Process Biochemistry 41 (2006) 17991805

www.elsevier.com/locate/procbio

Bioreactor design for enhanced carrier-assisted surfactin

production with Bacillus subtilis
Mao-Sung Yeh a, Yu-Hong Wei b, Jo-Shu Chang a,*
a
Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan
b
Graduate School of Biotechnology and Bioinformatics, Yuan-Ze University, Chung-Li, Taiwan
Received 12 January 2006; received in revised form 16 March 2006; accepted 24 March 2006

Abstract
Bacillus subtilis ATCC 21332 was used to produce a lipopeptide-type biosurfactant (namely, surfactin) in an innovative bioreactor tailored to
solve the problems of severe foaming arising from production of the biosurfactant. To cope with the rapid foam generation, a conventional jar
fermentor was integrated with a foam collector, a cell recycler, and a surfactin precipitation unit. Meanwhile, solid carriers (e.g., activated carbon)
were added into the fermentation broth to increase cell mass concentration and surfactin yield. The proposed bioreactor allowed stable and efficient
surfactin fermentation under intensive foaming conditions without the need of adding antifoam agents. The effect of oxygen transfer rate and mass
transfer efficiency on surfactin production was also explored by employing various combinations of aeration and agitation rates. The best
combination was 1.5 vvm and 300 rpm, giving an excellent maximum production rate, overall production rate, surfactin concentration, and
surfactin yield of 190 mg L
1 h
1, 106 mg L
1 h
1, 6.45 g L
1, and 161 mg surfactin (g glucose)
1, respectively. It was also found that the
oxygen volumetric mass transfer coefficient (KLa) was highly correlated with the performance of surfactin production. The highest surfactin
productivity was achieved when the fermentation was carried out at a KLa value of 0.0132 s
1.
# 2006 Elsevier Ltd. All rights reserved.

Keywords: Surfactin; Biosurfactant; Bioreactor design; Aeration rate; Agitation rate; Volumetric oxygen transfer coefficient; Bacillus subtilis

1. Introduction or bioreactor operation and design [6,7], as well as using

Surface-active molecules produced by bacteria, yeasts, and
fungi are known as biosurfactants, which are characteristic of
high surface activity, low toxicity, high biodegradability and
ecological acceptability [13]. These favorable features make
biosurfactants potential alternatives of chemically synthesized
surfactants in a variety of applications. In fact, biosurfactants
have been widely utilized in industries like cosmetics, specialty
chemicals, food, pharmaceutics, agriculture, cleansers,
enhanced oil recovery, and bioremediation of oil-contaminated
sites [35]. However, the high production cost of biosurfactants
has been the major obstacle for commercial applications.
Consequently, numerous efforts have been made on cost
reduction of biosurfactant production. The concepts of cost-
effective biosurfactant production include enhancement of
production yield by strain improvement, medium optimization,
* Corresponding author. Tel.: +886 6 2757575x62651;
fax: +886 6 2357146/886 6 2344496.
E-mail address: changjs@mail.ncku.edu.tw (J.-S. Chang).
cheaper and renewable substrates, such as sugars, oils, alkanes,
and wastes to lower the cost of fermentation substrate [1,810].
The target biosurfactant product in this study was surfactin
produced by various strains of Bacillus subtilis [1113].
Surfactin is recognized as one of the most effective
biosurfactants available [14], but the high cost and low yield
involved in surfactin production appeared to limit its
applications [4,15]. Thus, development of strategies for
high-throughput and low-cost production of surfactin is of
urgent demand. In our previous work, an iron-enriched medium
was developed to achieve high surfactin yield from a B. subtilis
strain [16,17]. Our recent finding also shows that addition of
solid carriers (such as activated carbon) into fermentation
culture could enhance surfactin production significantly [18]. It
is also found that the supply of sufficient dissolved oxygen and
mechanical agitation played an important role in the efficiency
of surfactin production [6,7,19]. However, in a carrier-assisted
surfactin fermentation system [18], the vigorous agitation and
aeration may lead to severe foaming and damages on solid
carriers, causing unstable and inefficient fermentor operation as
well as the requirement of antifoam agent addition [6,7,1921].

