Enzyme and Microbial Technology 30 (2002) 747751

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Importance of specific growth rate for subtilisin expression in fed-batch

cultivation of Bacillus subtilis spoIIG mutant
Jeong Seok Oh, Byung-Gee Kim, Tai Hyun Park*
School of Chemical Engineering, Institute of Chemical Processes, Seoul National University, Kwanak-Gu Shilim-Dong San 56 1, Seoul 151744, Korea
Received 15 July 2001; received in revised form 13 October 2001; accepted 25 October 2001

Abstract
A feeding strategy was developed for controlling the specific growth rate in a Bacillus subtilis fed-batch operation, and the importance
of the specific growth rate was investigated for subtilisin expression. The ratio of the glucose and peptone concentrations in the feeding
medium was critical in controlling the specific growth rate, and the appropriate ratio was found to be 1:2. The specific growth rate was well
controlled in fed-batch operations including an exponential feeding of a 200 g/liter glucose and 400 g/liter peptone solution. Controlling the
value of the specific growth rate had a significant effect on the subtilisin expression, even though the subtilisin expression was only enhanced
after the feeding ended. This would appear to be due to cell components, such as sigma factors, produced in the growth phase that influenced
the aprE gene expression. The optimal specific growth rate for the maximum expression rate and longest production period was 0.35 h1.
2002 Elsevier Science Inc. All rights reserved.
Keywords: Bacillus subtilis spoIIG mutant; Fed-batch cultivation; Specific growth rate; Peptone concentration

1. Introduction
Bacillus subtilis is widely used as a host cell for the
production of industrial enzymes since it is safe, noninfectious for humans, does not produce endotoxin, and can
easily secrete recombinant products into a medium. The
expression of the aprE gene encoding subtilisin in B. subtilis is minimal during the growth phase, then enhanced
during the sporulation process, particularly in the initial
stage [1], and almost terminated during stage III of sporulation [2]. Accordingly, since subtilisin production is limited to the period between stage 0 and stage II of sporulation, asporogeneous mutants offer an advantage over the
wild-type in prolonging the enzyme production phase. Various asporogeneous mutants have been developed and the
spo IIG mutant of Bacillus subtilis DB104 (npr
apr)(Emr) spoIIG(Blmr)::pMK101(Cmr) has exhibited a
subtilisin yield that is about ten times higher than that from
the wild-type DB104 (npr apr)(Emr)::pMK101 (Cmr)
[3].

* Corresponding author. Tel.: 82-2-880-8020; fax: 82-2-875-9348.

E-mail address: thpark@plaza.snu.ac.kr (T. H. Park).

A fed-batch operation is one of the most popular operation modes for the production of recombinant proteins.
Various feeding strategies for fed-batch operations have
been proposed to obtain a high cell density and high concentration of recombinant proteins. These include feeding
strategies using pH-stat [4], DO-stat [5], measuring the
glucose concentration [6 8], controlling the amino acid [8],
and controlling the specific growth rate [9 13]. The specific
growth rate is one of the most important process parameters
representing the dynamic behavior of microorganisms during fermentation. Therefore, manipulating the specific
growth rate at an appropriate value in a fed-batch operation
can provide a desirable metabolic condition, resulting in
maximum productivity. This strategy has been successfully
applied to recombinant Escherichia coli fermentation [10,
13]. This article presents a strategy for a B. subtilis fedbatch operation in which the specific growth rate is controlled at a set point, and the effect of the specific growth
rate on the subtilisin expression and production period is
investigated using the spo IIG mutant. A discussion is also
included on why the specific growth rate in the growth
phase influences the aprE gene expression that is only
activated in the early sporulation phase.

