentation profile for Ped. acidilactici NRRL B-5627 in MPW
medium with glucose feeding. Comparison of model predicted (full lines) and exper-
imental (symbols) profiles. Time courses are shown for pH, biomass (), nitrogen
(♦), pediocin (䊉), lactic acid (), butane-2,3-diol (), ethanol () and total sugars
(). [N0 ] is the experimental nitrogen concentration (g/l), which determines the
end of the first nitrogen consumption phase and the beginning of the second.
A shift from homolactic to mixed acid fermentation started after
42 h of incubation with the accumulation of butane-2,3-diol in the
culture medium. Ethanol production was detected in the culture
medium after 74 h of fermentation. Due to the joint effect of con-
sumption and dilution (which occurred due to the combined effect
of sampling and nutrient feeding), the nitrogen concentration in the
culture medium progressively decreased, thus becoming limiting
for the growth of Ped. acidilactici at the end of the incubation.
In the second culture in MPW medium (fermentation 2), which
was fed with a concentrated MPW medium (Table 2), two phases
of growth, bacteriocin production and nitrogen consumption were
again observed (Fig. 2). Remarkably, the Pediococcus strain only pro-
duced lactic acid and did not show loss of the capacity to recover
the acidic pH at the end of the cultivation. This was probably due to
the feeding with CMPW medium, which contains more nutrients
(nitrogen, phosphorous, vitamins, minerals) than the concentrated
glucose used as the feeding medium in the first fermentation.
467
Fig. 2. Fed-batch fermentation profile for Ped. acidilactici NRRL B-5627 in MPW
medium with CMPW medium feeding. Comparison of model predicted (full lines)
and experimental (symbols) profiles. Time courses are shown for pH, biomass (),
nitrogen (♦), pediocin (䊉), lactic acid () and total sugars (). [N0 ] is the experi-
mental nitrogen concentration (g/l), which determines the end of the first nitrogen
consumption phase and the beginning of the second.
3.2. Re-alkalized fed-batch fermentations in DWYE2 medium
using different feeding substrates (fermentations 3–5)
Three re-alkalized fed-batch fermentations were carried out in
DWYE2 medium with intermittent cycles (each 12 h) of feeding and
re-alkalizations. Since the Pediococcus species more readily ferment
glucose than lactose [14,15], in the first culture in DWYE2 medium
(fermentation 3), the fermentor was fed with a 400 g/l concen-
trated glucose. Fig. 3 shows the variations of biomass, pH, nutrients
(total nitrogen and sugars) and products (lactic acid, acetic acid,
ethanol and pediocin) with the time in fermentation 3. In this cul-
ture, the pediocin-producing strain was able to bring about the
decrease of pH by a period of time of 192 h. Consequently, higher
amounts of biomass, pediocin, lactic acid, acetic acid and ethanol
were obtained as compared to the batch culture of Ped. acidilac-
tici in DW medium [14]. However, the growth stopped after 144 of
incubation, when the nitrogen concentration in the fermentation
468 N.P. Guerra et al. / Biochemical Engineering Journal 40 (2008) 465–472
Fig. 3. Fed-batch fermentation profile for Ped. acidilactici NRRL B-5627 in DWYE2
medium with glucose feeding. Comparison of model predicted (full lines) and exper-
imental (symbols) profiles. Time courses are shown for pH, biomass (), nitrogen
(♦), pediocin (䊉), lactic acid (), acetic acid (), ethanol () and total sugars ().
[N0 ] is the experimental nitrogen concentration (g/l), which determines the end of
the first nitrogen consumption phase and the beginning of the second.
medium was 0.35 g/l. This represents a mean reduction of 85.5%,
when the initial nitrogen concentration in the medium (2.42 g/l) is
taken as 100%. The nitrogen source was almost completely depleted
at the end of the fermentation as it was observed in the previous
cultures in MPW medium (Figs. 1 and 2).
In order to remove the nitrogen limitation observed in fermenta-
tion 3, a third re-alkalized fed-batch culture in DWYE2 medium was
carried out (fermentation 4) using a mixture of a 400 g/l concen-
trated glucose and CWYE2 medium as feeding substrates (Table 2).
Due to the joint feeding of glucose and nitrogen, the active period
in fermentation 4 was higher than that observed in fermentation
3 (Fig. 4) and consequently, increased amounts of biomass and
antimicrobial metabolites were obtained. Interestingly, the two
phases of growth and nitrogen consumption were more clearly
observed in fermentation 4 than in the previous fed-batch culture
in DWYE2 medium (Fig. 3).
The results obtained from the comparison between fermenta-
tions 3 and 4 clearly showed that the use of glucose and yeast extract
Fig. 4. Fed-batch fermentation profile for Ped. acidilactici NRRL B-5627 in DWYE2
medium with feeding with CWYE2 medium and a 400 g/l concentrated glucose.
Comparison of model predicted (full lines) and experimental (symbols) profiles.
Time courses are shown for pH, biomass (), nitrogen (♦), pediocin (䊉), lactic acid
(), acetic acid (), ethanol () and total sugars (). [N0 ] is the experimental nitrogen
concentration (g/l), which determines the end of the first nitrogen consumption
phase and the beginning of the second.
in the feeding substrates is an effective way to enhance both the
biomass and pediocin.
Since the yeast extract has growth and pediocin-promoting
properties [19], the use of a CW medium with a high yeast
extract concentration would contribute to further enhancements of
pediocin production. For this reason, a third fed-batch culture was
carried out in DWYE2 medium (fermentation 5), using a mixture
of a 400 g/l concentrated glucose and CW medium supplemented
with a yeast extract concentration (4% (w/v) YE) twice higher than
that (2% (w/v) YE) used in the previous fed-batch experiment. The
growth, bacteriocin concentration and the active period observed
in fermentation 5 (Fig. 5) were higher than those of the previ-
ous fed-batch fermentation. From a kinetic point of view, it can
be noted that the concentrations of nitrogen, biomass and pediocin
also described the above-mentioned biphasic profiles (Fig. 5).
 N.P. Guerra et al. / Biochemical Engineering Journal 40 (2008) 465–472 469