1359-5113/$ see front matter # 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2006.03.027
1800 M.-S. Yeh et al. / Process Biochemistry 41 (2006) 17991805
Therefore, the agitation and aeration strategies need to be
optimized not only to meet the requirement of sufficient oxygen
and mass transfer, but also to minimize the side effects of
intensive foaming and breaking of solid carriers. Meanwhile,
the bioreactor should also be tailored to cope with the foaming
problems, avoiding massive addition of costly and probably
cell-growth-inhibiting antifoam agents.
In this work, a special design of a batch bioreactor was
proposed as a conventional fermentor was modified by
integrating with a foam collector from its gaseous outlet. In
addition, a cell recycler and a surfactin precipitator were also
connected in proper position. Meanwhile, surfactin fermenta-
tion was also carried out under different combinations of
aeration and agitation rates to identify the optimal condition for
surfactin production. The correlation between oxygen volu-
metric mass transfer coefficient (KLa) and surfactin production
was also revealed. The objective of this work was to develop
feasible, cost-effective, and commercially viable fermentation
technology for surfactin production.
2. Materials and methods
2.1. Bacterial strain and culture medium
Bacillus subtilis ATCC 21332 was used to produce surfactin. The strain was
grown on an iron-enriched minimal salt (MSI) medium [16] consisting of
glucose (40 g L
1), NH4NO3 (50 mM), Na2HPO4 (30 mM), KH2PO4 (30 mM),
CaCl2 (7 mM), MgSO47H2O (800 mM), sodium EDTA (4 mM), and FeS-
O47H2O (2.0 mM). This medium composition, including a glucose concentra-
tion of 40 g L
1, was found to be efficient in batch surfactin production
according to our previous studies [1618,22].
2.2. Solid carriers
Activated carbon (AC) carriers (China Carbon, Inc., Taipei, Taiwan) were
used to stimulate surfactin production in batch cultures [18]. The AC carrier was
of cylindrical shape with a diameter of 34 mm, a height of 9 mm, and a specific
surface area of 1200 m2 g
1. The carriers were sterilized prior to use.
2.3. Bioreactor configuration
The bioreactor was a modified version of a conventional 5-L jar fermentor
(Firstek Scientific, Taipei, Taiwan) using impellers for agitation (Fig. 1). A foam
collector with a volume of 3 L was connected to the gas effluent of the
fermentor. A cell recycler was design to pump a portion of liquid culture back
to the fermentor from the bottom of the foam collector (Fig. 1). The overflowed
Fig. 1. Schematic description of the modified bioreactor module.
foams were introduced to an acidic tank, where the concentrated surfactin
carried by the foam was precipitated at a pH of 2.0 for preliminary isolation of
surfactin product. The purity of precipitated surfactin was around 55% judged
by using a 98% pure surfactin standard (Sigma).
2.4. Carrier-assisted surfactin fermentation
The frozen stock culture of B. subtilis ATCC 21332 was revived for 24 h and
grown in MSI medium at 30 8C with 200 rpm of agitation. Cultures from late
exponential growth phase were harvested as the inoculum for surfactin fermenta-
tion experiments. The seed cultures (100 mL each) were inoculated into the
modified fermentor containing 2 L of MSI medium with the addition of 25 g L
1
of solid carriers. Batch cultures were incubated at 30 8C with an agitation rate of
200350 rpm and an aeration rate of 0.51.5 vvm (1.03.0 L min
1). The foams
generated in the culture were introduced into the foam collector. The liquid culture
accumulated on the bottom of the foam collector was completely recycled into the
fermentor with a peristaltic pump, while the overflowed foams with a typical
volume of 50500 mL were introduced into the acidic tank to allow precipitation
of surfactin. No antifoam agent was added during the course of fermentation. The
cell growth, pH, dissolved oxygen, and surfactin concentration were monitored
with respect to time.
2.5. Measurement of oxygen volumetric mass transfer coefficient
The oxygen volumetric mass transfer coefficient (KLa) representing the
oxygen transfer efficiency from gas bubble to bulk liquid is an important index
for scaling up of a fermentation process. In this work, the KLa values resulting
from designated combinations of aeration and agitation rates were measured by
the gassing-in method proposed by Trible et al. [23]. The equation used for KLa
calculation is shown below:
 