0141-0229/02/$ see front matter 2002 Elsevier Science Inc. All rights reserved.
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J.S. Oh et al. / Enzyme and Microbial Technology 30 (2002) 747751

2. Materials and methods

2.1. Bacterial strain and growth conditions
Bacillus subtilis DB104 (npr apr)(Emr)spoIIG(Blmr)
::pMK101(Cmr) was used for this research. The plasmid,
pMK101, was an integrational vector combined with aprE
and pCP115, and contained a chloramphenicol resistance
marker [14]. The plasmid amplification was carried out with
30 g/mL chloramphemicol. An LB medium with 30
g/mL chloramphemicol was inoculated with 1% (v/v) of
glycerol stock and incubated for 12 h. This seed culture,
which was 5% (v/v) of the working volume of the main
culture, was then transferred to a fermentor. One liter of the
main culture medium contained 5 g glucose, 5 g peptone
(Difco), 4 g KH2PO4, 4 g K2HPO4, 7 g NaHPO4 12H2O,
1.2 g (NH4) 2SO4, 0.2 g NH4Cl, 0.01 g MnSO4 4H2O,
0.004 g CoCl2 6H2O, 0.002 g NaMoO4 6H2O, 0.002 g
ZnSO4 7H2O, 0.001 g AlCl3 6H2O, 0.001 g CuCl2 H2O,
0.0005 g H3BO4, 1 g MgSO4 7H2O, 0.04 g FeSO4 7H2O,
0.04 g CaCl2, and 0.5 g glutamic acid. The temperature and
pH were maintained at 37C and 6.8, respectively.
2.2. Fed-batch operation
The seed culture (100 ml) was transferred into 2 L of the
medium in a 7 L fermentor. No feed was supplied during the
early phase of fermentation. The feeding medium, containing 200 g/liter glucose and 400 g/liter peptone, began to be
supplied when the glucose concentartion decreased to 2
g/liter. The specific growth rate () was controlled at a
constant value based on an exponential feeding of the feeding medium. The feeding rate was determined using a mass
balance equation of the cells and the glucose, represented by
F

X 0V 0 t
e
YS F S

where is the set point of the specific growth rate and SF

is the glucose concentration in the feeding medium. The
growth yield (Y) was calculated by measuring the glucose
consumption and cell growth at various specific growth
rates and various peptone concentrations. The difference in
Y at different specific growth rates or peptone content was
not significant and Y 0.28 (g dry cell/g glucose) was used
in this formula. The calculated value of the flow rate was
converted to a signal which then operated the feeding pump
(Masterflex L/S drive, 7550 90, Cole-Parmer). During the
fed-batch operation the dissolved oxygen in the medium
was maintained at a concentration above 20% by either
regulating the agitation speed or using oxygen-enriched air.
2.3. Analytical procedures
The cell concentration was measured at 600 nm by a
spectrophotometer (GenisysTM 5, Spectronic). The dry cell

Fig. 1. Cell growth and glucose consumption with various concentrations

of peptone: (F) without peptone, (f) 1 g/liter peptone, () 2 g/liter
peptone, () 5 g/L peptone, () cell concentration, () glucose
concentration.

weight was obtained by drying washed cells at 90C for

12 h. The glucose concentration was measured using a
glucose kit (A-510, Sigma). To analyze the subtilisin activity, 0.2 mg of N-succiny-Ala-Ala-Pro-Phe-p-nitroanilide (S7388, Sigma) in 1.8 ml of a 0.1 M Tris buffer was mixed
with 0.2 ml of the sample. The increase in absorbance at 420
nm was measured for 1 min at 25C [15]. One unit of
subtilisin activity was defined by the amount of the enzyme
hydrolyzing 1 mol substrate for 1 min.