limitation by the carbon source during the five fed-batch fermen-

tations. However, the nitrogen concentration decreased throughout
the fermentations, concomitant with the increase in biomass pro-
duction. Thus, the progressive decrease in nitrogen concentration
influenced the growth of Ped. acidilactici. In addition, biomass
production stopped when the cultures reached a low nitrogen con-
centration, even though the total sugars concentration was still suf-
ficiently available (Figs. 1–5). These observations suggest that the
nitrogen source was the growth limiting substrate in these cultures.
Therefore, the specific growth rate ( in h−1 ) was expressed as
the sum of the specific growth rates on each nitrogen consump-
tion phase (1 and 2 ) multiplied by a dimensionless inhibition
function (ıi ):
 · ıi = (1 + 2 ) · ıi (1)
where:
max1 · ([N] − [N0 ])
1 =
KN1 + ([N] − [N0 ])

max2 · ([N0 ] − [N])

2 =
KN2 + ([N0 ] − [N])
The numbers 1 and 2 in Eq. (1) refer to each of the two growth
phases. [N] is the remaining nitrogen concentration (g/l) in the cul-
ture medium and [N0 ] is the critical nitrogen concentration (g/l),
which determines the end of the first nitrogen consumption phase
and the beginning of the second. KN is the Monod constant (g of N/l)
for nitrogen and max is the maximum specific growth rate (h−1 ).
The inhibition function ıi accounts for the self-inhibition of
the cells due to the production of antimicrobial products and the
decrease in medium pH [12,14,15,20], so that:
ıi = ıLA · ıAA · ıEt · ı2,3B · ıpHt (2)
where ıLA , ıAA , ıEt and ı2,3B are inhibition functions which account
for the inhibition of cell growth by the synthesis of lactic acid (LA),
acetic acid (AA), ethanol (Et) and butane-2,3-diol (2,3B), which
could be expressed as follows [12]:
 KiLA

ıLA =
KiLA + [LA]
 KiAA

Fig. 5. Fed-batch fermentation profile for Ped. acidilactici NRRL B-5627 in DWYE2 ıAA =
medium with feeding with CWYE4 medium and a 400 g/l concentrated glucose. KiAA + [AA]
Comparison of model predicted (full lines) and experimental (symbols) profiles.
Time courses are shown for pH, biomass (), nitrogen (♦), pediocin (䊉), lactic acid  KiEt

(), acetic acid (), ethanol () and total sugars (). [N0 ] is the experimental nitrogen ıEt =
concentration (g/l), which determines the end of the first nitrogen consumption KiEt + [Et]
phase and the beginning of the second.
 
