This document summarizes a study on optimizing culture conditions to increase Bacillus subtilis spore production. The researchers investigated the effects of pH, dissolved oxygen concentration, and media composition in batch cultures. They achieved a maximum spore concentration of 5.6 × 109 spores/mL by maintaining pH at 7.5 and optimizing other conditions. They then developed a fed-batch process using an exponential feeding profile, achieving a maximum spore concentration of 7.4 × 109 spores/mL, representing a 3-fold increase over previous studies.

1. A Procedure for High-Yield Spore Production by Bacillus subtilis
Sandra M. Monteiro,†,‡ Joa˜o J. Clemente,† Adriano O. Henriques,‡ Rui J. Gomes,†
Manuel J. Carrondo,†,‡,§ and Anto´nio E. Cunha*,†,‡
Instituto de Biologia Experimental e Tecnolo´gica (IBET), Apartado 1, P-2781-901 Oeiras, Portugal, Instituto de
Tecnologia Quı´mica e Biolo´gica (ITQB), Universidade Nova de Lisboa, Apartado 127, P-2781-901 Oeiras,
Portugal, and Laborato´rio de Engenharia Bioquı´mica, Faculdade de Cieˆncias e Tecnologia, Universidade Nova de
Lisboa, Monte da Caparica, P-2829-516 Caparica, Portugal
Bacillus subtilis spores have a number of potential applications, which include their
use as probiotics and competitive exclusion agents to control zoonotic pathogens in
animal production. The effect of cultivation conditions on Bacillus subtilis growth and
sporulation was investigated in batch bioreactions performed at a 2-L scale. Studies
of the cultivation conditions (pH, dissolved oxygen concentration, and media composi-
tion) led to an increase of the maximum concentration of vegetative cell from 2.6 ×
109 to 2.2 × 1010 cells mL-1 and the spore concentration from 4.2 × 108 to 5.6 × 109
spores mL-1. A fed-batch bioprocess was developed with the addition of a nutrient
feeding solution using an exponential feeding profile obtained from the mass balance
equations. Using the developed feeding profile, starting at the middle of the exponential
growth phase and finishing in the late exponential phase, an increase of the maximum
vegetative cell concentration and spore concentration up to 3.6 × 1010
cells mL-1
and
7.4 × 109 spores mL-1, respectively, was obtained. Using the developed fed-batch
bioreaction a 14-fold increase in the concentration of the vegetative cells was achieved.
Moreover, the efficiency of sporulation under fed-batch bioreaction was 21%, which
permitted a 19-fold increase in the final spore concentration, to a final value of 7.4 ×
109
spores mL-1
. This represents a 3-fold increase relative to the highest reported
value for Bacillus subtilis spore production.
Introduction
Under conditions of extreme nutrient limitation, Bacil-
lus subtilis undergoes a differentiation process that
converts the rod-shaped bacterial cell into a dormant
spore, highly resistant to physicochemical stresses (1).
The spore is released at the end of the developmental
process upon lysis of the mother cell; later, if ap-
propriately stimulated, it can initiate germination, which
leads again to vegetative growth (1, 2). Recent studies
have demonstrated that spores of Bacillus spp. (3-5) and
in particular those of the nonpathogenic species B.
subtilis (6-8) may be successfully used as competitive
exclusion agents. The use of probiotics and competitive
exclusion agents in animal husbandry has gained in-
creased attention because of the rise of multiple antibiotic
resistant bacterial pathogens, in association with the
extensive use of antibiotics as growth promoters. The use
of spores as probiotics presents several advantages
including the ease with which spores can be produced,
their long storage life, and survival to the gastric barrier
(4, 5, 9).