kp KL a
Cp C  1
e
KL at e
kp t (1)
kp
K L a kp
K L a
where, Cp is a predicted value of dissolved oxygen (%), C* is the equilibrium
DO concentration (%), kp is DO probe constant (s
1), and t is operating time (s).
After sparging with N2 (representing a dissolved oxygen (DO) level of zero), the
tested solution was aerated under designated aeration and agitation rates. The
DO values (Cp) was recorded as a function of time until saturation was reached
(i.e., DO = C*). The kp value was determined as the inverse of time required to
reach Cp/C* = 0.632. The KLa value was then estimated by simulating the time-
course Cp data based on Eq. (1).
2.6. Analytical methods
Cell concentration was measured by detecting optical density of each
sample at 600 nm after serial dilution (OD600). The surfactin concentration
was determined by reverse phase HPLC (LC-10AT, Shimadzu, Tokyo, Japan)
equipped with a Merck C18 column (5 mm, Merck, Germany) using a UV
detector. Detailed procedures for HPLC measurement were described in our
recent work [5]. A commercially available surfactin compound (98% pure;
Sigma, USA) was used as the authentic standard.
3. Results and discussion
3.1. Time-course profile of batch surfactin fermentation
using the modified bioreactor
The modified bioreactor module depicted in Fig. 1 was
tailored for an antifoam-free surfactin fermentation. A typical
time-course profile for batch surfactin fermentation is shown in
Fig. 2. With an initial glucose concentration of 40 g L
1 and an
agitation and aeration rate of 300 rpm and 1.5 vvm, respec-
tively, the surfactin concentration produced along with the cell
growth (Fig. 2), indicating that surfactin was essentially a
 M.-S. Yeh et al. / Process Biochemistry 41 (2006) 17991805 1801

cell growth (2060 h). The DO level rapidly increased back to

Fig. 2. Time-course profiles of cell growth, dissolved oxygen level, pH,
residual glucose concentration, and surfactin production during batch fermen-
tation of Bacillus subtilis ATCC 21332 under an aeration rate of 1.5 vvm and an
agitation rate of 300 rpm.
growth associated product [19,20,24]. According to the growth
associated kinetics, qp = am (where, qp is the specific surfactin
production rate, mg g dry cell
1 h
1; m is specific growth rate,
h
1; a is the cell-based yield coefficient, mg g dry cell
1), the a
value was calculated as 735 mg g dry cell
1 (r2 = 0.942) using
the data indicated in Fig. 2. Surfactin production reached
maximum (6.45 g L
1) after cultivation for nearly 60 h, at
which cell growth creased. Fig. 2 also shows that the dissolved
oxygen level (DO) drastically dropped from 70 to 2% at early
cell growth and maintained at nearly zero during exponential
nearly 70% when cell growth stopped (at about 60 h). Surfactin
production started when the culture pH sharply decreased from
6.7 to 6.0, while the pH gradually increased to nearly 6.5 at the
end of fermentation. The initial pH drop at the onset of surfactin
production might be due to formation of acidic metabolites
during surfactin fermentation as mentioned in our recent work
[22]. Davis et al. [7] applied a similar foam-collecting device
during fermentative production of surfactin. However, since
their system did not include a cell-recycling device, the
fermentation broth was rapidly carried away by the overflowing
foams and thus the fermentation needed to be terminated in a
very short period of time (1136 h if agitation rate > 200 rpm).
This limitation resulted in a lower overall production of
surfactin [7,19,25]. In contrast, using the bioreactor designed
from this work, with few exceptions, over 90% of culture broth
was remained in the bioreactor at the end of fermentation
(Table 1), allowing a near complete utilization of carbon source
(e.g., glucose) and an elevated production of surfactin (Table 1,
Fig. 2). Using a larger foam collection vessel or a faster cell
recycle rate may also be helpful to avoid the loss of broth due to
rapid foam formation. Moreover, most of surfactin-producing
system utilized a variety of antifoam agents to suppress the
intensive foaming [7,19,20,24,26]. However, the antifoam
agents are either costly, harmful to cell growth, or cause
problems with downstream product separation. To avoid severe
foaming problems arising from vigorous air sparging, Lee and
Kim [21] utilized alternative oxygenation methods, such as
addition of H2O2 or other oxygen releasing compounds in
replacement of air aeration. Although their approach seemed to
facilitate surfactin production, the operational expense was still
relatively high. Hence, the bioreactor module designed in this
work appeared to possess a great benefit of enabling stable
operation under severe foaming conditions without the need of
adding any antifoam agents or oxygen-releasing compounds.
Under the optimal operating condition (agitation rate =
300 rpm; aeration rate = 1.5 vvm), the maximum surfactin