Ki2,3B
ı2,3B =
The biphasic growth pattern observed in the re-alkalized fed- Ki2,3B + [2, 3B]
batch cultures of Ped. acidilactici in MPW and DWYE2 media, has
where [LA], [AA], [Et] and [2,3B] are respectively, the concentrations
also been observed before for L. lactis [9,10,16], S. thermophilus [11]
of lactic acid, acetic acid, ethanol and butane-2,3-diol (g/l). KiLA ,
and Ped. acidilactici [16] strains in different culture media. This
KiAA , KiEt and Ki2,3B are the inhibition constants (g/l) for lactic acid,
diauxic growth was attributed to a biphasic nitrogen metabolism
acetic acid, ethanol and butane-2,3-diol, respectively.
[9–11]. Thus, the first exponential growth phase relies on the use of
The inhibition function ıpHt which accounts for the inhibition of
the free amino acids and utilizable oligopeptides present in the cul-
cell growth by the decrease in medium pH, could be expressed by
ture media and the second relies on the use of proteins (like caseins
using the expression proposed by Presser et al. [21]:
in milk) previously cleaved by specific proteases which produces
the net liberation of free amino acids [10,11]. ıpHt = (10−pH min − 10−pHt )

where pHmin is the minimum pH value beyond the growth stopped

3.3. Mathematical modelling
and pHt is the pH over time.
Biomass production in the fermentor is hence given by the fol-
In all the re-alkalized fed-batch fermentations, the feeding sub-
lowing overall differential equation:
strates were added to bring the cultures up to the initial total sugars
concentrations (∼5.3 g/l in MPW medium and ∼23 g/l in DWYE2 d[X]
rX = =  · ıi · [X] (3)
medium) in each feeding cycle. For these reasons, there was no dt
470 N.P. Guerra et al. / Biochemical Engineering Journal 40 (2008) 465–472

where [X] is the biomass concentration (g/l) and t is the being:

time (h). 1  ˛
i
In order to take into account the effect of dilution, which K1 = +
YX/TS YPi /TS
occurred due to the extraction of samples and the feeding of nutri-
ents, mass balance was carried out across the fermentor and the  ˇ
i
biomass accumulation rate (rX ) was expressed as: K2 = + mS
YPi /TS
dX ([X(tn )] − [X(tn−1 )] · (1 − (VS /VF )))
rX = = =  · ıi · [X] (4) where [TS(tn )] and [TS(tn−1 )] are the total sugars concentrations
dt dt
(g/l) inside the fermentor at the times (in hours) t = tn and t = tn−1 ,
where VS is the sampling volume (in litres) and VF is the volume respectively. YX/TS and YPi /TS are the yield coefficients of biomass
(in litres) of fermentation medium. [X(tn )] and [X(tn−1 )] are the (X) and products (Pi ) for the total sugars (in grams of cell dry mass
biomass concentrations (g/l) inside the fermentor at the times (in or products per gram of total sugars). Other terms are as previously
hours) t = tn and t = tn−1 , respectively. described.
The total product (Pi as lactic acid, acetic acid, ethanol or The model parameters were obtained by using the nonlinear
butane-2,3-diol) accumulation rates (rPi ) in the fed-batch model curve-fitting software of SigmaPlot (version 9.0, Systat Software,
was represented to contain growth (˛i ) and non-growth (ˇi ) asso- Inc., 2004), which minimised the deviations between model pre-
ciated constants as follows: dictions and experimental data according to the sum of squares of
([Pi (tn )] − [Pi (tn−1 )] · (1 − (VS /VF )) errors (SSE) of the model fit:
rPi = = ˛i · rX + ˇi · [X] (5)
dt