In the agricultural industry spores are receiving in-
creasing attention as potential alternatives to antibiotics
as growth promoters (10). Probiotics and competitive
exclusion agents are thought to enhance the gut micro-
flora by preventing the colonization of the gastrointes-
tinal tract by pathogenic bacteria (11, 12). There are four
basic ways in which this might be achieved: (i) immune
exclusion of pathogenic bacteria; (ii) exclusion of a
pathogen by competitive adhesion, (iii) synthesis of
antimicrobial substances that impair colonization of the
gastrointestinal tract by pathogens, and (iv) depletion of
or competition for essential nutrients (4-6, 13).
Industrial exploitation of spores requires high cell
density bioreaction and good sporulation efficiency. At
laboratory scale, sporulation is normally induced by
growth and nutrient depletion in media such as Difco
Sporulation Medium (DSM). The end of the exponential
phase of growth is defined as the onset of sporulation,
and the production of heat-resistant spores takes ap-
proximately 8 h to be completed. Under ideal conditions,
the culture will initiate sporulation at a cell density of
about 108
cells mL-1
, and typical sporulation efficiencies
will be in the range of 30-100% (14). One reasonable way
* To whom correspondence should be addressed. Tel: + 351 21
446 94 80/3. Fax: + 351 21 446 93 90. E-mail: cunha@itqb.unl.pt.
† Instituto de Biologia Experimental e Tecnolo´gica.
‡ Instituto de Tecnologia Quı´mica e Biolo´gica.
§ Faculdade de Cieˆncias e Tecnologia, Universidade Nova de
Lisboa.

2. to increase spore production is to achieve high cell density
cultivation and subsequently allow sporulation to occur
(15, 16). Although fed-batch bioreactions have been
frequently used to increase cell densities (17, 18), this
technique has not been applied for B. subtilis spore
production. The concentration of spores reported in the
literature covers a wide range, depending mainly of the
used strain: 1.0 × 105
spores mL-1
(19), 6.4 × 108
spores
mL-1
(20), 1.0 × 109
and 2.0 × 109
spores mL-1
(21, 22
respectively); so far the highest reported value is 3.0 ×
109
spores of B. subtilis per mL-1
(23).
In this work, the effect of the dissolved oxygen level,
pH, and nutrient concentration on the extent of B. subtilis
growth and sporulation has been investigated in batch
bioreactions. A fed-batch bioprocess was developed, with
the addition of a nutrient feeding solution. The nutrient
feed started before the complete depletion of the nutrients
present in the media, at the middle of the exponential
growth phase, thus before start of the sporulation process
(24). The feeding solution was added using an exponential
feeding profile obtained from the mass balance equations
(17). The overall biomass balance equation was expressed
in terms of specific glucose consumption, and the feeding
profile was directly proportional to the glucose consump-
tion.
Under controlled fed-batch conditions the maximum
spore concentration achieved was 7.4 × 109
spores mL-1
,
corresponding to a 20-fold increase when compared to the
results obtained with this strain under batch cultivation
and being 2.5 times higher than the best results previ-
ously reported (23).
Materials and Methods
Strain. The wild-type Spo+
B. subtilis strain MB24
(trpC2 metC3) (25) was used for all the experiments. A
spore stock of this strain was prepared, divided in 1-mL
aliquots and stored with 30% of glycerol in liquid
nitrogen.
Culture Media. Luria-Bertani (LB) medium was used
for the measurements of vegetative cell and spore con-
centrations, its composition being yeast extract 5 g L-1
,
peptone 10 g L-1
, and NaCl 10 g L-1
. Difco sporulation
medium (DSM) [bacto nutrient broth 8 g L-1
, KCl 1 g
L-1
, and MgSO4 0.25 g L-1
(14)], used for inocula
preparation and batch and fed-batch cultures, was steril-
ized at 121 °C for 30 min. To 1 L of this solution were
added 1 mL of each of the following filter-sterilized
solutions: Ca(NO3)2 1 M, MnCl2 10 mM, and FeSO4 1
mM (14).
The 2x-Difco sporulation medium (2 DSM) contains
double strength of all the components of DSM.