3. Results and discussion

In a fed-batch operation where the specific growth rate is
controlled by the feeding rate, determining the growthlimiting substrate in the medium is essential. In most bacterial fermentations, glucose is usually used as the growthlimiting substrate; however, peptone is widely used as an
organic nitrogen source in Bacillus fermentation. When
peptone was not added to the medium, the cell growth was
relatively slow, as shown in Fig. 1, indicating that peptone
is also a growth-limiting substrate if its concentration is too
low. Accordingly, in order to use glucose as the growthlimiting substrate, the peptone concentration should be sufficiently high relative to the glucose concentration. In other
words, the ratio of glucose and peptone in the medium is
important. Fig. 2 shows the cell growth and glucose consumption at various concentrations of glucose in the medium containing 5 g/liter peptone. In every case, the cell
growth rate decreased significantly after 4 h, thereby indicating that a certain component became growth-limiting
from this point onwards. At this time, there was still some
glucose remaining in those media containing 5 g/liter or
higher concentrations of initial glucose. This implies that
glucose was not the growth-limiting substrate under these
conditions. Therefore, it would appear the ratio of glucose
peptone should be less than 1 for glucose to be the growthlimiting substrate.

J.S. Oh et al. / Enzyme and Microbial Technology 30 (2002) 747751

749

Fig. 2. Cell growth and glucose consumption with various concentrations of glucose in medium containing 5 g/liter peptone: (F) 3.5 g/liter glucose, (f) 4.5
g/liter glucose, () 5 g/liter glucose, () 6 g/liter glucose, () 8 g/liter glucose, () cell concentration, () glucose concentration.

For a fed-batch operation, in which the specific growth

rate is controlled by the glucose concentration, the ratio of
glucose peptone concentrations needs to be determined
for the feeding solution. Fig. 3 shows the effect of the
peptone concentration in the feeding solution on the control
of the specific growth rate in a fed-batch operation when the
glucose concentration in the feeding solution was 200 g/liter. In this experiment, the set value was 0.6 h1, since this
value was considered high enough to cover the range of
specific growth rates which will be used in this study. The
specific growth rate became closer to the set point with an
increased concentration of peptone, and reached a plateau at
400 g/liter peptone. This shows that peptone as well as

Fig. 3. Effect of peptone concentration in feeding solution on control of

specific growth rate. The glucose concentration in the feeding solution was
200 g/liter. The set point of the specific growth rate was 0.6 h1.

glucose is a growth-limiting substrate if the peptone concentration is less than 400 g/liter. The resulting maximum
specific growth rate was 0.53 h1, which was still less than
the set point ( 0.6 h1), even when the peptone concentration was 400 g/liter or higher. Nonetheless, these
results suggest that the specific growth rate can be controlled at the set point, if the set point is less than 0.53 h1
and the peptone concentration is 400 g/liter or higher.
Therefore, a feeding solution containing 200 g/liter glucose
and 400 g/liter peptone was used in all the subsequent
fed-batch operations so that glucose would be the only
growth-limiting substrate.
Fed-batch fermentations were carried out at various controlled specific growth rates along with the exponential
feeding of the glucose and peptone solution. Fig. 4 shows
the cell growth in the fed-batch fermentations. The time
indicated by the X-axis is the time after the start of feeding.
The set points of the specific growth rates were 0.2, 0.3, 0.4,
and 0.5 h1 in each fermentation. Since V is not constant in
fed-batch fermentation, the specific growth rate is not (1/X)
(dX/dt) but rather (1/XV) (d(XV)/dt) (d[ln(XV)]/dt).
Therefore, the slopes (d[ln(XV)]/dt) of the cell mass curves
represent the specific growth rates. ln(XV) was found to be
linear with time for each fermentation, as shown in Fig. 4,
indicating that the specific growth rate was well controlled
at the set point in each fermentation. However, cell growth
deviated slightly from an exponential mode at the end of the
fermentation when the set point was 0.5 h1. This might be
caused by accumulated metabolites as in E. coli fermentation [10].

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J.S. Oh et al. / Enzyme and Microbial Technology 30 (2002) 747751

Fig. 4. Control of specific growth rate relative to exponential feeding: (F)

0.2 h1, (f) 0.3 h1, () 0.4 h1, () 0.5 h1. The
X axis represents the time after feeding started.