Table 1
Surfactin producing performance in batch fermentation of Bacillus subtilis ATCC 21332 under different combinations of aeration and agitation rate
Aeration rate Agitation KLa (s
1) Maximum Overall Maximum surfactin Surfactin yield Residual culture
rate (rpm) production production concentration (mg per volume at the end
rate (mg L
1 h
1) rate (mg L
1 h
1) (mg L
1) g glucose) of fermentation (L)*
vvm L min
1
1.5 3 200 0.0086 95 61 2658 66 1.75
1.5 3 250 0.0105 100 76 4436 111 1.72
1.5 3 300 0.0132 190 106 6450 161 1.51
1.5 3 350 0.0139 81 45 1010 25 0.95
1 2 200 0.0061 35 17 1368 34 1.80
1 2 250 0.0074 114 50 2875 72 1.80
1 2 300 0.0093 128 90 3831 96 1.70
1 2 350 0.0105 70 54 1993 50 1.45
0.5 1 200 0.0036 31 20 1385 35 1.92
0.5 1 250 0.0052 59 33 2233 56 1.90
0.5 1 300 0.0063 76 62 2806 70 1.85
0.5 1 350 0.0082 77 56 2299 57 1.70
All the values presented are mean value of duplicates (standard deviations were essentially within 211%).
*
Initial culture volume = 2 L.
1802 M.-S. Yeh et al. / Process Biochemistry 41 (2006) 17991805
production rate (190 mg L
1 h
1), overall production rate
(106 mg L
1 h
1), and the maximum surfactin concentration
(6450 mg L
1) were obtained (Table 1).
3.2. Effect of aeration and agitation rates on surfactin
production rate
The influence of aeration and agitation rates on maximum
vmax and overall voverall surfactin production rate was
investigated in experiments conducted under a series of
combinations of aeration and agitation rates. The vmax value
was estimated from the steepest slope of the time-course
surfactin production profile (such as the one shown in Fig. 2),
representing the fastest production rate of surfactin during the
course of fermentation. The voverall value took into account the
lag time for surfactin production and was calculated from
dividing maximum surfactin production by the time required to
Fig. 3. Effect of agitation and aeration rate on (a) maximum surfactin production rate, (b) overall surfactin production rate, and (c) maximum surfactin concentration
during batch fermentation of Bacillus subtilis ATCC 21332.
achieve that maximum value. Thus, voverall is virtually a
measure of the overall surfactin production activity of that
batch fermentation process. As indicated in Fig. 3a and b, there
was a general trend that both vmax and voverall tended to increase
with increasing the aeration rate from 0.5 to 1.5 vvm (i.e., 1.0
3.0 L min
1) as well as with increasing the agitation rate from
200 to 300 rpm. Apparently, sufficient oxygen supply and mass
transfer efficiency played major roles in the kinetics of surfactin
production. However, further increase in agitation rate to
350 rpm did not enhance the surfactin production rates (Fig. 3).
On the contrary, both vmax and voverall dropped significantly
when agitation rate getting too high (i.e. 350 rpm). This
phenomenon can be attributed to the vigorous and persistent
foaming arising from the high agitation rate, causing ineffective
cell recycling and thus poor surfactin production efficiency.
This is confirmed by a lower residual culture volume at the end
of fermentation (Table 1). Similarly, further increasing aeration
 M.-S. Yeh et al. / Process Biochemistry 41 (2006) 17991805 1803