Fed-batch bioreactions were performed using a solution
with the following composition: Bacto nutrient broth 120
g L-1
, KCl 1 g L-1
, MgSO4‚7H2O 7.7 g L-1
, and glucose
52.5 g L-1
, sterilized at 121 °C for 30 min. To 1 L of this
solution, 15 mL of each of the following filter sterilized
solutions were added: Ca(NO3)2 1 M, MnCl2 10 mM, and
FeSO4 1 mM.
Inocula Preparation. A 100-mL Erlenmeyer flask
containing 20 mL of DSM was inoculated with one
cryovial of MB24 from the frozen stock. The seeded
culture was incubated at 37 °C and 150 rpm on a rotary
shaker for 16 h to a final optical density of approximately
2.0. The cells were then used to inoculate the 2-L
bioreactor at an inoculum size of 1% (v/v).
Batch Bioprocess. A 2-L bioreactor (Biostat B, B.
Braun, Germany) was inoculated with 20 mL of seed
culture for a final volume of 2 L. Cultivation temperature
and aeration rate were maintained constant at 37 °C and
2 L min-1
, respectively. The dissolved oxygen concentra-
tion was maintained above the required value for each
experiment by varying the agitation rate between 100
and 200 rpm. When required, cultivation pH was con-
trolled at the desired values for each experiment with
the addition of NaOH 2 N or H2SO4 2 N. Whenever
necessary an antifoaming agent (SAG-471, 0.5 g L-1
) was
automatically added to the bioreactor.
Fed-Batch Bioprocess. A 2-L bioreactor (Biostat B,
B. Braun, Germany) was inoculated with 20 mL of seed
culture. Cultivation conditions were controlled as de-
scribed for the batch bioprocess, and the experiment was
initiated with 1.3 L of DSM containing 3.5 g L-1
of
glucose. The bioreaction was conducted in three stages:
batch culture for the first 5 h, fed-batch for the next 2 h,
and finally batch culture until the end of the run for a
total of approximately 45 h. At the middle of the
exponential growth phase, the nutrient feeding was
initiated. Approximately 300 mL of the feeding solution
was added using an exponential feeding profile obtained
from the mass balance equations as earlier indicated.
The feeding rate profile was determined using simple
mass balances based on a Monod-type kinetic model:
where X is the biomass concentration (g L-1
), X0 is the
biomass concentration at the beginning of the fed-batch
phase (g L-1
), µ is the specific growth rate (h-1
), and t is
the time length of the bioreaction .
Considering constant specific glucose consumption, a
constant glucose concentration in the cultivation medium
was achieved by feeding the concentrated glucose solution
according to the following equation:
where Q is the feeding solution flowrate, and Q0 is the
feeding solution flowrate at the beginning of the fed-batch
phase (g h-1
).
Glucose feeding solution addition to the bioreactor was
controlled using a weight control loop. A balance was
used to measure the addition flask weight, and a weight
profile over time was defined using the following equa-
tion:
where Q is the feeding solution flowrate (g h-1
), W is the
weight of the feeding solution (g), Q0 is the feeding
solution flowrate at the beginning of the fed-batch phase
(g h-1
), µ is the specific growth rate (h-1
), and t is the
time length of the bioreaction (h).
By analytical integration of the previous equation, the
following formula was obtained and used to control the
glucose feeding rate to the bioreactor:
This fed-batch strategy was designed with two main
objectives: avoid glucose limitation during the vegetative
growth phase, as this would initiate sporulation during
this stage of culture, and keep glucose concentration
below 3.5 g L-1
, as higher concentrations reduce spore
production.
X ) X0 eµ‚t
(1)
Q ) Q0 eµ‚t
(2)
∆W ) ∫0
t
Q0 eµ‚t
(3)
∆W )
Q0
µ
(eµ‚t
- 1) (4)

3. The necessary kinetic parameters as Q0 ) 16 g h-1
and
µ ) 1.04 h-1
were determined from batch experiments.
Optimization of these parameters was not performed as
the used ones where able to fulfill the above glucose
concentration criteria.