Fig. 5 shows the profiles of the cell, glucose, and subtilisin concentrations, when the specific growth rate was
controlled at 0.4 h1. During the exponential feeding, the
glucose concentration was maintained at a relatively constant level; however, glucose was accumulated at the late
phase of feeding. This might be caused by accumulated
metabolites as in E. coli fermentation [10]. Subtilisin began
to be produced after the feeding ended. The subtilisin expression per unit cell mass is shown in Fig. 6 at various
controlled specific growth rates, which also indicates an
optimal specific growth rate for maximum expression and
the longest production period. The subtilisin expression was
highest when the specific growth rate was 0.35 h1. The
production periods were 6, 16, 12, and 9 h at specific growth
rates of 0.3, 0.35, 0.4, and 0.45 h1, respectively. The

Fig. 6. Effect of specific growth rate on subtilisin expression: (F) 0.3

h1, (f) 0.35 h1, () 0.4 h1. () 0.45 h1. The X axis
represents the time after feeding ended.

longest production period was 16 h when the specific

growth rate was controlled at 0.35 h1. These production
periods were much longer than ca. 4 h obtained in a previous DO-stat for fed-batch fermentation [3]. The effect of
specific growth rate on the final concentration and productivity of subtilisin is shown in Table 1. The productivity was
maximum when specific growth rate was 0.35 h1.
Peptone is usually expensive as medium component,
therefore, its content must be minimized as possible in
practical application. The optimal specific growth rate, 0.35
h1 would be maintained even using the medium with lower
peptone content as shown in Fig. 3. If the subtilisin expression is regulated only by specific growth rate, it is more
beneficial to use the medium with lower peptone content.
However, with the lower peptone content, the available
amino acids will decrease as well during the production
phase. This may in turn affect the expression of subtilisin.

Fig. 5. Fed-batch fermentation with controlled specific growth rate. The specific growth rate was controlled at 0.4 h1: (F) cell concentration, (f) glucose
concentration, () subtilisin expression.

J.S. Oh et al. / Enzyme and Microbial Technology 30 (2002) 747751

Table 1
Effect of specific growth rate on the final concentration and productivity
of subtilisin

(h1)

Final
concentration
(103 unit L1)

Productivity
(103 unit L1 h1)

Total
operation
time (h)

0.3
0.35
0.4
0.45

1486
6190
4889
916.5

67.6
221.1
188.0
39.9

22
28
26
23

Glutamic acid and glycine are major amino acid components in peptone which might cause the effect on the subtilisin expression. Further investigation is required for the
possibility of saving peptone in this sense.
The specific growth rate dependence of subtilisin expression gives rise to an interesting question. Since subtilisin is
only produced after the feeding ends, i.e. after cell growth
stops, why does the specific growth rate in the growth phase
have an effect on subtilisin expression? The expression of
the aprE gene encoding subtilisin is induced by the sporulation process and controlled by a complex mechanism
including early sporulation gene products and sigma factors
produced in the growth phase.
Park et al. demonstrated that the aprE gene can be
transcribed by a RNA polymerase holoemzyme reconstituted from the core and A factor in vitro [16]. However,
even though the reconstituted holoenzyme is present in
abundance during the growth phase, the aprE gene is not
strongly expressed in this phase [17]. This may be explained by the notion that the promoter structure of the
aprE gene in the sporulation phase is somehow different
from its structure in the growth phase [16], such that the
holoenzyme requires other regulatory factors for efficient
transcription during the early sporulation phase [18].
These other factors may be gene products expressed in
the growth phase or early sporulation phase. Accordingly, even these factors are early sporulation gene products, their expression can still be controlled by the growth
phase gene products. For example, the phosphorylated
form of a spoOA gene product triggers entry into sporulation by activating transcription by A RNA polymerase
and H RNA polymerase already present in the growth
phase [19]. Therefore, such cell components produced in
the growth phase, including A, can influence the aprE
expression, thus the specific growth rate during the exponential feeding in the fed-batch operation can still have
an effect on the subtilisin expression even after the feeding ends. Not only the sigma factors but also the nutrient
availability during the production phase would be important to the aprE expression.