rate to 2.0 vvm also caused severe foaming and unstable
operation (data not shown). Hence, the aeration rate used in this
study was within the range of 0.51.5 vvm. Fig. 3 shows that
operation at an agitation rate of 300 rpm and an aeration rate of
1.5 vvm gave the best vmax and voverall of 190 and
106 mg L
1 h
1, respectively (Table 1). The maximum
surfactin production rate of 190 mg L
1 h
1 is higher than
most of the values reported in the literature [6,19,26], indicating
that the bioreactor strategies used in this study was technically
feasible.
3.3. Effect of aeration and agitation rates on surfactin
yield and concentration
In addition to the production rate, the surfactin yield and
final surfactin concentration are also crucial for assessing the
performance of surfactin production, as these parameters are
highly correlated with the production cost and the efficiency of
downstream processing. Here, the surfactin production yield
(Ys) was defined as the amount of surfactin produced per the
amount of carbon substrate (glucose) introduced. As shown in
Table 1 and Fig. 3c, except for the runs with an agitation rate of
350 rpm, the surfactin yield (Ys) and surfactin concentration
(Cmax) increased with the increase in both agitation rate and
aeration rate. However, like what occurred to surfactin
production rate, operation at an agitation rate of 350 rpm also
gave poor Cmax and Ys no matter what aeration rates were used.
This is also due to the problems with rapid and severe foaming
at 350 rpm, resulting in poor cell recycle efficiency and
operational instability. The highest Cmax and Ys occurred at
300 rpm agitation and 1.5 vvm aeration, achieving a value of
6.45 g L
1 and 161 mg surfactin (g glucose)
1, respectively.
These values are also higher than those obtained from
comparable studies [6,19,26], suggesting the potential of
commercial applications of this surfactin-producing system.
transfer coefficient (KLa), on surfactin productivity. Most
importantly, KLa is often used as a pivotal design parameter for
scale-up of the bioprocess [27] and thereby its optimal value
needs to be determined. The KLa value can be manipulated by
the rate of aeration and agitation. Table 1 shows that that
increasing aeration rate from 0.5 to 1.5 vvm led to increases in
KLa regardless of the agitation rate used. Meanwhile, at a fixed
aeration rate, KLa also increased gradually when the agitation
rate was increased from 200 to 350 rpm. In particular, the KLa
enhancement by increasing agitation rate became more
significant when the aeration rate was small (e.g., 0.5 vvm).
The results indicated in Table 1 appeared to provide valuable
information regarding how the oxygen transfer efficiency in the
fermentor was quantitatively correlated with the two operation
parameters (i.e., aeration and agitation rate).
From the perspective of bioreactor design, it would be of great
importance to understand how the efficiency of oxygen transfer
(represented by KLa value) affected the performance of surfactin
production. In Fig. 4a, all the KLa values resulting from different
combinations of agitation and aeration rates were plotted against
the corresponding surfactin production rate. It shows a general
trend that surfactin production rate essentially increased with
increasing KLa values, except for the two runs using a high
agitation rate of 350 rpm combining with 1.01.5 vvm of