Glucose Determination. One milliliter of culture
medium was clarified by centrifugation at 14 000g for 5
min. Glucose concentration in the supernatant was
determined using a glucose dehydrogenase based kit as
described by the manufacturer (Glucose HK, Sigma
Diagnostics).
Cell Growth Determination. Optical density (OD)
measurement at 595 nm was used for cell growth
monitoring. Whenever necessary samples were diluted
to a final OD value lower than 0.5.
Determination of Titers of Vegetative Cells and
Spores. Serial dilutions of the cell suspension to be
tested were prepared and 10 µL of each dilution was
inoculated to a 96-well plate containing 180 µL of LB
media. For each dilution 10 replicates were prepared.
Plates were incubated at 37 °C for 24 h and cell
concentration was determined using the Reed and Muench
method (26). Spores were counted using the same method,
but the plates were heated to 80 °C for 20 min before
incubation.
Sporulation Efficiency and Spore Fraction. Sporu-
lation efficiency was defined as the percentage of the
vegetative cells that undergo a complete sporulation
process yielding heat-resistant spores and was calculated
as the ratio between the final spore titer and the
maximum of the vegetative cell titer reached during the
bioreaction (14). The spore fraction was defined as the
percentage of spores at a given time and was calculated
as the ratio between spore concentration and total cell
concentration (spores and vegetative cells) in each sample.
Results
Effect of pH. Under a noncontrolled pH bioreaction
a high pH variation was observed: at the beginning of
the exponential growth phase the pH decreased from 6.7
to 6.5, then a sharp increase to 8.1 was observed until
the end of the exponential growth phase, and a slow
increase to pH 9.0 occurred during the sporulation
process. In this experiment the maximum vegetative cell
concentration achieved was 2.6 × 109
cells mL-1
but the
sporulation efficiency was low (approximately 16%),
leading to a final spore concentration of 4.2 × 108
spores
mL-1
.
To determine the effect of the pH on B. subtilis growth
and sporulation, batch cultures at various pH were
performed; the results depicted in Figure 1A and B show
that when the pH was maintained at a constant value
during the whole experiment, a significant increase of
the sporulation efficiency was achieved. This suggests
that if the pH is kept constant a higher synchronizm of
sporulation is achieved. Within the pH range of 6.0-9.0,
the sporulation efficiency did not depend of the pH value,
being approximately constant at 50%, whereas a decrease
in pH to 5.0 reduced the sporulation efficiency to 6%. At
pH 7.5 the spore fraction at the end of the run was
slightly higher than in all other experiments, with an
increase in the maximum vegetative cells concentration
up to 7.5 × 109
cells mL-1
and a sporulation efficiency of
approximately 50%. This led to a final spore concentra-
tion of 3.6 × 109
spores mL-1
, which corresponds to a
9-fold increase when compared to the batch performed
without pH control.
Effect of Dissolved Oxygen Concentration. To
investigate the effect of dissolved oxygen concentration
on B. subtilis growth and sporulation, a 2-L batch
cultivation with dissolved oxygen concentration above
10%, 30%, and 50% of air saturation was carried out
(Table 1). These results indicate that this parameter did
not influence the microorganism growth, although a
slightly higher spore concentration was reached control-
ling the dissolved oxygen concentration above 30% of air
saturation.
Effect of DSM and Glucose Concentration. The
effect of DSM concentration was investigated in 2-L batch
cultivations. The results depicted in Table 1 indicate that
with the duplication of DSM concentration (2 DSM),
although the maximum vegetative cell concentration
reached approximately the same value, a significant
increase of the sporulation efficiency was achieved from
48% to 77%. This effect led to a 50% increase in the final
spore concentration, reaching 4.8 × 109
spores mL-1
.