751

Acknowledgment
The authors wish to acknowledge the financial support of
the Korea Science & Engineering Foundation through the
Nano Bio-Electronic & System Center.
References
[1] Valle F, Ferrari E. Subtilisin: a redundantly temporally regulated gene
In: Smith I, Slepecky RA, Setlow P, editors. Regulation of procayotic
development. Washington, DC: ASM, 1989. p. 131 46.
[2] Prestidge, L. Gage, V. And Spizizen, J. Protease activation during the
course of sporulation in B. subtilis. J Bacteriol 1971;107:81523.
[3] Oh MK, Kim BG, Park SH. Importance of spore mutants for fedbatch and continuous fermentation of Bacillus subtilis. Biotechnol
Bioeng 1995;47:696 702.
[4] Robbins JW, Tayler KB. Optimization of Escherichia coli growth by
controlled addition of glucose. Biotechnol Bioeng 1989;34:1289 94.
[5] Mori H, Yano T, Kobayahi T, Shimizu S. High density cultivation of
biomass in fed-batch system with DO-stat. J Chem Eng Jpn 1979;12:
3139.
[6] Halst O, Hakanson H, Miyabayashi A, Mattiasson B. Monitoring of
glucose in fermentation processes using a commercial glucose analyzer. Appl Microbiol Biotechnol 1988;28:32 6.
[7] Mitzutani S, Iijima J, Morikawa M, Shimizu K, Matsubara M, Ogawa
Y, Izumi R, Matsumoto K, Kobayashi T. On-line control of glucose
concentration using an automatic glucose analyzer. J Fermet Technol
1987;65:32531.
[8] Park YS, Kai K, Iijima S, Kobayashi T. Enhanced -galactosidase
production by high cell density culture of recombinant Bacillus subtilis with glucose concentration control. Biotechnol Bioeng 1992;40:
686 96.
[9] Allen BR, Luli GW. A gradient-feed process for E. coli fermentations. Biopham 1987;11:38 41.
[10] Koo TY, Park TH. Increased production of recombinant protein by
Escheichia coli deficient in acetic acid formation. J Microbiol Biotechnol 1999;9:789 93.
[11] Paalme T, Tiisma K, Kahru A, Vanataln K, Vilu R. Glucose-limited
fed-batch cultivation of Escherichia coli with computer-controlled
fixed growth rate. Biotechnol Bioeng 1990;35:3129.
[12] Riesenberg D, Schulz V, Knorre WA, Pohl HD, Korz D, Sanders EA,
Ross A, Deckwer WD. High-cell density cultivation of Eschrichia
coli at controlled specific growth rate. J Biotechnol 1991;20:1728.
[13] Yoon SK, Kang WK, Park TH. Fed-batch operation of recombinant
Escherichia coli containing trp promoter with controlled specific
growth rate. Biotechnol Bioeng 1994;43:9959.
[14] Oh MK, Park SH, Kim BG. Development and characterization of
sporulation mutant for overexpression of recombinant protein of B.
subtilis. Kor J Biotechnol Bioeng 1994;9:16 25.
[15] Delmar EG, Largman C, Brodrick JW, Geokas MC. A sensitive new
substrate for chymotrypsin. Anal Biochem 1979;99:316 20.
[16] Park SS, Wong SL, Wang LF, Doi RH. Bacillus subtilis subtilisin
gene (aprE) is expressed from a A (43) promoter in vitro, and in
vivo. J Bacteriol 1989;171:2657 65.
[17] Doi RH. Role of ribonucleic acid RNA polymerase in gene selection
in procayotes. Bacteriol Rev 1977;41:568 94.
[18] Jan J, Valle F, Bolivar F, Merio E. Characterization of the 5subtilisin
(aprE) regulatory region from Bacillus subtilis. FEMS Microbiol Lett
2001;183:9 14.
[19] Kroos L, Zhang B, Ichikawa H, Yu YTN. Control of factor activity
during Bacillus subtilis sporulation. Mol Microbiol 1999;31:128594.