n

m

where [Pi (tn )] and [Pi (tn−1 )] are the product concentrations (g/l) SSE = 2i,j
inside the fermentor at the times (in hours) t = tn and t = tn−1 , respec- i=1 j=1
tively.
For describing pediocin production, Eq. (5) was modified by where i,j represents the difference between the model and the
including a term for the specific effect that the pH drop rate (rpH) experimental value, n and m represent the number of experimental
produces on bacteriocin production [14,15,22–25]. Hence, rate of data points and the number of variables, respectively.
pediocin production (rPed ) was described as: The coefficients of the models with P values lower than
0.05 were considered statistically significant. Parameters were
([Ped(tn )] − [Ped(tn−1 )] · (1 − (VS /VF )) removed from the models when their asymptotic interval of con-
rPed =
dt fidence included zero. The goodness of fit of the model was
= (˛i · rX + ˇi · [X]) · (1 + e−K·(rpH max−rpH) ) (6) checked by determining the values of the determination coefficient
(R2 ).
where [Ped(tn )] and [Ped(tn−1 )] are the pediocin concentra- The values of the model parameters are presented in
tions (BU/ml) inside the fermentor at the times (in hours) t = tn Tables 3 and 4. Excellent agreement was found between model
and t = tn−1 , respectively. K is a constant of proportionality to predictions and experimental results for biomass, pediocin, nitro-
be experimentally determined, rpH is the pH drop rate over gen, and total sugars (Figs. 1–5). In addition, the high values of
time and rpHmax is the maximum pH drop rate, from which the determination coefficients for biomass (RX 2 ) and pediocin (R2 )
Ped
bacteriocin production stopped. Other terms are as previously (Tables 3 and 4) have strengthened the usefulness of the proposed
described. model for describing growth and bacteriocin production by Ped.
Nitrogen is consumed for the growth of the catalytically active acidilactici in the re-alkalized fed-batch fermentations in MPW and
biomass and for the maintenance functions of the cell. Therefore,
the nitrogen consumption rate (rN ) in the fermentor was described
by the following equation: Table 3
Model parameters for the two re-alkalized fed-batch fermentations in MPW
d[N] −([N(tn )] − [N(tn−1 )] · (1 − (VS /VF )) + [NFS ] · VFS /VF
rN = − = medium
dt dt
  Culture medium and fermentation number
 · ıi
=− + mN · [X] (7) Parameters MPW (1) MPW (2)
YX/N
max1 (h−1 ) 0.467 ± 0.0002 0.823 ± 0.0006
where YX/N is the yield coefficient of biomass for nitrogen (in KN1 (g/l) 0.0003 ± 0.0000001 0.014 ± 0.0005
N0 (g/l) 0.45 ± 0.0000002 0.26 ± 0.0003
grams of cells per gram of nitrogen), mN is the maintenance coef- max2 (h−1 ) 0.087 ± 0.000008 0.090 ± 0.0002
ficient (h−1 ), [N(tn )] and [N(tn−1 )] are the nitrogen concentrations KN2 (g/l) 0.071 ± 0.000006 0.059 ± 0.0003
(g/l) inside the fermentor at the times (in hours) t = tn and t = tn−1 , pHmin 3.80 ± 0.00005 3.48 ± 0.005
respectively. VFS is the volume (in litres) of feeding substrates (con- KLA (g/l) 0.72 ± 0.003 0.31 ± 0.003
KB (g/l) 14.96 ± 1.600 –
centrated glucose, CMPW, CWYE2 or CWYE4 media) added to the
KEt (g/l) 2.06 ± 0.935 –
fermentation medium in each re-alkalization cycle. [NFS ] is the RX2 0.994 0.979
nitrogen concentration (g/l) in the feeding substrates. Other terms K1 (g/g) 2.05 ± 1.106 5.55 ± 2.122
are as previously described. K2 (g/g/h) 0.29 ± 0.020 0.23 ± 0.055
The carbon source was probably consumed to synthesise cell YX/N (g/g) 5.81 ± 0.121 3.93 ± 0.121
mN (h−1 ) NS NS
material and products and to provide energy for cell maintenance ˛ (BU/mg) 346.50 ± 2.934 439.53 ± 4.700
(mS proportional to [X]). The total sugars consumption rate (rTS ) ˇ (BU/mg/h) NS NS
was then expressed as:  21.54 ± 0.727 10.82 ± 3.902
rpHmax 0.83 ± 0.001 0.84 ± 0.006
d[TS] −([TS(tn )] − [TS(tn−1 )] · (1−(VS /VF ))+ [TSFS ] · VFS /VF RPed
2
0.991 0.983
rTS = − =
dt dt Statistically significant coefficients (P < 0.05) are expressed as means ± standard
= −(K1 ·  · ıi + K2 ) · [X] (8) errors. NS: no significant (P > 0.05).
 N.P. Guerra et al. / Biochemical Engineering Journal 40 (2008) 465–472 471