The effect of glucose on B. subtilis growth and sporu-
lation was also evaluated in 2-L batch bioreactions by
varying the initial glucose concentration between 3.5 and
20 g L-1
(Figure 2A and B). The maximum vegetative
cell concentration increased with the increase in glucose
concentration up to 5 g L-1
, remaining constant for higher
concentrations, as shown in Figure 3A. However, a
decrease in sporulation efficiency with the increase of
glucose concentration was observed. As shown in Figure
2 up to 5 g L-1
all the glucose initially added to the
medium was consumed before the end of the exponential
growth, while for higher initial glucose concentrations,
there was still glucose consumption during the stationary
phase.
Fed-Batch Bioreactions. A fed-batch cultivation for
B. subtilis spore production was developed at 2-L biore-
action scale. The cells were initially grown, in batch
mode, in 1.3 L of DSM containing 3.5 g L-1
of glucose;
then a nutrient feed was started at the middle of the
exponential growth phase, before the complete depletion
Figure 1. Effect of pH on Bacillus subtilis growth and
sporulation under batch cultivation. A: (b) spore concentration
(spores mL-1); (O) vegetative cell concentration (cells mL-1). B:
([) sporulation efficiency (%) and (0) spore fraction at the end
of the run (%).

4. of the nutrients present in the media, thus before the
beginning of the sporulation process. This feeding strat-
egy permitted to extend the exponential growth phase
(10 h after inoculation), leading to a maximum vegetative
cell concentration of 3.6 × 1010
cells mL-1
at the end of
the growth phase (Figure 4C). During this exponential
growth phase the agitation rate was increased from 100
to approximately 1000 rpm to compensate for the oxygen
consumption rate (Figure 4A). At the end of the fed-batch
phase, glucose was completely depleted from the medium
causing a spike in the dissolved oxygen concentration,
indicating the onset of the sporulation process. Although
a higher cell lyses occurred at this stage when compared
to the batch experiments, an increase of heat resistant
spores concentration was achieved due to the higher cell
growth.
Discussion
The optimization of the cultivation parameters (pH,
dissolved oxygen concentration, and media composition)
for B. subtilis growth and sporulation was performed in
controlled batch cultivations at 2-L scale. The results
indicate that the sporulation efficiency was almost inde-
pendent of pH values within the range 6.0-9.0, better
results being achieved at pH 7.5.
The dissolved oxygen concentration within the studied
range (10-50% of the oxygen saturation concentration)
did not significantly influence the microorganism growth,
although a slightly higher spore concentration was
achieved when controlling the dissolved oxygen concen-
tration above 30% of air saturation. Recently, it was
shown that B. subtilis, previously thought to be a strict
aerobe, could also grow anaerobically (27). However,
under anaerobic conditions sporulation efficiency is highly
reduced (28). The need for aerobic conditions for efficient
sporulation is in agreement with our observation that the
concentration of dissolved oxygen is important for ef-
ficient spore production, a value above 30% being optimal.
An increase in glucose concentration up to 5 g L-1
led
to an increase of the maximum vegetative cell and spore
concentration, while initial glucose concentrations higher
than 5 g L-1
inhibited sporulation. The complete glucose
Table 1. Summary of the Batch and Fed-Batch
Cultivations of Bacillus subtilisa
PH
min
pO2
(%)
DSM
concn
glucose
concn
(g L-1)
vegetative
cells
(109 mL-1)
spores
(109 mL-1)
sporulation
efficiency
(%)
Effect of pH (Batch)
ncc 30 1x 0 2.6 0.4 15.3
5.0 30 1x 0 1.0 0.1 10.0
6.0 30 1x 0 4.5 2.2 48.8
6.5 30 1x 0 4.8 2.3 47.9
7.0 30 1x 0 6.9 3.5 50.7
7.5b 30 1x 0 7.5 3.6 48.0
8.0 30 1x 0 6.1 2.8 45.9
9.0 30 1x 0 1.7 1.0 58.8
Effect of pO2 (Batch)
7.5 10 1x 0 6.1 2.8 45.2
7.5 30 1x 0 6.6 3.5 53.2
7.5 50 1x 0 6.8 3.2 46.4
Effect of DSM (Batch)
7.5 30 1x 0 7.5 3.6 48.0
7.5 30 2x 0 6.2 4.8 77.0
Effect of Glucose (Batch)
7.5 30 1x 3.5 10.5 4.3 40.9
7.5b 30 1x 5.0 21.9 5.6 25.6
7.5 30 1x 10 19.9 4.7 23.6
7.5 30 1x 15 22.2 3.7 16.7
7.5 30 1x 20 20.0 3.4 17.0
Fed-Batch
7.5b 30 1x 3.5 36.0 7.4 20.5
a Results obtained at the end of the bioreaction. b Optimal
results. c Not controlled.