3.4. Effect of oxygen volumetric mass transfer coefficient

on surfactin production

In general, aerobic bacteria, like B. subtilis, need sufficient

oxygen supply for cell growth and metabolite formation. As the
oxygen supply to the culture often acted as a limiting factor for
cell growth and production of target products [6,19,21], the
oxygen transfer rate should be properly adjusted to match the
oxygen consumption rate of cells to avoid poor cell growth and
inefficient metabolic function for the formation of target
product. However, previous finding in the literature pointed out
that excessive oxygen supply may not be favorable to surfactin
production [6,19], because the high rate of aeration and
agitation under O2-sufficient conditions promoted cell growth
and foam production in a short time. Without addition of a large
amount of antifoam agent, the rapid foam production resulted in
overflow of the culture broth and a short fermentation time due
to a swift decrease in culture volume. This appeared to lead to a
low level of biomass yield and biosurfactant production. Fig. 4. Effect of KLa on (a) maximum surfactin production rate and (b)
Therefore, it is of great interest to identify the effect of oxygen maximum surfactin concentration during batch fermentation of Bacillus subtilis
transfer efficiency, represented by oxygen volumetric mass ATCC 21332.
1804 M.-S. Yeh et al. / Process Biochemistry 41 (2006) 17991805
Table 2
Comparison of surfactin producing performance of this work with comparable studies
Foaming control strategy Fermentation time (h)
Foam collection 2540
Antifoam addition 3060
None 72
Foam collection 1150
Foam collection and cell recycling 4872
aeration. The reason for the two exceptions can also be ascribed
to the problems associated with severe and rapid foaming (as
mentioned earlier) that caused a decrease in surfactin production
rate despite a sufficient and efficient oxygen transfer. Likewise,
the effect of KLa on surfactin concentration (or yield) also
followed a similar trend to that on production rate (Fig. 4b and
Table 1). Therefore, an efficient and stable surfactin-producing
bioreactor operation should not only consider the oxygen transfer
efficiency, but also needs to control the foaming speed to avoid
the problem arising from overwhelming foam formation.
Nevertheless, a controllable foaming is desirable for surfactin
fermentation because surfactin product preferentially distributed
toward the foam fraction [6,7,19,24] and then was concentrated
in the foams. This would be beneficial to downstream isolation
and purification of surfactin products by collecting the foams.
Also, the majority of surfactin products could be continuously
removed from culture broth by the foams, leading to a condition
that is kinetically favorable for surfactin production and is free of
product inhibition. However, severe foaming could lead to a
harsh challenge in operation stability and may result in the loss of
extracellular signal peptides triggering the expression of gene
operons responsible for metabolism of surfactin production [28],
thereby being unfavorable from the aspect of genetic regulation.
In this study, although no antifoam agents were added, the
foaming situation was virtually controllable in most of the
operation conditions, except for some of the runs with 350 rpm
agitation. The best surfactin production performance occurred
when KLa = 0.0132 s
1, resulting from an optimal combination
of agitation (300 rpm) and aeration (1.5 vvm) (Table 1).
4. Conclusions
An innovative bioreactor developed in this work was able to
handle the foaming problem associated with production of
surfactin without the addition of any antifoam agent. The
performance of surfactin production was shown to strongly
correlate with oxygen volumetric mass transfer efficiency (KLa)
under various combinations of agitation and aeration rate.
Although the KLa value increased with increasing agitation and
aeration rates, the best surfactin producing performance was
obtained at a KLa of 0.132 s
1, resulting from an aeration rate
of 1.5 vvm and an agitation rate of 300 rpm. Under this optimal
condition, the system achieved the highest maximum surfactin
production rate vmax , concentration, and yield of
190 mg L
1 h
1 (106 mg L
1 h
1 for the best voverall ),
6.45 g L
1, and 161 mg surfactin (g glucose)
1, respectively.
Carbon source Maximum surfactin Reference
concentration (mg L
1)
Glucose 4500 [19]
Glucose 439 [6]
Potato starch 400 [29]
Glucose 4580 [7]
Glucose 6450 This study
Applying a higher agitation rate of 350 rpm with 1.5 vvm
aeration could further lift the KLa value to 0.139 s
1, but caused
a considerable decrease in surfactin production rate and
production yield, due primarily to the operational instability
arising from severe and rapid foam formation. The surfactin
producing performance obtained from this work is highly
competitive when compared with the results from relevant
studies in the literature (Table 2). Meanwhile, the optimal value
of a key scale-up parameter, namely KLa, was also identified
successfully. Thus, the proposed surfactin producing biopro-
cess seems to possess the potential for commercial applications.
Moreover, as batch cultures were the main focus of this study,
more favorable bioreactor modes (such as fed-batch operation)
may need to be identified in the future to further improve the
surfactin production rate and yield.
Acknowledgements
The authors gratefully acknowledge the financial support by
Taiwans Ministry of Economical Affairs (Grant nos. 91-EC-
17-A-10-S1-0013, 92-EC-17-A-10-S1-0013, and 93-EC-17-A-
10-S1-0013).
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