Table 4
Model parameters for the three re-alkalized fed-batch fermentations in DWYE2 medium and a re-alkalized fermentation in a synthetic medium (SM) [16]

Parameters Culture medium and fermentation number

DWYE2 (3) DWYE2 (4) DWYE2 (5) SM

max1 (h−1 ) 0.452 ± 0.0004 0.471 ± 0.0005 0.307 ± 0.0005 0.011 ± 0.0018
KN1 (g/l) 0.109 ± 0.0015 0.116 ± 0.0003 0.095 ± 0.0007 0.207 ± 0.0244
N0 (g/l) 1.26 ± 0.0019 1.16 ± 0.0003 1.16 ± 0.0002 1.66 ± 0.0011
max2 (h−1 ) 0.014 ± 0.0002 0.010 ± 0.0003 0.009 ± 0.0002 0.002 ± 0.0009
KN2 (g/l) 0.306 ± 0.0010 0.352 ± 0.0030 0.319 ± 0.0002 0.008 ± 0.0002
pH min 4.57 ± 0.010 4.61 ± 0.009 4.80 ± 0.003 5.12 ± 0.006
KLA (g/l) 2.50 ± 0.020 13.11 ± 0.168 17.24 ± 0.190 2.36 ± 0.126
KAA (g/l) 0.269 ± 0.0012 0.098 ± 0.0004 0.146 ± 0.0009 NS
KEt (g/l) NS NS NS –
K2,3-B (g/l) – – – 0.37 ± 0.068
RX2 0.986 0.989 0.990 0.991
K1 (g/g) 6.55 ± 0.173 7.88 ± 0.944 7.62 ± 1.445 9.65 ± 0.324
K2 (g/g/h) 0.24 ± 0.001 0.03 ± 0.013 0.04 ± 0.011 NS
YX/N (g/g) 2.29 ± 0.148 1.97 ± 0.056 2.08 ± 0.038 2.53 ± 0.129
mN (h−1 ) 0.001 ± 0.0004 NS 0.0002 ± 0.00007 NS
˛ (BU/mg) 48.17 ± 0.603 44.64 ± 0.420 54.88 ± 0.664 4.82 ± 0.169
ˇ (BU/mg/h) 0.36 ± 0.006 0.11 ± 0.007 0.01 ± 0.007 0.06 ± 0.006
 6.43 ± 0.006 44.68 ± 0.727 33.96 ± 1.617 118.55 ± 8.193
rpHmax 0.27 ± 0.0062 0.24 ± 0.0005 0.24 ± 0.0011 0.24 ± 0.0007
RPed
2
0.990 0.989 0.983 0.980

Statistically significant coefficients (P < 0.05) are expressed as means ± standard errors. NS: no significant (P > 0.05).