Figure 2. Effect of initial glucose concentration on Bacillus
subtilis growth and sporulation under batch cultivation. A:
optical density. B: glucose concentration (g L-1). Initial glucose
concentration (g L-1): (b) 0, ([) 3.5, (2) 5, (4) 10, (]) 15, (O)
20.
Figure 3. Effect of initial glucose concentration on Bacillus
subtilis growth and sporulation under batch cultivation. A: (2)
spore concentration (spores mL-1), (O) vegetative cell concentra-
tion (cells mL-1). B: ([) sporulation efficiency (%).

5. depletion at the end of the vegetative cell growth phase
made it possible to synchronize the sporulation by
creating the optimal conditions for sporulation to occur
(16). Spo0A, the product of the spo0A gene, is a response
regulator activated by phosphorylation in response to
several internal and external stimuli and is the master
regulator for entry into sporulation (2, 29, 30). Phospho-
rylated Spo0A stimulates its own synthesis and hence
entry into sporulation, by promoting switching of spo0A
transcription from a promoter active during vegetative
growth to a promoter active at the onset of sporulation
(29). Promoter switching is sensitive to catabolite repres-
sion (reviewed in ref 29), and thus it may be that under
our conditions, excess of glucose inhibits sporulation by
repressing transcription of the spo0A gene.
A controlled bioprocess comprising an initial and final
batch phases and an intermediate fed-batch operation
was developed to accommodate the physiology of the
bioreaction and sporulation activity. This may be related
to sporulation being controlled by catabolite repression
(31), as glucose may be involved in induction inhibition
of several enzymes at least partially responsible for
sporulation. The fed-batch strategy applied had two main
objectives: avoid glucose limitation during the vegetative
growth phase, as this would induce sporulation, and
avoid also concentrations higher than 3.5 g L-1
to achieve
increased spore production. As nutrient depletion is the
main stimulus for sporulation, it is very important to
achieve glucose depletion at the end of the exponential
growth phase. This fed-batch process conduced to an
increase in spore production up to 7.4 × 109
spores mL-1
,
which is 2.5 times higher than the highest earlier
reported value for B. subtilis spore production.
Recently, strains of B. subtilis have been isolated from
the gut of various animals and characterized in view of
their potential application as probiotics (32, 33). Being
indigenous to the gut, spores of these strains may result
in better probiosis. In preliminary experiments using new
B. subtilis strains isolated from the gut of healthy
animals the sporulation efficiency was almost 100% (data
not shown). Precise regulation of growth and sporulation
parameters are of great importance for obtaining repro-
ducible and homogeneous spore batches. To fully under-
stand the nutrient requirements for growth and sporu-
lation, determination of the carbon source mass balance
should be performed for both stages, growth and sporu-
lation. Development of a chemically defined media would
allow the optimization of a fed-batch process where
sporulation efficiency could also be increased by defining
feeding profiles to cope with the nutrient requirements
for the sporulation process.
The methodology herein described will likely be ap-
plicable to the high efficiency production of spores from
these as well as other strains of B. subtilis whenever high
yields of spores are desirable.
Acknowledgment
This work was supported by the European Commission
project “Spore Probiotics: An Alternative to Antibiotics”
(QLK-CT-2001-01729).
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Accepted for publication June 6, 2005.
BP050062Z