DWYE2 media. However, modelling the product formation (lactic In addition, the resulting growth model containing a large num-
acid, acetic acid, ethanol and butane-2,3-diol) was not always satis- ber of parameters would be difficult to interpret. For this reason,
factory probably due to the above-mentioned shift from homolactic it seems more adequate to describe the cell growth of this strain,
to mixed acid fermentation. by including the effect of the total nitrogen concentration in the
In the five re-alkalized fed-batch fermentations, the calculated growth model rather than using the individual concentrations of
maximum specific growth rates (max ) corresponding to the first each amino acid.
growth phase were higher than those calculated in the second
growth phase (Tables 3 and 4). In addition, the estimated values
for [N0 ] in fermentations 1 (0.45 g/l), 2 (0.26 g/l), 3 (1.26 g/l), 4
(1.16 g/l) and 5 (1.16 g/l) are in perfect agreement with the actual
critical nitrogen concentrations (0.49, 0.28, 1.26, 1.18 and 1.17 g/l)
observed in each culture (Tables 3 and 4 and Figs. 1–5). On the
other hand, the mean values obtained for pHmin (Tables 3 and 4)
are in agreement with the observed minimum pH values that
permitted growth in the batch cultures of Ped. acidilactici in
MPW (pHmin = 3.46) [15] and whey-based media (pHmin = 4.70)
[14].
With regard to bacteriocin production, it can be noted that the
optimum pH drop rates obtained from Eq. (6) were dependent on
the fermentation medium (Tables 3 and 4). Thus, the rpHmax values
were determined as 0.83 (fermentation 1) and 0.84 (fermentation
2) in MPW medium, and 0.27 (fermentation 3), 0.24 (fermentation
4) and 0.24 (fermentation 5) in DWYE2 medium. These values are
in perfect agreement with the maximum rpH values (0.87, 0.85,
0.24, 0.27 and 0.24) that provided the highest pediocin produc-
tion rates in each re-alkalized fed-batch fermentation (Figs. 1–5).
These observations support the proposal about the need of higher
pH drop values for high pediocin productions [14,15,22–25]. Since
the values obtained for ˛ were much higher than the values of ˇ
(Tables 3 and 4) and pediocin production was dependent on the
pH drop rate (K = 0), it can be concluded that this bacteriocin was
produced as a pH-dependent primary metabolite in the five re-
alkalized fed-batch cultures.
In order to further test the proposed growth model, another set
Fig. 6. Modelling of the experimental data obtained from Vázquez et al. [16] in a
of experimental data on the growth of Ped. acidilactici NRRL B-5627 re-alkalized cultivation of Ped. acidilactici NRRL B-5627 in a synthetic medium. Com-
in a synthetic medium supplemented with a mixture of amino acids parison of model predicted (full lines) and experimental (symbols) profiles. Time
were obtained from Vázquez et al. [16]. Although these authors fol- courses are shown for biomass (), nitrogen (♦), pediocin (䊉), lactic acid (), acetic
lowed the time-course of each amino acid, modelling the individual acid (), butane-2,3-diol () and total sugars (). The experimental total nitrogen
data (♦) were obtained according to Eq. (9). [N0 ] is the experimental nitrogen con-
effect of these nitrogen sources on the growth of Ped. acidilactici is centration (g/l), which determines the end of the first nitrogen consumption phase
a tedious process due to the high numbers of variables implicated. and the beginning of the second.
472 N.P. Guerra et al. / Biochemical Engineering Journal 40 (2008) 465–472
Therefore, the total nitrogen concentration ([N] in g/l) at each
sampling time was previously calculated as follows:

p [3] Y. Doleyres, C. Lacroix, Technologies with free and immobilised cells for pro-
[N] = Wi · [Aai ]
i=1
where Wi is the nitrogen concentration in each amino acid (g of
N/g of amino acid), p is the number of amino acids and [Aai ] is the
concentration of each amino acid (g/l) in the culture medium at
each sampling time.
The dynamics of diauxic cell growth based on the two phases
of nitrogen consumption, as well as the time courses for the prod-
ucts (lactic acid, acetic acid, butane-2,3-diol and bacteriocin), total
nitrogen and sugars concentrations were fitted by using the Eqs.
(4)–(8), respectively. The values obtained for the model parameters
are summarised in Table 4. Fig. 6 shows a remarkable agreement
between the model predictions and the published data obtained
from Vázquez et al. [16]. For example, the pHmin value predicted by
the model (Table 4) is in perfect agreement with the experimental
value of 5.22 observed in the batch culture of Ped. acidilactici in the
same synthetic medium [16]. In the same way, the calculated val-
ues for [N0 ] and rpmax (1.66 g/l and 0.24, respectively) are in good
agreement with the actual experimental values of 1.78 g/l and 0.25
observed in the re-alkalized culture [16]. From the results obtained,
it can be pointed out that the model developed in this work suc-
cessfully described diauxic growth, bacteriocin production, as well
as the total nitrogen (the limiting nutrient) and sugars time-course
in different culture media, which are usually difficult to describe.
4. Conclusion
The developed growth model offers a satisfactory fit and a real-
istic description of the experimental growth data by taking into
account the growth inhibition due to the depletion of nitrogen
source, product formation and medium pH decrease. It appeared to
provide a reasonable description for each parameter during the two
growth phases. In the same way, Eq. (6) clearly allowed pediocin to
be classified as a pH-dependent primary metabolite. Therefore, the
development of the unstructured mathematical based on the two
phases of nitrogen consumption may allow a better understanding
and control of the growth and pediocin production by Ped. acidilac-
tici NRRL B-5627 in repeated re-alkalized fed-batch fermentations
in different culture media.
Acknowledgements
The research presented in this paper was financially supported
by the Instituto Nacional de Investigación y Tecnologı́a Agraria y
Alimentaria (INIA), Spain (project CAL01-045-C2-2) and The Xunta
de Galicia, Spain (project PGIDT00BIO1E). We thank COREN, S.C.L.
for their collaboration in the elaboration of this work.
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